The Typhoid Bacilli Was First Spotted

Published Date: 02 Nov 2017

The typhoid bacilli was first spotted by Eberth (1880), in mesenteric lymph node and spleen section of typhoid patients and Gaffky (1884) successfully isolated the organism, it was then called Eberth Gaffky bacillus or Eberthella typhi. The genus Salmonella was named after Daniel Elmer Salmon, an American veterinary pathologist. While Theobal d Smith was the actual discoverer of the type bacterium (Salmonella enterica var. choleraesuis) in 1885. Dr. Salmon was the administrator of the USDA research program, and thus the organism was named after him by Smith (FDA/CFSAN, 2008). Smith and Salmon had been searching for the cause of common hog cholera and proposed this organism as the causal agent. Later research, however, would show this organism (now known as Salmonella enterica) rarely causes enteric symptoms in pigs (http://www.cgmh.org.tw/chldhos/intr/c4a00/academy/bugs/salcho, 2009) and was thus not the agent they were seeking (which was eventually shown to be a virus). However, related bacteria in the genus Salmonella were eventually shown to cause other important infectious diseases.

The genus Salmonella was finally formally adopted in 1900 by J. Lignières for the many species of Salmonella, after Smith's first type-strain Salmonella cholerae suis.. In 1896, Pfeiffer and Kalle made the first typhoid vaccine with heat-killed organisms. In the same year Widal and others demonstrated that convalescent sera from typhoid patients caused the organisms to "stick together in large balls and lose their motility." Georges Fernand Isidore Widal, a French physician coined the term agglutinin to describe this observation. Today the antigenic classification or stereotyping of Salmonella used which is a result of years of study of antibody interactions with bacterial surface antigens by Kauffman and White during the 1920s to 1940s. In 1948, Theodore Woodward and colleagues reported the successful treatment of Malaysian typhoid patients with Chloromycetin, (Woodward et al., 1948; Chakraborty, 2001). The most famous outbreak of enteric fever is Typhoid Mary. Mary Mallon, a New York City hired household cook, transmitted typhoid fever to at least 22 individuals causing 3 deaths between 1900 and 1907. After being apprehended by public health officials in 1907, she was isolated for 3 years. Even though she was released with the stipulation that she never cook again, she broke the promise and consequently caused at least 25 more cases of typhoid fever at Manhattan maternity hospital when she was employed as a cook in 1915. She was finally isolated until her death in 1938 (Parry, 2006).

2.2. Classification and Taxonomy

Salmonella is a genus of rod-shaped, Gram-negative, non-spore forming, predominantly, motile enterobacteria with diameters around 0.7 to 1.5 µm. The length ranges from 2 to 5 µm, and flagella that grade in all directions (i.e., peritrichous). They are chemo-organotrophs, obtaining their energy from oxidation and reduction reactions using organic sources, and are facultative anaerobes. Most species produce hydrogen sulfide (Clarke and Barret, 1987) which can readily be detected by growing them on media containing ferrous sulfate, such as TSI. Most isolates exist in two phases: a motile phase I and a non-motile phase II. Cultures that are non-motile upon primary culture may be switched to the motile phase using a Cragie tube.

Salmonella is closely related to the Escherichia genus and are found worldwide in cold- and warm-blooded animals (including humans), and in the environment. They cause illnesses such as typhoid fever, paratyphoid fever, and foodborne illness (Ryan and Ray, 2004). The genus Salmonella is divided into two species, each with multiple subspecies and serotypes. The two species are Salmonella is a genus of the family of Enterobacteriaceae.

Initially each Salmonella species was named according to clinical considerations (Kauffmann, 1941) e.g., Salmonella typhi-murium (mouse typhoid fever), S. cholerae-suis (hog cholera). After it was recognized that host specificity did not exist for many species, new strains (orserovar, short for serological variants) received species names according to the location at which the new strain was isolated. Later, molecular findings led to the hypothesis that Salmonella consisted of only one species (Le Minor et al., 1987), S. enterica, and the serovar were classified into six groups (Reeves et al., 1989) two of which are medically relevant. But as this now formalized nomenclature (Kauffmann and Edwards, 1952; Tindall et al., 2005) is not in harmony with the traditional usage familiar to specialists in microbiology and infectologists, the traditional nomenclature is common. Currently, there are three recognized species: S. enterica, S. bongori and S. subterranean, with six main subspecies: enetrica (I), salamae (II), arizonae (IIIa), houtenae (IV) and indica (VI) (Janda and Abbott, 2006). Historically, serotype (V) was bongori, which is now considered its own species. (Table.2.1).

The serovar (i.e. serotype) is a classification of Salmonella into subspecies based on antigens that the organism presents. It is based on the Kauffman-White classification scheme that differentiates serological varieties from each other. Serotypes are usually put into subspecies groups after the genus and species, with the serovars/sertypes capitalized but not italicized: an example is Salmonella enterica serovar Typhimurium. Newer methods for Salmonella typing and subtyping include genome-based methods such as pulsed field gel electrophoresis (PFGE), Multiple Loci VNTR Analysis (MLVA), Multilocus sequence typing (MLST) and (multiplex-) PCR-based methods (Porwolik, 2011; Achtman, 2012).

Before 1983 the existence of multiple Salmonella enteric and Salmonella bongori where the former acquired the SPI-2 cluster (Salmonella pathogenicity island) that is absent in S. bongori serotypes. S. enterica is postulated to have branched into several distinct phylogenetic groups which by current nomenclature are considered subspecies. The host adaptation of S. enterica subspecies I to warm-blooded vertebrates characterized a phase in the evolution of virulence in the genus members of the six Salmonella subspecies can be serotyped into one of over 2400 serotypes (serovars) according to somatic (O), surface (Vi), the surface Vi antigen (restricted to S. typhi and S. paratyphi C), and flagellar (H) antigen, and habitats (Brenner et. al., 2000).

Le Minor et al., (1982) carried out 90 carbon source utilization tests and 41 biochemical tests by using 88 Salmonella reference strains including the so-called subgenus I (20 strains), subgenus II (21 strains), subgenus III ( Arizona, 21 strains), subgenus IV (20 strains) and 6 atypical strains (bongor group). They carried out a cluster analysis (Jaccard coefficient, clustering according to the variance) that yielded 7 phenons; 35 strains from these 7 phenons were studied by DNA relatedness (S1 nuclease method with DE81 filters). Six hybridization groups, largely concordant with the phenon, were distinguished. Comparison of phenetic and genomic criteria allowed us to subdivide the genus Salmonella into 6 taxa corresponding to (1) subgenus I, subdivided phenotypically into an adapted group and a ubiquitous group, (2) subgenus II, (3) monophasic serovars of subgenus III, (4) diphasic serovars of subgenus III, (5) subgenus IV, and (6) bongor group. The taxonomic level of each of the 6 taxa is not that of a subgenus, but that of a subspecies.

Table.1. Classification of Salmonella species & subspecies (Old et. al., 2006).

Subspecies

No of serotypes within subspecies

Salmonella enterica subspecies enterica (I)

1531

Salmonella enterica subspecies salmae (II)

505

Salmonella enterica subspecies arizonae (IIIa)

99

Salmonella enterica subspecies diarizonae (IIIb)

336

Salmonella enterica subspecies houtenae (IV)

73

Salmonella enterica subspecies indica (VI)

13

Salmonella bongori (Formerly subgenera V)

22

Bacteria can be classified on the basis of phylogeny. A phylogenetic tree can be descended from the comparison of 16S rRNA or other gene sequences. There are 2463 Salmonella serotypes that are now placed under 2 species due to the difference in 16S rRNA sequence analysis: Salmonella enterica (2443 serotypes) and Salmonella bongori (20 serotypes). The system is currently used by World Health Organization (WHO) Collaborating Centre, Centers for Disease Control and Prevention (CDC) and some other organizations. Salmonella enterica is further divided into six subspecies, designated by Roman numerals. Salmonella enterica subspecies I is mainly isolated from warm-blooded animals and accounts for more than 99% of clinical isolates whereas remaining subspecies and S. bongori are mainly isolated from cold-blooded animals and account for less than 1% of clinical isolates. As an example, the Kauffmann species Salmonella typhimurium is now designated as Salmonella enterica subspecies I serotype typhimurium. Under the modern nomenclature system, the subspecies information is often omitted and culture is called S. enterica serotype Typhimurium and in subsequent appearance, it is written as S. typhimurium. This system of nomenclature is used nowadays to bring uniformity in reporting (Andrews and Baumler, 2005; Parry, 2006; Bhunia, 2008). (Fig. 2.1).

Selander et al., (1990) estimated the genetic diversity and relationships among 123 strains of Salmonella paratyphi B (serotype 1,4,[5],12:b:[1,2]) from an assessment of electrophoretically demonstrable allelic variation at 24 chromosomal enzyme gene loci. Fourteen electrophoretic types, marking clones, were distinguished; the phylogeny of the clonal lineages was reformed

Figure 2.1. Diagram showing the classification of Salmonella genus and its diseases. (Wain and Nair, 2009)

and biotype and other phenotypic characters were mapped onto this structure. Most d-tartrate-negative strains are members of an abundant, globally distributed clone (Pb 1) that is polymorphic for many biotype characters (including d-tartrate utilization), bacteriophage type, rRNA pattern, and colicin M and phage ES18 sensitivity. This clone is largely responsible for S. paratyphi B enteric fever in humans. In contrast, d-tartrate-positive strains (formerly known as S. java) occurred in all seven of the clonal lineages identified by population genetic analysis, although most d-tartrate-positive isolates belong to only two clones (Pb 3 and Pb 4), which vary in frequency geographically. Monophasic strains represent four closely related clones forming a distinctive phylogenetic lineage. The Kauffmann hypothesis of convergence in serotype among distantly related cell lineages through recombination (via phage transduction or other means) may account for the considerable genotypic diversity among clones of S. paratyphi B. Pb 4, Pb 6, and Pb 7 are more closely allied with clones of S. typhimurium and S. saintpaul than with other clones of S. paratyphi B. Sensitivity or resistance to colicin M and phage ES18 and the electrophoretic pattern of the rRNA, which were incorporated into a recently proposed scheme for the identification of types of S. paratyphi B, individually or in combination fail to mark clones or other meaningful phylogenetic subdivisions.

Christensen et al., (1998) established the phylogenetic relationships between the subspecies of Salmonella enterica (official name Salmonella choleraesuis), Salmonella bongori and related members of Enterobacteriaceae, by sequence comparison of rRNA using maximum-likelihood

analysis. The two Salmonella species were separated by 16S rRNA analysis and found to be closely related to the Escherichia coli and Shigella complex by both 16S and 23S rRNA analyses. The diphasic serotypes S. enterica subspp. I and VI were separated from the monophasic serotypes subspp. IIIa and IV, including S. bongori, by 23S rRNA sequence comparison.

Christensen and Olsen (1998) determined DNA sequences covering 57% of atpD encoding the β subunit of ATP synthase for 16 strains of Salmonella enterica, two strains of S. bongori, and one strain each of Citrobacter freundii and Yersinia enterocolitica, and comparison was made with the published Escherichia coli and Enterobacter aerogenes sequences. The phylogenetic tree based on maximum-likelihood analysis showed separation of the subspecies of S. enterica except for two serotypes of subspecies II which were unsupported by a common node. The two serotypes of S. bongori were separated from S. enterica and related to the serotypes of subspecies II. A tight relationship was found between S. enterica subspecies IIIa consisting of monophasic serotypes and subspecies IIIb consisting of diphasic serotypes. This is in conflict with results obtained for most other housekeeping genes and the 23S rRNA gene separating mono- from diphasic subspecies.

Paradis et al., (2005) studied the phylogeny of enterobacterial species commonly found in clinical samples by comparing partial sequences of their elongation factor Tu gene (tuf) and of their F-ATPase β-subunit gene (atpD). An 884 bp fragment for tuf and an 884 or 871 bp fragment for atpD were sequenced for 96 strains representing 78 species from 31 enterobacterial genera. The atpD sequence analysis exhibited an indel specific to Pantoea and Tatumella species, showing, for the first time, a tight phylogenetic affiliation between these two genera. Comprehensive tuf and atpD phylogenetic trees were constructed and are in agreement with each other.Monophyletic genera are Cedecea,Edwardsiella, Proteus, Providencia, Salmonella, Serratia, Raoultella andYersinia. Analogous trees based on 16S rRNA gene sequences available from databases were also reconstructed. The tuf and atpD phylogenies are in agreement with the 16S rRNA gene sequence analysis, and distance comparisons revealed that the tuf and atpD genes provide better discrimination for pairs of species belonging to the family Enterobacteriaceae. In conclusion, phylogeny based on tuf and atpD conserved genes allows discrimination between species of the Enterobacteriaceae.

McQuiston et al., (2008) characterized the phylogenetic relationships of the species and subspecies of Salmonella, and analyzed four housekeeping genes, gapA, phoP, mdh and recA, comprising 3,459 bp of nucleotide sequence data for each isolate sequenced. Sixty-one isolates representing the most common serotypes of the seven subspecies of Salmonella enterica and six isolates of Salmonella bongoriwere included in this study. We present a robust phylogeny of the Salmonellaspecies and subspecies that clearly defines the lineages comprising diphasic and monophasic subspecies. Evidence of inter-subspecies lateral gene transfer of the housekeeping gene recA, which has not previously been reported, was obtained.

2.3 Culture Characters and Growth Requirements

Salmonellae are non-fastidious as they can multiply under various environmental conditions outside the living hosts. They do not require sodium chloride for growth, but can grow in the presence of 0.4 to 4%. Most Salmonella serotypes grow at a temperature range of 5 to 47°C with

optimum temperature of 35 to 37°C but some can grow at temperatures as low as 2 to 4°C or as high as 54°C (Gray and Fedorka-Cray, 2002).

Schaechter et al., (1958) studied cell mass, the average number of nuclei/cell and the content of RNA and DNA in Salmonella typhimurium during balanced (steady state) growth in different media. These quantities could be described as exponential functions of the growth rates afforded by the various media at a given temperature. The size and chemical composition characteristic of a given medium were not influenced by the temperature of cultivation. Thus, under conditions of balanced growth, this organism exists in one of a large number of possible stable physiological states. The variations in mass/cell are due to changes in the number of nuclei/cell as well as in mass/nucleus. An increase in the number of ribonucleoprotein particles at higher growth rates could, it appears, largely account for the increase in mass/nucleus. Calculations indicated that the rate of protein synthesis per unit RNA is nearly the same at all growth rates.

Kjeldgaard et al., (1958) observed a definite pattern of rate changes in the cultures of Salmonella typhimurium undergoing balanced growth when shifted from one medium to another. Shifts from a low to a high growth rate resulted in a strict succession of events: RNA synthesis was immediately affected and its rate rapidly increased to that characteristic of the new medium; the increase in optical density showed a lag of a few minutes before the new rate was attained; DNA synthesis and cell division, on the other hand, continued at the old rate for appreciable periods of time and then abruptly shift to the new rates. The times at which these shifts took place were, at 37°, invariably 20 and 70 min., regardless of the actual growth rates before and after the shift. This rate maintenance effect on DNA synthesis and cell division was discussed in terms of specific rate-controlling mechanisms.

Iino (1969) investigated the phenomenon whereby Salmonella cells produce curly flagella in media containing p-fluorophenylalanine. Salmonella typhimurium LT2 in logarithmic growth in broth was transferred to minimal medium or saline, or their flagella shortened by mechanical breaking. Then, after 2 to 3 hour at 37° in a medium containing p-fluorophenylalanine the distributions of number, length and shape of their flagella were observed. Curly waves appeared at the distal portions of flagella. The growth rate of a flagellum decreased as its length increased, reaching zero at approximately five normal wave-numbers (about 15μ). The growth rate of flagella shortened by mechanical breaking was not less than that of intact ones of similar length. The decline is attributed to decrease in transport efficiency with increase in length rather than to ageing of the flagellum-forming apparatus. A normal flagellar strain and a curly mutant strain of Salmonella abortusequiwere grown together in broth. Numbers of both types of rods and flagella increased to 1·5-fold in 3 hr. At this stage, neither heteromorphous rods nor single flagella having both normal and curly waves were detected. Hence, flagellin molecules reach the top of a growing flagellum without being excreted into the culture medium.

Chung and Goepfert (1970) observed the growth of salmonellae at pH values as low as 4.05 × 0.05. The growth-limiting pH was dependent on several factors, most important the acid molecule itself. Additionally, the effect of temperature, relative oxygen supply and level of inoculum was studied. The salmonellae could not be "trained" to grow at a lower pH by sequential transfer at near optimum pH values.

Ferreira and Lund (1987) investigated the ability of 13 strains of Salmonella, representing 12 serotypes, to grow in a tryptone-yeast extract-glucose medium, acidified with HC1 to pH values between 3.80 and 5.60 at intervals of 0.20 units. During incubation at 30°C, growth occurred at minimum pH values of 3.8–4.0 in 1–3 d. At 20°C, growth occurred at minimum pH values of 3.8–4.2 in 3–5 d. In tests incubated at 10°C, growth occurred at minimum pH values of 4.4–4.8 in between 10 and 19 d.

Kim et al., (1989) examined the influence of time and temperature on Salmonella enteritidis multiplication in experimentally injected eggs. There was an increase in the number of S. enteritidis with the increase in temperature of egg storage. There was less increase of S. enteritidis in eggs stored at 4 C than in eggs held at temperatures higher than 4 C (P < 0.05). These results suggest a possible method for monitoring commercial eggs for the presence of S. enteritidis. It was concluded that the chances of recovery of S. enteritidis can be increased 106-fold or more by holding the eggs at temperatures of 21 or 27 C for more than 20 days and culturing their contents

Clay and Board (1991) investigated the effect of some factors on the growth of Salmonella enteritidis phage type 4 in artificially contaminated shell eggs. Salmonella enteritidis was found to be resistant to the antimicrobial properties of the albumen. Growth occurred on storage at 25 °C but not at 4 or 10 °C. The rate and extent of infection was influenced by the size of inoculum, the site of contamination relative to yolk movement, and the presence of iron in the inoculum.

Lee and Falkow (1990) examined the effect of different growth conditions on the ability of Salmonella to interact with Madin-Darby canine kidney cells. Two growth conditions that affect the expression of Salmonella adherence and invasiveness have been identified. First, bacteria lose their invasiveness in the stationary phase of growth. Second, bacteria growing in oxygen-limited growth conditions are induced for adherence and invasiveness, whereas those growing aerobically are relatively nonadherent and noninvasive. Salmonella from cultures aerated with gas mixtures containing 0% or 1% oxygen were 6- to 70-fold more adherent and invasive than those from cultures aerated with a gas mixture containing 20% oxygen. The Salmonella typhimurium oxrA gene that is required for the anaerobic induction of many proteins is not involved in the regulation of Salmonella invasiveness. They speculate that oxygen limitation might be an environmental cue that triggers the expression of Salmonella invasiveness within the intestinal lumen and other tissues.

Schiemann and Shope (1991) found that growth of Salmonella typhimurium under anaerobic conditions resulted in its greater ability to invade Henle 407 epithelial cells and in greater uptake by mouse peritoneal cells in vitro. Anaerobic growth also resulted in the repression of at least one major outer membrane protein.

Golden et al., (1993) investigated the ability of Salmonella spp. to grow on the interior tissues of cantaloupe, watermelon, and honeydew melons. Pieces of rind-free melons (pH 5.90-6.67) and tryptic soy broth (TSB, pH 5.90) were inoculated with a mixed culture (approximately 100 CFU/g or ml) containing equal proportions of five species of Salmonella (S. anatum, S. chester, S. havana, S. poona, and S. senftenberg). Inoculated melon pieces and TSB were incubated for 24 h at 5 or 23 degreesC. Viable populations of salmonellae were determined by surface plating test portions on Hektoen enteric agar. Results indicated that Salmonella growth was rapid and prolific on the melons and in TSB at 23 degrees C incubation. Final populations on watermelons were approximately 1.0 log10 greater than populations on cantaloupe and honeydew and in TSB. Although viable Salmonella populations on melons and in TSB did not increase during the 24-h incubation at 5 degrees C, little or no decrease in viable populations was observed

McKay and Peters (1995) followed the growth of Salmonella typhimurium colonies on a model food system (agar solidified culture medium). Colony radius, determined using computer image analysis (IA) techniques, and viable cell number per colony were measured as indices of colony growth, and the effect of [NaCl] (0.5–3.5% (w/v)) and pH (7.0–5.0) on colony growth at 30°C was observed; colonies were point inoculated from serial dilutions. Colony growth (between 13 and 26 h after inoculation) was linear when expressed in terms of radius, and exponential when expressed in terms of viable cell number per colony. Overall, both increasing the [NaCl] and decreasing the pH had little effect on colony growth, other than to delay the onset of linear radial growth. Initial specific growth rate (μ) ranged from 0.73 to 0.87 h−1. Thin films of agar medium on microscope slides allowed the growth of microcolonies to be observed after just 4 h incubation. A greater understanding of the growth kinetics of bacterial colonies, and the effects of environment on such data, may enable better control of foodborne bacterial pathogens, and consequently an improvement in food product safety.

Tsolis et al., (1996) examined the role of iron(II) and iron(III) uptake, mediated by FeoB and TonB, respectively, in infection of the mouse by Salmonella typhimurium. The S. typhimurium feoB gene, encoding a homolog of an Escherichia coli cytoplasmic membrane iron(II) permease, was cloned, and a mutant was generated by allelic exchange. In addition, an S. typhimurium tonB mutant was constructed. Together these two mutations inactivate all known iron uptake systems of S. typhimurium. We examined the abilities of these mutants to grow in vitro and in different compartments of the host. Mutants in feoB were outcompeted by the wild type during mixed colonization of the mouse intestine, but the feoB mutation did not attenuate S. typhimurium for oral or intraperitoneal infection of mice. The tonB mutation attenuated S. typhimurium for infection of mice by the intragastric route but not the intraperitoneal route, and the mutant was recovered in lower numbers from the Peyer's patches and mesenteric lymph nodes than the wild type. These results indicate that TonB-mediated iron uptake contributes to colonization of the Peyer's patches and mesenteric lymph nodes but not the liver and spleen of the mouse. The tonB feoB double mutant, given intraperitoneally, was able to infect the liver and spleen at wild-type doses, indicating that additional iron acquisition systems are used during growth at systemic sites of infection.

Baron et al., (1997) designed a study to investigate the growth potential of Salmonella enteritidis in liquid egg white at 30°C and to examine the mechanism of egg white resistance to Salmonella growth. They observed a low and variable growth in whole egg white: Salmonella cell counts rose by 2 log units during the 4 to 6 days of incubation. Treatments to render the egg white components more homogeneous and to facilitate the circulation of nutrients had no effect on the low and variable growth of Salmonella cells. They also investigated whether a lack of nutrients or the presence of inhibitory factors could explain this low growth, the growth of various strains at 30°C in egg white filtrate (egg white without protein). Growth was fast and comparable with growth observed in optimum medium (tryptic soy broth). The addition of 10% egg white to the filtrate decreased the growth of Salmonella enteritidis to the same level observed in egg white, leading to conclude that inhibitory factors, probably proteins, inhibit the growth of S. enteritidis. To determine the role of the different egg white proteins and to identify which of these inhibit S. enteritidis growth, the effect of each protein added to the filtrate was evaluated. To test the inhibitory potency of three binding proteins, supplementation with their corresponding ligands was also studied. Their study showed that ovotransferrin, or iron deficiency resulting from iron binding to ovotransferrin, was the major protein or mechanism implicated in the inhibition of the growth of S. enteritidis in egg white.

Moncrief* and Maguire (1998) found that Salmonella typhimurium has three distinct transport systems for Mg2+: CorA, MgtA, and MgtB. The mgtCB operon encodes two proteins, MgtC, a hydrophobic protein with a predicted molecular mass of 22.5 kDa, and MgtB, a 102-kDa P-type ATPase Mg2+ transport protein. The mgtCB locus has been identified as part of a new Salmonella pathogenicity island, SPI-3. Transcription of mgtCB is regulated by extracellular Mg2+ via the two-component PhoPQ regulatory system important for virulence. To elucidate MgtC’s role in a low-Mg2+ environment, they looked at growth and transport in strains lacking the CorA and MgtA Mg2+transporters but expressing MgtB, MgtC, or both. mgtC mgtB+ and mgtC+mgtB+strains exhibited growth in N minimal medium without added Mg2+ with a 1- to 2-h lag phase. AnmgtC+ mgtB strain was also able to grow in N minimal medium without added Mg2+ but only after a 24-h lag phase. In N minimal medium containing 10 mM Mg2+, all strains grew after a short lag phase; the mgtC+mgtB strain grew to a higher optical density at 600 nm than a nmgtC+ mgtB+ strain and was comparable to wild type. The lengthy lag phase before growth in anmgtC+mgtB strain was not due to lack of expression of MgtC. Western blot analysis indicated that substantial MgtC protein is present by 2 h after suspension in N minimal medium. Surprisingly, in an mgtC+mgtB+ strain, MgtC was undetectable during Mg2+ starvation, although large amounts of MgtB were observed. The lack of expression of MgtC is not dependent on functional MgtB, since a strain carrying a nonfunctional MgtB with a mutation (D379A) also did not make MgtC. Since, during invasion of eukaryotic cells, S. typhimurium appears to be exposed to a low-pH as well as a low-Mg2+ environment, the growth of an mgtC+ mgtB strain was tested at low pH with and without added Mg2+. While significant quantities of MgtC could be detected after suspension at pH 5.2, the mgtC+ mgtB strain was unable to grow at pH 5.2 whether or not Mg2+ was present. Finally, using63Ni2+ and 57Co2+ as alternative substrates for the unavailable28Mg2+, cation uptake could not be detected in an mgtC+ mgtB strain after Mg2+starvation. They conclude that MgtC is not a Mg2+ transporter and that it does not have a primary role in the survival of S. typhimurium at low pH.

Skandamis et al., (2000) studied the growth of Salmonella typhimurium in liquid culture and in a gel matrix system at two pH values (5.0 and 7.0) with (0.03% w/v) or without oregano essential oil. It was shown that the type of growth media (liquid or gel) influenced significantly both the type of end-product formation and growth of bacteria as well as the inhibitory efficacy of the essential oil. The oil inhibited S. typhimurium more strongly in the liquid medium than in the gelatin matrix. In particular, the addition of essential oil in gelatin matrix delayed the initiation of growth and caused a slight suppression of the maximum population level, while in liquid culture, growth was prevented completely in identical conditions. Structure also was found to affect the rate of consumption of glucose and the rate of production of end products. Formic and acetic acids were produced in both systems, while an unidentified peak was formed only in broth samples.

Palacios et al., (2003) identified propionyl coenzyme A (propionyl-CoA) as the common intermediate in the 1,2-propanediol and propionate catabolic pathways of Salmonella enterica serovar Typhimurium LT2. Growth on 1,2-propanediol as a carbon and energy source led to the formation and excretion of propionate, whose activation to propionyl-CoA relied on the activities of the propionate kinase (PduW)/phosphotransacetylase (Pta) enzyme system and the CobB sirtuin-controlled acetyl-CoA and propionyl-CoA (Acs, PrpE) synthetases. The different affinities of these systems for propionate ensure sufficient synthesis of propionyl-CoA to support wild-type growth of S. enterica under low or high concentrations of propionate in the environment. These redundant systems of propionyl-CoA synthesis are needed because the prpE gene encoding the propionyl-CoA synthetase enzyme is part of the prpBCDE operon under the control of the PrpR regulatory protein, which needs 2-methylcitrate as a coactivator. Because the synthesis of 2-methylcitrate by PrpC (i.e., the 2-methylcitrate synthase enzyme) requires propionyl-CoA as a substrate, the level of propionyl-CoA needs to be raised by the Acs or PduW-Pta system before 2-methylcitrate can be synthesized and prpBCDE transcription can be activated.

Brinsmade et al., (2005) found that during growth on ethanolamine, Salmonella enterica 

synthesizes a multimolecular structure that mimics the carboxysome used by some photosynthetic bacteria to fix CO2. In S. enterica, this carboxysome-like structure (hereafter referred to as the ethanolamine metabolosome) is thought to contain the enzymatic machinery needed to metabolize ethanolamine into acetyl coenzyme A (acetyl-CoA). Analysis of the growth behavior of mutant strains of S. enterica lacking specific functions encoded by the 17-gene ethanolamine utilization (eut) operon established the minimal biochemical functions needed by this bacterium to use ethanolamine as a source of carbon and energy. The data obtained support the conclusion that the ethanolamine ammnonia-lyase (EAL) enzyme (encoded by the eutBC genes) and coenzyme B12 are necessary and sufficient to grow on ethanolamine. We propose that the EutD phosphotransacetylase and EutG alcohol dehydrogenase are important to maintain metabolic balance. Glutathione (GSH) had a strong positive effect that compensated for the lack of the EAL reactivase EutA protein under aerobic growth on ethanolamine. Neither GSH nor EutA was needed during growth on ethanolamine under reduced-oxygen conditions. GSH also stimulated growth of a strain lacking the acetaldehyde dehydrogenase (EutE) enzyme. The role of GSH in ethanolamine catabolism is complex and requires further investigation. Our data show that the ethanolamine metabolosome is not involved in the biochemistry of ethanolamine catabolism. They propose the metabolosome is needed to concentrate low levels of ethanolamine catabolic enzymes, to keep the level of toxic acetaldehyde low, to generate enough acetyl-CoA to support cell growth, and to maintain a pool of free CoA.

Uesugi et al., (2006) carried out a study to traceback the investigation of a 2000 to 2001 outbreak of salmonellosis associated with consumption of raw almonds led to isolation of the outbreak strain Salmonella enterica serovar Enteritidis phage type (PT) 30 on three geographically linked almond farms. Interviews with these growers revealed that significant rain fell during the 2000 harvest when many almonds were drying on the ground. The objectives of this study were to document weather conditions during the 2000 harvest, determine the potential for growth of Salmonella Enteritidis PT 30 in hull or shell slurries, and evaluate survival of Salmonella Enteritidis PT 30 on wet almond hulls during drying. Dry almond hulls and in-shell kernels wetted for 24 h increased in weight by 250 to 300% and 100%, respectively. Both hull and shell slurries supported rapid growth of Salmonella Enteritidis PT 30 at 24°C; slurries containing hulls also supported growth at 15°C. Maximum Salmonella Enteritidis PT 30 concentrations of 6.2 and 7.8 log CFU/ml were observed at 15 and 24°C, respectively. Salmonella Enteritidis PT 30 grown in wet hulls that were incubated at 24°C survived drying at either 15 or 37°C. Reductions of 1 to 3 log°CFU/g of dry hull were observed during drying; reductions generally declined as incubation time increased from 2 to 7 days. Evaluation of shipping records revealed that approximately 60% of outbreak-associated almonds had not been exposed to rain, eliminating this factor as the sole Cause of the outbreak. However, the data provide evidence that wet almonds may be a greater risk for high Concentrations of Salmonella, and specific guidelines should be established for harvesting and processing almonds that have been exposed to rain or other water sources.

Brooks et al., (2007) reported that Salmonella characteristically, possess flagella and are motile. They ferment glucose and mannose without producing gas but fail to ferment lactose and sucrose. They are sensitive to heat and are often killed at a temperature of 70°C or above. Salmonellae grow in at a pH range of 4 to 9 with the optimum lying between 6.5 and 7.5. They require high water activity (aw) between 0.99 and 0.94 (pure water aw=1.0) and yet can survive at aw <0.2 such as in dried foods. Complete inhibition of growth occurs at temperatures <7°C, pH <3.8 or water activity <0.94 (Hanes, 2003; Bhunia, 2008).

Sant’Ana et al., (2011) aimed a study for detecting and enumerating Salmonella spp. in MPV marketed in the city of São Paulo, Brazil. A total of 512 samples of MPV packages collected in retail stores were tested for Salmonella spp. and total coliforms and Escherichia coli as indication of the hygienic status. Salmonella spp. was detected in four samples, two using the detection method and two using the counting method, where the results were 8.8 × 102 CFU/g and 2.4 × 102 CFU/g. The serovars were Salmonella Typhimurium (three samples) andSalmonella enterica subsp. enterica O:47:z4,z23:- (one sample). Fourteen samples (2.7%) presented counts of E. coli above the maximum limit established by the Brazilian regulation for MPV (102 CFU/g). Therefore, tightened surveillance and effective intervention strategies are necessary in order to address consumers and governments concerns on safety of MPV.

2.4. Biochemical Reactions of Salmonella

Ewing has brought to bear his wide experience in the field of the Enterobacteriaceae. Salmonella species are Gram negative rods. On blood agar, colonies are 2-3 mm in diameter. Colonies are generally lactose non-fermenters. Salmonella species are motile (with a few exceptions), facultatively anaerobic, produce acid from glucose usually with the production of gas, and are oxidase-negative (Le Minor, 1984) (Table 2.2). Most produce hydrogen sulphide except Salmonella paratyphi A and Salmonella typhi, which is a weak producer. They are identified with a combination of serological and biochemical tests. Salmonella species are classified and identified into serotypes according to the Kauffmann White scheme (The Salmonella Subcommitte of the Nomenclature Committee of the International Society for Microbiology, 1900), which currently contains in excess of 2000 serotypes. Primary subdivision is into ‘‘O’’ serogroups (those which share a common somatic antigen), and these are then subdivided on the basis of ‘‘H’’ (flagella) antigens (The Salmonella Subcommitte of the Nomenclature Committee of the International Society for Microbiology, 1900). Strains of Salmonella typhi may produce Vi antigen, which is an acidic polysaccharide layer outside the cell wall. When fully developed it renders the bacteria agglutinable with Vi antiserum and inagglutinable by ‘‘O’’ antiserum. Antigens similar to Vi may also be found in some strains of Salmonella paratyphi C and Salmonella dublin. (Fig. 2.2)

Table.2.2. Biochemical reactions of Salmonella (Old et. al., 2006).

Figure. 2.2. Flowchart showing identification of Salmonella species.

Lüderitz (1970) studied that lipopolysaccharides, which are located in the cell wall of gram-negative bacteria, are characterized by their biological versatility. They represent the O antigens of the bacteria, they are potent endotoxins, and they often function as the receptor sites for bacteriophages. The study of the mode of action of lipopolysaccharides and the search for structures in the macromolecules that are responsible for biological activity became promising when principles of the chemical fine structure of lipopolysaccharides were identified. He summarized the results of recent investigations regarding the structure of lipopolysaccharides, their biosynthesis and its genetic determination.

Anderson et al., (1980) reported some biochemical characteristics of a second L-proline transport system in Salmonella typhimurium. In the accompanying paper, R. Menzel and J. Roth (J. Bacteriol. 141:1064--1070, 1980) have identified this system by showing that it is inactivated by mutations at the locus proP. They found that it is an active transport system with an apparent Km for L-proline of 3 x 10(-4) M and a strict specificity for L-proline and some of its analogs. Unlike the L-proline transport system encoded in putP, this second system is induced by amino acid limitation.

Morgan et al., (1986) studied that hydrogen peroxide treatment induces the synthesis of 30 proteins in Salmonella typhimurium. Five of these proteins are also induced by heat shock, including the highly conserved DnaK protein. The induction of one of these five proteins by heat shock is dependent on oxyR, a positive regulator of hydrogen peroxide-inducible genes, while the induction of the other four by heat shock is oxyR independent. Five of the 30 hydrogen peroxide-inducible proteins have been identified, and their structural genes have been mapped. Other stresses such as nalidixic acid, ethanol, or cumene hydroperoxide treatment also induce subsets of the 30 hydrogen peroxide-inducible proteins as well as additional proteins. Hydrogen peroxide-inducible proteins are shown to be largely different from those proteins induced by aerobiosis. In addition, the expression of the katG (catalase) gene is shown to be regulated by oxyR at the level of mRNA.

Poolis and Ellis (1996) overexpressed and purified two components, AhpF and AhpC, of the Salmonella typhimurium alkyl hydroperoxide reductase enzyme from Escherichia coli for investigations of their catalytic properties. Recombinant proteins were isolated in high yield (25−33 mg per liter of bacterial culture) and were shown to impart a high degree of protection against killing by cumene hydroperoxide to the host E. coli cells. They developed quantitative enzymatic assays for AhpF alone and for the combined AhpF/AhpC system which have allowed us to address such issues as substrate specificity and inhibition by thiol reagents for each protein. All assays gave identical results whether overexpressed S.typhimurium proteins from E. coli or proteins isolated directly from S. typhimurium were used. Anaerobic hydroperoxide reductase assays have demonstrated that cumene hydroperoxide, ethyl hydroperoxide, and hydrogen peroxide can all be reduced by the combined enzyme system. AhpF possesses multiple pyridine nucleotide-dependent activities [5,5‘-dithiobis(2-nitrobenzoic acid) (DTNB) reductase, oxidase, transhydrogenase, and, in the presence of AhpC, peroxide reductase activities]. Although AhpF can use either NADH or NADPH as the electron donor for these activities, NADH is the preferred reductant (Km,app of AhpF for NADH was more than 2 orders of magnitude lower than that for NADPH when analyzed using DTNB reductase assays). Thiol-modifying reagents react readily with each reduced protein, leading to complete loss of hydroperoxide and DTNB reductase activities. In contrast, thiol modification of reduced AhpF does not affect transhydrogenase or oxidase activities. These data provide the first direct evidence for a catalytic mechanism for peroxide reduction involving redox-active disulfides within each protein.

Shi et al., (2001) identified that Salmonella enterica serovar Typhimurium requires Mn2+, but only a few Mn2+-dependent enzymes have been from it. To characterize Mn2+-dependent enzymes from serovar Typhimurium, two putative PPP-family protein phosphatase genes were cloned from serovar Typhimurium and named prpA andprpB. Their DNA-derived amino acid sequences showed 61% identity to the corresponding Escherichia coli proteins and 41% identity to each other. Each phosphatase was expressed in E. coliand purified to near electrophoretic homogeneity. Both PrpA and PrpB absolutely required a divalent metal for activity. As with other phosphatases of this class, Mn2+ had the highest affinity and stimulated the greatest activity. The apparentKa of PrpA for Mn2+ of 65 μM was comparable to that for other bacterial phosphatases, but PrpB had a much higher affinity for Mn2+ (1.3 μM). The pH optima were pH 6.5 for PrpA and pH 8 for PrpB, while the optimal temperatures were 45 to 55°C for PrpA and 30 to 37°C for PrpB. Each phosphatase could hydrolyze phosphorylated serine, threonine, or tyrosine residues, but their relative specific activities varied with the specific substrate tested. These differences suggest that each phosphatase is used by serovar Typhimurium under different growth or environmental conditions such as temperature or acidity.

Hume et al., (2003) found an essential early event in Shigella and Salmonella pathogenesis is

invasion of non-phagocytic intestinal epithelial cells. Pathogen entry is triggered by the delivery of multiple bacterial effector proteins into target mammalian cells. The Shigella invasion plasmid antigen B (IpaB), which inserts into the host plasma membrane, is required for effector delivery and invasion. To investigate the biochemical properties and membrane topology of IpaB, we purified the native full-length protein following expression in laboratory Escherichia coli. Purified IpaB assembled into trimers via an N-terminal domain predicted to form a trimeric coiled-coil, and is predominantly α-helical. Upon lipid interaction, two transmembrane domains (residues 313–333 and 399–419) penetrate the bilayer, allowing the intervening hydrophilic region (334–398) to cross the membrane. Purified IpaB integrated into model, erythrocyte and mammalian cell membranes without disrupting bilayer integrity, and induced liposome fusion in vitro. An IpaB-derived 162 residue α-helical polypeptide (IpaB418−580) is a potent inhibitor of IpaB-directed liposome fusion in vitro and blocked Shigella entry into cultured mammalian cells at 10−8 M. It is also a heterologous inhibitor of Salmonella invasion protein B (SipB) activity and Salmonella entry. In contrast, IpaB418−580 failed to prevent the contact-dependent haemolytic activity of Shigella. These findings question the proposed direct link between contact-dependent haemolysis and Shigella entry, and demonstrate that IpaB and SipB share biochemical properties and membrane topology, consistent with a conserved mode of action during cell entry.

Campos-Bermudez et al., (2007) examined that glyoxalase II is a hydrolytic enzyme part of the glyoxalase system, responsible for detoxifying several cytotoxic compounds employing glutathione. Glyoxalase II belongs to the superfamily of metallo-β-lactamases, with a conserved motif able to bind up to two metal ions in their active sites, generally zinc. Instead, several eukaryotic glyoxalases II have been characterized with different ratios of iron, zinc, and manganese ions. We have expressed a gene coding for a putative member of this enzyme superfamily from Salmonella typhimurium that we demonstrate, on the basis of its activity, to be a glyoxalase II, named GloB. Recombinant GloB expressed in Escherichia coli was purified with variable amounts of iron, zinc, and manganese. All forms display similar activities, as can be shown from protein expression in minimal medium supplemented with specific metal ions. The crystal structure of GloB solved at 1.4 Å shows a protein fold and active site similar to those of its eukaryotic homologues. NMR and EPR experiments also reveal a conserved electronic structure at the metal site. GloB is therefore able to accommodate these different metal ions and to carry out the hydrolytic reaction with similar efficiencies in all cases. The metal promiscuity of this enzyme (in contrast to other members of the same superfamily) can be accounted for by the presence of a conserved Asp residue acting as a second-shell ligand that is expected to increase the hardness of the metal binding site, therefore favoring iron uptake in glyoxalases II. (Fig. 2.3)

Figure. 2.3 Structure of glyoxalase II of Salmonella

2.4.1. Enteric Fever

Enteric fever is a systemic illness, characterized by prolonged fever. Salmonella typhi causes typhoid fever whereas Paratyphi A, B and C cause paratyphoid fever with symptoms which are milder and a mortality rate that is lower for the latter. Both serotypes are solely human pathogens. Infection typically occurs due to ingestion of food or water contaminated with human waste. In recent years, antibiotic-resistant strains have been isolated in most endemic areas, particularly Southeast Asia, India, Pakistan and Middle East (Scherer and Miller, 2001).

Salmonellosis can be spread by chronic carriers who potentially infect many individuals, especially those who work in food-related industries. Factors contributing to the chronic carrier state have not been fully explained. On average, nontyphoidal serotypes persist in the gastrointestinal tract from 6 weeks to 3 months, depending on the serotypes. Only about 0.1% of nontyphoidal Salmonella cases are shed in stool samples for periods exceeding 1 year. About 2 to 5% of untreated typhoid infections result in a chronic carrier state. Up to 10% of untreated convalescent typhoid cases will excrete S. typhi in feces for 1 to 3 months and between 1 to 4% become chronic carriers excreting the microorganism for more than one year (Scherer and Miller, 2001; Parry, 2006).

Roughly 10% of patients may relapse, die or encounter serious complications such as typhoid encephalopathy, gastrointestinal bleeding and intestinal perforation. Relapse is the most common occurrence probably due to persisting organisms within reticuloendothelial system (RES). Typhoid encephalopathy, often accompanied by shock, is associated with high mortality. Slight gastrointestinal bleeding can be resolved without blood transfusion but in 1 to 2% of cases can be fatal if a large vessel is involved. Intestinal perforation may present with abdominal pain, rising pulse and falling blood pressure in sick people. Hence, it is very serious in 1 to 3% of hospitalized patients (Hu and Kopecko, 2003; Parry, 2006).

Initially, fever is low grade and rises by the second week of illness to 39° to 40° C. Patients with typhoid fever usually appear acutely ill, although those previously exposed to S. typhi or who seek early medical attention can present with a milder illness. Relative bradycardia is neither a sensitive nor a specific sign of typhoid fever, occurring in fewer than 50% of patients. Approximately 30% of patients will have rose spots a faint salmoncolored maculopapular rash on the trunk, Hematologic abnormalities associated with typhoid includes leukopenia, anemia, and subclinical disseminated intravascular coagulopathy. Leukocytosis is seen most often in children and within the first 10 days of illness. Some patients develop thrombocytopenia and clotting abnormalities that usually resolve spontaneously.

2.4.2. Typhoid Fever .

The world health organization (WHO) estimates that 16 to 33 million case of typhoid fever occur each year, with 500.000 to 600,000 deaths (a case fatality rate of between 1.5 and 3.8%), Primarily in South East Asia, Africa and Latin America attributed to rapid population growth (Edelman and Levine, 1986; Beninger et. al., 1988).

Typhoid fever may occur at any age but it is considered to be a disease mainly of children and young adults, reported that persons who excrete the bacilli for more than a year after a clinical attack are called chronic carriers. A chronic carrier state can be expected to develop in about 3% of cases (Singh, 2001). The highest attack rate occurs in children aged 8-13 years. Older people appear to be relatively immune, presumably because of frequently reinforced acquired immunity through numerous sub-clinical exposures to S. typhi (Parry et al., 2005).

The disease is characterized by the onset of prolonged high fever, severe headache, malaise and abdominal pain. The illness often causes diarrhea, especially in younger children, whereas constipation is common in older children and adults. Serious complications occur in up to 10% of typhoid fever patients, especially those who have been ill for more than two weeks and have not received proper treatment. Almost half of the treated patients continue to excrete the pathogen for about one month after the symptoms have been disappeared and 5% still continue up to five months. Approximately 3% become chronic carriers and continue to excrete the organism lifelong. The encounters of S. typhi to humans are generally caused through fecal-oral route from infected individuals to healthy ones (Dutta et. al., 2000, WHO, 2003; Parry, 2006). The patient carriers usually excrete S. typhi up to 6-8 weeks, by the end of one year, 3-4% of cases continue to excrete typhoid bacilli (Betty et al., 2007).

Padmini et al., (2007) conducted a study in Uzbekistan revealed that drinking untreated or unboiled water especially outside the home is a major risk factor in getting infected with S. typhi. Typhoid fever caused by the bacteria Solmonella typhi it passed in the faeces and urine of infected people, people become infected after eating food or drinking beverages that have been handled by a person who is infected or by drinking water that has been contaminated by swage containing the bacteria once the bacteria enter the person’s body they multiply and spread from the intestines in to the blood stream (WHO, 2008).

2.4.3. Paratyphoid fever

Similar to the tyhphoid fever but possibly less severe disease, paratyphoid fever is caused predominantly by S. paratyphi A , although previously estimated to cause approximately a quarter of the incidence of typhoid fever (Crump and Mintz, 2004). Studies from India and Nepal suggest that paratyphoid fever can contributed up to half of all cases of enteric fever (Bhan and Bhatnagar, 2005). In some regions , surveillance has reveald an incidence of S. paratyphi A ranging from 14% of enteric fever episodes in Indonesia to 15% in Pakistan, 24% in India and in China S. paratyphi A is more common (64%) than S. thphi (Ochiai et al., 2005).

Widal test for enteric fever

Widal test detects agglutinating antibodies against the O & H antigens of S. Typhi & H antigens of S. paratyphi A & B, widal agglutination was introduced as a serologic technique to aid in diagnosis of typhoid fever. Widal test becomes positive at end of the first week and peak by third week of disease. Antibodies against the O (somatic) antigen are predominantly IgM, rise early in the illness & disappear early. H (flagellar) antigens of S. Typhi, S. paratyphi A & B, begin to appear towards the end of the first week, increases to a maximum during 3rd week, & persist for month or years afterwards. Usually O antibodies appear on day 6-8 & H antibodies on days 10-12 after the onset of the disease. The test can be done in tubes or on slides (Olopoenia et al., 2000; Old, 2006).

There are two types of agglutination techniques are available the slide test and the tube test. The slide test is rapid and is used as a screening procedure. Using commercially available antigens of S. typhi a drop of the suspend antigen is added to an equal amount of previously prepared serum. An initial positive screening test requires the determination of the strength of the antibody. This is done by adding together equal amounts of antigen suspension and serially diluted serum form the suspected patients. Agglutinations may require an adequate light source for proper visualized as clumps. The tube agglutination test requires much more technical work than the rapid slide tests, and is a macroscopic test (Olopoenia et al., 2000; Old, 2006).

Incidence of Salmonella species

Worldwide, typhoid fever affects roughly 17 million people annually, causing nearly 600,000 deaths. The causative agent, Salmonella enterica typhi (referred to as Salmonella typhi from now on), is an obligate parasite that has no known natural reservoir outside of humans. Little is known about the historical emergence of human S. typhi infections, however it is thought to have caused the deaths of many famous figures such as British author and poet Rudyard Kipling, the inventor of the airplane, Wilbur Wright, and the Greek Empire’s Alexander the Great. The earliest recorded epidemic occurred in Jamestown, VA where it is thought that 6,000 people died of typhoid fever in the early 17th Century. This disease is rare in the United States and developed nations, but always poses the risk of emergence.

Salmonella paratyphi causes bacterial enteric fever which is characterized by an abrupt onset, continued fever, malaise, headache, anorexia, enlargement of spleen, bradycardia, rose spots on trunk occur on approximately 25% of Caucasians, constipation is more common than diarrhea in adults; complications include perforation/hemorrhage/ulceration of the intestines, less frequently psychosis, hepatitis, cholecystitis, pneumonitis, and pericarditis. It is clinically similar to typhoid fever but milder with lower fatality rate. Common enterocolitis may result without enteric fever this is characterised by headache, abdominal pain, nausea, vomiting, diarrhea and dehydration. 

Salmonella paratyphi occurs sporadically or in limited outbreaks and is restricted to humans. It is transmitted by direct or indirect contact with faeces or rarely the urine of a patient or carrier, contaminated food, especially milk, milk products and shellfish, it may be contaminated by the hands of a carrier or flies may be a possible vector. A few outbreaks related to water supplies have been documented. The incubation period is 1 to 3 weeks.Salmonella are excreted in the faeces of infected humans or animals. Faeces contamination of ground water or surface waters, as well as insufficiently treated and inadequately disinfected drinking water, are the main causes of epidemic waterborne caused by Salmonella species. 

Eduardo Gotuzzo et al., (1991) identified eight cases of typhoid and paratyphoid fever during a 4-year period in a cohort of 117 patients who were positive for human immunodeficiency virus in Lima, Peru. Asymptomatic patients with human immundeficiency virus infection and patients with the lymphadenopathy syndrome had a typical clinical presentation and response to therapy. Patients with the acquired immunodeficiency syndrome who were culture positive for Salmonella typhi or Salmonella paratyphi presented with fulminent diarrhea and/or colitis; the two patients for whom at least 2 months of follow-up were available relapsed. In our cohort there were 0.06 cases of typhoid or paratyphoid per patient year of observation; this rate is approximately 60 times that in the general population in Lima, and 25 times that in the 15- to 35-year-old age group. Their data indicate that patients who are positive for human immunodeficiency virus are at significantly increased risk for infection with S typhi and S paratyphi, and suggest that the clinical presentation of these diseases in patients with the acquired immunodeficiency syndrome differs from that seen in immunocompetent hosts.

El-Newihi et al., (1996) aimed to characterize the clinical picture, biochemical features, and prognosis of Salmonella hepatitis. Retrospective case-control analysis of medical records included 27 patients with Salmonella hepatitis and 27 in patients with acute viral hepatitis from 1973 to 1993. Travel history, clinical picture, a standard battery of 18 biochemical tests, complete blood counts, disease complications, duration of hospital admission, and final outcome were analyzed. Eleven patients with Salmonella hepatitis (40%) travelled abroad within 1 month of illness. A greater proportion of Salmonella hepatitis patients developed fever > 104 degrees (44% vs. 4%, respectively; P < .0001), and had relative bradycardia (42% vs. 4%, respectively; P < .002) than viral hepatitis patients. Salmonella hepatitis was associated with lower peak serum alanine transaminase (ALT), aspartate transaminase, and higher peak serum alkaline phosphatase (296 vs. 3,234 U/L, 535 vs. 2,844 U/L, and 500 vs. 228 U/dL, respectively; P < .0001, <.0003, and <.004). The admission ALT/lactic dehydrogenase (LDH) ratio, when levels of both enzymes were expressed as multiples of upper limit of normal value for each, was significantly lower in Salmonella hepatitis. All Salmonella hepatitis cases had a ratio < 4, and all viral hepatitis cases had a ratio > 5, P < .0001. Left shift of white blood cells was more common in Salmonella hepatitis (83% vs. 37%; P < .004). Patients with Salmonella hepatitis had a longer hospitalization (14.8 vs. 6.5 days, respectively; P < .0001). All 54 patients survived their illness. The clinical picture of Salmonella hepatitis is frequently indistinguishable from viral hepatitis. The admission ALT/LDH ratio is the best discriminator between both entities.

Hanes (2003) referred that the World Health Organization (WHO) estimates 16 to 17 million cases occur annually, resulting in about 600,000 deaths. The mortality rates differ from region to region, but can be as high as 5 to 7% despite the use of appropriate antibiotic treatment. On the other hand, nontyphoidal cases account for 1.3 billion cases with 3 million deaths. In the United States, approximately 2 to 4 million cases of Salmonella gastroenteritis occur with about 500 deaths per year. A more accurate figure of salmonellosis is difficult to determine because normally only large outbreaks are investigated whereas sporadic cases are under-reported. Data

on salmonellosis are scarce in many countries of Asia, Africa and South and Central America

where only 1 to 10% of cases are reported.

Vugia et al., (2004) analyzed population-based data collected during 1996–1999 by the Foodborne Diseases Active Surveillance Network (Food Net), to determine the incidences, infecting serotypes, and outcomes of invasive Salmonella infection. We found that the mean annual incidence of invasive salmonellosis was 0.9 cases/100,000 population and was highest among infants (7.8 cases/100,000). The incidence was higher among men than women (1.2 vs. 0.7 cases/100,000; P < .001) and higher among blacks, Asians, and Hispanics than among whites (2.5, 2.0, and 1.3 cases/100,000 population, respectively, vs. 0.4 cases/100,000; all P < .001). Seventy-four percent of cases were caused by 8 Salmonella serotypes: Typhimurium, Typhi, Enteritidis, Heidelberg, Dublin, Paratyphi A, Choleraesuis, and Schwarzengrund. Of 540 persons with invasive infection, 386 (71%) were hospitalized and 29 (5%) died; 13 (45%) of the deaths were among persons aged ⩾60 years. Invasive Salmonella infections are a substantial health problem in the United States and contribute to hospitalizations and deaths.

Brooks et al., (2005) confirmed a bacteremic typhoid fever incidence of 3.9 episodes/1,000 person-years during fever surveillance in a Dhaka urban slum. The relative risk for preschool children compared with older persons was 8.9. Our regression model showed that these children were clinically ill, which suggests a role for preschool immunization.

Ogunleye et, al., (2005) identified that Salmonella septicaemia was most common (56%) among children of age 5-11 years bracket and below bracket followed by 1-5 years and below group (36%; while those within 0-1 year group showed of frequency of (8%).

Ochiai et al., (2005) examined that in China and India, countries with the largest populations in the world, S. paratyphi A is the causal agent for a substantial proportion of enteric fever episodes that cannot be distinguished clinically from typhoid fever episodes. While similar treatment strategies may work for both organisms, future enteric fever prevention strategies in Asia must focus on S. paratyphi A as well as on S. typhi, especially when considering the emergence of drug-resistant strains (13–15). Future vaccination strategies should include bivalent vaccines that protect against S. typhi as well as S. paratyphi A. Otherwise, the protective effectiveness of typhoid fever vaccines (Vi, Ty21a) against enteric fever may diminish, which could result in a loss of public confidence and decrease public willingness to be vaccinated.

Siddiqui et. al., (2006) revealed in a study in Pakistan that the highest burden of disease has been observed in children, the incidence of enteric fever in children is estimated at 170 per 100,000 of the population, while serology based incidence is estimated at 710 per 100,000 of the population.

Scherer and Miller (2001) and Parry (2006) examined that typhoid fever is endemic throughout Africa and Asia as well as persists in the Middle East, some eastern and southern European countries and central and South America. In the US and most of Europe, typhoid is predominantly a disease of the returning traveler. Typhoid incidence in endemic areas is typ