Causative agents of enteric infections: escherichiosis, klebsiellosis, typhoid fever, paratyphoid, salmonellosis. Enterobacteria – causative agents of oral cavity diseases

II. Microbiological diagnosis and prophylaxis of  bacterial dysentery (shigellosis)

III. Microbiological diagnosis and prophylaxis of cholera. Campylobacteriosis and aeromonas infection. Helycobacter

pylory infection – causative agent of the gastroduodenal diseases.

 

ENTEROBACTERIACEAE

The Enterobacteriaceae contain gram negative rods which, if motile, are peritrichously flagellated.

 

Because members of this family are morphologically and metabolically similar, much effort has been expended to develop techniques for their rapid identification. In general, biochemical properties are used to define a genus, and further subdivision frequently is based on sugar fermentation andantigenic differences. Yet, many paradoxes exist, for example, more than 2600 species of Salmonella have beennamed, whereas the equally complex species Escherichiacoli is divided into more than 1000 serotypes. Over the yearse, many taxonomists with different ideas have been involved in the classification of these bacteria, and disagreement still exists concerning family and genericnames. Table 1 gives an outline of the taxonomic scheme proposed by Ewing and Martin for the Enterobacteriaceae, compared with that proposed in Bergey’s Manual of Systematic Bacteriology. As shown, Bergey’s has elimimated all tribes in the taxonomic division of this large family. Both schemes are used in various diagnostic laboratories, but this chapter adheres more closely to the Bergey classification.

Table 1

Classification of the Enterobacteriaceae

 

Biochemical Properties Used for Classification

Early taxonomic schemes relied heavily on the organism’s ability to ferment lactose, and numerous differential andselective media have been devised to allow one to recognize a lactose fermenting colony on a solid medium. The effectiveness of such differential media is based on the fact that organisms fermenting the lactose form acid, whereas nonlactose fermenters use the peptones present and donot form acids m these media. The incorporation of anacid base indicator into the agar medium thus causes acolor change around a lactose fermenting colony. Thus has been a valuable technique for selectingthe major nonlactose fermenting pathogens that causesalmonellosis or shigellosis, under special conditions, however, many lactose fermenters also cause a variety of infectious diseases.

Furthermore, many enterics ferment lactose onlyslowly, requiring several days before sufficient acid isformed to change the indicator. They all synthesize beta galactosidase, (the enzyme that splits lactose into glucoseand galactose) but lack the specific permease necessary for the transport of lactose into the cell One can easilydetermine whether an organism is a slow lactose or nonlactose fermenter by mixing a loopful of bacteria with orthonitrophenol beta galactoside (ONPG) dissolved ina detergent. The linkage of the galactose in ONPG is thesame as its linkage m lactose, inasmuch as the ONPG canenter the cell in the absence of a permease, an organism possessing beta galactosidase will hydrolyze ONPG to yield galactose and the bright yellow compound, orthonitrophenol. Thus, only the absence of a specific lactose permease differentiates the slow lactose fermenters fromthe normal lactose fermenters.

In addition, a number of selective media have been devised that contain bile salts, dyes such as brilliant greenand methylene blue, and chemicals such as selenite and bismuth. The incorporation of such compounds into thegrowth of medium has allowed for the selective growth of the enterics while inhibiting the growth of gram positive organisms.

Some other biochemical properties used to classify members of the Enterobacteriaceae include the ability to form H₂S; decarboxylate the ammo acids lysine, ornithine,or phenylalanine, hydrolyze urea into CO₂ and NH₃, form indole from tryptophan; grow with citrate as a sole source of carbon; liquefy gelatin; and ferment a large variety of sugars.

 

Serologic Properties Used for Classification

No other group of organisms has been so extensively classified on the basis of cell surface antigens as the Enterobacteriaceae. These antigens can be divided into threetypes, designated O, K, and H antigens.

O ANTIGENS. All gram-negative bacteria possess a lipopolysaccharide (LPS) as a component of their outer membrane. This toxic LPS (also called endotoxin) is composed of three regions, lipid A, core, and arepeating sequence of carbohydrates called the O antigen. Based on different sugars, alpha- or beta-glycosidic linkages, and the presence or absence of substituted acetyl groups, Escherichia coil can be shown to possess at least 173 different 0 antigens, and 64 have beendescribed in the genus Salmonella.

Sometimes, after continuous laboratory growth,strains will, through mutation, lose the ability to synthesize or attach this oligosaccharie O antigen to the coreregion of the LPS. This loss results in a change from a smooth colony to a rough colony type, and it is referred to as an S to R transformation Interestingly, the R mutants have lost the ability to produce disease.

K ANTIGENS. K antigens exist as capsule or envelope polysaccharidesand cover the O antigens when present, inhibiting agglutinarion by specific 0 antiserum. Most K antigens can be removed by boiling the organisms in water.

H ANTIGENS. Only organisms that are motile possess H antigens because these determinants are in the proteins that makeup the flagella. However, to complicate matters, members of the genus Salmonella alternate back and forth to formdifferent H antigens. The more specific antigens are called phase 1 antigens and are designated by lower-case letters (a, b, c, and so on), whereas the less-specific phase 2 H antigens  are giveumbers. The mechanism of this phase variation reveals an interesting way in which a cell canregulate the expression of its genes. In short, Salmonella possesses two genes. H1 encoding for phase 1 flagellar antigens, and H2 encoding for phase 2 flagellar antigens.The transcription of H2 results in the co-ordinate expression of gene rhl, which codes for a repressor that preventsthe expression of H 1. About every 10³ to 10^s generations, a 900-base-pair region, containing the promoter for the H2 gene, undergoes a site-specific inversion, stopping the transcription of both H2 and rhl. In the absence of the rhl gene product, the H1 gene is then transcribed until the 900-base pair region in the H2 promoter is again inverted, resulting in the expression ofH2 and rhl.

After obtaining the serologic data, an antigenic formula can be written, such as E.coli  O111:K-58:H6, meaning this E. coli possesses O antigen 111, K antigen 58, and H antigen 6. The formula Salmonella togo 4,12:1,w:1,6 indicates this serotype of Salmonella possesses O antigens 4 and 12, phase 1 H antigens  1 and w, and phase 2 H antigens  1 and 6.

 

Escherichia coli. The organism was isolated from faeces in 1885 by T. Escherich.  E. coli is a common inhabitant of the large intestine of humans and mammals. It is also found in the guts of birds, reptiles, amphibians, and insects. The bacteria are excreted in great numbers with the faeces and are always present in the external environment (soil, water, foodstuffs, and other objects).

Morphology. E coli are straight rods measuring 0.4-0.7 in breadth and 1-3 in length. They occur as individual organisms or in pairs and are marked by polymorphism. There are motile and non-motile types. The G+C content in DNA is 50-51 per cent. The cell surface has pili on which certain phages are adsorbed. The microcapsule is not always clearly defined.

Cultivation. E. coli is a facultative anaerobe. The optimum temperature for growth is 30-37 °C and the optimum pH value of medium up 7.2-7.5. The organism also grows readily on ordinary media at room temperature and at 10 and 45 °C, growth becomes visible in the first two days. E. coli from cold-blooded animals grows at 22-37° C but not at 42-43° C.

On meat-peptone agar E. coli produces slightly convex semitransparent, greyish colonies, and in meat broth it forms diffuse turbidity and a precipitate. The organism produces colonies which are red on Ploskirev’s medium, red with a metallic hue on Endo’s medium, and dark-blue on Levin’s medium.

Fermentative properties. E. coli does not liquefy gelatin. It produces indole and hydrogen sulphide, and reduces nitrates to nitrites; ferments glucose, levulose, lactose, maltose, mannitol, arabinose, galactose, xylose, rhamnose, and occasionally saccharose, raffinose, dulcitol, salycin, and glycerin, with acid and gas formation. It also coagulates milk. There are varieties of the bacteria which ferment saccharose, do not produce indole, have no flagella, and do not ferment lactose.

 

Endo’s medium

 

Toxin production. Certain strains of E. coli are conditionally pathogenic They contain a gluco-lipo-protein complex with which their toxic, antigenic, and immunogenic properties are associated. Some strains possess haemolytic properties (O124 and others) determined by plasmids. Pathogenic cultures possess endotoxins and thermolabile neurotropic exotoxins. The latter accumulate in broth cultures on the second-fourth day of cultivation, while the endotoxins appear only after the twentieth day. Haemotoxins and pyrogenic substances, proteinases, deoxyribonucleases, urease, phosphatase, hyaluronidase, amino acid decarboxylases have been obtained from pathogenic strains.

Antigenic structure. The antigenic structure of E. coli is characterized by variability and marked individuality. Along with the H- and O-antigens, the presence of other antigens has been shown m some strains, i.e. the surface somatic (membranous, capsular) K-antigens which contain the thermolabile L- and B-antigens and the thermostable A- and M-antigens.

Each antigen group in its turn is composed of a number of antigens designated by Arabic numbers, e.g. the O-group has 173 antigens, the K-subgroup 90, the H-subgroup 50, etc. On the basis of antigenic structure an antigenic formula is derived which fully reflects the antigenic properties of the strain For example, one of the most widely spread serotypes is designated 0111 : K58 : H2. Under the effect of transformation, lysogenic conversion, transduction, and conjugation E. coli may change its antigenic properties.

Numerous varieties of the organism are produced on cultivation under artificial conditions. Such varieties are not only of theoretical interest, but also of great practical importance in laboratory diagnosis of enteric infections.

Classification. Genus Escherichia includes one E. coli species consisting of several biotypes and serotypes. They are differentiated according to cultural, biochemical, and serological properties. The genus Escherichia includes E. coli, E. freundi, E. intermedia, and others. E. coli comprises several varieties which are differentiated by their cultural and biochemical properties. F. Kauffmann has detected 25 O-groups responsible for various diseases in humans.

About 50 phage variants have been revealed among E. coli organisms. They are used in laboratory diagnosis as confirmatory characteristics of the isolated serotypes.

Resistance. E. coli survives in the external environment for months. It is more resistant to physical and chemical factors of the external environment than the typhoid and dysentery bacteria. E. coli is killed comparatively rapidly by all methods and preparations used for disinfection. At 55° C the organism perishes in 1 hour, and at 60° C in 15 minutes. E. coli is sensitive to brilliant green.

E. coli is used as a test microbe in the assay of disinfectants and methods of disinfection and also in titration of certain antibiotics.

Pathogenicity for animals. The pathogenic serovars of E. coli cause severe infections in calf sucklings giving rise to an extremely high mortality. A parenteral injection of the culture into rabbits, guinea pigs, and white mice results in a fatal toxico-septical condition.

Pathogenesis and diseases in man. Definite E. coli serogroups are capable of causing various acute intestinal diseases in humans: (1) the causative agents of colienteritis in children are O-groups-25, -26, -44,   -55, -86, -91, -111, -114, -119, -125, -126, -127, -128, -141, -146, and others (they cause diseases in infants of the first months of life and in older infants); (2) the causative agents of dysentery-like diseases are E. coli of the O-groups-23, -32, -115, -124, -136, -143, -144, -151, and others; (3) the causative agents of cholera-like diarrhoea are the O-groups-6, -15, -78, -148, and others, they produce thermolabile and thermoresistant enterotoxins.

Colienteritis begins acutely with high temperature (38-39 °C), and frequently with severe meteorism, vomiting, diarrhoea, and general toxicosis. The disease usually occurs in infants of the first year of life.

The infection is acquired from sick children or carriers. Pathogenic E. coli serovars are found on various objects. It is assumed that colienteritis is transmitted not only by the normal route for enteric infections but also through the respiratory tract by the droplets and dust.

The pathogenesis of colienteritis depends entirely on the organism’s condition. In prematurely born infants and in infants during the first months of life the bactericidal activity of blood is considerably lower in respect to the pathogenic E. coli serovars in comparison to the nonpathogenic types. The reactivity of the child’s body at the time of infection plays an important role in the mechanism of resistance to the pathogenic strains. The pathological process develops mainly in the small intestine. Most probably, the mucous membrane of the small intestine in particular is exposed to the action of thermolabile toxic substances. Serovars O-124, O-151 and others cause diseases which are similar to dysentery.

E. coli may cause colibacillosis in adults (peritonitis, meningitis, enteritis, toxinfections, cystitis, pyelitis, pyelonephntis, angiocholitis, salpingooophontis, appendicitis, otitis, puerperal sepsis, etc.). Over-strain, exhaustion, and conditions following infectious diseases facilitate the onset of various E. coli infections. In a number of cases the organism is responsible for food poisoning.

Immunity. In individuals who had suffered from diseases caused by pathogenic E. coli serovars, cross immunity is not produced owing to which re-infection may occur. Over 85 per cent of E. coli strains contain inhibiting substances, colicins, marked by antagonistic properties in relation to pathogenic microbes of the enteric group, they are used as therapeutic and preventive agents, e.g. colibacterin (E. coli M 17, etc.).

Besides this, E colt as well as other common inhabitants of the intestine are capable of synthesizing various vitamins (K₂, E, and group B) which are indispensable to the human organism. The ability of various E. coli serovars to suppress the growth of Mycobacterium tuberculosis has also been observed. The suppression of E. coli and other members of the biocoenosis may result in a chronic disease known as dysbacteriosis.

Laboratory diagnosis. The patients’ faeces, throat and nasal discharges, material obtained at autopsy (blood, bile, liver, spleen, lungs, contents of the small and large intestine, pus), water, foodstuffs, and samples of washings from objects and hands of staff of maternity hospitals, hospitals, and dairy kitchens are all used for laboratory examination during colienteritis. If possible, faecal material should be seeded immediately after it has been collected. The throat and nasal discharges are collected with a sterile swab. Specimens of organs obtained at autopsy are placed in separate sterile jars.

The tested material is inoculated onto solid nutrient media (Endo’s, Levin’s) and, simultaneously, onto Ploskirev’s media and bismuth-sulphite agar for isolation of bacteria of the typhoid-paratyphoid and dysentery group. BIood is first inoculated into broth and then subcultured on solid media when development of a septic process is suspected. Pus is collected for examination in suppurative lesions. It is placed into a dry sterile vessel and then inoculated onto the differential media of Endo or Levin. The pure culture isolate is identified by its morphological, cultural, biochemical, serological, and biological properties.

T

he corresponding 0-group to which an enteropathogenic-serovars belong is determined by means of the agglutination reaction after the K-antigen of the culture that is being studied has been destroyed by boiling.

Besides, the immunofluorescence method employing type specific labelled sera is also used. It yields a preliminary answer in one to two hours.

In serological diagnosis of colienteritis beginning with the third to fifth day of the disease the indirect haemagglutination reaction is used which excels the agglutination reaction in sensitivity. It is positive when the antibody titre grows in the course of the diseased.

Treatment. Patients with colienteritis are prescribed antibiotics (tetracycline with vitamins C, B₁ and B₂) and biopreparations (coli autovaccine, coli bacteriophage, colicin, bacterin, lactobacterin, bificol, bifidumbacterin). Physiological solutions with glucose are injected for controlling toxicosis.

Prophylaxis. To prevent diseases caused by pathogenic serovars of E. coli, special attention is given to early identification of individuals suffering from colienteritis, and also to their hospitalization and effective treatment. Regular examination of personnel is necessary in children’s institutions as well as of mothers whose children are suffering from dyspepsia. Considerable importance is assigned to observation of sanitary regulations in children’s institutions, infant-feeding centres, maternity hospitals, and children’s nurseries. Protection of water and foodstuff’s from contamination with faeces, the control of flies, and gradual improvement of standards of hygiene of the population are also particularly important.

Sanitary significance of E. coli. This organism is widely spread iature. It occurs in soil, water, foodstuff’s, and on various objects. For this reason E. coli serves as an indicator of faecal contamination of the external environment.

Detection of E. coli is of great importance in estimating the sanitary index of faecal contamination of water, foodstuff’s, soil, beverages, objects, and hand-washings. The degree of contamination of water, soil and foodstuff’s is determined by the coli titre or coli index (these terms have been discussed in the chapter concerning the spread of microbes iature). Faecal contamination of articles of use is estimated by qualitative determination of the presence of E. coli.

Additional materials

Pathogenicity of Escherichia coli. Although E. coli is part of the normal flora of the intestinaltract, it is also the most common gram-negative pathogen responsible for nosocomially acquired septic shock, meningitis ieonates, cystitis and pyelonephritis in women, and for several distinct forms of diarrheal disease and dysentery affecting populations throughout the world. Strains of E coli capable of causing such diseases possess one or more virulence factors that are not found in E. coli strains comprising the normal flora. Such virulence factorscan be characterized as follows, the capacity to adhere to specific mammalian cells; the ability to invade and grow intracellularly in intestinal epithelial cells; the secretion of one or more enterotoxins that cause fluid loss, resulting mdiarrhea; the formation of a cytotoxin that blocks protein synthesis, causing a hemorrhagic colitis; and the possession of an antiphagocytic capsule that is responsible, at least in part, for the bacteremia and meningitis caused by E. coli. In addition, the ability to obtain iron from transferrin or lactoferrin by the synthesis of iron-binding siderophores markedly enhances the virulence of such strains through their ability to grow in host tissues. No one strain of E. coli possesses all of these properties but, as is discussedlater, all pathogenic strains must have one or more virulence factors to produce disease.

Diarrheal Diseases. It is estimated that during the American Revolutionary War there were more deaths from diarrhea than from English bullets, and during the American War between the states, over 25% of all deaths were because of diarrheaand dysentery. Diarrhea kills more people worldwide than AIDS and cancer, with about five million diarrheal deaths occurring annually primarily because of dehydrationMost of these occur in neonates and young children, anda large number are caused by pathogenic E. coli. Thedisease in adults, known by many names such as traveller’sdiarrhea or Montezuma’s revenge, may vary from a milddisease with several days of loose stools to a severe andfatal cholera-like disease. Such life-threatening E. coli infections occur throughout the world but are most com-mon in developing nations.

The virulence factors responsible for diarrheal diseaseare frequently encoded in plasmids, which may be spreadfrom one strain to another either through transduction^: or by recombination. As a result, various combinations of virulence factors have occurred, which has been used to place the diarrhea-producing strains of E. coli into variousgroups based on the mechanism of disease production

Enterotoxigenic Escherichia coli. Enterotoxin-producing E coli, called enterotoxigenic E.coli (ETEC), produce one or both of two different toxins – a heat labile toxin called LT and a heat-stable toxin called ST. The genetic ability to produce both LT and ST is controlled by DNA residing in transmissible plasmids called ent plasmids. Both genes have been cloned, and the ST gene has been shown to possess the characteristics of a transposon.

HEAT-LABILE TOXIN. The heat-labile toxin LT, which is destroyed by heating at 65 °C for 30 minutes, has been extensively purified, and its mode of action is identical to that described for cholera toxin (CT). LT has a molecula rweight of about 86,000 daltons and is composed of twosubunits, A and B Subunit A consists of one moleculeof A_i (24,000 daltons) and one molecule of A₂ (5000daltons) linked by a disulfide bridge. Each A unit is joinednoncovalently to five B subunits.

Like CT, LT causes diarrhea by stimulating the activity of a membrane-bound adenylate cyclase.This results in the conversion of ATP to cyclic AMP (cAMP):  ATP ® cAMP + PPi

Minute amounts of cAMIP induce the active secretion of Cl^– and inhibit the absorption of NaCI, creating an electrolyte imbalance across the intestinal mucosa, resulting in the loss of copious quantities of fluid and elec-trolytes from the intestine.

The mechanism by which LT stimulates the activityof the adenylate cyclase is as follows: (1) The B subunit of the toxin binds to a specific cell receptor, GM₁ ganglioside, (2) the A₁ subunit is released from the toxin and enters the cell; and (3) the A₁ subunit cleaves nicotinamide-adenic dinucleonde (NAD) into nicotinamide and ADP-ribose and, together with a cellular ADP-ribosylating factor, transfers the ADP-ribose to aGTP-binding protein. The ADP-ribosylation of the GTP-binding protein inhibits a GTPase activity of the binding protein, leading to increased stability of the catalytic cornplex responsible for adenylate cyclase activity. This results in an amplified activity of the cyclase and a corresponding increase in the amount of cAMP produced.

Two antigenically distinct heat labile toxins are produced by various strains of E. coli. LT-I is structurally andantigenically related to CT to an extent that anti-CT will neutralize LT I LT-II has, on rare occasions, beenisolated from the feces of humans with diarrhea, but it is most frequently isolated from feces of water buftalos and cows LT-II is biologically similar to LT-I, but it is notneutralized by either anti-LT-I or anti-CT.

LT will bind to many types of mammalian cells, and its ability to stimulate adenylate cyclase can be assayed incell cultures.

A report has also shown that CT stimulated an increase in prostaglandin E (PGE), and that PGE1 and PGE2 caused a marked fluid accumulation in the ligated lumen of rabbit intestinal segments. The mechanism whereby CT induces PGE release is unknown.

HEAT-STABLE TOXIN. The heat-stable toxin STa consists of afamily of small, heterogeneous polypeptides of 1500 to 2000 daltons that are not destroyed by heating at 100 °Cfor 30 minutes. STa has no effect on the concentrationof cAMP, but it does cause a marked increase m thecellular levels of cyclic GMP (cGMP). cGMP causes aninhibition of the cotransport of NaCI across the intestinal wall, suggesting that the action of STa may be primarily antiabsorptive compared with that of LT, which is both antiabsorptive and secretory.

STa stimulates guanylate cyclase only in intestinal cells, indicating that such cells possess a unique receptorfor Sta. The cell receptor for STa is known to be either tightly coupled to, or a part of, a particulate form of guanylate cyclase located in the brush border membranes of intestinal mucosal cells. Also, intimately associated with this complex is a cGMP-dependent protein kinase that phosphorylates a 25,000 dalton protein in the brush border. It has been proposed that this phosphorylated protein might be the actual mediator for the toxin-induced iontransport alterations that lead to fluid loss. The usual assay for STa is to inject the toxin intragastrically into a 1 – to 4-day old suckling mouse and measure intestinal fluid accumulation (as a ratio of intestinal/remaining body weight) after 4 hours. STa may also be assayed directly by measuring its effect on the increase in guanylate cyclasein homogenized intestinal epithelial cells.

A second heat stable toxin that is produced by somestrains of E. coli has been termed STb. This toxin is inactivein suckling mice but will produce diarrhea in weaned piglets. STb producers have not been isolated from humans. It does not seem to increase the level of adenylate or guanylate cyclase in intestinal mucosal cells, but maystimulate the synthesis of prostaglandin E2. The end resuit is to enhance net bicarbonate ion secretion.

COLONIZATION FACTORS. Animals also are subject to infections by their own strains of ETEC, and such infections in newborn animals may result in death from the loss of fluids and electrolytes. Extensive studies of strains infecting newborn calves and piglets (as well as humans) have revealed that, in addition to producing an enterotoxin, such strains possess one of several fimbriate surface structures that specifically adhere to the epithelial cellslining the small intestine. These antigens (K-88 for swine strains, and K-99 for cattle) usually are fimbriate struc-tures that cause the toxin-producing organisms to adhere to and colonize the small intestine. The need for this colonizing ability is supported by the fact that antibodies directed against the colonizing fimbriae are protective.

Analogous human ETEC strains also possess fimbriate structures that have been designated as colonizationfactors (CFA). At least five such serologicallydifferent factors, CFA/I, CFA/II, CFA/III, E8775, andCFA/V, have been described. Interestingly, these colonization factors also are plasmid mediated, and single plasmids have been described that carry genes for both CFA/I and STa.

Interestingly, during the Gulf War in 1990, there were about 100 cases of diarrhea per week per 1000 personnel. Of these, 55% resulted from ETEC.

Enterohemorrhagic Escherichia coli. The enterohemorrhagic E. coli (EHEC) were first described in 1982 when they were shown to be the etiologic agent of hemorrhagic colitis, a disease characterized bysevere abdominal cramps and a copious, bloody diarrhea. These organisms are also known to cause a condition termed hemolytic-uremic syndrome (HUS), which is manifested by a hemolytic anemia, thrombocytopenia (decrease in the number of blood platelets), and acuterenal failure. HUS occurs most frequently in children.

Although most initially recognized EHEC belong to serotype O157:H7, other EHEC serotypes such as O26, O111, O128, and O143 have been recognized. These organisms are not invasive, but they do possess a 60-megadalton plasmid that encodes for a fimbrial antigen that adheres to intestinal epithelium. In addition, the EHEC are lysogenic for one or more bacteriophages that encode for the production of one or both of two antigenically distinct toxins. These toxins are biologically identical and antigenically similar to the toxins formed by Shigella dysenteriae (Shiga’s bacillus), and are designated as Shiga-like toxin I (SLT-I) and Shiga-like toxin II (SLT II). Because the Shiga-like toxins initially were characterized by their ability to kill Vero cells, a cell line developed from African green monkey kidney cells, they also arecalled Verotoxin I and Verotoxin II.

SLT I consists of an A subunit and five B subunits. The sequence of the B subunit from S. dysenteriae type 1 is identical to that of the B subunit of SLT I. The B subunit binds specifically to a glycolipid in microvillus membranes, and the released A subunit stops protein synthesis by inactivating the 60S ribosomal subunit. This inactivation results from the N-glycosidase activity of the toxin, which cleaves off an adenine molecule (A-4324) from the 28S ribosomal RNA, causing a structural modification of the 60S  subunit, resulting in a reduced affinity for EF-1 and, thus, an inhibition of aminoacyl- tRNA binding. The consequence of toxin action is a cessation of protein synthesis, the sloughing off of dead cells, anda bloody diarrhea. Notice that SLT 1 carries out the same reaction as the plant toxins ricin and abrin.

SLT II is biologically similar to SLT I, but because only a 50% to 60% homology exists between the two toxins, it is not surprising that they are antigenically distinct. Interestingly, both STL I and STL-II can be transferred to nontoxin producing strains of E. coli by transduction.

Outbreaks of hemorrhagic colitis have been traced to contaminated food as well as to person to person transmission iursing homes and day care centres. Contaminated, undercooked hamburger meat seems to be the most frequently implicated source of food borne illnesses followed by contaminated milk and water, indicating thatcattle are a common reservoir for EHEC. Of note is that E. coli  0157:H7 has been shown to survive up to 9 months at -20°C in ground beef.

Thus, the EHEC are able to cause hemorrhagic colitis as a result of their ability to adhere to the intestinal mucosa, and they presumably destroy the intestinal epithelial lining through their secretion of Shiga like toxins. The mechanism whereby the EHEC cause HUS is unclear but seems to follow bloodstream carriage of SLT II to the kidney. Experimental results have shown that humanrenal endothelial cells contain high levels of receptor for SLT-2. Moreover, in the presence of interleukin (IL)1/b, the amount of receptor increases, enhancing the internalization of the toxin and the death of the cell.

The section, “A Closer Look,” describes several epidemics of hemorrhagic colitis that have occurred in the United States and techniques that are used for the identification of this serotype

Enteroinvasive Escherichia coli

The disease produced by the enteroinvasive E. coli (EIEC) is indistinguishable from the dysentery produced by members of the genus Shigella, although the shigellae seem to be more virulent because considerably fewer shigellae are required than EIEC to cause diarrhea. The key virulence factor required by the EIEC is the ability to invade the epithelial cells.

EIEC INVASION. The specific property that provides these organisms with their invasive potential is far from understood. It is known, however, that this ability is encoded in a plasmid and that the loss of the plasmid results m aloss of invasive ability and a loss of virulence. Moreover, the shigellae seem to possess the same plasmid, because Western blots show that shigellae and EIEC plasmids express polypeptides that are similar in molecular weight and antigenicity.

EIEC TOXINS. Although the primary virulence factor of EIEC strains is the ability to invade intestinal epithelial cells, they also synthesize varying amounts of SIT I and SLT II. Based on the severity of the disease, however, it could assumed that the amount of toxin produced is considerably less than that formed by the highly virulent shigellae or the EHEC. Other enterotoxic products produced by the EIEC are under study.

EIEC can be distinguished from other E. coli by their ability to cause an inflammatory conjunctivitis in guinea pigs, an assay termed a Sereny test. A DNA probe thathybridizes with colony blots of EIEC and all species of Shigella also has been used to identify organisms producing Shiga-like toxins.

Enteropathogenic Escherichia coli. The enteropathogenic E. coli (EPEC) are diffusely adherent organisms that are particularly important in infantdiarrhea occurring in developing countries, where they may cause a mortality rate as high as a 50%. They comprise a mixture of organisms that seem to produce diarrhea by a two step process. The classic EPEC exist among a dozen or so different serotypes, all of which are characterizedby the possession of a 55 to 65-megadalton plasmid that encodes for an adhesin termed EPEC adherence factor (EAF). EAF causes a localized adherence of the bacteria to enterocytes of the small bowel, resulting m distinct microcolonies. This is followed by the formation ofunique pedestal-like structures bearing the adherent bacteria. These structures have been termed attaching and effacing lesions. The ability to form the effacing lesion resides in an attaching and effacing gene (eae). The lesions are characterized by a loss of microvilli and a rearrangement of the cytoskeleton, with a proliferation of filamentous actin beneath are as of bacterial attachment.

Thus, the ability of the EPEC to cause diarrhea involves two distinct genes, EAF and eae. The end result is anelevated intracellular Ca⁺² level in the intestinal epithelialcells and the initiation of signal transduction, leading to protein tyrosine phosphorylation of at least two eucaryotic proteins.

EPEC strains routinely have been considered noninvasive, but data have indicated that such strains can invadeepithelial cells in culture. However, EPEC strains do not typically cause a bloody diarrhea, and the significance of cell invasion during infection remains uncertain.

Other Diairhea-Producing Escherichia coli. All possible combinations, deletions, or additions of the various virulence factors responsible for intestinal fluid loss result in diarrhea producing strains that do not fitthe categories already described. Such has been found tobe the case.

The most recent of these has been termed the enteroaggregative E. coli. These strains seem to cause diarrhea through their ability to adhere to the intestinal mucosa and possibly by yet a new type of enterotoxin. It seems possible that the acquisition of other virulence factors may result in the discovery of additional pathogenic strains of E. coli.

E. coli Urinary Tract Infections. Escherichia coli is the most common cause of urinary tract infections of the bladder (cystitis) and, less frequently, of the kidney (pyelonephritis). In either case, infections usually are of an ascending type (enter the bladder fromthe urethra and enter the kidneys from the bladder). Many infections occur in young female patients, in persons with urinary tract obstructions, and in persons requiring urinary catheters, and they occur frequently in otherwise healthy women. Interestingly, good data support the postulation that certain serotypes of E. coli are more likely to cause pyelonephritis than others. Thus, the ability to produce P-fimbriae (so called because of their ability to bind to P blood group antigen) has been correlated withthe ability to produce urinary tract infections, seemingly by mediating the adherence of the organisms to human uroepithelial cells. Of note is that the rate of nosocomial urinary tract infection per person-day was significantly greater in patients with diarrhea, particularly in those with an indwelling urinary catheter.

In addition to fimbrial adhesins, a series of afimbrial adhesins has been reported. Their role in disease is not yet firmly established, but it has been demonstrated that at least one afimbrial adhesins mediated specific binding to uroepithelial cells.

Recurrent urinary tract infections in premenopausal, sexually active women frequently can be prevented by the postcoital administration of a single tablet of an antibacterial agent such as trimethoprim-sulfamethoxazole, cinoxacin, or cephalexin.

E. coli Systemic Infections. About 300,000 patients in United States hospitals develop gram-negative bacteremia annually, and about 100,000 of these persons the of septic shock. As might be guessed, E. coli is the most common organism involved in such infections. The ultimate cause of death in these cases is an endotoxin-induced synthesis and release of tumor necrosis factor-alpha and IL-1, resulting in irreversible shock.

The newborn is particularly susceptible to meningitis, especially during the first month of life. A survey of 132 cases of neonatal meningitis occurring in the Netherlands reported that 47% resulted from E. coli and 24% from group B streptococci. Notice that almost 90% of all cases of E. coli meningitis are caused by the K1 strain, which possesses a capsule identical to that occurring on group B meningococci.

Table  summarizes the virulence factors associated with pathogenic E. coli.

Table

Escherichia coif Virulence Factors

 

A Closer Look

In January 1993, the Childrens Hospital in Seattle notified the Washington State Health Department ofan outbreak of hemorrhagic colitis. This epidemiceventually involved over 500 persons living in the Pacific Northwest, of whom 125 were hospitalized, 41developed acute renal failure, and 4 died. The causeof this outbreak was quickly linked to Escherichia coli O157:H7, an enterohemorrhagic strain of E. coli that was acquired by eating undercooked hamburgers obtained from a fast food chain.

Symptoms of hemorrhagic colitis are characterizedby diarrhea that is frequently bloody and a kidney involvement, which is termed hemolytic uremic syndrome (HUS). HUS occurs in 3% to 7% of these infections, particularly in children younger than 5 years of age. In about 90% of cases, HUS results in renal failure that often requires dialysis; approximately 10% will require a kidney transplant, and 3% to 5% will die.

Such infections were initially described in 1982 when epidemics of E. coli O157:H7 occurred in Oregon and Michigan. These also were attributed to the ingestion of undercooked hamburger from fast foodrestaurants. Another outbreak, occurring in a nursinghome during 1984, resulted in 34 cases; 14 patients were hospitalized and 4 died. This also was traced to undercooked hamburger, as have a number of similar outbreaks that have been reported throughout the last decade.

Not all such epidemics, however, are traced to undercooked beef. One major outbreak involving 243 cases—32 patients were hospitalized and 4 died—was traced to a municipal water supply whose distribution system had become contaminated with sewage. Others are thought to result from person-to-person spread because, after recovery, one becomes an asymptomatic carrier for 3 weeks to 2 months.

The primary reservoir of E. coli O157:H7 is the intestinal tract of animals, particularly cattle, where  it may exist completely asymptomatically. Thus, it is not surprising that outbreaks of this infection have been traced to unpasteurized milk and undercooked beef that had been contaminated in the slaughter house. If such contaminated beef is cooked in a single piece (steaks or roasts), it probably can be safely eaten rare because contamination by E. coli is limited to the surface of the meat. After grinding to make hamburger, however, any contaminating organisms are spread throughout the meat, and cooking to 68 °C (155°F) is the only safe procedure to avoid ingesting viable organisms.

There is considerable pressure for meat inspectors to sample all beef at the slaughter houses to detect contamination with E. coli 0157:H7. This is accomplished by growing a sample on sorbitol-MacConkey agar plates. Because E. coli 0157:H7 usually does notferment sorbitol, colonies not producing add can be selected for a serologic determination of O or H antigens. A single nonsorbitol-fermenting colony can be mixed with commercially available latex beads to which anti-0157 antibodies have been absorbed. There is also a polymerase chain reaction (PCR) available to test for the genes encoding Shiga-like toxins I and II.

All in all, it seems to require an incredible amountof work to ensure that rare hamburger is safe to eat. An alternative might be to subject all beef to sufficient gamma-irradiation to eliminate all contaminating organisms.

 

Klebsiellae

The family Enterobacteriaceae, genus Klebsiella, include bacteria which are capable of producing capsules when present in the host’s body or outrient media.

Morphology. The Klebsiella organisms are thick short bacilli 0.6-6.0 mcm in length and 0.3-1.5 mcm in breadth. They have rounded ends, are non-motile and devoid of spores. They occur mainly in pairs but may be seen frequently as single organisms, and are normally surrounded by a capsule. They stain readily with all aniline dyes and are Gram-negative. K. pneunoniae and K. ozaenae have fimbriae. The G + C content in DNA ranges from 52 to 56 per cent.

Cultivation. The klebsiellae are facultative anaerobes, which grow readily on commoutrient media at pH 7.2 and at a temperature of 35-37 ° C. No growth is shown below 12 °C or above 37 °C. The organisms are capable of synthesizing all ammo acids essential for their growth. They form turbid mucilaginous colonies on agar and produce intense turbidity in broth. After 2 or 4 hours the capsulated bacteria show a characteristic arrangement in the young colonies (Fig. 78). The young colonies are studied with a dry lens (lens No. 7) in pieces of agar taken from Petri dishes. The agar-microscopy  method is used for differentiation of capsulated bacteria.

The klebsiellae may lose their capsules by prolonged subculture on 50 per cent bile broth, and acquire them again by passage through white mice. The organisms dissociate into S- and R-forms when they are exposed to the action of low temperatures, bacteriophage, chemical substances, bile, and antiserum or when they are frequently subcultured.

Fermentative properties. The Klebsiella organisms do not liquefy gelatin and produce no indole or hydrogen sulphide. They reduce nitrates to nitrites and decompose urea. Milk is not always curdled. The organisms ferment carbohydrates, producing both acid and gas or, sometimes, only acid. Glucose and urea fermentation is usually a constant property.

Toxin production. K. pnewnoniae produce thermostable exotoxm, their toxicity being associated with an endotoxm.

Antigenic structure. Capsulated bacteria contain three types of antigens: capsular (K-antigen), smooth somatic (0-antigen), and rough somatic (R-antigen). The K- and 0-antigens are carbohydrates, and the R-antigen is a protein The O-antigen is subdivided mto three groups: O-group 1, O-group 2, and O-group 3. The O-group 1 and the cohbacilli possess common antigens. Bactenocines and phages have been discovered in Klebsiella organisms.

The organisms are differentiated by the presence of O- and K-antigens. An agglutination reaction with the non-capsulated strain which contains antigens and the complement-fixation reaction with the capsular antigen are performed for antibody detection.

Resistance. Klebsiella organisms survive at room temperature for weeks and even months. When heated to a temperature of 65 °C they are destroyed in one hour. The organisms are susceptible to treatment with solutions of chloramine, phenol, citral, and other disinfectants.

Pathogenicity for animals. Among the experimental animals white mice are most susceptible They die in 24-48 hours following inoculation, displaying symptoms of septicaemia. Severe inflammation and enlargement of the spleen and liver are found at autopsy. Capsulated bacteria are found in abundance in smears made from organs and blood. The pathogenicity of capsulated bacteria is associated with the capsule, and bacteria which have lost their ability to produce capsules become non-pathogenic and are rapidly exposed to the action of phage when injected into the animal body.

Pathogenesis and diseases in man. Three species of capsulated bacteria play a most important role in human pathology: the causative agents of pneumonia, ozaena, and rhinoscleroma.

Klebsiella pneumoniae grow readily on solid media, producing opaque mucilaginous colonies. In young colonies grown on agar they occur in loops and are serologically heterogeneous. Infected guinea pigs and white mice exhibit septicaemia. The causative agents are found in the blood and tissues, types A and B being most virulent.

K. pneunoniae is responsible for pneumonia. Pneumonia (broncho-pneumonia) involves one or several lung lobes, sometimes producing fused foci and lung abscesses. The death rate is quite high. In some cases the organisms may be responsible for meningitis, appendicitis, pyaemia, mastoiditis, and cystitis. They may also cause inflammation in cases of mixed infections.

Klebsiella ozaenae — the morphological characteristics are given above. In young colonies the organisms are concentrically scattered. It is assumed that they are responsible for rhinitis which is characterized by an offensive nasal discharge. K. ozaena affects the mucous membranes of the nose, nasal sinuses, and conchae. This results in production of a viscid discharge which dries up and forms thick scabs with an offensive odour. These scabs make breathing difficult.

Ozaena is mildly contagious disease and is transmitted by the air-droplet route. It is possible that other factors (trophic and endocrine disturbances, etc.) also contribute to its development. The disease is revalent in Spain, India, China, and Japan and occurs sporadically in the USSR.

Klebsiella rhinoscleromatis are differentiated by their growth on agar and other properties. In young colonies they are arranged concentrically.

The rhinoscleroma bacteria occur in tissue nodes (infectious granulomas) in the form of short capsulated microbes. They are localized intra- and extracellularly.

The organisms are responsible for chronic granulomatosis of the skin and mucous membranes of the nose, pharynx, larynx, trachea, and bronchi, with the formation of granulomas. Rhinoscleroma is a mildly contagious disease. It prevails in Austria and Poland and occurs sometimes in Belorussia, the Ukraine, Siberia, and Central Asia. Treatment is a matter of great difficulty and involves complex therapeutic measures which must be carried out over a long period of time.

Immunity. Diseases caused by capsulated pathogenic bacteria leave low-grade immunity. Agglutinins and complement fixing antibodies are present in the blood of ozaena and rhinoscleroma patients, but their defence role is negligible. The absence of an infectious immunity isprobably the reason for the chronic nature of these diseases.

Laboratory diagnosis includes the following methods. 1. Microscopic examination of smears made from sputum (from patients with pneumonia), nasal mucus discharge (from patients with ozaena), and tissue specimens (from patients with rhinoscleroma). Pathohystological examination of infiltrates reveals a great number of peculiar giant Mikulicz’s cells which contain capsulated bacteria in a gelatin-like substance. The material is collected with a loop or cotton- wool swab, having previously scarified the mucosa surface.

2. Isolation of the pure culture and its identification by cultural, biochemical, phagocytolytic, and serological properties.

3. Complement-fixation reaction with patients’ sera and capsular antigen. This reaction yields positive results most frequently. Sera diluted in ratios from 1:5 to 1:400 are used for the agglutination reaction with a non-capsulated strain.

4. The allergic skin test is employed as an additional test, but is less specific than the agglutination reaction or the complement-fixation reaction.

Treatment Patients are treated with streptomycin, chloramphenicol, tetracycline, and antimony preparations (solusurmin). Vaccine therapy is also employed. The vaccine is prepared from capsulated bacteria strains which have been killed by heat treatment.

Prophylaxis is ensured by recognition of the early stages of ozaena and rhinoscleroma, active antibiotic therapy, and prevention of healthy individuals from being infected by the sick.

 

ADDITIONAL MATERIAL ABOUT DIAGNOSIS OF DISEASES

INFECTION CAUSED BY ESCHERICHIA COLI (ESCHERICHIOSIS)

Presumptive Test for Escherichia coli. To determine the presence of E colt in water, test tubesof nutrient broth containing lactose are inoculated withmeasured quantities of water samples. These tubes also contain an inverted vial to trap gas produced and anacid-base indicator to show acid production (Fig.). Because E. coli ferments lactose, the presence of acid and gas in the inoculated tubes after 24 hours of incubationis presumptive evidence for its presence. If the lactose isnot fermented, it is concluded that E. coli is not presentand that the water is free from recent fecal contamination. The fermentation of lactose may, however, result fromnonenteric organisms and, for a positive conclusion of fecal contamination, it is necessary to show that the lac-tose-fermenting organisms are of fecal origin.

FIGURE. Fermentation tubes with inverted vials and acid indicator to test for Escherichia coli. Notice the light colour (actually yellow) in the right hand rube, because of the presence of acid, and the bubble in the vial, because of the evolution of gas.

 

Confirmed Test. Because fecally derived E. coli can grow at 44.5 °C and nonfecal coliforms cannot, differential agar media (contaming lactose as a carbon source) can be streaked with known amounts of water and incubated at both 37 °C and 44.5 °C. A comparison of the number of lactosefermenting colonies growing at 37 °C and 44.5 °C will provide information concerning the origin of the coli-forms. More precise results, however, can be obtained by carrying out the following test: eosin methylene blue agaris streaked from the positive lactose broth fermentation grown at 37 °C. E. coli grows as a characteristic small, flat colony that has a definite metallic green sheen.

Completed Test. Colonies from the confirmed test that show a metallic green sheen are reinoculated into lactose broth  and, if acid and gas are produced, die organism is identified as E. coli.

Membrane filters also can be used to detect the presence of bacteria in water. The membrane of cellulose acetate permits water to pass easily, but it traps bacteriaon its surface. Coliform counts can be made by filtering a known volume of water through the membrane followed by incubation of the membrane in a special differential medium. Colonies growing on the surface of the membrane can be counted and observed for the characteristic appearance of  E. coli on the differential medium.

Escherichia coli is a permanent inhabitant of the human gut. Yet, in some cases Escherichia coli may induce various pyoinflaminatory diseases, while their pathogenic serovars may be responsible for colienteritis in infants under 1 year of age and dysentery-like disease and cholera-like gastroenteritis in both children and adults.

Bacteriological examination. The material to be studied is -faeces collected from the patient three times at 3-4-hour interval within the first day of the disease manifestation, as well as vomit, washings of the stomach and intestines, blood (in sepsis), pus (in pyoinflam­matory processes), faucial and nasal mucosa secretions, and urine. In lethal cases contents of the intestines, blood, pieces of the spleen, lungs, and other organs are examined.

In cases of toxi-infection, the remains of the food, washings from the hands of the attending staff, air of the wards, and peroral drugs are also tested.

To isolate Escherichia, Endo’s and Ploskirev’s media are utilized. The blood is inoculated into meat-peptone broth. Culturing should preferably be done at the patient’s bedside.

After 18-24-hour incubation at 37 °C examine the nature of col­onies on solid media.Pathogenic serovars of Escheri­chia do not differ from commensal Escherichia microorganisms which are always present in the human gut by their cultural properties or their ability to ferment lactose. Therefore, use the agglutination test to determine whether the isolated microorganisms belong to pathogenic serogroups. For this purpose mark on any ten lactose-positive colonies in the medium and induce agglutination on a glass slide, using a mixture of OK-sera of pathogenic serogroups. The mixture is prepared in such a way that the final dilution of each serum is 1:10. A negative agglutination reaction points to the absence of enteropathogenic E. coli.

If the agglutination reaction is positive, the remaining portion of the colony is subcultured onto Olkenitsky’s medium (100 ml of meat-peptone agar, 1 g of lactose, 1 g of sucrose, 0,1 g of glu­cose. 1 g of urea, 0.02 g of Moor’s salt, 0.003 g of sodium thiosulphate, 0.4 ml of phenol red (0.4 per cent solution); pH 7.2-7.4).

The next day, the triple sugar agar is examined for changes. Fermentation of glucose and lactose with the formation of acid and gas is recognized by alteration in the colour of the medium (yellowing of the entire surface and column of the agar) and impairment of its integrity. For­mation of a black ring on the borderline between the column and a slant surface suggests the elaboration of hydrogen sulphide by E. coli.

Using the slide agglutination test with a set of OK-diagnostic sera (1:10) against pathogenic serovars of E. coli, the isolated cul­ture is presumptively referred to a definite serovar. Then, a standard agglutination test is made to demonstrate the O- and K-antigens of bacteria. The diagnostic serum is diluted in two rows of test tubes to the titre indicated on the label of an ampule for the 0- and K-agglutinins. To demonstrate the K-antigen, introduce unheated culture washed off the agar slant into one row of test tubes, whereas to detect the 0-antigen, employ a bacterial suspension boiled for 1-2 hrs. Boiling brings about destruction of the K-antigen which overlies the 0-antigen and inhibits the latter. The test tubes are placed into a 37 °C incubator for 24 hrs. Strains homologous to the serum are agglutinated to the titre or to half the titre.

The bacterial culture, which has produced a routine agglutina­tion reaction, is inoculated into media to study its sugarlytic and proteolytic properties and mobility.

For the purpose of faster identification of the isolated cultures or the test material, direct or indirect immunofluorescence is employed, which makes it possible to obtain the presumptive result within 1-2 hrs.

Determination of the phagovar and colicinogenovar of the isolated strain is important in identifying the source of infection.

Serological diagnosis. Demonstration of antibodies in the agglu­tination reaction with an autostrain is helpful in the diagnosis of escherichiosis and its differentiation from a bacterial carrier-state.

The value of serological diagnosis is enhanced with differential grouping of antibodies into the classes of immunoglobulins, e.g.. change of specific antibodies of the IgM to the IgG class indicates an acute infectious process, whereas the presence in the sera of antibod­ies of the IgG class only is typical of bacterial carriers.

 

 

INFECTION CAUSED BY KLEBSIELLAE

The genus Klebsiella of the Enterobacteriaceae family includes causative agents of pneumonia (Klebsiella pneumoniae), rhinoscleroma (Klebsiella rhinoscleromatis), and ozaenae (Klebsiella ozaenae).

Representatives of this type of bacteria can cause variable clinical syndromes, inflammatory processes of various localization, and sepsis.

Material for the study includes sputum in pneumonia, secretion of the nasal mucosa in ozaena, tissue of infiltrates in rhinoscleroma, as well as secretion, blood, and pus from inflammatory foci.

Bacterioscopic examination. Examination of smears stained by the Gram technique reveals Gram-negative bacteria arranged in pairs and surrounded by a common capsule. Examination of histological preparations from scleroma infiltrates demonstrates large numbers of giant Mikulicz’s cells filled with gelatin-like substance which contains capsular bacteria of scleroma.

Bacteriological examination. The material to be studied is inocu­lated into differential media, bromthymol and bromcresolpurpur agar; one can also use meat-peptone (pH 7.2) or glycerol agar. The dishes are allowed to stand at room temperature for 2-4 hrs and then young colonies are examined by dark-field microscopy. All capsular bacteria are characterized by a definite structure of colonies, which permits their differentiation. Following 24-hour cultivation at 37 °C, mucosal colonies are subcultured to an agar slant for pure culture isolation. For their identification the isolated cultures are inoculated into Hiss’s media, bile broth, or agar.

Differentiation from other enteric bacteria is based on fermentative properties and the absence of motility. Klebsiella microorganisms break down urea, malonate, inosite, possess decarboxylase activity, give a positive Voges-Proskauer reaction, and do not produce hydro­gen sulphide.

For the serological identification of Klebsiella, capsular slide agglutination and 0-agglutination tests are employed. To perform the capsular agglutination test, immune anti-capsular sera and a drop of 0.5 per cent sodium chloride solution are placed on a glass slide. A loopful of the culture to be examined is comminuted in the immediate vicinity to a drop of serum and both are mixed. If the reaction is positive, agglutination is observed in the form of gross threads or strands.

For O-agglutination the test culture is sterilized for 2.5 hrs at a pressure of 202.6 kPa (2 atm). As a result, the bacteria lose the capsule and the ability to interact with K-sera but retain the capacity to agglutinate with 0-sera. After the sterilization the culture is washed two times with isotonic sodium chloride solution, using centrifugation, and the sediment is used to perform the slide agglu­tination reaction.

Additional methods of identification of cultures include determi­nation of their virulence (inoculation of white mice) and sensitivity to bacteriophages. Phages of capsular bacteria have a strictly deter­mined species specificity.

Phagotyping of scleroma cultures may be utilized for ascertaining epidemiological links in scleroma foci. Comparative characteristics of pathogenic Klebsiella microorganisms are presented in Table 4.

Table 4

 Differential-Diagnostic Criteria of Pathogenic Klebsiella

 

Note. A –  Acid, AG – acid and gas

 

Serological diagnosis. The CF test with patients’ serum and capsular antigen is employed. To diagnose rhinoscleroma, the antigen usually used presents suspension of a 24-hour culture of Kl. rhinoscleromatis containing 500 million microorganisms per ml, which is heated for 1 h at 60 °G. Fresh antigen is prepared for each reaction or antigen preserved with 0.25 per cent phenol may be used.

The results are more specific when the CF test is conducted in the cold. Both fresh and dried patient’s serum can be used for the CF test. Dried serum is prepared in the following manner: two 0.5 ml drops of serum are dripped on filter paper and dried at room temperature for 24 hours. Paper with dried drops is put in an envelope and sent to the laboratory. A spot of blood is cut off, comminuted   together   with   paper,   and   placed   into 1 ml of isotonic sodium  chloride solu­tion which is later pipetted off to run the reaction. The results of the CF test with fresh and dried sera are identical. To recover agglutinins in the serum of patients with rhinoscleroma, one can use the reaction of agglutination with an acapsular strain. For this purpose diagnosticums treated with formalin may be utilized. Diagnostically signifi­cant is a positive reaction in a 1:1600 dilution and over.

 

SALMONELLA

Enteric Fever and Paratyphoid Salmonellae. The causative agent of enteric (typhoid) fever, Salmonella typhi was discovered in 1880 by K. Eberth and isolated in pure culture in 1884 by G. Gaffky.

In 1896 the French scientists C. Archard and R. Bensaude isolated paratyphoid B bacteria from urine and pus collected from patients with clinical symptoms of typhoid fever. The bacterium responsible for  paratyphoid A (Salmonella paratyphi) was studied in detail m 1902 by the German bacteriologists A. Brion and H. Kayser, and the causative agent of paratyphoid B {Salmonella schottmuelleri) was studied in 1900 by H. Schottmueller.

Morphology. The morphology of the typhoid salmonella corresponds with the general characteristics of the Enterobacteriaceae family. Most of the strains are motile and possess flagella, from 8 to 20 in number. It is possible that the flagella form various numbers of bunches.

The paratyphoid salmonellae do not differ from the typhoid organisms in shape, size, type of flagella, and staining properties.

The typhoid salmonellae possess individual and intraspecies variability. When subjected to disinfectants, irradiation, and to the effect of other factors of the external environment they change size and shape. They may become coccal, elongated (8-10 mcm), or even threadlike. The G+C content in DNA ranges between 45 and 49 per cent.

Cultivation. The typhoid and paratyphoid organisms are facultative anaerobes. The optimum temperature for growth is 37° C, but they also grow at temperatures between 15 and 41°C. They grow on ordinary media at pH 6.8-7.2. On meat-peptone agar S. typhi forms semitransparent fragile colonies which are half or one-third the size of E. coli colonies. On gelatin the colonies resemble a grape leaf in shape Cultures on agar slants form a moist transparent film of growth without a pigment and in meat broth they produce a uniform turbidity.

On Ploskirev’s and Endo’s media S. typhi and S. paratyphi form semitransparent, colourless or pale-pink coloured colonies. On Levin’s medium containing eosin and methylene blue the colonies are transparent and bluish in colour, on Drigalski’s medium with litmus they are semitransparent and light blue, and on bismuth-sulphite agar they are glistening and black. The colonies produced by S. paratyphi A on nutrient media (Ploskirev’s, Endo’s, etc.) are similar to those of S. typhi. 

Salmonella on Ploskirev’s mrdium

 

Colonies of S. schottmuelleri have a rougher appearance and after they have been incubated for 24 hours and then left at room temperature for several days, mucous swellings appear at their edges. This is a characteristic differential cultural property.

Salmonella on bismuth-sulphite agar

 

 

Fermentative properties. S. typhi does not liquefy gelatin, nor does it produce indole. It produces hydrogen sulphide, and reduces nitrates to nitrites.

 

Figure. Colonies of Salmonella paratyphi (1); colonies of Salmonella schottmuelleri (2); smear from Salmonella enteritidis culture (3)

 

The organisms do not coagulate milk, but they give rise to a slightly pink colouration in litmus milk and cause no changes in Rotberger’s medium. They ferment glucose,      mannitol, maltose, levulose, galactose, raffinose, dextrin, glycerin, sorbitol and, sometimes, xylose, with acid formation.

S. paratyphi ferments carbohydrates, with acid and gas formation, and is also distinguished by other properties. Two types of S. typhi occur iature: xylose-positive and xylose-negative. They possess lysin decarboxylase, ornithine decarboxylase and oxidase activity.

 

Differentiating Characteristics of Salmonella typhi, Salmonella paratyphi and Food-poisoning  Salmonella

 

In the process of dissociation S. typhi changes from the S-form to the R-form. This variation is associated with loss of the somatic 0-antigen (which is of most immunogenic value) and, quite frequently, with loss of the Vi-antigen.

Toxin production. S. typhi contains gluco-lipo-protein complexes. The endotoxin is obtained by extracting the bacterial emulsion with trichloracetic acid. This endotoxin is thermostable, surviving a temperature of 120° C for 30 minutes, and is characterized by a highly specific precipitin reaction and pronounced toxic and antigenic properties. Investigations have shown the presence of exotoxic substances in S. typhi which are inactivated by light, air, and heat (80° C), as well as enterotropic toxin phosphatase, and pyrogenic substances.

Note: A, acid formation;  AG, acid and gas formation; +, hydrogen sulfide formation;  —, absence of carbohydrate fermentation and hydrogen sulphide formation;      ±, arabinose fermentation and hydrogen sulfide formation do not always occur.

 

Antigenic structure. S. typhi possesses a flagellar H-antigen and thermostable somatic 0- and Vi-antigens. All three antigens give rise to the production of specific antibodies in the body, i. e. H-, 0-, and Vi-agglutinins. H-agglutinins bring about a large-flocculent agglutination, while 0- and Vi-agglutinins produce fine-granular agglutination.

The antigens differ in their sensitivity to chemical substances. The O-antigen is destroyed by formalin but is unaffected by exposure to weak phenol solutions. The H-antigen, on the contrary, withstands formalin but is destroyed by phenol.

S. typhi, grown on agar containing phenol in a ratio of 1:1000, loses the H-antigen after several subcultures. This antigen is also destroyed on exposure to alcohol. These methods are employed to obtain the 0-antigen in its pure form. The H-antigen is isolated by treating the bacterial emulsion with formalin or by using a broth culture which contains a large number of flagellar components. Immunization with H-and 0-antigens is employed for obtaining the corresponding agglutinating sera.

The discovery of the Vi-antigen isolated from virulent S. typhi is of great theoretical interest and practical importance.

Vi- and 0-antigens are located within the micro-organism, on the surface of the bacterial cell. It is assumed that the Vi-antigen occurs in isolated areas and is nearer to the surface than the 0-antigen. The presence of Vi-antigens hinders agglutination of salmonellae by 0-sera, and the loss of the Vi-antigen restores the 0-agglutinability. S. typhi, which contains Vi-antigens, is not agglutinated by 0-sera. Vi-agglutinating serum is obtained by saturation of S. typhi serum of animals inoculated with freshly isolated salmonellae, employing H- and O-antigens. The Vi-antigen is a labile substance. It disappears from the culture when phenol is added to the medium and also when the temperature is low (20 °C) or high (40 ° C). It is completely destroyed by boiling for 10 minutes and by exposure to phenol. Exposure to formalin and to temperature of 60° C for 30 minutes produces partial changes in the antigen.

Together with H-, O-, and Vi-antigens, other more deeply located antigens have been revealed. The latter are detected during the change transformation of the bacterial cell to the R-form when the superficial 0- and Vi-antigens are lost. The deeply located antigens are non-specific. Later, salmonellae were found to possess an M-mucous antigen (polysaccharide).

It has been ascertained that the Vi-antigen content of cultures varies, some serovars possessing a large quantity of this antigen, while others only a small quantity. F. Kauffmann subdivides all salmonellae containing Vi-antigens into three groups: (1) pure V-forms with a high Vi-antigen content; (2) pure W-forms which contaio Vi-antigens; (3) transitional V-W-forms which possess Vi-antigens and are agglutinated by O-serum. S. paratyphi have been found to have antigens in common with isoantigens of human erythrocytes.

Classification. The salmonellae of typhoid fever and paratyphoids together with the causative agents of toxinfections have been included in the genus Salmonella (named after the bacteriologist D. Salmon) on the basis of their antigenic structure and other properties. At present, about 2000 species and types of this genus are known.

F. Kauffmann and P. White classified the typhoid-paratyphoid salmonellae into a number of groups according to antigenic structure and determined 65 somatic 0-antigens. For instance, S. typhi (group D) contains three different 0-antigens — 9, 12, and Vi.

Serological Classification of Bacteria of the Genus Salmonella

 

S. paratyphi A alone constitutes  group A, and S. schottmuelleri belongs to group B. It has been proved by F. Andrewes that the flagellar H-antigen is not homogeneous but is composed of two phases: phase 1 is specific and agglutinable by specific serum, phase 2 is non-specific and agglutinable not only by specific, but also by group sera. Salmonellae, which possess two-phase H-antigens, are known as diphasic, while those which possess only the specific H-antigen are monophasic.

Resistance. Typhoid and paratyphoid A and B salmonellae survive in ice for several months, in soil contaminated with faeces and urine of patients and carriers for up to 3 months, in butter, cheese, meat and bread for 1-3 months, in soil, faecal masses, and water for several weeks, and in vegetables and fruits for 5-10 days. They remain unaffected by desiccation and live for a  long time in dry faeces. Salmonellae survive for only a short time (3-5 days) in polluted water owing to the presence of a large number of saprophytic microbes and substances harmful to pathogenic microorganisms.

S. typhi and S. paratyphi A are susceptible to heat and are destroyed at 56° C in 45-60 minutes, and when exposed to the usual disinfectant solutions of phenol, calcium chloride, and chloramine, perish in several minutes. The presence of active chlorine in water in a dose of 0.5-1 mg per litre provides reliable protection from S. typhi and S. paratyphi A.

Pathogenicity for animals. Animals do not naturally acquire typhoid fever and paratyphoids. Therefore, these diseases are anthroponoses. A parenteral injection of the Salmonellae organisms into animals results in septicaemia and intoxication, while peroral infection produces no disease. E. Metchnikoff and A. Bezredka produced a disease similar to human typhoid fever by enteral infection in apes (chimpanzee).

Pathogenesis and diseases in man. The causative agent is primarily located in the intestinal tract. Infection takes place through the mouth (digestive stage).

Cyclic recurrences and development of certain pathophysiological changes characterize the pathogenesis of typhoid fever and paratyphoids.

There is a certain time interval after the salmonellae penetrate into the intestine, during which inflammatory processes develop in the isolated follicles and Peyer’s patches of the lower region of the small intestine (invasive stage).

As a result of deterioration of the defence mechanism of the lymphatic apparatus in the small intestine the organisms enter the blood (bacteriemia stage). Here they are partially destroyed by the bactericidal substances contained in the blood, with endotoxin formation. During bacteraemia typhoid salmonellae invade the patient’s body, penetrating into the lymph nodes, spleen, bone marrow, liver, and other organs (parenchymal diffusion stage). This period coincides with the early symptoms of the disease and lasts for a week.

During the second week of the disease endotoxins accumulate in Peyer’s patches, are absorbed by the blood, and cause intoxication. The general clinical picture of the disease is characterized by status typhosus, disturbances of thermoregulation, activity of the central and vegetative nervous systems, cardiovascular activity, etc.

On the third week of the disease a large number of typhoid bacteria enter the intestine from the bile ducts and Lieberkuhn’s glands. Some of these bacteria are excreted in the faeces, while others reenter the Peyer’s patches and solitary follicles, which had been previously sensitized by the salmonellae in the initial stage. This results in the development of hyperergia and ulcerative processes. Lesions are most pronounced in Peyer’s patches and solitary follicles and may be followed by perforation of the intestine and peritonitis (excretory and allergic stage).

The typhoid-paratyphoid salmonellae together with products of their metabolism induce antibody production and promote phagocytosis. These processes reach their peak on the fifth-sixth week of the disease and eventually lead to recovery from the disease.

Clinical recovery (recovery stage) does not coincide with the elimination of the pathogenic bacteria from the body. The majority of convalescents become carriers during the first weeks following recovery, and 3-5 per cent of the cases continue to excrete the organisms for many months and years after the attack and, sometimes, for life. Inflammatory processes in the gall bladder (cholecystitis) and liver are the main causes of a carrier state since these organs serve as favourable media for the bacteria, where the latter multiply and live for long periods. Besides this, typhoid-paratyphoid salmonellae may affect the kidneys and urinary bladder, giving rise to pyelitis and cystitis. In such lesions the organisms are excreted in the urine.

In one, two, or three weeks following marked improvement in the patient’s condition, relapses may occur as a result of reduced immunobiological activity of the human body and hence a low-grade immunity is produced.

Due to the wide range in the severity of typhoid fever from gravely fatal cases to mild ambulant forms it cannot be differentiated from paratyphoids and other infections by clinical symptoms. Laboratory diagnosis of these diseases is of decisive importance. In recent years typhoid fever has changed from an epidemic to a sporadic infection, being milder iature and rarely producing complications. In the USSR typhoid fever mortality has diminished to one hundredth that in 1913. Diseases caused by S. paratyphi are similar to typhoid fever. The period of incubation and the duration of the disease are somewhat shorter in paratyphoid infections than in typhoid fever.

Immunity. Immunity acquired after typhoid fever and paratyphoids is relatively stable but relapses and reinfections sometimes occur. Antibiotics, used as therapeutic agents, inhibit the immunogenic activity of the pathogens, which change rapidly and lose their O– and Vi-antigens.

Laboratory diagnosis. The present laboratory diagnosis of typhoid fever and paratyphoids is based on the pathogenesis of these diseases.

1.     Isolation of haemoculture.

Blood collection

 

Bacteraemia appears during the first days of the infection. Thus, for culture isolation 10-15 ml of blood (15-20 ml during the second week of the disease and 30-40 ml during the third week) are inoculated into 100, 150 and 200 ml of 10 per cent bile broth, after which cultures are incubated at 37° C and on the second day subcultured onto one of the differential media (Ploskirev’s, Endo’s, Levin’s) or common  meat-peptone agar.

The isolated culture is identified by inoculation into a series of differential media and by the agglutination reaction. The latter is performed by the glass-slide method using monoreceptor sera or by the test-tube method using purified specific sera.

2. Serological method. Sufficient number of agglutinins accumulate in the blood on the second week of the disease, and they are detected by the Widal reaction. Diagnostic typhoid and paratyphoid A and B suspensions are employed in this reaction. The fact that individuals treated with antibiotics may yield a low titre reaction must be taken into consideration. The reaction is valued positive in patient’s serum in dilution 1 : 200 and higher.

The Widal reaction may be positive not only in patients but also in those who had suffered the disease in the past and in vaccinated individuals. For this reason diagnostic suspensions of O– and H-antigens are employed in this reaction. The sera of vaccinated people and convalescents contain H-agglutinins for a long time, while the sera of patients contain O-agglutinins at the height of the disease.

In typhoid fever and paratyphoids the agglutination reaction may sometimes be of a group character since the patient’s serum contains agglutinins not only to specific but also to group antigens which occur in other bacteria. In such cases the patient’s blood must be sampled again in 5-6 days and the Widal reaction repeated. Increase of the agglutinin titre makes laboratory diagnosis easier. In cases when the serum titre shows an equal rise with several antigens, 0-, H-, and Vi-agglutinins are detected separately.

The Vi-agglutination reaction is employed for identification of S. typhi carriers. This reaction is performed with sera (inactivated at 56° C for 30 minutes and diluted in the ratio of 1:10-1:80) and diagnostic Vi-suspensions. Individuals who give a positive Vi-agglutination reaction are subjected to microbiological examination for isolation of S. typhi from the bile, faeces, and urine. The best results are obtained when Vi-haemagglutination is employed.

For quick serological diagnosis of typhoid fever and paratyphoids Nobel’s agglutination method and agglutination on glass by the Minkevitch-Brumpt method are carried out. In the latter case the bacterial emulsion is agglutinated in a drop of undiluted blood placed on a slide.

3. A pure culture is isolated from faeces and urine during the first, second, and third weeks of the disease. The test material is inoculated into bile broth, Muller’s medium, Ploskirev’s medium, or bismuth sulphite agar.

Isolation and identification of the pure culture are performed in the same way as in blood examination.

Selective media are recommended for isolation of the typhoid-paratyphoid organisms from water, sewage, milk, and faeces of healthy individuals. These media slightly inhibit the growth of pathogenic strains of typhoid-paratyphoid organisms and greatly suppress the-growth of saprophytic microflora.

A reaction for the detection of a rise in the phage titre is employed in typhoid fever and paratyphoid diagnosis. This reaction is based on the fact that the specific (indicator) phage multiplies only when it is in contact with homologous salmonellae. An increase in the number of phage corpuscles in the test tube as compared to the control tube is indicative of the presence of organisms homologous to the phage used. This reaction is highly sensitive and specific and permits to reveal the presence of the salmonellae in various substrates in 11-22 hours without the necessity of isolating the organisms in a pure culture. The reaction is valued positive if the increase in the number of corpuscles in the tube containing the test specimen is not less than 5-10 times that in the control tube.

When unagglutinable cultures of the typhoid and paratyphoid organisms are isolated, the agglutination reaction is performed using Vi-sera. If the latter are not available, the tested culture is heated for 30 minutes at 60° C or for 5 minutes at 100° C. The agglutination reaction is carried out with a suspension of this heated culture.

In some cases a bacteriological examination of duodenal juice (in search for carriers), bone marrow, and material obtained from roseolas is conducted.

Phage typing of typho-paratyphoid organisms is sometimes employed. The isolated culture is identified by type-specific O– and Vi-phages. Sources of typhoid and paratyphoid infections are revealed by this method.

Water is examined for the presence of typho-paratyphoid bacteria by filtering large volumes (2-3 litres) through membrane filters and subsequent inoculation on plates containing bismuth sulphite agar. If the organisms are present, they produce black colonies in 24-48 hours. The reaction of increase in phage titre is carried out simultaneously.

Treatment. Patients with typhoid fever and paratyphoids are prescribed chloramphenicol, oxytetracycline, and nitrofuran preparations. These drugs markedly decrease the severity of the disease and diminish its duration. Great importance is assigned to general non-specific treatment (dietetic and symptomatic). Treatment must be applied until complete clinical recovery is achieved, and should never be discontinued as soon as the bacteria disappear from the blood, urine, and faeces since this may lead to a relapse. Mortality has now fallen to 0.2-0.5 per cent (in 1913 it was 25 per cent).

The eradication of the organisms from salmonellae carriers is a very difficult problem.

Prophylaxis. General measures amount to rendering harmless the sources of infection. This is achieved by timely diagnosis, hospitalization of patients, disinfection of the sources, and identification and treatment of carriers. Of great importance in prevention of typhoid fever and paratyphoids are such measures as disinfection of water, safeguarding water supplies from pollution, systematic and thorough cleaning of inhabited areas, fly control, and protection of foodstuff’s and water from flies. Washing of hands before meals and after using the toilet is necessary. Regular examination of personnel in food-processing factories for identification of carriers is also extremely important.

In the presence of epidemiological indications specific prophylaxis of typhoid infections is accomplished by vaccination. Several varieties of vaccines are prepared: typhoid vaccine (monovaccine), typhoid and paratyphoid B vaccine (divaccine).

Good effects are obtained also with a chemical associated adsorbed vaccine which contains 0- and Vi-antigens of typhoid, paratyphoid B, and a concentrated purified and sorbed tetanus anatoxin. All antigens included in the vaccine are adsorbed on aluminium hydroxide.

A new areactogenic vaccine consisting of the Vi-antigen of typhoid fever Salmonella organisms has been produced. It is marked by high efficacy and is used in immunization of adults and children under seven years of age. When there are epidemiological indications, all the above-mentioned vaccines are used according to instructions and special directions of the sanitary and epidemiological service.

 

ADDITIONAL MATERIALS FOR DIAGNOSIS

INFECTION CAUSED BY SALMONELLAE OF TYPHOID AND PARATYPHOID FEVERS

Typhoid fever and paratyphoid infections A and B are acute hu­man infectious diseases attended by bacteremia, intoxication, and characteristic ulcerous-necrotic damage to the lymphatic apparatus of the small intestine. They can be distinguished by laboratory methods only. The causative agent of abdominal typhoid is Salmo­nella typhi, of paratyphus A, Salmonella paratyphi A, of paratyphus B,  Salmonella schottmuelleri {paratyphi B).

Bacteriological examination is of the key significance in the diagnosis of typhoid-paratyphoid diseases since it allows both iso­lation and typing of the causal organism. The material to be studied for diagnostic purposes may include blood, faeces, urine, bile, secre­tions from scarified roseolas, and, occasionally, a puncture sample of bone marrow, cerebrospinal fluid, pus from septic foci, necrosis-affected tissues, etc.

The earliest and most reliable technique of bacterial diagnosis is the isolation of the causal organisms from the blood, the haemoculture method. Salmonellae of typhoid and paratyphoid fevers persist in the blood throughout the febrile period and even during the first days of temperature normalization (particularly in vaccinated sub­jects with typhoid fever). At an early stage of the disease, the in­tensity of bacteremia is higher than at the end of the pyrexial pe­riod. This explains why 10 ml of blood is sufficient to perform exami­nation at the onset of the disease, whereas at later stages 15-20 ml of blood is required. The sample of blood aseptically obtained at the patient’s bedside is inoculated into 10 per cent bile broth or Rapoport’s medium (10 per cent bile broth supplemented with 1 per cent of mannitol or 2 per cent of glucose, and 1 per cent of Andrade’s indicator; a float is placed into the vial with the medium to capture the gas formed), with the blood-medium ratio being 1:10.

The inoculated vials are incubated at 37 °C for 18-24 hrs. Prolif­eration of salmonellae as a result of mannitol or glucose splitting with the formation of acid is signalled by an alteration in the pH of the medium (it acquires red colour). Propagation of paratyphoid salmonellae is accompanied by the formation of gas and acid.

From enrichment media the material is transferred onto an agar slant or Olkenitsky’s medium and into a plate with Endo’s medium (the second day of examination). Inoculation into a plate with En­do’s medium allows the isolation of a pure culture in cases where the enrichment medium has been  contaminated by air flora (non-sterile syringe, inadequate disinfection of the skin prior to blood taking, etc.).

The inoculated cultures are examined after 18-24-hour incubation in a heating block . The growth of salmonellae on Endo’s medium is characterized by the appearance of colourless lactose-negative colonies.

On Olkenitsky’s medium the typhoid salmonellae ferment glucose with the formation of acid (yellowing of the agar column), do not split lactose (the colour of the slanted portion of the agar does not change), and produce hydrogen sulphide (blackening of the medium at the  borderline between the agar column and the slanted surface). The paratyphoid salmonellae ferment glucose with the formation of acid (yellowing) and gas (rupture of the agar column).

After the nature of the growth has been evaluated and the purity of the culture determined, it is time to identify the culture.

Inoculate the isolated culture (the third day of the investigation) onto Hiss’ media and perform the agglutination test with aggluti­nating adsorbed sera (abdominal typhoid, paratyphoid A. and pa­ratyphoid B), and make wet-mount or hanging drop preparations to estimate motility of the microorganisms.

The hemoculture of the salmonellae of typhoid and paratyphoid fevers may contain the Vi-antigen. If there is no agglutination with O-sera, introduce Vi-sera or destroy the Vi-antigen. For this purpose, heat the obtained culture at 60 °C for 30 min or at 100 °C for 5 min.

On the fourth day of the study, read changes in the Hiss’ media (Table 3) and the final results of the agglutination reaction, and make conclusive report.

The enrichment medium with a blood inoculum is left in the incu­bator for several days since in some cases propagation of the causa­tive agent in bile media may be slow. Subculturing to an agar slant, Olkenitsky’s and Endo’s media, is performed every 24-48 hrs over a period of 7 to 10 days.

Salmonellae may be isolated from the blood in chronic bacteria carriers when they present a dramatic change in immunoreactivity (helminthiasis, malignant tumours, etc.).

The faeces to be tested are collected in sterile test tubes or jars in an amount of 5-10 g. In taking the material, care should be exer­cised to exclude the action of disinfectants. The samples are inoculated into one plate with Ploskirev’s medium. Salmonellae in the faeces may be found by the direct and indirect immunofluorescence tests.

On the second day (following 18-24-hour incubation) in a heating block, inspect for colourless, lactose-negative colonies, study their morphology and structure, and subculture to Olkenitsky’s medium.

On the third day of investigation, pure culture of the isolated bacteria is transferred onto Hiss’ media, and presumptive and then standard agglutination tests are conducted.

On the fourth day of the investigation, consider changes in the Hiss’ media and the agglutination test results, and make the report. Thus, identification of pure culture isolated from faeces (coproculture) does not differ from identification of a hemoculture.

To detect possible bacteria carriers, examination is performed in individuals with a history of typhoid and paratyphoid fevers, as well as among the staff of child-caring institutions, food-catering and water supply services. Prior to collection of faeces, those examined are to drink on a fasting stomach 100ml of 30 per cent solution of magnesium or sodium sulphate that possess bile-expelling action. Faecal matter to be tested is collected 2-4 hours after ingestion of a purgative. Inoculate onto Ploskirev’s medium and simultaneously onto an enrichment medium (bile broth, selenite broth, Kauffmann’s medium-tetrathionate, etc.). After a 6-hour incubation, subculture the latter to Ploskirev’s medium. Further investigation is conducted in the aforementioned manner.

Following centrifugation, urine and its deposit are inoculated onto Ploskirev’s medium and into bile broth for enrichment. In the presence of the characteristic growth identification is performed, using the same procedure as in the case of a hemo- and coproculture.

Duodenal contents, scraping of roseolas, and section material are studied and identified in the like manner.

Typing of phages and colicins of Salmonellae isolated from patients and carriers is helpful in establishing the source of contamination.

In serological diagnosis Widal’s reaction is employed. Antibodies to the causative agents of typhoid, paratyphoid A and paratyphoid B fevers can be recovered in the patient’s blood serum beginning from the 8th-10th day of the disease. To perform the Widal test, draw 2-3 ml of blood from a vein or 1 ml of blood from  a finger or an ear lobe and obtain serum.

Schematic Description of the Widal Reaction

 

 

Successively dilute the serum in three parallel rows of test. tubes from 1:100 to 1:1600 and introduce 0-diagnosticums (usual or erythrocyte ones) of Salmonella typhi into test tubes of the first row, of Salmonella paratyphi A into test tubes of the second row, and of Salmonella paratyphi B into test tubes of the third row. The use of 0-diagnosticums makes it possible to reveal 0-antibodies which appear in the blood during the second week of the disease and disappear by the end of the illness. The diagnostic titre of antibodies in the Widal test ion-immunized subjects is 1:100 and higher.

Demonstration of the H-antibodies is of no diagnostic value since they are detected during convalescence, and also in vaccinated individuals and those with a history of the disease.

In some cases O-antibodies may be recovered in vaccinated persons. Hence, it is necessary that the Widal test be performed over time to look for an increase in its titre.

If the patient’s blood serum agglutinates two or three types of diagnosticums simultaneously, the titre of agglutination should be taken into account. Typically, the specific agglutination occurs at larger and the group one at lower serum dilutions.

Currently, the onset of antibiotic treatment at early stages of the disease poses difficulty in evaluating the results of the agglutina­tion reaction since the antibody titre in patients is small and cannot be considered diagnostically significant.

The indirect haemagglutination test with erythrocyte monoreceptor diagnosticums O₉, O₁₂, and Vi is a more sensitive test, yielding-positive results in a greater number of cases. Antibodies to the 0-an-tigens are detected beginning from the second week of the disease. Antibodies to the Vi-antigens are recovered at later stages. Vi-anti-bodies occur most commonly in carriers of Salmonella typhi. To identify bacteria carriers, indirect haemagglutination with demon­stration of antibodies belonging to Immunoglobulins G is employed (a signal method).

Examination, of water. The causal organisms of typhoid and para­typhoid fevers are contained in water in minute quantities. To obviate this problem, one utilizes techniques allowing for their concentration in water. The best of these methods is examination with the help of membrane filters.

Two litres of water or more is poured through No 2 or No 3 membrane fil­ters. If the water contains a large number of suspended particles which hinder filtration, it is first passed through a No 6 nitrocellulose filter which arrests gross particles. Filters with a deposit are immersed in a bile broth or placed on a bis­muth-sulphite agar. After a 6-10-hour incubation, subculture from the bile broth to Ploskirev’s medium. Further procedures are the same as in examination of faeces. After a 48-hour incubation in a heating block, harvest black colonies from the bismuth-sulphite agar and identify them in the way salmonellae are identified.

Examination of water for the presence of typhoid and paratyphoid phages is employed in cases where bacteria evade detection. The sewage is passed through a bacterial filter. Into a sterile Petri dish place 1-2 ml of the filtrate test­ed, pour in l5-20ml of meat-peptone agar cooled to 45 °C, and mix thoroughly. After the agar has solidified, streak (in sectors) cultures of salmonellae causing typhoid, paratyphoid A and paratyphoid B fevers. The appearance of negative colonies confirms the presence of the corresponding phages.

To examine drinking water, introduce it into a concentrated peptone solu­tion. To 100 ml of the water to be tested add 10 ml of peptone and 5 g of sodium chloride. Place the inoculated cultures in an incubator for 24 hours and then examine the filtrate for the presence of the phage.

Along with bacteriological and serological methods, an intra-cutaneous allergy test with the Vi-typhine of typhoid bacteria is used. This test becomes positive during recovery and may be utilized for retrospective diagnosis.

Salmonellae — Causative Agents of salmonellosis.  The genus Salmonella comprises many species and types of bacteria which possess properties similar to those of S. schottmuelleri. In 1885 in America D. Salmon isolated the bacterium S. cholerae-suis, which was long  considered the causative agent of plague in pigs. Later it was shown to be in association with the causative agent of this disease and the cause of human toxinfections. In 1888 during a large-scale outbreak of toxinfections in Saxony A. Gartner isolated S. enteritidis bacteria from the flesh of a cow which had to be killed, and also from the spleen of a dead person. The organisms proved to be pathogenic for mice, guinea pigs, rabbits, sheep, and goats. In 1896 in Breslau K. Kensche and in 1898 in Ertike G. Nobel discovered S. typhimurium (Bacillus Breslau) in cases of food poisoning and isolated a pure culture of the organism. It is now known that among the large number of organisms which comprise the salmonella group, about 440 species and types are pathogenic for humans and are the cause of food poisoning (toxinfections).

Morphology. Morphologically Salmonella organisms possess the general characteristics of the family Enterobacteriaceae. They are motile and peritrichous.

Cultivation. The organisms are facultative aerobes, the optimum temperature for growth being 37° C. They grow readily on ordinary nutrient media.

Fermentative properties. Salmonellae do not liquefy gelatine and do not produce indole. The majority of species produce hydrogen sulphide and ferment glucose, maltose, and mannitol, with acid and gas formation.

Toxin production. Salmonellae produce no exotoxin. Their ability to cause diseases in animals and humans is associated with an endotoxin which is a gluco-lipo-protein complex and is characterized by its high toxicity.

Antigenic structure. As was mentioned above, all salmonella® are divided into 65 groups according to their serological properties (see Table 4, Methodological Instructioo 35). Thus, according to the Kauffmann-White Scheme, S. enteritidis belongs to group D, S. typhimurium to group B, and S. cholerae-suis to group C.

Classification. The organisms are classified according to their antigenic, cultural, and biological properties (see Methodological Instructioo 35).

Virulence Factors of Salmonella Organisms. It is surprising that virulence factors for organisms that have caused so much disease still arc largely unknown. However, the ability to invade and grow inside of non-phagocytic cells undoubtedly comprises the major virulence determinant of the Salmonella because this intracellular location provides a compartment where they can replicate and avoid host defences. The mechanism whereby these bacteria accomplish this invasion is complex and only beginning to unfold.

Using various mutants of Salmonella typhimurium, John Pace and colleagues at the State University of New York determined that invasion of a host cell occurs in two separable steps: (1) adhesion to the host cell, and (2) invasion of the host cell. Furthermore, they found that invasion required that the organisms activate a growth factor receptor on the host cell known as epidermal growth factor receptor (EGFR). Mutants that could adhere, but not invade, were unable to activate EGFR. However, if EGF was added to the host cell-bacterium mixture, the EGFR was activated and the noninvasive mutant was internalized.

When EGFR is activated, a signal transduction process occurs, which results in at least two major events: (1) a rapid rise in the internal Ca²⁺ level occurs, and (2) enzymes are activated that lead to the synthesis of leukotriene D₄ (LTD₄). It is unclear how these events trigger the entry of Salmonella into the cell, but it is known that the Ca²⁺ level increase is essential because the addition of Ca²⁺ chelators blocked entry of the bacterium into the cell. It is also known that the addition of LTD₄ to cultured cells causes an increase in intracellular Ca²⁺ levels, permitting the internalization of an invasion-deficient mutant.

One can postulate, therefore, that the mediation of Ca²⁺ influx by LTD4 results in the opening of a Ca²⁺ channel, which, in turn, causes a reorganization of the host cell cytoskeleton, permitting entry of the bacterium.

It is also of note that the inflammatory diarrhea produced by the Salmonella may result from its ability to induce leukotriene synthesis because leukotrienes are well-known mediators of inflammation.

It is also known that a number of Salmonella, serotypes carry plasmids that greatly increase virulence in experimentally infected mice. Although many of these plasmids are distinct, all have a highly conserved 8-kb region that has beeamed the spv regulon. Interestingly, spv genes are not expressed during logarithmic growth in vitro but seem to enhance the growth of salmonellae within host cells. In experimentally infected mice, the expression of spv by intraccelular salmonellae in vivo has been postulated to lead to an increased rate of bacterial growth, resulting in early bacteremia and death before the infected mice can develop immunity.

The general types of infections that may be caused by the salmonellae usually are grouped into three categories: enterocolitis, enteric fevers, and septicemia.

Resistance. Salmonellae are relatively stable to high temperatures (60-75 °C), high salt concentrations, and to certain acids. They with stand 8-10 per cent solution of acetic acid for 18 hours, and survive for 75-80 days at room temperature. The endotoxins remain active within large pieces of meat for long periods (even after the meat has been cooked) as well as in inadequately fried rissoles and other foods.

A characteristic feature of foodstuffs contaminated by Salmonellae is that they show no changes which can be detected organoleptically.

Pathogenicity for animals. Salmonellae, the causative agents of toxinfections, are pathogenic micro-organisms which may give rise to paratyphoid in calves, typhoid and paratyphoid iewly-born pigs, typhoid in fowls and pullorum disease in chickens, typhoid in mice and rats, and enteritis in adult cattle.

Among laboratory animals, white mice are most susceptible to the organisms (S. typhimurium, S. enteritidis, S. cholerae-suis, etc.). Enteral and parenteral inoculations result in septicaemia in these animals.

Pathogenesis and diseases in man. Ingestion of food contaminated by salmonellae is the main cause of disease. Most frequently food poisoning is due to meat prepared from infected animals and waterfowls without observance of culinary regulations. Eggs of infected waterfowls are also sources of infection. Seabirds are frequent Salmonellae carriers. Meat may be infected while the animal is alive or after its death.

As distinct from typhoid fever and paratyphoids A and B, salmonellae toxinfections are anthropo-zoonotic diseases. S. typhimurium, S. cholerae-suis, S. Heidelberg, S. enteritidis, S. anatum, S. newport. S. derby, and others cause clinically manifest forms. Intoxication develops in a few hours following infection. Masses of microbes ingested with the food are destroyed in the gastro-intestinal tract and m me blood. This results in the production of large amounts of endotoxin which, together with the endotoxin entering the body with the ingested food, gives rise to intoxication. Salmonellae are known to be highly infestive. Bacteremia usually becomes manifest in the first hours after the onset of the disease.

The disease course is characterized by clinical manifestation of toxinfectional, gastroenteric, and typhoid- and cholera-like symptoms.

Along with typical zoonotic salmonella diseases, there are salmonelloses which occur as a result of infection from sick people and carriers. Such cases are predominant iewborn and prematurely born children, convalescents, and individuals with chronic diseases. In children’s institutions, maternity hospitals, somatic departments of pediatric clinics, and among children suffering from dysentery in departments for contagious diseases the main sources of infection are sick children and bacteria carriers. Children suffering from salmonelloses display symptoms of dyspepsia, colitis (enterocolitis), and typhoid fever, and often these conditions are accompanied by septicaemia and bacteremia. The diseases are of long duration or become chronic and are sometimes erroneously diagnosed as chronic dysentery.

Immunity acquired after salmonellosis is of low grade and short duration. Low titres of agglutinins (from 1:50 to 1 :400 and, rarely, up to 1:800) appear in the blood of convalescents during the second week.

Laboratory diagnosis. Specimens of food remains, washings from objects, stools, vomit, lavage water, blood, urine and organs obtained at autopsy are carefully collected and examined systematically. In the beginning, the specimens are inoculated into nutrient media employed for diagnosis of typhoid fever and paratyphoids A and B. Then the cultural, serological, and biological properties of the isolated cultures are examined (Table 3, Methodological Instructioo 35).

In some cases the biological test is performed not only with the cultures, but also with remains of the food which caused the poisoning.

For retrospective diagnosis blood of convalescents is examined for the presence of agglutinins on the eighth-tenth day after the onset of disease. This is performed by the Widal reaction with suspensions of the main diagnostic bacterial species which cause food toxinfections.

Table 4 (Methodological Instructioo 35) shows that differential laboratory diagnosis between S. typhimurium and S.  schottmuelleri is particularly difficult since they have group, somatic, and flagellar phase 2 antigens in common. Pathogenicity for white mice and appearance of mucous swellings and daughter colonies on agar serve as differential criteria.

Treatment. Therapeutic measures include antibiotics (chloramphenicol, oxytetracycline and tetracycline). Good effects are also obtained with stomach lavage, injections of glucose and physiological solution, and cardiac drugs.

Prophylaxis of salmonellae toxinfections is ensured by veterinary and sanitary control of cattle, slaughter-houses, meat factories and fish industries, laboratory control of meat intended for sale, and sterilization of meat which otherwise may not be sold. The medical hygiene service identifies carriers among people working in food factories, catering houses, and other food-processing establishments and controls the sanitary regulations at food enterprises, shops, store-houses, and in catering houses.

 

Diagnosis OF SALMONELLAL GASTROENTERITIS

(FOOD POISONING)

The primary reservoir for the salmonellae is the intestinal tracts ot many animals, including birds, farm animals, and reptiles. Humans become infected through the ingestionot contaminated water or food. Water, of course, becomes polluted by the introduction of feces from any animal excreting salmonellae. Infection by food usually results either from the ingestion of contaminated meat or by way of the hands, which act as intermediates in the transfer of salmonellae from an infected source. Thus, the handling of an infected – although apparently healthy – dog or cat can result in contamination with salmonellae. An-other major source of Salmonella infections has been pet turtles. In the early 1970s, almost 300,000 cases of turtle-associated salmonellosis were estimated to occur annually in the United States and, as a result, it is illegal to import turtles or turtle eggs or even to ship domestic turtles with shells less than 4 inches in diameter across state lines.

In the United States, poultry and eggs increasingly comprise the most common source of salmonellae for humans T his occurs because a large percentage of chickens routinely are infected with salmonellae. Thus, humans can acquire these organisms through direct contact with uncooked chicken or by the ingestion of undercooked chicken. And, because the organisms may occur both on the outer shell and in the yolk and egg white, consuming anything containing raw eggs (caesar salad, hollandaise sauce, mayonnaise, homemade ice cream) could result in a Salmonella infection. The CDC even cautions agains teating eggs sunny-side up and recommends that eggs be boiled for 6 to 7 minutes before being served.

On an industrial scale, slaughterhouse workers are faced with salmonellosis as an occupational hazard, primarily from poultry and pigs. Because humans can become asymptomatic carriers of Salmonella, infected food handlers also are responsible for the spread of these organisms.

Salmonella enterocolitis is one of the most frequent cause of food-borne outbreaks of gastroenteritis in the United States. It may be caused by any one of the hundreds of serotypes of Salmonella, and it is characterized by the fact that organisms do not cause an appreciable bacteremia. The hallmark of all Salmonella infections lies in the ability of the Salmonella to invade the intestinal epithelial cells, which are normally nonphagocytic. Those species involved in gastro-enteritis may reach the bloodstream early in the disease but arc rapidly taken up and killed by phagocytic cells. In general, bacteremia occurs only in persons having an impaired phagocyte system, AIDS, or chronic granulomatous disease. In the average case, symptoms of diarrhea may occur 10 to 28 hours after ingesting contaminated food, and the headache, abdominal pain, nausea, vomiting, and diarrhea may continue for 2 to7 days.

A search for salmonella toxins has not been as conclusive as one might wish, but there are multiple reports that many Salmonella species secrete a cholera-like enterotoxin that induces increased levels of cAMP, and that some strains produce a heat-stable enterotoxin. In addition, a cytotoxin that inhibits protein synthesis in intestinal epithelial cells has been described This toxin, characterized by its ability to kill Vero cells, is immunologically distinct from both Shiga toxin and the Shiga-like toxins produced by strains of E. coli and Shigella. The observation that those species of Salmonella causing the more severe enteric symptoms and inflammatory diarrhea also produce the highest levels of cytotoxin suggests that this toxin may be of paramount importance in the pathologic manifestations of gastrointestinal salmonellosis. Neither the molecular structure, its specific mechanisms of blockingprotein synthesis and causing cell death, nor the number of such Salmonella cytotoxins is known

Most cases of Salmonella enterocolitis formerly had not been treated with antibiotics because such treatment did not seem to shorten the duration of the infection. There have been reports, however, that the fluoroquinolones do decrease the period of illness but, interestingly, they do not eradicate the organisms from the intestinal tract.

At present, there are over 400 serovars of salmonellae known to be pathogenic for man and capable of inducing acute gastroenteritis. The most important of them are S. typhimurium, S. enteritidis, S. cholerae-suis, S. gallinarium, etc. (about 60 serovars). The material subject to laboratory examination includes vomited matter, waters from stomach lavage, bile, urine, cerebrospinal fluid, puncture sam­ple of the bone marrow, blood (in the first hours of the disease, for isolat­ing a haemoculture, and then in two weeks, usually after recovery, for demonstrating antibodies).

To identify carriers among the staff of food-catering or child-caring institutions, etc., samples of faeces are collected following the ad­ministration of a purgative. At autopsy one collects the contents of the stomach and intestines, heart blood, pieces of parenchymatous organs, and mesenteric lymph nodes.

In food toxinfections one should examine the remains of the food, foodstuffs from which it has been prepared, washings off from the tables, preparation boards, hands of the catering personnel, etc.

The material is collected in sterile jars and test tubes in the fol­lowing amounts: faeces and vomit, 50-100 ml; lavage waters, 100-200 ml; meat and meat products, several pieces weighing about 500 g; semi-solid and liquid foodstuffs (cream, milk, etc.), 100-200 ml.

Before being sent to the laboratory, the above materials are packed and sealed. Integrity of the packing or otherwise is noted in a special register. Before the examination, material that cannot be inoculated without preliminary treatment (e.g., solid faecal and vomited mat­ter, food remains, etc.) is ground in a porcelain mortar and suspend­ed in isotonic saline. The surface of the tested meat, sausage, and cheese is sterilized by applying to it a red-hot metallic spatula, and samples are cut from the depth, placed into a porcelain mortar with glass sand, and isotonic salt solution is added.

Bacteriological examination. To isolate a haemoculture of sal­monellae, the blood is introduced into a bile broth. The vomit, faeces, section material, pus, cerebrospinal fluid, foodstuffs, and washings off are inoculated into plates with Ploskirev’s medium and in enrichment media (bile broth and selenite medium) from which subinoculation is made into Ploskirev’s medium in 6-10 hrs. The inoculated cultures are incubated at 37 “C for 24 hrs. after which they are examined, colourless lactose-negative colonies are selected and transferred to OIkenitsky’s triple sugar medium or to an agar slant to enrich for pure culture. On the third day of the in­vestigation, the isolated pure cultures are identified: they are inocu­lated into Hiss’ cultures and the agglutination test with adsorbed group sera (A, B, C, D, E) is performed. If a positive result has been obtained with one of serum groups, one makes the agglutination test with the adsorbed 0-sera typical for the given group and then with monoreceptor H-sera (non-specific and specific phases) in order to determine the species and serovars of bacteria. For example, if the studied culture has agglutinated with a group B-serum, it is nec­essary to perform the agglutination test with sera against O, and OB antigens, which are typical of this group. If agglutination has been positive, the H-monoreceptor sera are utilized.

On the fourth day of the investigation, changes in Hiss’ media are assessed. The causative agents of salmonellal gastroenteritis, similar to the salmonellae responsible for paratyphus A and B, do not ferment lactose and sucrose, split glucose, mannitol, and maltose with the formation of acid and gas, do not form indol and, with minor excep­tions, release hydrogen sulphide.

Salmonella cultures can most frequently be isolated from patients’ faeces, somewhat less commonly, from vomit and stomach washings, and even less often from blood, urine, and bile. The results of bacte­riological examination of various biosubstrates are of varying diag­nostic significance. Isolation of salmonellae from the blood, bone mar­row, cerebrospinal fluid, vomit, and waters from the stomach lavage is a definite confirmation of the diagnosis. On the other hand, de­tection of salmonellae in the faeces, urine, and bile may be related to a bacteria carrier-state. The aetiological role of salmonellae in the development of gastroenteritis is corroborated by an increased titre of specific antibodies in an agglutination reaction with an autestrain.

Biological examination. Salmonellae of food poisoning, in con­trast to salmonellae of paratyphi A, are pathogenic for white mice. This property is used for the differentiation between the two types. On the first day of examination, along with inoculation of the patho­logical material and foodstuffs, white mice are infected per os. One-two days later the mice die of septicaemia. Post-mortem examination demonstrates a sharply enlarged spleen and, occasionally, liver, while inoculation of the blood from the heart and samples from the internal organs permits isolation of salmonella culture.

The agglutination reaction and indirect haemagglutination test are employed for serological diagnosis. These may be carried out from the first days of the disease and should be repeated in 7-10 days to determine whether the titre of specific antibodies tends to in­crease. In conducting these tests, salmonellal polyvalent and group (group A, B, C, D, E) diagnosticums (corpuscular and erythrocyte) are utilized.

A two-four-order elevation of the antibody titre is of diagnostic importance.

Salmonella Septicemia

Septicemia caused by Salmonella is a fulminating blood infection that does not involve the gastrointestinal tract. Most cases are caused by S. choleraesuis and are characterized by suppurative lesions throughout the body. Pneumonia, osteomyelitis, or meningitis may result from such an infection. Salmonella osteomyelitis is especially prevalent in persons who have sickle cell anemia, and focal infections, particularly on vascular prosthesis, also are common.

 

ShigellaE. LABORATORY DIAGNOSIS of SHIGELLOSIS. 

LABORATORY DIAGNOSIS of CHOLERA. CAmpYlobacter, helicobacter and aeromonas infections

 

SHIGELLAE

The causative agent of dysentery was described in 1888 by A. Chantemesse and in 1891 by A. Grigoryev and F. Widal. In 1898 this organism was studied in detail by K. Shiga in Japan and in 1900-1901 by V. Kruse in Germany (Shiga bacillus). In 1900 S. Flexner and R. Strong in the Philippines isolated dysentery organisms (Flexner bacillus) which possessed properties different to those of the above-mentioned bacillus. In 1904 P. Hiss and F. Russel described a dysentery bacterium which became to be known as the Hiss-Russel bacillus. At present this organism is included in the Flexner species. In 1904 K. Duval, in 1907 V. Kruse and others, and in 1915 K. Sonne recognized a dysentery bacillus which ferments lactose. In 1917 M. Stutzer in Russia and K. Schmitz in Rumania simultaneously isolated another species of dysentery bacilli (Stutzeri-Schmitzii bacillus). Later other bacilli causing dysentery were discovered. According to the current International Nomenclature, all dysentery bacilli are grouped together in one genus known as Shigella.

Morphology. Morphologically dysentery bacilli correspond to the organisms of the family Enterobacteriaceae. Dysentery bacilli have no flagella and this is one of the differential characters between these organisms and bacteria of the coli-typhoid-paratyphoid group. Some strains of Flexner bacilli are found to possess cilia.

Cultivation. Dysentery bacilli are facultatively aerobic and grow readily on common media at pH 6.7-7.2, the optimum temperature for growth being 37° C, they do not grow at 45° C. On solid media they form small (1-1.5 mm in diameter), fragile, semitransparent colonies which are similar to those of the typhoid bacteria. In meat broth dysentery bacilli produce a diffuse turbidity.

 

 

Fermentative properties. None of the species of dysentery bacilli liquefy gelatior produce hydrogen sulphide. They ferment glucose, with acid formation, with the exception of the Newcastle subspecies which sometimes produce both acid and gas during this reaction. With the exception of the Sonne bacilli, none of them ferment lactose.

Table

Shigellae Biochemical Properties

Note: “+”, fermentation of carbohydrates, formation of undol and catalase; “–”, the absence of carbohydrate fermentation and indol and ornithine formation; “±”,  weak for­mation of indol and ornithine and weak carbohydrate fermentation.

 

Toxin production. S. dysenteriae produce thermolabile exotoxin which displays marked tropism to the nervous system and intestinal mucous membrane. This toxin may be found in old meat broth cultures, lysates of a 24-hour-old agar culture, and in desiccated bacterial cells.

An intravenous injection of small doses of the exotoxin is fatal to rabbits and white mice. Such an injection produces diarrhoea, paralysis of the hind limbs, and collapse.

The dysentery exotoxin causes the production of a corresponding antitoxin. The remaining types of dysentery bacilli produce no soluble toxins. They contain endotoxins, which are of a gluco-lipo-protein nature, and occur in the smooth but not in the rough variants.

Thermolabile substances exerting a neurotropic effect were revealed in some S. sonnei strains. They were extracted from old cultures by treating the latter with trichloracetic acid.

Antigenic structure. Dysentery bacilli are subdivided into 4 subgroups within which serovars may be distinguished. The antigenic structure of shigellae is associated with somatic 0-antigens and surface K-antigens.

Classification. Dysentery bacilli are differentiated on the basis of the whole complex of antigenic (Table) and biochemical (Table 2) properties. S. sonnei have four fermentative types which differ in the activity of ramnose and xylose and in sensitivity to phages and colicins.

Table

International Classification of Shigellae

 

 

Resistance. Dysentery bacilli live in the external environment for a period of 5-10 days (in soil, foodstuffs and water, and on objects, plates and dishes). Direct sunlight and a 1 per cent phenol solution destroy the organisms in 30 minutes and at a temperature of 60° C the organisms perish in 10 minutes. The bacilli are easily killed by treatment with chloramine and calcium chloride solutions. The Shiga bacilli are most sensitive to physical and chemical factors, while the Sonne bacilli are relatively resistant to them. Dysentery bacilli may acquire resistance to drugs (sulphonamides, antibiotics) and to ionizing radiation.

Pathogenicity for animals. Monkeys are susceptible to dysentery bacilli. They contract the infection from sick people or carriers in the nurseries. In some cases they become sources of contamination of personnel iurseries and zoological gardens.

Parenteral infection causes fatal toxicosis in rabbits. An intravenous injection of a S. dysenteriae culture exerts a highly toxic effect. The resulting infection constitutes diarrhoea, paresis or paralysis of the limbs, followed by collapse and death. Autopsy reveals hyperaemia of the intestinal mucous membrane, haemorrhages, necrosis, and ulcerations. Infected white mice die within the first four days.

When cultures of virulent dysentery bacilli are introduced into the respiratory tract of white mice, the organisms multiply. However, attempts to reproduce dysentery in white mice are of no success. Kittens and puppies are more susceptible. Guinea pigs display low susceptibility to dysentery bacilli, but infection through the eye conjunctiva results in keratoconjunctivitis which is assumed to be a specific lesion.

Epidemiology and Pathogenesis of Shigellosis. Humans seem to be the only natural hosts for the shigellae, becoming infected after the ingestion of contaminated food or water. Unlike Salmonella, the shigellae remain localized in the intestinal epithelial cells, and the debilitating effects of shigellosis are mostly attributed to the loss of fluids, electrolytes, and nutrients and to the ulceration that occurs in the colon wall.

It has been known for many years that Shigella dysenteriae type 1 secreted one or more exotoxins (called Shiga toxins), which would cause death when injected into experimental animals and fluid accumulation when placed in ligated segments of rabbit ileum. These toxins are essentially identical to the Shiga-like toxins produced by the EIEC and the EHEC. Thus, Shiga toxin consists of one A subunit and five B subunits and seems to kill an intestinal epithelial cell by inactivating the 60S ribosomal subunit, halting all protein synthesis. Moreover, although all virulent species of Shigella produce Shiga toxins, there seems to be a wide variation in the amount of toxin formed.

The mechanism whereby Shiga toxin causes fluid secretion is thought to occur by blocking fluid absorption in the intestine. In this model, Shiga toxin kills absorptive epithelial cells, and the diarrhea results from an inhibition of absorption rather than from active secretion.

Of note is that, like the EHEC, Shigella species can cause HUS. Moreover, Shiga-like toxins have been detected in certain strains of Vibrio cholerae and Vibrio parahaemolyticus that were associated with HUS, indicating an important role of Shiga toxin in this malady. There has also been a report indicating that tumor necrosisfactor-alpha acts synergistically with Shiga toxin to induce HUS.

To cause intestinal disease, shigellae must invade the epithelial cells lining of the intestine. After escaping from the phagocytic vacuole, they multiply within the epithelial cells in a manner similar to that described for EIEC strains. Thus, Shigella virulence requires that the organisms invade epithelial cells, multiply intracellularly, and spread from cell to cell by way of finger-like projections to expand the focus of infection, leading to ulceration and destruction of the epithelial layer of the colon. Interestingly, for Shigella to be fully invasive, both plasmid and chromosomally encoded products seem to be required. The invasion plasmids is identical for the Shigella and the EIEC and contains at least four genes, IpaA, IpaB, IpaC, and IpaD that encode for a series of proteins termed invasion-plasmid antigens, which arc involved in the virulence of these organisms. Interestingly, IpaB acts both as an invasin that triggers phagocytosis of the bacterium and as a cytolysin that allows escape from the phagocytic vacuole. The elaboration of toxic products causes a severe local inflammatory response involving both polymorphonuclear leukocytes and macrophages, resulting in a bloody, mucopurulent diarrhea.

During 1990, over 27,000 cases of shigellosis were reported to the Centres for Disease Control (CDC) and, of these, the most prevalent species in the United States was S sonnei. The disease produced by this species is transmitted by a fecal-oral route, and most of patients are preschool-age children, particularly those in day-care centres.

Immunity. Immunity acquired after dysentery is specific and type-specific but relatively weak and of a short duration. For this reason the disease may recur many times and, in some cases, may become chronic. This is probably explained by the fact that Shigella organisms share an antigen with human tissues.

Laboratory diagnosis. Reliable results of laboratory examination depend, to a large extent, on correct sampling of stool specimens and its immediate inoculation onto a selective differential medium. The procedure should be carried out at the patient’s bedside, and the plate sent to the laboratory.

In hospital conditions the stool is collected on a paper plate or napkin, placed into a bedpan. The latter should be washed previously with running water or, better still, with boiling water, be dry, and should contaio disinfectants. It is best to collect the faeces directly from the rectum by means of a rectal tube or rectal swab. The specimen should be sown in the isolation department immediately after collection. Portions of the stool, containing pus and mucus, are picked out with a swab and plated on Ploskirev’s medium. The plates are incubated at 37°C for 24 hours The isolated pure culture is identified by its biochemical and serological properties.

An accelerated method of dysentery diagnosis is employed to shorten the examination period. In some cases an agglutination reaction, similar to the Widal reaction, is used. This test is relevant to retrospective diagnosis.

The nature of the isolated culture may be determined m some cases by its lysis by a polyvalent dysentery phage and by the reaction of passive haemagglutmation as well as by the method of immunofluorescence. This method is used for demonstrating antigens of Shigella organisms in smears from faeces or in colonies by means of specific sera treated with fluorochromes.

An allergic test consisting in intracutaneous injection of 0.1 ml of dysenterin is applied in the diagnosis of dysentery in adults and children. Hyperaemia and a papule 2 to 3.5 cm in diameter develop at the site of the injection in 24 hours in a person who has dysentery. The test is strictly specific.

Treatment and Control of Shigellosis. Intravenous replacement of fluids and electrolytes plus antibiotic therapy are used for severe cases of shigellosis. Ampicillin frequently is not effective, and alternative therapies include sulfamethoxazole/trimethoprim and, with increasing sulfamethoxazole/trimethoprim resistance, the quinolone antibiotics such as nalidixic acid and ciprofloxacin. In the Far East, India, and Brazil where shigellosis is more common than in the United States, multiple antibiotic resistance because of the acquisition of plasmids has become common. Shigellosis also is common in Latin America.

Efforts to control the disease usually are directed toward sanitary measures designed to prevent the spread of organisms. This is particularly difficult in view of the fact that many persons remain asymptomatic carriers after recovery from an overt infection. Such individuals provide a major reservoir for the spread of the shigellae.

The injection of killed vaccines is worthless, because humoral IgG does not seem to be involved in immunity to the localized intestinal infection. Live vaccines that cannot grow in the absence of streptomycin (ie, streptomycin-dependent vaccines) have been developed and used in clinical trials, but success has been equivocal. It seems that the organisms must invade and colonize the intestine to induce a local immunity. An engineered vaccine designed to induce this type of immunity used an avirulent E coli K12 into which was transferred a 140-megadalton plasmid obtained from a virulent strain of Shigella flexneri. The transfected plasmid endowed the E. coli K12 strain with the ability to invade intestinal epithelial cells, and its use as an oral vaccine in monkeys conferred significant protection against oral challenge with virulent S flexneri. Acquired immunity seems to result from both a cell-mediated immune response and an IgA antibody production.

Interestingly, human milk contains a globotriaosylceramide that binds to Shiga and Shiga-like toxins. This suggests that human milk could contribute to a protective effect by preventing these toxins from binding to their intestinal target receptors.

Thus, dysentery control is ensured by a complex of general and specific measures; (1) early and a completely effective clinical, epidemiological, and laboratory diagnosis; (2) hospitalization of patients or their isolation at home with observance of the required regimen; (3) thorough disinfection of sources of the disease; (4) adequate treatment of patients with highly effective antibiotics and use of chemotherapy and immunotherapy; (5) control of disease centres with employment of prophylaxis measures; (6) surveillance over foci and the application of prophylactic measures there; (7) treatment with a phage of all persons who were in contact with the sick individuals; (8) observance of sanitary and hygienic regimens in children’s institutions, at home and at places of work, in food industry establishments, at catering establishments, in food stores.

 

Additional materials for diagnosis

Dysentery is an infectious disease with the predominant involve­ment of the large intestine and general intoxication caused by bacteria of the genus Shigella: S. dysenteriae, S. sonnei, S. flexneri, S. boydii.

Material used for isolating the causal organism of dysentery in­cludes faeces of patients, convalescents, and carriers, less frequently, vomited matter and waters from stomach and intestine lavage. Shigellae may be recovered in washings off hands, cutlery and crockery, and various other objects (toys, door handles, etc.) as well as in milk and other foodstuffs. The  results of laboratory examina­tion depend to a large degree on the correct procedure of material collection. The following rules should be strictly adhered to: (1) carry out bacteriological examination of faeces before aetiotropic therapy has been initiated; (2) collect faecal samples (mucus, mucosal admixtures) from the bedpan and with swabs (loops) directly from the rectum (the presence in the bedpan of even the traces of disin­fectants affects the results of examination); (3) inoculate without delay the collected material onto enrichment media, place them into an incubator or store them in preserving medium in the cold; (4) send the material to the laboratory as soon as possible.

Bacteriological examination. Faecal samples are streaked onto plates with Ploskirev’s medium and onto a selenite medium con­taining phenol derivatives, beta-galactosides, which retard the growth of the attendant flora, in particular E. coli. The inoculated cultures are placed into a 37 °C incubator for 1S-24 hrs. The nature of tile colonies is examined on the second day.

Colourless lactose-negative colonies are subcultured to Olkenitsky’s medium or to an agar slant to enrich for pure cultures. On the third day, examine the nature of the growth on Olkenitsky’s medium for changes in the colour of the medium column without gas formation. Subculture the material to Hiss’ media with malonate, arabinose, rhamnose, xylose, dulcite, salicine, and phenylalanine. Read the re­sults indicative of biochemical activity on the following day. Shigel­lae ferment carbohydrates with the formation of acid (Table 2).

For serological identification the agglutination test is performed first with a mixture of sera containing those species, and variants of Shigellae that are prevalent in a given area, and then the slide ag­glutination test with monoreceptor species sera.

To determine the species of Shigellae, one can employ the following tests:

1. Direct and indirect immunofluorescence test.

2. The coagglutination test which allows to determine the specificity of the causative agent by a positive reaction with protein A of staphylococci coated with specific antibodies. On a suspected colony put a drop of specific sensitized protein A of Staphylococcus aureus, then rock the dish and 15 min later examine it microscopically for the appearance of the agglutinate (these tests may also be carried out on the second day of the investigation with the material from lactose-negative colonies).

3. Another test, which is highly specific for dysentery, is ELISA. For the epidemiological purpose the phagovar and colicinovar of Shigellae are also identified.

To determine whether the isolated cultures belong to the genus Shigella, perform the keratoconjunctival test on guinea pigs. In contrast to causal organisms of other intestinal infections, the dy­sentery Shigellae cause marked keratitis.

Depending on the findings obtained, the presence of Shigella bac­teria in the test material is either confirmed or ruled out.

For the serological diagnosis of dysentery the indirect haemagglutination (IHA) test with erythrocyte diagnosticums with the titre of 1:160 and higher is performed. The test. is repeated after at least seven days. Diagnostically important is a four-fold rise in the anti­body litre, which can be elicited from the 10th-12th day of the disease. To distinguish between patients with subclinical forms of the disease and Shigella carriers, identify immunoglobulins of the G class.

An allergy intracutaneous test with Tsuverkalov’s dysenterine is of supplementary significance. It becomes positive in dysentery patients beginning with the fourth day of the disease. The result is read in 24 hrs by the size of the formed papula. The test is consid­ered markedly positive in the presence of oedema and skin hyperaemia 35 mm or more in diameter, moderately positive if this diameter is 20-34 mm, doubtful if there is no papula and the diameter of skin hyperaemia measures 10-15 mm, and negative if the hyperaemic area is less than 10 mm.

Another technique that can be employed is determination of the indicator of neutrophil damage in the presence of dysenterine.

Examination of water, milk, and washings off various objects for Shigellae is conducted utilizing the above mentioned techniques. Of especial importance for examination of these objects is the test aimed at determining the increase in the phage litre, which is also employed for demonstration of Shigella bacteria in the patient’s faeces.

To carry out this test, the indicator phages and reference strains of Flexner’s and Sonne’s Shigella bacteria are used. A rise in the phage titre by 3-5 orders (4-) is considered as weak positive reaction, by 5-7 orders (++) and 7-10 orders (+++), positive, and by over 10 orders (++++), markedly positive.

The immunofluorescence test for Shigella recovery is employed in examining objects containing minor amounts of the causative agents and for rapid laboratory diagnosis of dysentery.

 

VIBRIO.  LABORATORY DIAGNOSIS of CHOLERA. CAmpYlobacter, helicobacter and aeromonas infections

 

The Cholera Vibrio.  The causative agents of cholera are the classical Vibrio cholera biovars discovered by R. Koch in 1883 and the El Tor vibrio biovar isolated from the cadaver of a pilgrim on the Sinai peninsula by Gotschlich in 1906, RO- and O139 strains.

Vibrio cholerae biovar Proteus (N. Gamaleya, 1888) and Vibrio cholerae biovar albensis were discovered” later. V. cholerae was described by F. Pacini in 1854.

 

Morphology. Cholera vibrios are shaped like a comma or a curved rod measuring 1-5 mcm in length and 0.3 mcm in breadth (Fig.).

 

Figure. Vibrio cholerae: 1-pure culture; 2- flagellate vibrios

 

They are very actively motile, monotrichous, nonsporeforming, noncapsulated, and Gram-negative.

 

 

The cholera vibrio is subject to individual variation when it is exposed to physical and chemical factors. On artificial media and in old cultures it occurs in the form of grains, globes, rods, threads, clubs or spirals. When it is re-inoculated into fresh media, the organism assumes its initial form.

Electron microscopy reveals flagella 25 nm thick and three-layer cell wall and cytoplasmatic membrane. Small vacuoles are seen  between the wall and the cytoplasmatic membrane. The formation of the vacuoles is assumed to be linked with exotoxin synthesis. The nucleoid is in the centre of the cell. No essential difference has beeoted between the structure of the classical cholera vibrion and that of the El  Tor vibrio. The G-C content in DNA ranges between 45 and 49 per cent.                            

Cultivation. Cholera vibrios are facultative (anaerobes). The optimum growth temperature is 37° C, and growth is arrested below 14 °C and above 42° C. The organisms grow readily on alkaline media at pH 6.0-9.0, and on solid media the colonies are transparent with a light-blue hue, forming domes with smooth edges. On gelatin the organisms produce transparent granular colonies which, when examined under a microscope, resemble broken glass. In 48 hours the medium surrounding the colonies becomes liquefied and the colonies sink into this area. Six-hour-old cultures on alkaline meat broth and peptone water produce a pellicle, which consists of cholera vibrios.

The organism is also subjected to cultural changes. It dissociates from the S-form to the R-form, this process being accompanied by profound changes in antigenic structure.

Fermentative properties. The cholera vibrio liquefies coagulated serum and gelatin; it forms indole and ammonia, reduces nitrates to nitrites, breaks down urea, ferments glucose, levulose, galactose, maltose, saccharose, mannose, mannite, starch, and glycerine (slowly) with acid formation but does not ferment lactose in the first 48 hours, and always coagulates milk. The cholera vibrio possesses lysin and ornithine decarboxylases and oxidase activity. B. Heiberg differentiated vibrios into biochemical types according to their property of fermenting mannose, arabinose, and saccharose. Eight groups of vibrios are known to date; the cholera vibrios of the cholerae and El Tor biovar belong to biochemical variant 1.

The haemolytic activity and haemagglutinating properties of the cholera vibrios in relation to different erythrocytes (sheep, goat, chick, and others) as well as the ability for forming acetylmethylcarbinol are not stable characteristics and are taken into account as less important data in differentiating microbes of the genus Vibrio.

Toxin production. The cholera vibrio produces an exotoxin (cholerogen) which is marked by an enterotoxic effect and plays an important role in the pathogenesis of cholera; the endotoxin also exerts a powerful toxic effect. The cholera vibrios produce fibrinolysin, hyaluronidase, collagenase, mucinase, lecithinase, neuraminidase, and proteinases.

V cholerae produces diarrhea as a result of the secretion of an enterotoxin, choleratoxin, which acts identically to E coli LT to stimulate the activity of the enzyme adenylcyclase. This, in turn, converts ATP to cAMP, which stimulates the secretion of Cl^– and inhibits the absorption of NaCl. The copious fluid that is lost also contains large amounts of bicarbonate and K⁺. Thus, the patient has both a severe fluid loss and an electrolyte imbalance.

The enterotoxin has been shown to bind specifically to a membrane ganglioside designated GM₁. Interestingly, V cholerae produces a neuraminidase that is unable to remove the y-acetylneuraminic acid from GM₁, but it is able to convert other gangliosides to GM₁, thus synthesizing even more receptor sites to which its enterotoxin can bind. Like the LT of E coli, choleragen is composed of five B subunits that react with the cell receptor, an Ai-active subunit that enters the cell and, together with a cellular ADP-ribosylating factor, carries out the ADP-ribosylation of the GTP-binding protein, and a small A; subunit that seems to link the Ai subunit to the B subunit. Interestingly, unlike LT, the DNA en-coding choleragen is not plasmid mediated but is on the chromosome of V cholerae.

 

CT (as well as the LT produced by E coli) can be quantitated by a number of in vivo, cell culture, or immunologic assay units. In one method, a segment of rabbit small intestine is tied to form a loop. Enterotoxin is serially diluted, and an aliquot of each dilution is injected into a loop. The highest dilution that stimulates fluid accumulation in the loop is recorded as the titer of the enterotoxin. A second method takes advantage of the fact that cAMP causes a morphologic response in cultured Chinese hamster ovary cells, and that enterotoxin will induce such cells to produce cAMP. To quantitate enterotoxin using this assay, a standard, curve is established (with purified enterotoxin) that can be used subsequently to assay an unknown enterotoxin from E coli or V. cholerae (Fig.).

 

FIGURE. A standard curve to equate Escherichia coli enterotoxin with purified cholera toxin. The percentage of Chinese hamster ovary cells that have elongated after growing 24 hours in the presence of cholera toxin in 1% fetal calf serum is plotted against the concentration of cholera toxin present in the culture. As shown, heated toxin or toxin preincubated with antitoxin (anti-CT) have no effect on the morphologic features of the cells.

 

As is true with essentially all diarrhea-producing bacteria, V cholerae must specifically colonize the intestinal epithelial cells to produce disease. In this case, however, the pili binding the bacteria to the host cells seem to be under the same regulator as choleragen production and, as a result, are termed toxin-coregulated pili. Mutants unable to bind to intestinal cells are avirulent in spite of their ability to produce choleragen. Moreover, antibody directed to toxin-coregulated pili are protective.

Remember that non-01 and non-0139 strains of V.cholerae also cause a wide spectrum of infections, ranging from mild diarrhea to one indistinguishable from classic cholera. Some of these serotypes are known to produce a choleratoxin that is identical to that of the classic biotypes, whereas other products a heat-stable enterotoxin analogous to the ST of E. coli.

Antigenic structure. The cholera vibrios have thermostable O-antigens (somatic) and thermolabile H-antigens (flagellar). The O-antigen possesses species and type specificity, the H-antigen is common for the genus Vibrio. According to the O-antigen content, the vibrios are separated into subgroups of which there are more than 140. The cholerae vibrios, El Tor biovars and biovars cholera belong to the O-1 subgroup. In the 0-1 subgroup there are three O-antigens (A, B, and C) according to the combination of which three serological variants, Ogawa (AB), Inaba (AC) and an intermediate variant Hikojima (ABC), are distinguished.

Classification. Vibrio cholerae belongs to family Vibrionaceae, genus Vibrio consisting of 5 species. The species Vibrio cholerae is subdivided into four biological variants: biovar cholerae, biovar El Tor, biovar Proteus, and biovar albensis.

Biovar cholerae and biovar El Tor of Vibrio cholerae are the causative agents of human cholera. Biovar Proteus of Vibrio cholerae causes diarrhoea in birds and gastroenteritis in humans; biovar albensis of Vibrio cholerae was revealed in fresh water and in human faeces and bile.

Resistance. The cholera vibrio survives for a long time at low temperatures. It lives in faeces for up to a month, in oysters, crabs, on the surface offish and in their intestines from 1 to 40 days, in water for several days, on foodstuffs from 1 to 10 days, and in the intestines of flies from 4 to 5 days. .

The El Tor vibrio is marked by high resistance. It lives more than four weeks in sea and river water, 1-10 days on foodstuff’s, and 4-5 days in the guts of flies. It is possible that under favourable conditions El Tor vibrio may reproduce in various water reservoirs.

The organism shows a low resistance to sunlight, X-rays, desiccation, and high temperatures. It is destroyed instantly at 100°C, and in 5 minutes at 80° C. Cholera vibrios are highly sensitive to disinfectants, particularly to acids (e. g. a 1 :10000 solution of hydrochloric acid kills them within one minute). The organism is also very sensitive to the action of gastric juice.

Pathogenicity for animals. Iature animals are not attacked by cholera, but an intraperitoneal injection of the culture into rabbits and guinea pigs gives rise to general toxicosis and peritonitis which is followed by death.

In his experiments with rabbit-sucklings E. Metchnikoff produced a disease similar to human cholera by oral infection. R. Koch reproduced the disease in guinea pigs previously alkalizing the gastric juice and introducing opium. An intravenous vibrio injection into rabbits and dogs gives rise to lethal toxaemia.

Pathogenesis and diseases in man.  Cholera is undoubtedly the most dramatic of the water-borne diseases. As far as is known, cholera was confined to India for the almost 2000 years between its first description by Hindu physicians in 400 b c and its spread to Arabia, Persia, Turkey, and Southern Russia in the early1800s. There were six major pandemics of cholera during the 1800s covering the entire world, killing millions wherever it struck. During one such outbreak in London during 1849, the famous physician, John Snow, traced the spread of the disease to a

Broad Street

pump from which area residents obtained their water. The spread of cholera in this area was stopped when Snow recommended that the handle of the pump be removed. This is particularly remarkable when one remembers that the germ-theory of disease had not yet been formulated.

The cholera vibrios are transmitted from sick persons and carriers by food, water, flies, and contaminated hands. Via the mouth the organisms gain entrance into the small intestine, where the alkaline medium and an abundance of products of protein catabolism furnish favourable conditions for their multiplication. When the cholera vibrios perish, a large amount of toxin is released. This toxin invades the patient’s blood owing to necrosis of the intestinal    epithelium and the resulting condition promotes disturbance of the intestinal vegetative nerve fibres, dehydration of the body, and a development of intoxication.                                                

Cholera is characterized by a short incubation period of several hours to up to 6 days (in a disease caused by the El Tor vibrio it lasts three to five days), and the disease symptoms include  general weakness, vomiting, and a frequent loose stool. The stools resemble rice-water and contain enormous numbers of torn-off intestinal epithelial cells and cholera vibrios. The major symptom of cholera is a severe diarrhea in which a patient may lose as much as 10 to 20 L or more of liquid per day. Death, which may occur in as many as 60% of untreated patients, results from severe dehydration and loss of electrolytes.

Three phases can be distinguished in the development of the disease.  1. Cholera enteritis (choleric diarrhoea) which lasts 1 or 2 days. In some cases the infectious process terminates in this period and the patient recovers. 2. Cholera gastroenteritis is the second phase of the disease. Profuse diarrhoea and continuous vomiting lead to dehydration of the patient’s body and this results in lowering of body temperature, decrease in the amount of urine excreted, drastic decrease in the number of mineral and protein substance, and the appearance of convulsions. The presence of cholera vibrios is revealed guite frequently in the vomit and particularly in the stools which have the appearance of rice water. 3.  Cholera algid which is characterized by severe symptoms. The skin becomes wrinkled due to the loss of water, cyanosis appears, and the voice becomes husky and is sometimes lost completely. The body temperature falls to 35.5-34° C. As a result of blood concentration cardiac activity is drastically weakened and urination is suppressed.

In severe cases the algid period is followed by the asphyctic phase characterized by cyanosis, dyspnoea, uraemia, azotaemia, and unconsciousness (cholera coma), which lead to prostration and death. Effective treatment and proper nursing care may induce a change of the algid period to the reactive phase during which urination becomes normal, intoxication decreases, and the patient recovers. Fulminate forms of cholera (dry cholera or cholera sicca) may occur in a number of cases.These forms are characterized by the absence of diarrhoea and vomiting and result in death due to severe intoxication. Atypical and latent forms of cholera are exhibited quite frequently, particularly in children, resembling mild cases of gastroenteritis.

Non-specific complications in cholera include pneumonia, erysipelas, phlegmons, abscesses, occasionally sepsis, etc. Among the specific complications cholera typhoid is the most menacing. It is accompanied by a rise in body temperature to 38-39° C, eruptions on the skin, vomiting and fetid loose stools. This condition causes a mortality rate of 80-90 per cent.

Erased and mild forms are observed in 80 to 90 per cent of cases caused by El Tor vibrio. Severe forms with a fatal outcome are encountered in individuals whose condition is aggravated by various somatic diseases which reduce the general body resistance, in those with hypoacidic gastric function, and in elderly persons.

Post-mortem examination of cholera cases reveals distinct hyperaemia of the peritoneum and serosa of the small intestine, which are covered with a sticky exudate. The mucous membrane of the small intestine is congested, peach-coloured, the intestinal epithelium is frequently desquamated, and there are haemorrhages in the submucosa. The vibrios are present in great abundance in the intestinal wall, particularly in Lieberkuhn’s glands, and, not infrequently, in the gall-bladder.

Cholera mortality was quite high in the past (50 to 60 per cent), but has markedly decreased with the application of aetiotropic and pathogenetic therapy. According to WHO, in 1969-1971 it was 17.7 per cent.

Immunity acquired after cholera is high-grade but of short duration and is of an anti-infectious (antibacterial and antitoxic) character. It is associated mainly with the presence of antibodies (lysins, agglutinins, and opsonins). The cholera vibrios rapidly undergo lysis under the influence of immune sera which contain bacteriolysins.

E. Metchnikoff attributed definite significance to phagocytosis following immunity. The normal activity of the stomach, whose contents are bactericidal to the cholera vibrio, plays an essential role in the natural defence mechanism.

Laboratory diagnosis. A strict regimen is established in the laboratory. Examinations are carried out in accordance with the general rules observed for particularly hazardous diseases.

 

Test specimens are collected from stools, vomit, organs obtained at autopsy, water, objects contaminated by patient’s stools, and, in some cases, from foodstuffs. Certain rules are observed when the material is collected and transported to the laboratory, and examination is made in the following stages.

1. Stool smears stained by a water solution of fuchsin are examined microscopically. In the smears, the cholera vibrios occur in groups similar to shoals of fish (Fig.).

 

    Figure. Vibrio cholerae (stool smear)

 

 

 

2. A stool sample is inoculated into 1 per cent peptone water and alkaline agar. After 6 hours incubation at 37°C the cholera vibrios form a thin pellicle in the peptone water, which adheres to the glass. The pellicle smears are Gram stained, and the culture is examined for motility. A slide agglutination reaction is performed with specific agglutinating 0-serum diluted in a ratio of 1 in  100.

The organisms are then transferred from the peptone water onto alkaline agar for isolation of the pure culture. If the first generation of the vibrios in peptone water is not visible, a drop taken from the surface layer is re-inoculated into another tube of peptone water. In some cases with such re-inoculations, an increase in the number of vibrios is achieved.

The vibrio culture grown on solid media is examined for motility and agglutinable properties. Then it is subcultured on an agar slant to obtain the pure culture.

3. The organism is identified finally by its agglutination reaction with specific 0-serum, determination of its fermentative properties (fermentation of mannose, saccharose, and arabinose), and its susceptibility to phagolysis (Table ).

Table

Differentiation of Biovars of Cholera vibrio

 

Note   “A” – carbohydrate fermentation with acid production; “+””– positive result; “–” – negative result; “+” – negative or positive result is not always observed.

 

The following procedures are undertaken for rapid diagnosis: (1) dark field microscopy of the stool; (2) stool culture by the method of  tampons incubated for 16-18 hours in an enrichment medium with repeated dark field microscopy; (3) agglutination reaction by the method of fluorescent antibodies;

 (4) bacterial diagnosis by isolation of cholera vibrios (the faecal mass is seeded as a thin layer into a dish containing non-inhibiting nutrient agar and grown for 4-5 hours, the vibrio colonies are detected with a stereoscopic microscope, and the culture is tested by the agglutination reaction with O-serum on glass; (5) since neuraminidase is discharged by the cholera vibrios and enters the intestine, a test for this enzyme is considered expedient as a means of early diagnosis (it is demonstrated in 66-76 per cent of patients, in 50-68 per cent of vibrio carriers, and occasionally in healthy individuals).

Treatment. The mortality rate of cholera can be reduced to less than1% by the adequate replacement of fluids and electrolytes. Antibiotics of the tetracycline group (tetracycline, sigmamycin), amphenicol, and streptomycin are prescribed at first intravenously and then by mouth.

Pathogenetic therapy is very important: control of dehydration, hypoproteinaemia, metabolic disorders, and the consequences of toxicosis, acidosis in particular, by infusion of saline (sodium and potassium) solutions, infusion of plasma or dry serum, glucose, the use of warm bath, administration of drugs which improve the tone of the heart and vessels.

Prophylaxis. Cholera patients and vibrio carriers are the source of the disease. Individuals remain carriers of the El Tor vibrio for a lengthy period of time, for several years. Vibrios of this biotype are widely distributed in countries with a low sanitary level. They survive in water reservoirs for a long time and have been found in the bodies of frogs and oysters. Infection may occur from bathing in contaminated water and fishing for and eating shrimps, oysters, and fish infected with El Tor vibrio.The following measures are applied in a cholera focus:

(1) detection of the first cases with cholera, careful registration of all sick individuals, immediate information of health protection organs;

(2) isolation and hospitalization, according to special rules, of all sick individuals and carriers, observation and laboratory testing of all contacts;

(3) concurrent and final disinfection in departments for cholera patients and in the focus;

(4) protection of sources of water supply, stricter sanitary control over catering establishments, control of flies; in view of the possibility of El Tor vibrio reproducing in water reservoirs under favourable conditions (temperature, the presence of nutrient substrates) systematic bacteriological control over water reservoirs has become necessary, especially in places of mass rest and recreation of the population in the summer;

(5) strict observance of individual hygiene; boiling or proper chlorination of water, decontamination of dishes, hand washing;

(6) specific prophylaxis: immunization with the cholera monovaccine containing 8 thousand million microbial bodies per 1 ml or with the cholera anatoxin. Chemoprophylaxis with oral tetracycline is conducted for persons who were in contact with the sick individual or for patients with suspected cholera.

Immunization with heat-killed cholera organisms seems to give some protection, and recovery from the disease imparts immunity of an unknown degree or duration. Killed whole cells of V cholerae given orally along with purified B subunits of the toxin induced immunity in about 85% of persons who received it. Another experimental engineered oral vaccine consists of a live attenuated V. cholerae El Tor Ogawa strain. This mutant no longer expresses the A subunit of the toxin but does produce B subunits. It seemed to provide good immunity in volunteers but it has not been used in large-scale field trials.

A experimental vaccine that induces toxin-neutralizing antibodies in mice uses. an ingenious technique in which a 45-base-pair oligonucleotide encoding an epitope of the B subunit of CT is inserted into the flagellin gene of an avirulent Salmonella. This 15-amino acid insert was expressed at the flagellar surface without abolishing flagellar function. The concept of placing an immunogen in a prominently displayed position on the bacterial surface could be used as a cholera vaccine as well as for inserting a number of other epitopes from both bacteria and viruses.

Remember, however, that none of these vaccines offer any protection against the newly described 0139 strain of V. cholera, and it is necessary to develop new vaccines for these organisms.

General epidemic measures play the principal role in cholera prophylaxis, whereas immunization is regarded as an auxiliary measure.

Cholera dates back to the most ancient times. Its endemic focus is India (Lower Bengal, and the deltas of the Ganges and Brahmaputra rivers).

There were six cholera pandemics between 1817 and 1926: in 1817-1823, 1826-1837, 1846-1862, 1864-1875, 1883-1896, and in 1900-1926. In 1961-1963 the seventh outbreak of cholera pandemic occurred, it was caused by the El Tor vibrio.

According to WHO, 668650 cholera cases were recorded between 1953 and 1961 in the countries of Asia and Africa and 348752 cases between 1961 and 1966.

Beginning with 1966, over 50 per cent of cholera cases in the countries of Asia were caused by the El Tor vibrio. According to WHO, however, the incidence of cholera induced by the classical Vibrio cholerae has doubled. There were cases with cholera in the southern regions of the Soviet Union (Astrakhan, Odessa, Kerch) in 1970. More than 464307 people sick with cholera were recorded in all countries in the period between 1970 and 1976. Cholera epidemic with high morbidity and
mortality rates occurred in the second half of 1977 in the Near East (Syria, Saudi Arabia, and other countries).

Vibrio Parahaemolyticus. Vibrio parahaemolyticus was discovered in 1963 by R. Sakazaki and colleagues. It was isolated from sea water, sea animals (fish) and the stool of humans sick with acute enteritis. Two biovars have been identified, biovar 1 (parahaemolyticus) and biovar 2 (alginolyticus). According to the 0-antigen, the Vibrio haemolyticus contains 12 serovars.

Vibrio parahaemolyticus is the causative agent of toxinfections. It produces haemolysin which has an enterotoxic effect. The ocean water along the coast of Japan is the natural reservoir of Vibrio parahaemolyticus. The sea fish and crustaceans are seeded with the organism but it reproduces within them only after they have been caught.

Vibrio parahaemolyticus strains isolated from humans cause lysis of erythrocytes and a cytopathic effect in human tissue cell cultures, whereas strains isolated from food and sea water are devoid of these properties. Fermentation of saccharose and arabinose is not a constant property.

Vibrio anguilarum is isolated from sea and fresh water and from sick fish. Vibrio fischeri from sea water and sea animals, and Vibrio costicola from canned meat and pickles.

The principles of therapy and prophylaxis are the same as those in other toxinfections.

Vibrio vulnificus is a halophilic organism that characteristically produces an overwhelming primary sepsis without an obvious source of infection, or an infection of a preexisting wound followed by a secondary sepsis. Theprimary sepsis seems to follow the ingestion of undercooked or raw seafood, particularly raw oysters. The number of V. vulnificus infections totals fewer than 100 per year in the United States, but the mortality rate is 45 % to 60 % particularly in individuals with liver disease, or thoses with diabetes, kidney disease and and other ailments affecting immune system. As a result, the CDC have strongly recommended: “Don’t eat raw oysters if you suffer from any kind of liver disease.” Secondary sepsis may also occur after the exposure of wounds to salt water or infectes shellfish.

Vibrio fluvialis is another halophilc that has been isolated from the diarrheal stools of many patients in Bangladesh. It has also been found in coastal waters shellfish on the east and west coasts of the United States. This organism has been reported to produce bott enterotoxin-like substances and an extracellular cytotoxin that kills tissue cells.

Vibrio mimicus, an organism similar to certaion-O1 V. choleras strains, also produces a cholera-like disease and reports indicate that it produces an entcrotoxin thatis indistinguishable from choleragen.

Campylobacter. Members of the genus Campyhbacter are gram-negative, curved, spiral rods possessing a single polar flagellum. Four acknowledged species of Campylobacter exist, and several additional species have been termed Campylobacter-like organisms. All seem to be inhabitants of the gastrointestinal tract of wild and domestic animals, including household pets. Transmission to humans occursby a fecal-oral route, originating from farm animals, birds, cats, dogs, and particularly processed poultry. Fifty percent to 70% of all human infections result from handlingor consuming improperly prepared chicken. Because the organisms often are found in unpasteurized milk, many epidemics of campylobacteriosis have been spread via milk. Some epidemics have occurred in school children who were given unpasteurized milk during field trips to dairies. The Food and Drug Administration has, therefore, specifically recommended that childreot be permitted to sample raw milk during such visits.

Campylobacter jejuni ranks along with rotaviruses and ETEC as the major cause of diarrheal disease in the world, particularly in developing countries. Clinical isolates of this organism have been shown to produce a heat-labile enterotoxin that raises intracellular levels of cAMP. Furthermore, the activity of this enterotoxin is partially neutralized by antiserum against E. coli LT and CT, demonstrating that Campylobacter enterotoxin belongs to this same group of adenylate cyclase-activating toxins. The production of this cholera-like toxin does not, however, explain the mechanism by which C. jejuni causes an inflammatory dysentery or bloody diarrhea. Analysis ofstrains producing such infections have revealed the presence of an additional cytotoxin that is biologically distinct from Shiga-like and Clostridium difficile toxins. The role of this toxin as a cause of inflammatory colitis, however, remains unknown. As is true for most intestinal pathogens, C jejuni has been shown to possess an adhesin for intestinal mucosa.

A number of reports have also indicated a close association between certain serotypes of C. jejuni and Guillain-Barre syndrome, but the nature of this relationship is completely unknown. In one study of 46 patients with Guillain-Barre syndrome, C. jejuni was isolated from 30% of patients compared with 1% of controls. Of these, 83% were serotype 19 and 17%  were serotype 2.

Campylobacter fetus also causes human diarrheal disease, but this species is more likely to progress to a systemic infection resulting in vascular necrosis.

The incubation period for the diarrheal disease usually is 2 to 4 days. The organisms can be grown readily on an enriched medium under microaerophilic conditions (6% O₂ and 10% CO₂). Gentamicin, erythromycin, and a number of other antibiotics may be used successfully for the treatment of Campylobacter infections.

Helicobacter. A. new species of gram-negative curved rods, named Helicobacter pylori, was first described in 1983. This organism was found growing in gastric epithelium, and it is accepted by most investigators that H. pylori is the primary etiologic agent of chronic gastritis and duodenal ulcers in humans. Symptoms of chronic gastritis include abdominal pain, burping, gastric distention, and halitosis. The disease can be reproduced in gnotobiotic piglets and in human volunteers after the ingestion of H. pylori. The observation that their eradication by antibacterial treatment results innormalization of the gastric histology and prevents there currence of peptic ulcers strongly supports the role of a this agent in chronic gastritis and peptic ulcer disease. Notice that Helicobacter mustelae can be routinely isolated from both normal and inflamed gastric mucosa of ferrets, and H. felis routinely colonizes the gastric mucosa of cats.

Surprisingly, H. pylori infection is widespread, particularly in developing countries where it occurs at a younger age than in developed countries. For example, the prevalence of H. pylori infection in Guangdong Province in China was 52.4%, and it has been suggested that earlya cquisition and, hence, long-term infection may be animportant factor predisposing to gastric cancer.

Adhesins, proteases, and cytotoxins all have been reported as virulence factors for H. pylori. One adhesin that has definitely been characterized is the blood group antigen, Lewis^b, (Le^b) which, if present, is found on the surface of gastric epithelial cells in the stomach. Gastric tissue lacking Le^b antigen or antibodies to the Le^b antigen inhibited bacterial binding. Thus, because Le^b is part of the antigen that determines blood group A, individuals with blood group O run a greater risk for developing gastric ulcers. A second adhesin reported to occur on the surface of H. pylori binds specifically to the monosacchande sialic acid, also found on glycoproteins on the surface of gastricepithelial cells.

The production of a cytotoxin that induces vacuolation of eucaryotic cells has been reported to occur inabout 50% of all isolates. Interestingly, one small study suggested that infection with toxin-producing strains was associated with increased antral inflammation.

All wild-type strains of H. pylori do produce the enzyme urease, and a number of reports have indicated that urease may protect the organisms from the acidic environment of the stomach by the release of ammonia from urea. Urease may also function as a cytotoxin, dc- stroying gastric cells that are susceptible to its activity.

Notice that over-the-counter medications containing bismuth salts have been used for years to treat gastritis (Pepto-Bismol, Procter & Gamble Pharm., Norwich, NY) and the fact that H. pylori is sensitive to bismuth may explain its efficacy for the relief of gastric symptoms.

Plesiomonas shigelloides. Plesiomonas shigelloides has been implicated as a cause of diarrhea in the United States as well as in tropical and subtropical countries. The mechanism by which this organism causes diarrhea is unknown, but a report indicated that sterile nitrates of growth medium obtained from 24 different strains of P. shigelloides induced the synthesis of cAMP in Chinese hamster ovary cells. Moreover, this effect was eliminated by either heating the filtrates or by preincubation of them with cholera antitoxin, suggesting that the diarrhea produced by P. shigelloides results from the formation of a cholera-like toxin.

These organisms have been isolated from surface waters, the intestines of fresh water fish, pet shop aquariums, and many animals, particularly dogs and cats. It is more common in tropical and subtropical areas, and isolations from Europe and the United States have been rare and usually associated with foreign travel or consumption of raw oysters.

Aeromonas. Aeromonas species are gram-negative, facultatively anaerobic bacteria that are found in soil, fresh and brackish water, and as pathogens of fish, amphibians, and mammals; symptoms range from diarrhea in piglets to fatal septiccmia in fish and dogs and abortion in cattle. Human infections are most commonly seen as a gastroenteritis but Aeromonas organisms have also been recovered from wounds and soft tissue abscesses that have been contaminated with soil or aquatic environments.

In 1988, California became the first state to make infections by Aeromonas a reportable condition and during that year 280 infections were reported, of which 81% were gastroenteritis and 9% were wound infections. Others were isolated from blood, bile, sputum, and urine, occurring mostly in persons with chronic underlying diseases.

Virulence factors that have been reported for Aeromonas include cholera-like and heat stable entcrotoxins andat least two hemolysins, one or both of which may be cytotoxic or enterotoxic. Aeromonas hydrophila and Aeromonas sobria probably are the only clinically important species.

 

Additional materials for diagnosis

CHOLERA

Cholera is a particularly dangerous infectious disease, caused by Vibrio cholerae and Vibrio El Tor biovars, which runs as gastroenteritis associated with dehydration.

The main method in the laboratory diagnosis of cholera is bacterio­logical examination. Some 10-20 ml of faeces and vomited matter from patients with suspected cholera are collected with a sterile metallic or wooden spoon, transferred into a sterile wide-mouthed vessel, and tightly stoppered with a-glass or cork plug.

The second portion of faeces and vomit (1-2 ml) is inoculated into 1 per cent peptone water (50 ml) at the patient’s bedside. Both vessels are sealed and immediately sent to the laboratory.

If the patient has no bowel movements at the moment of material collection, cut off soiled samples of the bed linen or underclothes and collect the contents of the rectum with a sterile wire loop inserted 5-8 cm deep. Following removal, put the loop with a faecal sample into a flask with a nutrient medium. At autopsy the material to be tested is obtained in the following manners mark off three sites in the area of the upper, middle and lower portions of the small in­testine and a site of the rectum some 10-15 cm long; then from each end of the marked section express the contents of the intestine side-wise, apply two ligatures, and cut an intestine between them. The gallbladder is removed with a part of the liver. The water (1 L) and foodstuffs (no less than 200 g) should also be examined.

In examining convalescents, individuals who have contacted with patients or carriers, it is recommended that a purgative or a cholagogue (25-30 g of magnesium sulphate, etc.) be preliminary given to them to obtain liquid faeces from the upper part of the intestines and the contents of the gallbladder.

The material to be studied is collected, packed, and sent to the laboratory with special measures of precaution. The glassware should not contain any traces of disinfectants, particularly of acids; it is sterilized or boiled for 15 min.

Jars and test tubes should be closed with glass or rubber stoppers. When cork plugs are used, cellulosic film is placed under them. After the  material has been collected, the plugs are sealed with paraffin and wrapped with double cel­lulosic film.

On each vessel stick on a slip of paper with the name and age of the patient, his or her home and office address, diagnosis, the dates of the onset of the disease and hospitalization, as well as the date and exact time of material collection, and also the name of the person who has sent in the analysis.

The material should be brought to the laboratory no later than six hours after its collection. If the delivery within this period is impossible, the samples are inoculated into 1 per cent peptone water with potassium tellurite and onto plates with alkaline agar. If the laboratory is a long way off, jars and test tubes with the specimens to be tested are put, packing them with saw dust, into a metallic container which, in turn, is packed into a wooden box. The latter is wrapped, sealed, signed “Top, fragile”, and is sent with a cou­rier.

The material should be exam­ined in a special laboratory. Yet, if no such laboratory is available, the samples are sent to any bacte­riological laboratory which may provide an isolated room with a separate entrance and exit. No other analyses are taken in this case and stricter measures of precau­tion are introduced. Personnel with special training only is allowed to do this kind of investigation. No operators on a fasting stomach should be allowed in the laboratory. The examination is carried out around the clock since the results should be available no later than 30-36 hrs later.

Bacteriological examination. Stage I. Using the material col­lected, prepare smears, dry them in the air, fix with alcohol or Nikiforov’s mixture, stain by the Gram technique, and examine under the microscope. Later on, if laboratory findings confirm the diagnosis of cholera in at least one case, stain the smears with Pfeiffer’s fuchsine only. Cholera vibrios appear as thin curved Gram-negative rods (Fig. 15). Because of great polymorphism the smear may, along with typical cells, contain coccal, rod-shaped, and spiral forms, which diminishes the value of this method.

The first preliminary answer is given after the microscopic exam­ination of the smear. It refers to the presence of vibrios and the nature of their Gram-staining.

At this stage of bacteriological examination, one can also perform the immunoftuorescence test, using specific labelled 0-cholera sera. Moreover, the cholera vibrio may be recovered by the immune Indian ink method. In the latter case smears fixed on a glass slide are treated for 2 min in a humid chamber with Indian ink mixed with immune serum, then washed with water and examined with a microscope. The vibrio is stained black by Indian ink: the walls of the cell are black-brown, the centre is slightly greyish. If the bacteria are few, they are preliminarily cultivated for 3-5 hrs in peptone water.

Simultaneously with bacterioscopy, the material tested is inoculat­ed onto liquid and solid nutrient media. Enrichment liquid media that are usually recommended for use include alkaline 1 per cent peptone water, 1 per cent peptone water with potassium tellurite in a ratio of 1 to 100 000, and alkaline taurocholate-tellurite-peptone medium (Monsur’s liquid medium), etc.

Solid nutrient media usually employed are alkaline meat-peptone agar and one of the selective nutrient media: Aronson’s medium, Monsur’s  alkaline  taurocholate-lellurite-gelatine-agar medium, TCBS, etc.

To isolate the vibrio from carriers or patients with subclinical forms of cholera, use media which improve the growth of vibrios and suppress the attendant flora (predominantly E. coli). All inoculat­ed cultures are placed in an incubator at  37 °C.

Aronson’s medium consists of 2-3 per cent of meat-peptone agar to which sucrose and destained fuchsine are added.

Monsur’s alkaline taurocholate-tellurite-gelatine-agar medium contains 10 g of trypticase, l0 g of sodium chloride, 50 g of sodium taurocholate, 30g of sod­ium carbonate, 1 g of gelatin, 15 g of agar-agar, and 1 L of distilled water.

TCBS (thiosulphate-citrate-bromthymol sucrose) is manufactured in the form ready for use; 69 g of the dry medium is taken per 1 L of distilled water.

Stage II. Some 5-6 hours after inoculation examine the film on the peptone water. To do it, tilt the test tube or the vial so that a delicate bluish film is attached to the wall. Prepare smears from the  film or the surface of the medium, stain them by the Gram method, evaluate motility, and conduct presumptive slide agglutination test with 0-cholera (0-1) serum diluted 1:100 or the reaction of cholera vibrio immobilization with 0-cholera serum. The results of the latter are estimated by phase-contrast microscopy. Inhibition of vibrios motility and the formation of agglutinate occur within 1-2 min.

On the basis of the results obtained give a second preliminary result referring to the motility of the vibrio and its relation to the agglutinating serum.

Subculture the material from the film onto plates with alkaline agar or selective medium and simultaneously onto the second peptone water and look for changes in 5-6 hrs.

Stage III. Some 10-16 hrs after inoculation, examine the growth in the second enrichment medium (peptone water) and on the plates with the culture of the native material. The film formed on the pep­tone water is examined as described above.

On an alkaline agar the cholera vibrio grows with the formation of round, smooth, flat, bluish, homogeneous colonies which are 1–2 mm in diameter, transparent in the transmitted light and have smooth edges. They are oily in consistence, are readily removed and emulsified. Examination of the material from convalescents, bac­teria carriers, and individuals treated with antibiotics may reveal atypical colonies.

On Aronson’s medium colonies of cholera vibrios are scarlet in the centre and pale-pink or colourless at the periphery. On Monsur’s medium colonies are transparent or semitransparent, or they may be of a greyish-black colour with turbid edges. On the TCBS medium they appear as flat and yellow against a bluish-grey background.

The selected colonies are introduced into test tubes with Oikenitsky’s medium or onto an agar slant for enrichment of pure culture and placed in an incubator.

Preliminary identification of cholera vibrios grown on plates with solid media is based on the study of cultural and morphological characteristics and on a presumptive slide agglutination test with 0-cholera serum diluted 1:100 and with Ogawa’s and Inaba’s sera in a 1 to 60 dilution, which is carried out to determine the serovar.

If the examination demonstrates signs typical of the cholera vib­rio, a third preliminary answer about the positive result of the investigation is issued. Some material from the typical colonies may be transferred to a broth; then, using a 3-4-hour old culture, perform a standard agglutination test, check fermentation of carbo­hydrates, and determine whether the isolated culture belongs to Group I according to Heiberg and whether it is liable to phagolysis by cholera phages C and El Tor 2. If the results are positive, an answer concerning the isolation of the causative agent is given within 18-24 hrs from the beginning of the study.

Stage IV. After the results of the standard agglutination test and the reaction of phagolysis and fermentation of carbohydrates by 3-4-hour broth culture have been analysed, a preliminary conclusion about the isolation of the cholera vibrio is made. Plates with the inoculated culture on the second peptone water are examined, using the scheme which is employed in examining the plates with the culture of the native material. On Oikenitsky’s medium the vibrio breaks down sucrose without gas formation and does not ferment lactose (reddening of the medium in the column without gas for­mation).

To distinguish vibrios from homogeneous species of microorganisms (Aeromonas, Pseudomonas, Plesiomonas), a number of tests may be employed: the oxidase test, glucose oxidation-fermentation reac­tion, the “strand” test (Table ).

Table

Differential-Diagnostic Signs of Vibrios and Related Types of Bacteria

 

 

The oxidase test consists of placing a solution of paraaminodimethyl–aniline and alpha-naphthol onto the culture in a Petri dish or onto a meat-peptone agar slant.

To carry out the oxidation-fermentation test, medium with the following composition (per 100 ml) is prepared: 2.0 g of peptone; 5.0 g of sodium chloride;   0.3 g of potassium hydrophosphate; 3.0 g of agar-agar; bromthymol blue (1 per- cent aqueous solution).

Dispense the medium in 3-4-ml portions into 13 X 100 mm test tubes and sterilize for 15 min at 120 °C. After that, add to the tubes 10 per cent glucose solution sterilized by nitration to adjust to the final concentration of 1 per cent. Inoculate the test culture into two tubes with the above mentioned medium- Into one of the tubes pour a layer (1.5-2 cm) of sterile petrolatum oil. Incubate the test tubes for four days and note acid and gas formation. Darkening of the medium in the open test tube indicates oxidation and in the tube with the oil, fermentation. Gas formation is sometimes observed.

The “strand” test. Onto a glass slide, place a drop of 0.5 per cent solution of sodium desoxycholate in buffer isotonic saline. Into this drop, introduce a loop-ful of the tested culture of vibrios grown on a solid nutrient medium and mix. If the result is positive, the mixture becomes transparent, acquires mucilaginous consistency, and trails the loop in the form of a strand in the first minutes after its preparation.

To differentiate between the classical cholera vibrio and the El Tor vibrio, utilize tests determining the sensitivity of cholera vibrios toward phage’s and polymixin and the ability of vibrios to agglutinate chick erythrocytes.

Sensitivity to diagnostic phages is determined by streaking onto a plate with a culture of whole cholera phages C and EI Tor 2, with ten-fold dilutions of the above. Phage C is active only toward the classic cholera vibrio, while the El Tor 2 phage is active toward El Tor biovar. The presence of lysis in the form of one “sterile” spot or a group of small spots in the place of phage introduction is assessed as a positive result.

Sensitivity to polymixin is determined by inoculating the isolated culture onto Petri dishes with nutrient agar containing 50 U of polymixin M or B in 1 ml of nutrient medium. El Tor vibrios are insensitive to antibiotics and show good growth on the dishes, un­like the classic cholera vibrios.

Haemagglutination of chicken erythrocytes is performed on a glass slide. In a drop of isotonic sodium chloride solution, comminute a loopful of 18-hour culture of the vibrio and add a drop of 2.5 per cent suspension of chicken erythrocytes. The cholera El Tor vibrio agglutinates the red blood cells within 1-3 min, whereas the classical biovar fails to induce any clumping.

Haemolysis of sheep erythrocytes (Greig’s test) occurs after their 2-hour incubation with broth culture of cholera El Tor vibrios at 37 °C. Yet, this sign is not stable and some strains of the El Tor biovar, similar to the classical cholera vibrio, display no haemolytic effect.

The Voges-Proskauer test is based on the ability of El Tor vibrios to form acetylmethylcarbinol, which is recognized by the fact that Clark’s glucose-phosphate broth becomes pink or ruby-red, following 1-3 day incubation of the inoculated cultures with addition of alpha-naphthol.

The hexamine test is performed with 24-hour broth culture of the vibrio a loopful of which is streaked onto 1 ml of a glucose-hexamine medium. Following incubation at 37 °C for 6-24 hrs, the El Tor vibrio alters the colour of the medium from green to yellow. The classical cholera vibrio induces no changes in the medium colour over this time.

Production of enterotoxin by the cholera vibrio is determined by means of a specific reaction of passive immune haemolysis. To carry out this reaction, re-suspend in 0.08 M phosphate buffer the erythrocytes from defibrinated sheep blood (after their triple washing). Prepare 10 per cent suspension of red blood cells in 0.02 M solution of the buffer. With a micropipette introduce 0.025 ml portions of 0.02 M phosphate buffer into agglutinating plates, then add two­fold dilutions of the antigen and 1 per cent suspension of erythrocytes. Cover the plates and place them into a 37 °C incubator for 30 min, add to each well (1.025 ml of antitoxic serum diluted 1:50 and 0.02 per cent bovine serum al­bumin, reincubate the culture for 30 min, then add 0.025 ml of complement and replace the culture into the incubator for 90 min. .Haemolysis is evaluated after 30 min of keeping the plates at room temperature. Simultaneously, one monitors non-immune haemolysis and the ingredients of the serum, antigen, and complement.

Isolation of non-agglutinating vibrios brings about the  necessity of studying their biochemical properties, namely: liquefaction of gelatine, splitting of 5tarch (Cadamot’s test), formation of indol from triptophane, reduction of nitrates into nitrites, as well as oxidase and decarboxylase activity. It is also necessary to classify the culture with one of the biochemical groups according to Heiberg.

Demonstration of cholera vibrios in water is of great importance for identifying the factors of infection transmission and conducting anti-epidemic measures. Using a saturated solution of sodium hydrocar-bonate, alkalize the water (900 ml) delivered to the laboratory to pH of 7.8-8.0, add 100 ml of basic peptone, pH 8.0 (peptone, 100 g, sodium chloride, 50 g, potassium nitrate, 1 g, sodium hydrocarbon-ate, 20 g, distilled water, 1000 ml), and dispense it into flasks or vials in 100-200-ml portions. Incubate the inoculated cultures at 37 °C for 5-8 hrs and then inspect them in the manner employed for studying other inoculated cultures in peptone water (vomited matter and faeces). The results are more reliable when the water tested is filtered through membrane filters. Large amounts of water (1.5-2.5 1) are examined and the deposit from the filters is transferred to peptone water (pH 8.0) and alkaline agar.

Rapid detection of cholera vibrios in drinking water. If water con­tamination with cholera vibrios is heavy (at least 100 vibrios per 1 ml), the agglutination reaction is utilized for their recovery. To the water to be assayed add weakly alkaline concentrated solution of peptone in a quantity sufficient to produce 1 per cent solution. With this mixture dilute the 0-cholera agglutinating serum from 1:100 to its titre. Use a mixture free of the serum as a control. Place the test tubes into an incubator and read the results of the test in 6 hrs. The reaction is considered positive it flocculation is observed upon serum dilution to half the litre or the litre.

Rapid recovery of the cholera vibrio in water may also be based on increase in the phage titre.

Rapid method of wide-scale screening for carriers. During an out­break of cholera wide-scale screening for carriers of the cholera vibrio is performed. When a large number of analyses is to be made in the laboratory, faeces from ten subjects are examined simultaneously. Faeces are collected with wire loops and placed into one flask con­taining 200 ml of peptone water and 0-cholera agglutinating serum which is diluted to half the titre. The flask is placed into a 37 “C incubator. In 3-4 hrs the multiplied cholera vibrios begin to agglu­tinate and fall to the bottom in the form of flakes. If this is the case, faecal material is taken from each of the ten individuals, and the examination is repeated with each sample.

Serological diagnosis of cholera is supportive and relies on de­tecting agglutinins and vibriocidal antibodies in the patient’s serum. It is recommended that paired sera obtained from the patients at a 6-8 day interval be used for these reactions. Titres of agglutinins and vibriocidal antibodies usually tend to increase simultaneously. The most sensitive test is demonstration of vibriocidal antibodies. The presence of agglutinating antibodies in the titre of 1:80-1:320 and vibriocidal ones in the titre of 1:1000 is considered diagnostically positive.