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VIT-Salmonella

June 14, 2024

Lectures 20

Causative agents of escherichiosis, enteric fever, parathyphoid fever and salmonellosis

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Escherichia coli

Escherichia coli Theodor Escherich first described E. coli in 1885, as Bacterium coli commune, which he isolated from the feces of newborns. It was later renamed Escherichia coli, and for many years the bacterium was simply considered to be a commensal organism of the large intestine. It was not until 1935 that a strain of E. coli was shown to be the cause of an outbreak of diarrhea among infants.

E. coli and its relatives are known to microbiologists as “enteric bacteria“, because they live in the intestinal tract of humans and other animals. The best known other enteric bacteria are Salmonella, which includes the agent of typhoid fever, and Shigella, which is the bacterial cause of dysentery.

E. coli is in the bacterial family Enterobacteriaceae, which is made up of Gram-negative, nonsporeforming, rod-shaped bacteria that are often motile by means of flagella. The majority of strains grow well on the usual laboratory media in both the presence and absence of oxygen, and metabolism can be either by respiration or fermentation.

The GI tract of most warm-blooded animals is colonized by E. coli within hours or a few days after birth. The bacterium is ingested in foods or water or obtained directly from other individuals handling the infant. The human bowel is usually colonized within 40 hours of birth. E. coli can adhere to the mucus overlying the large intestine. Once established, an E. coli strain may persist for months or years. Resident strains shift over a long period (weeks to months), and more rapidly after enteric infection or antimicrobial chemotherapy that perturbs the normal flora. The basis for these shifts and the ecology of Escherichia coli in the intestine of humans are poorly understood despite the vast amount of information on almost every other aspect of the organism’s existence. The entire DNA base sequence of the E. coli genome has been known since 1997.

E. coli is the head of the large bacterial family, Enterobacteriaceae, the enteric bacteria, which are facultatively anaerobic Gram-negative rods that live in the intestinal tracts of animals in health and disease. The Enterobacteriaceae are among the most important bacteria medically. A number of genera within the family are human intestinal pathogens (e.g. Salmonella, Shigella, Yersinia). Several others are normal colonists of the human gastrointestinal tract (e.g. Escherichia, Enterobacter, Klebsiella), but these bacteria, as well, may occasionally be associated with diseases of humans.

Physiologically, E. coli is versatile and well-adapted to its characteristic habitats. It can grow in media with glucose as the sole organic constituent. Wild-type E. coli has no growth factor requirements, and metabolically it can transform glucose into all of the macromolecular components that make up the cell. The bacterium can grow in the presence or absence of O₂. Under anaerobic conditions it will grow by means of fermentation, producing characteristic “mixed acids and gas” as end products. However, it can also grow by means of anaerobic respiration, since it is able to utilize NO₃, NO₂ or fumarate as final electron acceptors for respiratory electron transport processes. In part, this adapts E. coli to its intestinal (anaerobic) and its extraintestinal (aerobic or anaerobic) habitats.

E. coli can respond to environmental signals such as chemicals, pH, temperature, osmolarity, etc., in a number of very remarkable ways considering it is a unicellular organism. For example, it can sense the presence or absence of chemicals and gases in its environment and swim towards or away from them. Or it can stop swimming and grow fimbriae that will specifically attach it to a cell or surface receptor. In response to change in temperature and osmolarity, it can vary the pore diameter of its outer membrane porins to accommodate larger molecules (nutrients) or to exclude inhibitory substances. With its complex mechanisms for regulation of metabolism the bacterium can survey the chemical contents in its environment in advance of synthesizing any enzymes that metabolize these compounds. It does not wastefully produce enzymes for degradation of carbon sources unless they are available, and it does not produce enzymes for synthesis of metabolites if they are available as nutrients in the environment.

E. coli is a consistent inhabitant of the human intestinal tract, and it is the predominant facultative organism in the human GI tract; however, it makes up a very small proportion of the total bacterial content. The anaerobic Bacteroides species in the bowel outnumber E. coli by at least 20:1. however, the regular presence of E. coli in the human intestine and feces has led to tracking the bacterium iature as an indicator of fecal pollution and water contamination. As such, it is taken to mean that, wherever E. coli is found, there may be fecal contamination by intestinal parasites of humans.

E. coli as an Indicator of Fecal Pollution

For most of the 20th century, E. coli has been used as the principal indicator of fecal pollution in both tropical and temperate countries. E. coli comprises about 1% of the total fecal bacterial flora of humans and most warm-blooded animals. Sewage is always likely to contain E. coli in relatively large numbers. In addition, E. coli, being a typical member enteric bacterium is presumed to have survival characteristics very similar to those of the well-known pathogens such as Salmonella and Shigella. Thus, E. coli has been used world-wide as an indicator of fecal microbiological contamination. As such an indicator organism, its value is significantly enhanced by the ease with which it can be detected. and cultured.

Tests to identify isolates as E. coli have, of necessity, been simple tests designed predominantly to differentiate them from organisms normally associated with uncontaminated water. Since full biochemical analyses are not generally performed, the term “coliform” has been coined to describe E. coli-like organisms that satisfy these limited tests. As a result, regulations are promulgated throughout the world defining standards for water based on the so-called “coliform count.” For example, in the U.S., according to a regulation published in the Federal Register (1986), there is a requirement that there be 0 coliforms/100 ml drinking water, as determined by any method for any sampling frequency.

Since not all organisms which meet the criteria of a coliform are associated with the intestinal tract (some may be free-living), a further distinction must be made between “fecal coliforms” (E. coli) and “nonfecal coliforms” (e.g. Klebsiella and Enterobacter). The nonfecal coliforms are regularly found in soil and water and in associations with plants, so that their occurrence does not necessarily indicate fecal pollution.

In order to distinguish E. coli from related species likely to be found naturally in the environment, a battery of tests called the IMViC reactions was developed in order to differentiate fecal coliforms from nonfecal coliforms. IMViC is an acronym in which the capital letters stand for Indole, Methyl red, Voges-Proskauer, and Citrate.) The IMViC set of tests examines: the ability of an organism to (1) produce Indole; (2) produce sufficient acid to change the color of Methyl red indicator; (3) produce acetoin, an intermediate in the butanediol fermentation pathway (a positive result of the Voges-Proskauer test); and (4) the ability to grow on Citrate as the sole source of carbon. E. coli is positive in the first two tests and negative in the second two; nonfecal coliforms are the opposite – negative in the first two tests and positive for the second two.

If E. coli is detected in water, it is an indication of fecal pollution. Most-likely the strain of E. coli is a harmless non pathogen, but the indication is that other pathogenic intestinal microbes could also be present. The pathogenic fecal coliforms (e.g. Salmonella and Shigella ) can be readily distinguished from strains of E. coli on the basis of a lactose fermentation test. All strains of E. coli ferment the sugar lactose while those of Salmonella and Shigella do not.

Detection of E. coli in Food

The International Commission on Microbiological Specifications for Foods (ICMSF, 1978) has adopted a set of standard techniques for the enumeration of E. coli in food products, accepted by the International Standards Organization (ISO, 1984). This method employs the use of lauryl sulfate tryptose broth at 35 or 37ўXC as a mildly selective-enrichment medium. This is followed by growth in EC broth containing 0.15% bile salts at 45ўXC as a second selective step. The ability to produce indole from tryptophan (in tryptone broth) at 45ўXC defines the strains as E. coli. These tests miss some types of E. coli, such as those most closely related to the Shigella group, but it is the detection of possible fecal contamination that is important in these tests rather than the presence of specific types.

Detection of E. coli in Water

There is no method for the detection of E. coli in water that is accepted throughout the world. In the US, a standard method using membrane filter enumeration for both total and thermotolerant coliforms has been established (American Public Health Association (1986). Further IMViC tests on selected isolates can then be performed as described above.

In the UK, the definition of E. coli in water microbiology is also based on the ability to produce gas from lactose and produce indole from tryptophan at 44ўXC. A method for enumeration employs a standard multiple tube test with a modified glutamate synthetic medium at 37ўXC as a first selective step, followed by further cultivation in standard media at 44ўXC.

Detection of E. coli in Clinical Specimens

While large numbers of E. coli will be found in fecal specimens or specimens contaminated with feces or intestinal contents, most other clinical specimens are usually not contaminated with E. coli. The major exception is urine, which requires special attention in the clinical situation. From those specimens in which E. coli is likely to be present in large numbers, direct plating on media such as MacConkey agar or Eosin Methylene Blue (EMB) agar is sufficient. If the number of E. coli is likely to be very low or the amount of specimen is limited, enrichment in a rich nutrient medium such as brain heart infusion broth may be used. A number of different commercially available kits are generally used to identify the isolates as E. coli.

From specimens likely to contain only a few viable E. coli cells, such as blood from patients suspected of having E. coli bacteremia, various enrichment procedures are used. Identification follows standard bacteriological techniques.

aRapid Methods for Detecting E. coli

A fluorogenic detection method has been developed based on the cleavage of methylumbelliferyl-D-glucuronide (MUG) to the free methylumbelliferyl moiety, which fluoresces a blue color after irradiation with long-wave ultraviolet radiation. Although strains of E. coli are generally positive in this test, some strains of Salmonella, Shigella, and Yersinia are also capable of splitting MUG; the latter two genera are usually not present in food. A disadvantage is that enterohemorrhagic E. coli (EHEC) strains are generally negative in this test. MUG can be added to various selective media, so there is a great potential in its use for detecting E. coli.

Automated or semi-automated systems are also being used for the detection of E. coli as part of the detection methods for Enterobacteriaceae. Techniques involving impedance measurements have shown promise. Other techniques such as immunoassays and nucleic acid hybridization studies can also be used to enumerate E. coli, and DNA probes directed at a number of genes have also been developed.

Physiology of E. coli

Physiologically, E. coli is versatile and well-adapted to its characteristic habitats. In the laboratory it can grow in media with glucose as the sole organic constituent. Wild-type E. coli has no growth factor requirements, and metabolically it can transform glucose into all of the molecular components that make up the cell. The bacterium can grow in the presence or absence of O₂. Under anaerobic conditions it will grow by means of fermentation, producing characteristic “mixed acids and gas” as end products. However, it can also grow by means of anaerobic respiration, since it is able to utilize NO₃ or fumarate as final electron acceptors for respiratory electron transport processes. In part, this adapts E. coli to its intestinal (anaerobic) and its extraintestinal (aerobic or anaerobic) habitats.

In the ecological niches that E. coli occupies and its abilities to grow both aerobically and anaerobically are important. E. coli is well adapted to its intestinal environment as it is able to survive on a relatively limited number of low-molecular weight substances, which may only be available transiently and at relatively low concentrations. The generation time for E. coli in the intestine is thought to be about 12 hours. The type of nutrients available there to E. coli consist of mucus, desquamated cells, intestinal enzyme secretions, and incompletely digested food. Given the absorption capacity and efficiency of the intestine, there are probably only small amounts free carbohydrates or other easily absorbable forms of nutrients, and there is competition from hundreds of other types of bacteria. A similar situation probably also applies to sources of nitrogen.

In its natural environment, as well as the laboratory, E. coli can respond to environmental signals such as chemicals, pH, temperature, osmolarity, etc., in a number of very remarkable ways considering it is a single cell organism. For example, it can sense the presence or absence of chemicals and gases in its environment and swim towards or away from them. Or it can stop swimming and grow fimbriae that will specifically attach it to a cell or surface receptor. In response to changes in temperature and osmolarity, it can vary the pore diameter of its outer membrane porins to accommodate larger molecules (nutrients) or to exclude inhibitory substances (e.g. bile salts). With its complex mechanisms for regulation of metabolism the bacterium can survey the chemical content its environment in advance of synthesizing any enzymes necessary to use these compounds. It does not wastefully produce enzymes for degradation of carbon sources unless they are available, and it does not produce enzymes for synthesis of metabolites if they are available as nutrients or growth factors in the environment.

Escherichia coli in the Gastrointestinal Tract

The commensal E. coli strains that inhabit the large intestine of all humans and warm-blooded animals comprise about 1% of the total bacterial biomass. This E. coli flora is in constant flux. One study on the distribution of different E. coli strains colonizing the large intestine of women during a one year period (in a hospital setting) showed that 52.1% yielded one serogroup, 34.9% yielded two, 4.4% yielded three, and 0.6% yielded four. The most likely source of new serotypes of E. coli is acquisition by the oral route. To study oral acquisition, the carriage rate of E. coli carrying antibiotic resistance (R) plasmids was examined among vegetarians, babies, and non vegetarians. It was assumed that non vegetarians might carry more E. coli with R factors due to their presumed high incidence in animals treated with growth-promoting antimicrobial agents. However, omnivores had no higher an incidence of R-factor-containing E. coli than vegetarians, and babies had more resistant E. coli in their feces thaon vegetarians. No suitable explanation could be offered for these findings. Besides, investigation of the microbial flora of the uninhabited Krakatoa archipelago has shown the presence of antibiotic-resistant E. coli associated with plants.

Infections Caused by Pathogenic E. coli

E. coli is responsible primarily for three types of infections in humans: urinary tract infections, neonatal meningitis, and intestinal diseases. These conditions depend on a specific array of pathogenic (virulence) determinants possessed by the organism. Pathogenic E. coli are discussed elsewhere in the text in more detail at

Pathogenic E. coli: Gastroenteritis, Urinary tract Infections and Neonatal Meningitis.

Urinary Tract Infections

Uropathogenic E. coli cause 90% of the urinary tract infections (UTI) in anatomically-normal, unobstructed urinary tracts. The bacteria colonize from the feces or perineal region and ascend the urinary tract to the bladder. Bladder infections are 14 times more common in females than males by virtue of the shortened urethra. The typical patient with uncomplicated cystitis is a sexually-active female who was first colonized in the intestine with a uropathogenic E. coli strain. The organisms are propelled into the bladder from the periurethral region during sexual intercourse. With the aid of specific fimbriae they are able to colonize the bladder.

The frequency of the distribution of the host cell receptor for the bacterial fimbriae plays a role in susceptibility and explains why certain individuals have repeated UTI caused by E. coli. Uncomplicated E. coli UTI virtually never occurs in individuals lacking the receptors.

Neonatal meningitis

Neonatal meningitis affects 1/2,000-4,000 infants. Eighty percent of E. coli strains involved synthesize K-1 capsular antigens (K-1 is only present 20-40% of the time in intestinal isolates).

E. coli strains invade the blood stream of infants from the nasopharynx or GI tract and are carried to the meninges.

Epidemiological studies have shown that pregnancy is associated with increased rates of colonization by K-1 strains and that these strains become involved in the subsequent cases of meningitis in the newborn. Probably, the infant GI tract is the portal of entry into the bloodstream. Fortunately, although colonization is fairly common, invasion and the catastrophic sequelae are rare.

Neonatal meningitis requires antibiotic therapy that usually includes ampicillin and a third-generation cephalosporin.

Intestinal Diseases

As a pathogen, E. coli, of course, is best known for its ability to cause intestinal diseases. Five classes (virotypes) of E. coli that cause diarrheal diseases are now recognized: enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), enterohemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), and enteroaggregative E. coli (EAggEC). Each class falls within a serological subgroup and manifests distinct features in pathogenesis.

Enterotoxigenic E. coli (ETEC)
ETEC are an important cause of diarrhea in infants and travelers in underdeveloped countries or regions of poor sanitation. The diseases vary from minor discomfort to a severe cholera-like syndrome. ETEC are acquired by ingestion of contaminated food and water, and adults in endemic areas evidently develop immunity. The disease requires colonization and elaboration of one or more enterotoxins. Both traits are plasmid-encoded.

Enterotoxins produced by ETEC include the LT (heat-labile) toxin and/or the ST (heat-stable) toxin, the genes for which may occur on the same or separate plasmids. The LT enterotoxin is very similar to cholera toxin in both structure and mode of action. It binds to the same intestinal receptors that are recognized by the cholera toxin, and its enzymatic activity is identical to that of the cholera toxin.

The ST enterotoxin is actually a family of toxins which are peptides of molecular weight about 2,000 daltons. Their small size explains why they are not inactivated by heat. ST causes an increase in cyclic GMP in host cell cytoplasm. This leads to secretion of fluid and electrolytes resulting in diarrhea.

Symptoms ETEC infections include diarrhea without fever. The bacteria colonize the GI tract by means of a fimbrial adhesin, e.g. CFA I and CFA II, and are noninvasive, but produce either the LT or ST toxin.

Enteroivasive E. coli (EIEC)
EIEC closely resemble Shigella in their pathogenic mechanisms and the kind of clinical illness they produce. EIEC penetrate and multiply within epithelial cells of the colon causing widespread cell destruction. The clinical syndrome is identical to Shigella dysentery and includes a dysentery-like diarrhea with fever. Like Shigella, EIEC are invasive organisms. but hey do not produce LT or ST toxin and, unlike Shigella, they do not produce the shiga toxin.

Enteropathogenic E. coli (EPEC)
EPEC induce a watery diarrhea similar to ETEC, but they do not possess the same colonization factors and do not produce ST or LT toxins. They produce a non fimbrial adhesin designated intimin, an outer membrane protein, that mediates the final stages of adherence. Although they do not produce LT or ST toxins, there are reports that they produce an enterotoxin similar to that of Shigella. Other virulence factors may be related to those in Shigella.

Adherence of EPEC strains to the intestinal mucosa is a very complicated process and produces dramatic effects in the ultrastructure of the cells resulting in rearrangements of actin in the vicinity of adherent bacteria. The phenomenon is sometimes called “attaching and effacing” of cells. EPEC strains are said to be “moderately-invasive” meaning they are not as invasive as Shigella, and unlike ETEC or EAggEC, they cause an inflammatory response. The diarrhea and other symptoms of EPEC infections probably are caused by bacterial invasion of host cells and interference with normal cellular signal transduction, rather than by production of toxins.

Some types of EPEC are referred to as Enteroadherent E. coli (EAEC), based on specific patterns of adherence. They are an important cause of traveler’s diarrhea in Mexico and in North Africa.

Enteroaggregative E. coli (EAggEC)
The distinguishing feature of EAggEC strains is their ability to attach to tissue culture cells in an aggregative manner. These strains are associated with persistent diarrhea in young children. They resemble ETEC strains in that the bacteria adhere to the intestinal mucosa and cause non-bloody diarrhea without invading or causing inflammation. This suggests that the organisms produce a toxin of some sort. Recently, a distinctive heat-labile plasmid-encoded toxin has been isolated from these strains, called the EAST (EnteroAggregative ST) toxin. They also produce a hemolysin related to the hemolysin produced by E. coli strains involved in urinary tract infections. The role of the toxin and the hemolysin in virulence has not been proven. The significance of EAggEC strains in human disease is controversial.

Enterohemorrhagic E. coli (EHEC)
EHEC are represented by a single strain (serotype O157:H7), which causes a diarrheal syndrome distinct from EIEC (and Shigella) in that there is copious bloody discharge and no fever. A frequent life-threatening situation is its toxic effects on the kidneys (hemolytic uremia).

EHEC has recently been recognized as a cause of serious disease often associated with ingestion of inadequately cooked hamburger meat. Pediatric diarrhea caused by this strain can be fatal due to acute kidney failure (hemolytic uremic syndrome [HUS]). EHEC are also considered to be “moderately invasive”. Nothing is known about the colonization antigens of EHEC but fimbriae are presumed to be involved. The bacteria do not invade mucosal cells as readily as Shigella, but EHEC strains produce a toxin that is virtually identical to the Shiga toxin. The toxin plays a role in the intense inflammatory response produced by EHEC strains and may explain the ability of EHEC strains to cause HUS. The toxin is phage encoded and its production is enhanced by iron deficiency.

Biotechnological Applications of E. coli

The advances in molecular biology, genetics and biochemistry during the past four decades have led to an enormous development in the field of biotechnology. Studies with E. coli have played a major role in these developments, and the bacterium has been in the forefront of many technological advances.

In the early days of biotechnology (1960s), emphasis was placed on improvements of established procedures of bioprocessing, such as the production of yeast, vaccines, and antibiotics. These investigations stimulated genetic research of microbes to increase their potential to produce a wide variety of products in the service of humanity. Although much was being learned about E. coli and its genetics, the direct use of the bacterium in the industry was limited. The industrial production of the amino acid threonine by E. coli mutants, begun in 1961, is an exception.
At this time, organisms were generally subjected to mutagenic agents, which produced a series of random mutations, from which the specifically required mutants were selected.

In the last two decades, procedures have evolved which permit the preparation of strains that have very specific productive capabilities. As the genetic structure of E. coli was well known, and it is an organism which can grow on simple media (mineral salts and glucose) under aerobic and anaerobic conditions, the bacterium became the basis for most developments in genetic manipulations leading to genetic engineering.

The basic principle of these genetic manipulations is gene cloning, which enables the isolation and replication of individual DNA fragments. This consists of a series of linked steps, involving the isolation of the desired gene as double-stranded DNA (dsDNA), insertion of the gene into a suitable vector, and using the vector to introduce the DNA into a cell which will express the desired genetic information. In the case of cloning a gene in E. coli, first the DNA of suitable character is isolated, then it is joined to the DNA of a suitable vector producing a series of recombinant molecules. Then the recombinant molecules are introduced into the bacterium in which the target gene becomes established. Recombinants are selected in various ways with the purpose of expressing the desired genetic information.

The source for DNA cloning can be genomic DNA fragments, cDNA fragments produced by the action of reverse transcriptase on mRNA molecules, chemically synthesized oligonucleotides, or amplified DNA from the products of the polymerase chain reaction (PCR). Plasmids, phages, and cosmids have all been successfully used as vectors, and transformation, transfection, and transduction have all been used to introduce the foreign DNA into the E. coli cell. Plasmids are among the most widely used vectors for the insertion of foreign DNA into an E. coli. Plasmids lend themselves very well as vectors since they are independent replicons which are stabily inherited in an extrachromosomal state and can be made to carry easily identifiable phenotypic markers such as antibiotic resistance or sugar fermentation.

An example of the use of plasmids to introduce a foreign gene into E. coli in order to produce a useful product is illustrated by the use of the E. coli plasmid pBR322 to clone the gene for production of the human growth hormone, somatostatin. In this case, the gene for the small polypeptide hormone was produced by synthetic means. The double-stranded DNA coding for the 15 amino acids of somatostatin was synthesized with the addition of a translation a stop signal at the end. The synthetic gene was then recombined with the plasmid within the beta-galactosidase structural gene and introduced into E. coli. In this way, the production of the somatostatin peptide could be controlled by the lac operon. In a similar manner, the genes for human insulin production were inserted into E. coli which was then able to synthesize the human hormone.

Such general techniques of molecular biology and bacterial genetics are now being applied within research laboratories and industry to produce a wide variety of strains of genetically engineered E. coli from which a number of useful products can be produced. Likewise, the problems associated with the expression of eukaryotic DNA by a procaryotic promoter in E. coli were solved by construction of a fusion gene. In this system, the control region and the N-terminal coding sequence of an E. coli gene are ligated to a eukaryotic sequence so that translation of the chimeric protein can occur. The only condition is that the eukaryotic sequence must be in the correct reading frame. The desired protein is then enzymatically or chemically cleaved from the E. coli product.

E. coli strains are been genetically engineered to produce a variety of mammalian proteins, especially products of medical or veterinary interest including enzymes and vaccine components. E. coli has also been used to manufacture other substances including enzymes that are useful in the degradation of cellulose and aromatic compounds and enzymes for ethanol production. There may be no limit to what E. coli can produce through recombinant DNA technology as long as the substance is a natural product for which a genetic sequence can be found.

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Salmonella and Salmonellosis

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Salmonella is a Gram-negative facultative rod-shaped bacterium in the same proteobacterial family as Escherichia coli, the family Enterobacteriaceae, trivially known as “enteric” bacteria. Salmonella is nearly as well-studied as E. coli from a structural, biochemical and molecular point of view, and as poorly understood as E. coli from an ecological point of view. Salmonellae live in the intestinal tracts of warm and cold blooded animals. Some species are ubiquitous. Other species are specifically adapted to a particular host. In humans, Salmonella are the cause of two diseases called salmonellosis: enteric fever (typhoid), resulting from bacterial invasion of the bloodstream, and acute gastroenteritis, resulting from a foodborne infection/intoxication.

Discovery of the Typhoid Bacillus

At the beginning of the 19th century, typhoid was defined on the basis of clinical signs and symptoms and pathological (anatomical) changes. However, at this time, all sorts of enteric fevers were characterized as “typhoid”.

In 1880s, the typhoid bacillus was first observed by Eberth in spleen sections and mesenteric lymph nodes from a patient who died from typhoid. Robert Koch confirmed a related finding by Gaffky and succeeded in cultivating the bacterium in 1881. But due to the lack of differential characters, separation of the typhoid bacillus from other enteric bacteria was uncertain.

In 1896, it was demonstrated that the serum from an animal immunized with the typhoid bacillus agglutinated (clumped) the typhoid bacterial cells, and it was shown that the serum of patients afflicted with typhoid likewise agglutinated the typhoid bacillus. Serodiagnosis of typhoid was thus made possible by 1896.

Salmonella Nomenclature

The genus Salmonella is a member of the family Enterobacteriaceae, It is composed of bacteria related to each other both phenotypically and genotypically. Salmonella DNA base composition is 50-52 mol% G+C, similar to that of Escherichia, Shigella, and Citrobacter. The bacteria of the genus Salmonella are also related to each other by DNA sequence. The genera with DNA most closely related to Salmonella are Escherichia, Shigella, and Citrobacter. Similar relationships were found by numerical taxonomy and 16S ssRNA analysis.

Salmonella nomenclature has been controversial since the original taxonomy of the genus was not based on DNA relatedness, rather names were given according to clinical considerations, e.g., Salmonella typhi, Salmonella cholerae-suis, Salmonella abortus-ovis, and so on. When serological analysis was adopted into the Kauffmann-White scheme in 1946, a Salmonella species was defined as “a group of related fermentation phage-type” with the result that each Salmonella serovar was considered as a species. Since the host-specificity suggested by some of these earlier names does not exist (e.g., S. typhi-murium, S. cholerae-suis are in fact ubiquitous), names derived from the geographical origin of the first isolated strain of the newly discovered serovars were next chosen, e.g., S. london, S. panama, S. stanleyville.

Susequently it was found that all Salmonella serovars form a single DNA hybridization group, i.e., a single species composed of seven subspecies, and thenomenclature had to be adapted. To avoid confusion with the familiar names of serovars, the species name Salmonella enterica was proposed with the following names for the subspecies:
enterica I
salamae II
arizonae IIIa
diarizonae IIIb
houtenae IV
bongori V
indica VI
Each subspecies contains various serovars defined by a characteristic antigenic formula.

Since this formal Latin nomenclature may not be clearly understood by physicians and epidemiologists, who are the most familiar with the names given to the most common serovars, the common serovars names are kept for subspecies I strains, which represent more than 99.5% of the Salmonella strains isolated from humans and other warm-blooded animals. The vernacular terminology seems preferred in medical practice, e.g., Salmonella ser. Typhimurium (not italicized) or shorter Salmonella (or S.) Typhimurium.

Discovery of the Typhoid Bacillus

Antigenic Structure

As with all Enterobacteriaceae, the genus Salmonella has three kinds of major antigens with diagnostic or identifying applications: somatic, surface, and flagellar.

Somatic (O) or Cell Wall Antigens
Somatic antigens are heat stable and alcohol resistant. Cross-absorption studies individualize a large number of antigenic factors, 67 of which are used for serological identification. O factors labeled with the same number are closely related, although not always antigenically identical.

Surface (Envelope) Antigens
Surface antigens, commonly observed in other genera of enteric bacteria (e.g., Escherichia coli and Klebsiella), may be found in some Salmonella serovars. Surface antigens in Salmonella may mask O antigens, and the bacteria will not be agglutinated with O antisera. One specific surface antigen is well known: the Vi antigen. The Vi antigen occurs in only three Salmonella serovars (out of about 2,200): Typhi, Paratyphi C, and Dublin. Strains of these three serovars may or may not have the Vi antigen.

Flagellar (H) Antigens
Flagellar antigens are heat-labile proteins. Mixing salmonella cells with flagella-specific antisera gives a characteristic pattern of agglutination (bacteria are loosely attached to each other by their flagella and can be dissociated by shaking). Also, antiflagellar antibodies can immobilize bacteria with corresponding H antigens.

A few Salmonella entericaserovars (e.g., Enteritidis, Typhi) produce flagella which always have the same antigenic specificity. Such an H antigen is then called monophasic. Most Salmonella serovars, however, can alternatively produce flagella with two different H antigenic specificities. The H antigen is then called diphasic. For example, Typhimurium cells can produce flagella with either antigen i or antigen 1,2. If a clone is derived from a bacterial cell with H antigen i, it will consist of bacteria with i flagellar antigen. However, at a frequency of 10^-3– 10^-5, bacterial cells with 1,2 flagellar antigen pattern will appear in this clone.

Habitats

The principal habitat of the salmonellae is the intestinal tract of humans and animals. Salmonella serovars can be found predominantly in one particular host, can be ubiquitous, or can have an unknown habitat. Typhi and Paratyphi A are strictly human serovars that may cause grave diseases often associated with invasion of the bloodstream. Salmonellosis in these cases is transmitted through fecal contamination of water or food. Gallinarum, Abortusovis, and Typhisuis are, respectively, avian, ovine, and porcine Salmonella serovars. Such host-adapted serovars cannot grow on minimal medium without growth factors (contrary to the ubiquitous Salmonella serovars).

Ubiquitous (non-host-adapted) Salmonella serovars (e.g., Typhimurium) cause very diverse clinical symptoms, from asymptomatic infection to serious typhoid-like syndromes in infants or certain highly susceptible animals (mice). In human adults, ubiquitous Salmonella organisms are mostly responsible for foodborne toxic infections.

The pathogenic role of a number of Salmonella serovars is unknown. This is especially the case with serovars from subspecies II to VI. A number of these serovars have been isolated rarely (some only once) during a systematic search in cold-blooded animals.

Salmonella in the Natural Environment

Salmonellae are disseminated in the natural environment (water, soil, sometimes plants used as food) through human or animal excretion. Humans and animals (either wild or domesticated) can excrete Salmonella either when clinically diseased or after having had salmonellosis, if they remain carriers. Salmonella organisms do not seem to multiply significantly in the natural environment (out of digestive tracts), but they can survive several weeks in water and several years in soil if conditions of temperature, humidity, and pH are favorable

Isolation and Identification of Salmonella

A number of plating media have been devised for the isolation of Salmonella. Some media are differential and nonselective, i.e., they contain lactose with a pH indicator, but do not contain any inhibitor for non salmonellae (e.g., bromocresol purple lactose agar). Other media are differential and slightly selective, i.e., in addition to lactose and a pH indicator, they contain an inhibitor for nonenterics (e.g., MacConkey agar and eosin-methylene blue agar).

The most commonly used media selective for Salmonella are SS agar, bismuth sulfite agar, Hektoen enteric (HE) medium, brilliant green agar and xylose-lisine-deoxycholate (XLD) agar. All these media contain both selective and differential ingredients and they are commercially available.

Media used for Salmonella identification are those used for identification of all Enterobacteriaceae. Most Salmonella strains are motile with peritrichous flagella, however, nonmotile variants may occur occasionally. Most strains grow outrient agar as smooth colonies, 2-4 mm in diameter. Most strains are prototrophs, not requiring any growth factors. However, auxotrophic strains do occur, especially in host-adapted serovars such as Typhi and Paratyphi A.

Table 1. Characteristics shared by most Salmonella strains belonging to subspecies I

Motile, Gram-negative bacteria
Lactose negative; acid and gas from glucose, mannitol, maltose, and sorbitol; no Acid from adonitol, sucrose, salicin, lactose
ONPG test negative (lactose negative)
Indole test negative
Methyl red test positive
Voges-Proskauer test negative
Citrate positive (growth on Simmon’s citrate agar)
Lysine decarboxylase positive
Urease negative
Ornithine decarboxylase positive
H₂S produced from thiosulfate
Do not grow with KCN
Phenylalanine and tryptophan deaminase negative
Gelatin hydrolysis negative

Genetics of Salmonella

The genetic map of the Salmonella Typhimurium strain LT2 is not very different from that of Escherichia coli K-12. The F plasmid can be transferred to Typhimurium, and an Hfr strain of Typhimurium may subsequently be selected. Conjugative chromosomal transfer may occur from Typhimurium Hfr to E. coli or from E. coli Hfr to Typhimurium. Chromosomal genes responsible for O, Vi, and H antigens can be transferred from Salmonella to Escherichia.

Also, Salmonella may harbor temperate phages and plasmids. Plasmids in Salmonella may code for antibiotic resistance (resistance plasmids are frequent due to the selective pressure of extensive antibiotic therapy), bacteriocins, metabolic characteristics such as lactose or sucrose fermentation, or antigenic changes of O antigen.

Pathogenesis of Salmomella Infections in Humans

Salmonella infections in humans vary with the serovar, the strain, the infectious dose, the nature of the contaminated food, and the host status. Certain serovars are highly pathogenic for humans; the virulence of more rare serovars is unknown. Strains of the same serovar are also known to differ in their pathogenicity. An oral dose of at least 10⁵Salmonella Typhi cells are needed to cause typhoid in 50% of human volunteers, whereas at least 10⁹ S. Typhimurium cells (oral dose) are needed to cause symptoms of a toxic infection. Infants, immunosuppressed patients, and those affected with blood disease are more susceptible to Salmonella infection than healthy adults.

In the pathogenesis of typhoid the bacteria enter the human digestive tract, penetrate the intestinal mucosa (causing no lesion), and are stopped in the mesenteric lymph nodes. There, bacterial multiplication occurs, and part of the bacterial population lyses. From the mesenteric lymph nodes, viable bacteria and LPS (endotoxin) may be released into the bloodstream resulting in septicemia Release of endotoxin is responsible for cardiovascular ï¿½collapsus and tuphosï¿½ (a stuporous stateï¿½origin of the name typhoid) due to action on the ventriculus neurovegetative centers.

Salmonella excretion by human patients may continue long after clinical cure. Asymptomatic carriers are potentially dangerous when unnoticed. About 5% of patients clinically cured from typhoid remain carriers for months or even years. Antibiotics are usually ineffective on Salmonella carriage (even if salmonellae are susceptible to them) because the site of carriage may not allow penetration by the antibiotic.

Salmonellae survive sewage treatments if suitable germicides are not used in sewage processing. In a typical cycle of typhoid, sewage from a community is directed to a sewage plant. Effluent from the sewage plant passes into a coastal river where edible shellfish (mussels, oysters) live. Shellfish concentrate bacteria as they filter several liters of water per hour. Ingestion by humans of these seafoods (uncooked or superficially cooked) may cause typhoid or other salmonellosis. Salmonellae do not colonize or multiply in contaminated shellfish.

Typhoid is strictly a human disease.The incidence of human disease decreases when the level of development of a country increases (i.e., controlled water sewage systems, pasteurization of milk and dairy products). Where these hygienic conditions are missing, the probability of fecal contamination of water and food remains high and so is the incidence of typhoid.

Foodborne Salmonella toxic infections are caused by ubiquitous Salmonella serovars (e.g., Typhimurium). About 12-24 hours following ingestion of contaminated food (containing a sufficient number of Salmonella), symptoms appear (diarrhea, vomiting, fever) and last 2-5 days. Spontaneous cure usually occurs.

Salmonella may be associated with all kinds of food. Contamination of meat (cattle, pigs, goats, chicken, etc.) may originate from animal salmonellosis, but most often it results from contamination of muscles with the intestinal contents during evisceration of animals, washing, and transportation of carcasses. Surface contamination of meat is usually of little consequence, as proper cooking will sterilize it (although handling of contaminated meat may result in contamination of hands, tables, kitchenware, towels, other foods, etc.). However, when contaminated meat is ground, multiplication of Salmonella may occur within the ground meat and if cooking is superficial, ingestion of this highly contaminated food may produce a Salmonellainfection. Infection may follow ingestion of any food that supports multiplication of Salmonella such as eggs, cream, mayonnaise, creamed foods, etc.), as a large number of ingested salmonellae are needed to give symptoms. Prevention of Salmonella toxic infection relies on avoiding contamination (improvement of hygiene), preventing multiplication of Salmonella in food (constant storage of food at 4°C), and use of pasteurized and sterilized milk and milk products. Vegetables and fruits may carry Salmonella when contaminated with fertilizers of fecal origin, or when washed with polluted water.

The incidence of foodborne Salmonella infection/toxication remains reletavely high in developed countries because of commercially prepared food or ingredients for food. Any contamination of commercially prepared food will result in a large-scale infection. In underdeveloped countries, foodborne Salmonella intoxications are less spectacular because of the smaller number of individuals simultaneously infected, but also because the bacteriological diagnosis of Salmonella toxic infection may not be available. However, the incidence of Salmonella carriage in underdeveloped countries is known to be high.

Salmonella epidemics may occur among infants in pediatric wards. The frequency and gravity of these epidemics are affected by hygienic conditions, malnutrition, and the excessive use of antibiotics that select for multiresistant strains.

Salmonella Enteritidis Infection
Egg-associated salmonellosis is an important public health problem in the United States and several European countries. Salmonella Enteritidis, can be inside perfectly normal-appearing eggs, and if the eggs are eaten raw or undercooked, the bacterium can cause illness. During the 1980s, illness related to contaminated eggs occurred mosy frequently in the northeastern United States, but now illness caused by S. Enteritidis is increasing in other parts of the country as well.

Unlike eggborne salmonellosis of past decades, the current epidemic is due to intact and disinfected grade A eggs. Salmonella Enteritidis silently infects the ovaries of healthy appearing hens and contaminates the eggs before the shells are formed. Most types of Salmonella live in the intestinal tracts of animals and birds and are transmitted to humans by contaminated foods of animal origin. Stringent procedures for cleaning and inspecting eggs were implemented in the 1970s and have made salmonellosis caused by external fecal contamination of egg shells extremely rare. However, unlike eggborne salmonellosis of past decades, the current epidemic is due to intact and disinfected grade A eggs. The reason for this is that Salmonella Enteritidis silently infects the ovaries of hens and contaminates the eggs before the shells are formed.

Although most infected hens have been found in the northeastern United States, the infection also occurs in hens in other areas of the country. In the Northeast, approximately one in 10,000 eggs may be internally contaminated. In other parts of the United States, contaminated eggs appear less common. Only a small number of hens seem to be infected at any given time, and an infected hen can lay many normal eggs while only occasionally laying an egg contaminated with Salmonella Enteritidis.

A person infected with the Salmonella Enteritidis usually has fever, abdominal cramps, and diarrhea beginning 12 to 72 hours after consuming a contaminated food or beverage. The illness usually lasts 4 to 7 days, and most persons recover without antibiotic treatment. However, the diarrhea can be severe, and the person may be ill enough to require hospitalization. The elderly, infants, and those with impaired immune systems (including HIV) may have a more severe illness. In these patients, the infection may spread from the intestines to the bloodstream, and then to other body sites and can cause death unless the person is treated promptly with antibiotics.

Exotoxins

Salmonella strains may produce a thermolabile enterotoxin that bears a limited relatedness to cholera toxin both structurally and antigenically. This enterotoxin causes water secretion in rat ileal loop and is recognized by antibodies against both cholera toxin and the thermolabile enterotoxin (LT) of enterotoxinogenic E. coli, but it does not bind in vitro to ganglioside GM1 (the receptor for E. coli LT and cholera ctx). Additionally, a cytotoxin that inhibits protein synthesis and is immunologically distinct from Shiga toxin has been demonstrated. Both of these toxins are presumed to play a role in the diarrheal symptoms of salmonellosis.

Salmonella and Salmonellosis

Antibiotic Susceptibility

During the last decade, antibiotic resistance and multiresistance of Salmonella spp. have increased a great deal. The cause appears to be the increased and indiscriminate use of antibiotics in the treatment of humans and animals and the addition of growth-promoting antibiotics to the food of breeding animals. Plasmid-borne antibiotic resistance is very frequent among Salmonella strains involved in pediatric epidemics (e.g., Typhimurium, Panama, Wien, Infantis). Resistance to ampicillin, streptomycin, kanamycin, chloramphenicol, tetracycline, and sulfonamides is commonly observed. Colistin resistance has not yet been observed.

Until 1972, Typhi strains had remained susceptible to antibiotics, including chloramphenicol (the antibiotic most commonly used against typhoid) but in 1972, a widespread epidemic in Mexico was caused by a chloramphenicol-resistant strain of S. Typhi. Other chloramphenicol-resistant strains have since been isolated in India, Thailand, and Vietnam. Possible importation or appearance of chloramphenicol-resistance strains in the United States is a real threat. Salmonella strains should be systematically checked for antibiotic resistance to aid in the choice of an efficient drug wheeeded and to detect any change in antibiotic susceptibility of strains (either from animal or human source). Indiscriminate distribution and use of antibiotics should be discouraged.

Vaccination Against Typhoid Fever

Three types of typhoid vaccines are currently available for use in the United States: (1) an oral live-attenuated vaccine; (2) a parenteral heat-phenol-inactivated vaccine; (3) a newly licensed capsular polysaccharide vaccine for parenteral use. A fourth vaccine, an acetone-inactivated parenteral vaccine, is currently available only to the armed forces.

1. Live oral vaccines. Although oral killed vaccines are without efficacy, vaccines using living avirulent bacteria have shown promise. A galactose-epimeraseless mutant of Typhi has given very good results in a field trials. Mutants of Typhimurium that have given a good protection in animals include mutants lacking adenylate-cyclase and AMP receptor protein, and mutants auxotrophic for p-aminobenzoate and adenine.Typhi with the same mutations does not cause adverse reactions and is immunogenic in human.

The Live Oral Typhoid Vaccine should not be given to children younger than 6 years of age. It is given in four doses, 2 days apart, as needed for protection. The last dose should be given at least 1 week before travel to allow the vaccine time to work. A booster dose is needed every 5 years for people who remain at risk.

2. The parenteral heat-phenol-inactivated vaccine has been widely used for many years. In field trials involving a primary series of two doses of heat-phenol- inactivated typhoid vaccine, efficacy over the 2- to 3-year follow-up periods ranged from 51% to 77% . Efficacy for the acetone- inactivated parenteral vaccine, available only to the armed forces, ranges from 75% to 94%.

Since the inactivated vaccines contain the O antigen (endotoxin), local and general reactions occur. Vi antigen extracted following the methodology used for the meningococcal vaccine seems to avoid reactions to endotoxin.

The inactivated Typhoid Vaccine should not be given to children younger than 2 years of age. One dose provides protection. It should be given at least 2 weeks before travel to allow the vaccine time to work. A booster dose is needed every 2 years for people who remain at risk.

3. The newly licensed parenteral vaccine [Vi capsular polysaccharide (ViCPS)] is composed of purified Vi (“virulence”) antigen, the capsular polysaccharide elaborated by S.Typhi isolated from blood cultures. In recent studies, one 25-ug injection of purified ViCPS produced seroconversion (i.e., at least a fourfold rise in antibody titers) in 93% of healthy U.S. adults. Two field trials in disease-endemic areas have demonstrated the efficacy of ViCPS in preventing typhoid fever. In one trial in Nepal, in which vaccine recipients were observed for 20 months, one dose of ViCPS among persons 5-44 years of age resulted in 74% fewer cases of typhoid fever. ViCPS has not been tested among children less than 1 year of age.

NOTE: No typhoid vaccine is 100% effective and is not a substitute for being careful about what you eat or drink.

Routine typhoid vaccination is not recommended in the United States, but typhoid vaccine is recommended for travellers to parts of the world where typhoid is common, people in close contact with a typhoid carriers, and laboratory workers who work with Salmonella Typhi bacteria.

VIT-Salmonella

Fast and reliable detection of Salmonella

VIT-Salmonella unequivocally detects all members of the genus Salmonella. In comparison with conventional methods VIT-Salmonella allows time savings of two to four days. You are thus provided with fast, clarifying results concerning Salmonella occurrence in your sample, so adequate counter measures may be taken punctually in case of contamination.

The analysis is easy to conduct – only few minutes hands-on time. The results are provided within 3 hours. Analysis is performed by using a fluorescence microscope. All essential expendable materials required for analysis are contained in the kit. Package size: 25 or 50 analysis.

Analysis can be performed with following samples: