Salmonella. Laboratory diagnosis of enteric fever and parathyphoids. Microbiologic diagnosis of salmonellosis.

Table

Classification of the Enterobacteriaceae

Ewing and Martin		Bergey's Manual
Tribe	Genera	Genera
Eschericheae	Escherichia Shigella	Escherichia Shigella
Edwardsielleae	Edwardsiella	Edwardsiella
Salmonelleae	Salmonella Arizona Citrobacter	Salmonella Citrobacter Klebsiella
Klebsielleae	Klebsiella Enterobacter Serratia	Enterobacter Hafnia Serratia
Proteeae	Proteus	Proteus Providencia Morganella
	Providencia	Yersinia
Erwineae	Erwinia Pectobacterium	Erwinia

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 given numbers. 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 in nature. 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 in nature). 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 in neonates, 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 in nursing 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

Diarrhea-producing E. coli	Virulence Factors
Enteroroxigenic E. coli	Heat-labile toxin (LT) Heat-stable toxin (ST) Colonization factors (fimbriae)
Enterohernorrhagic E. coli	Shiga like toxin (SLT-I) Shiga like toxin II (SLF-II) Colonisation factors (fimbriae)
Enteroinvasive E. coli	Shiga like toxin (SLT-I) Shiga like toxin II (SLF-II) Ability to invade epithelial cells
Enteropathogenic E. coli	Adhesin factor for epithelial cells
Urinary trace infections	P- fimbriae
Meningitis	K-1 capsule

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 on nutrient 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 common nutrient 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.

Classification of K ozaenae and K rhinoscleromatis is presented in Table .

Table

Differentiation of Klebsiellae Organisms

Bacteria		Microscopical structure of colonies on agar	Growth in bile or in 50% bile broth	Fermentation of carbochydrates
				lactose	glucose	dulcitol	urease
K. pneunoniae	Form loops		+	AG	AG	A	+
K. ozaenae	Concentrically scattered		+	A	A	–	+
K. rhinoscleromatis	Concentrical		–	–	A	–	–

Note: “A” indicates acid; “AG” indicates acid and gas; “+” indicates growth in bile, fermnentation of urea; “–” indicates absence of fermentation and growth.

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.

Table

Differential-Diagnostic Criteria of Pathogenic Klebsiella

Type of Klebsiella	Microscopic structure of young colonies	Fermentation				Subculture to agar after a 4-day vegetation on a bile
		lactose	glucose	sucrose	urea
K. pneumoniae	Loop-like	AG	AG	AG	+	Abundant growth
Kl. rhinoscleromatis	Concentric	–	A	A	–	No growth
Kl. ozaenae	Diffuse concentric	A	A	A	+	Abundant growth

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 in nature: 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

Species	Antigenic formula	Fermentation				Hydrogen sulfide formation
		arabinose	glucose	mannite	maltose
S. typhi	9, 12(Vi):d–	A+	A	A	A	+
S. paratyphi A	1, 2, 12:a:–	AG	AG	AG	AG	–
S. schotlmuelleri	1, 4. 5, 12:b:l,2	AG	AG	AG	AG	+
S. typhimurium	1,4,5,12:i:l,2	AG	AG	AG	AG	+
S. cholerae-suis	6,7:c:l,5	–	AG	AG	AG	+
S. enteritidis	1,9,12:g,m:–	AG+	AG	AG	AG	+
S. hirschfeldii	6,7(Vi):c:l,5	AG+	AG	AG	AG	+

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 contain no 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

Group and species (type)	Antigenic structure
	somatic antigen	flagella antigen
		phase I	phase II
Group A
S. paratyphi A	1, 2, 12	a
Group B
S. schottmuelleri	1, 4, 5, 12	b	1, 2
S. abony	1, 4, 5, 12	b	e, n, x
S. typhimurium	1, 4, 5, 12	i	1, 2
S. stanley	4, 5, 12	d	1, 2
S. heidelberg	4, 5, 12	r	1, 2
S. abortivoequina	4, 12	–	e, n, x
S. abortus ovis	4, 12	c	1, 6
S. abortus bovis	1. 4, 12, 27	b	e, n, x
Group C (1, 2)
S. hirschfeldii	6, 7, Vi	c	1, 5
S. cholerae-suis	6, 7	c	1, 5
S. typhi-suis	6, 7	c	1, 5
S. thomson	6, 7	k	1, 5
S. duesseldorf	6, 8	Z4, Z24,	–
S. newport	6, 8	e, h	1,2
S. albany	(8), 20	Z4, Z24,	–
Group D1
S. typhi	12. Vi	d	–
S. enteritidis	9, 12	g, m	–
S. dublin	9, 12	g, p	–
S. rostock	9, 12	g, p, u	–
S. moscow	12	g, q	–
S. gallinarum and oth.	9, 12	i	–
Group E (1, 3)
S. london	10	i, v	1. 6
S. anatum	10	e, h	1. 6
S. harrisonburg	(3) (15), 34	z10	1, 6

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 in nature 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

Ingredient	Number of test the tubes
	1	2	3	4	5	6	7
Isotonic sodium chloride solution, ml	–	1,0	1,0	1,0	1,0	–	1,0
Patient's serum in1:100 dilution, ml	1,0	1,0	®	®		1,0	–
Diagnosticum, drops	1,0	1,0	1,0	1,0	1,0	1,0	–
Serum dilution obtained	1:100	1:200	1:400	1:800	1:1600	1:100	–
Results

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 in non-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 Instruction no 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 Instruction no 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 been named 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 in newly-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 in newborn 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 Instruction no 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 Instruction no 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.

References:

1. Essential of Medical Microbiology /Wesley A. Wolk and al. / Lippincott-Raven Publishers, Philadelphia-Ney-York, 1995, 725 p.

2. Hadbook on Microbiology. Laboratory diagnosis of Infectious Disease/ Ed by Yu.S. Krivoshein, 1989, P. 88–96.

3. Review of Medical Microbiology /E. Jawetz, J. Melnick, E. A. Adelberg/ Lange Medical Publication, Los Altos, California, 2002, P. 217-223, 225-228,