Prevalence and characterization of motile Salmonella in commercial layer poultry farms in Bangladesh

Salmonella is a globally widespread food-borne pathogen having major impact on public health. All motile serovars of Salmonella enterica of poultry origin are zoonotic, and contaminated meat and raw eggs are an important source to human infections. Information on the prevalence of Salmonella at farm/holding level, and the zoonotic serovars circulating in layer poultry in the South and South-East Asian countries including Bangladesh, where small-scale commercial farms are predominant, is limited. To investigate the prevalence of Salmonella at layer farm level, and to identify the prevalent serovars we conducted a cross-sectional survey by randomly selecting 500 commercial layer poultry farms in Bangladesh. Faecal samples from the selected farms were collected following standard procedure, and examined for the presence of Salmonella using conventional bacteriological procedures. Thirty isolates were randomly selected, from the ninety obtained from the survey, for serotyping and characterized further by plasmid profiling and pulsed-field gel electrophoresis (PFGE). Results of the survey showed that the prevalence of motile Salmonella at layer farm level was 18% (95% confidence interval 15-21%), and Salmonella Kentucky was identified to be the only serovar circulating in the study population. Plasmid analysis of the S. Kentucky and non-serotyped isolates revealed two distinct profiles with a variation of two different sizes (2.7 and 4.8 kb). PFGE of the 30 S. Kentucky and 30 non-serotyped isolates showed that all of them were clonally related because only one genotype and three subtypes were determined based on the variation in two or three bands. This is also the first report on the presence of any specific serovar of Salmonella enterica in poultry in Bangladesh.     

Transposition of the heat-stable toxin astA gene into a gifsy-2-related prophage of Salmonella enterica serovar Abortusovis

The horizontal transfer and acquisition of virulence genes via mobile genetic elements have been a major driving force in the evolution of Salmonella pathogenicity. Serovars of Salmonella enterica carry variable assortments of phage-encoded virulence genes, suggesting that temperate phages play a pivotal role in this process. Epidemic isolates of S. enterica serovar Typhimurium are consistently lysogenic for two lambdoid phages, Gifsy-1 and Gifsy-2, carrying known virulence genes. Other serovars of S. enterica, including serovars Dublin, Gallinarum, Enteritidis, and Hadar, carry distinct prophages with similarity to the Gifsy phages. In this study, we analyzed Gifsy-related loci from S. enterica serovar Abortusovis, a pathogen associated exclusively with ovine infection. A cryptic prophage, closely related to serovar Typhimurium phage Gifsy-2, was identified. This element, named Gifsy-2AO, was shown to contribute to serovar Abortusovis systemic infection in lambs. Sequence analysis of the prophage b region showed a large deletion which covers genes encoding phage tail fiber proteins and putative virulence factors, including type III secreted effector protein SseI (GtgB, SrfH). This deletion was identified in most of the serovar Abortusovis isolates tested and might be dependent on the replicative transposition of an adjacent insertion sequence, IS1414, previously identified in pathogenic Escherichia coli strains. IS1414 encodes heat-stable toxin EAST1 (astA) and showed multiple genomic copies in isolates of serovar Abortusovis. To our knowledge, this is the first evidence of intergeneric transfer of virulence genes via insertion sequence elements in Salmonella. The acquisition of IS1414 (EAST1) and its frequent transposition within the chromosome might improve the fitness of serovar Abortusovis within its narrow ecological niche.     

Epidemiological analysis of Salmonella enterica ssp. enterica serovars Hadar, Brancaster and Enteritidis from humans and broiler chickens in Senegal using pulsed-field gel electrophoresis and antibiotic susceptibility

AIMS: Salmonella Hadar, Salmonella Brancaster and Salmonella Enteritidis are the main Salmonella enterica ssp. enterica serovars isolated from poultry in Senegal. Our objective was to analyse the pulsed-field gel electrophoresis (PFGE) and antibioresistance patterns of strains belonging to these serovars and to assess the significance of broiler-chicken meat as a source of human infection. METHODS AND RESULTS: A total of 142 Salmonella isolates were analysed: 79 were isolated from Senegalese patients with sporadic diarrhoea (11 S. Hadar, nine S. Brancaster and 59 S. Enteritidis) and 63 from poultry (30 S. Hadar, 17 S. Brancaster and 16 S. Enteritidis). The PFGE of XbaI- and SpeI-digested chromosomal DNA gave 20 distinct profiles for S. Hadar, nine for S. Brancaster and 22 for S. Enteritidis. Each serovar was characterized by a major pulsotype which was X3S1 in 42% of S. Hadar, X8S1 in 53.8% of S. Brancaster and X1S2 in 43% of S. Enteritidis isolates. Human and poultry isolates of Salmonella had common PFGE patterns. Antibiosensitivity tests showed multiresistance (more than two drugs) was encountered in 14.5% of S. Hadar and in 5% of S. Enteritidis isolates. Resistance to quinolones was considered to be of particular importance and 14.5% of S. Hadar isolates were found to be resistant to nalidixic acid. CONLCUSIONS: The sharing of similar PFGE profiles among isolates from humans and poultry provided indirect evidence of Salmonella transmission from contaminated broiler meat. But most of the Salmonella isolates remained drug sensitive. SIGNIFICANCE AND IMPACT OF THE STUDY: Efforts are needed to eliminate Salmonella from poultry meat intended for human consumption. This study has also highlighted the importance of continuous surveillance to monitor antimicrobial resistance in bacteria associated with animals and humans.     

Antimicrobial resistance and virulence determinants in European Salmonella genomic island 1-positive Salmonella enterica isolates from different origins

Salmonella genomic island 1 (SGI1) contains a multidrug resistance region conferring the ampicillin-chloramphenicol-streptomycin-sulfamethoxazole-tetracycline resistance phenotype encoded by bla(PSE-1), floR, aadA2, sul1, and tet(G). Its increasing spread via interbacterial transfer and the emergence of new variants are important public health concerns. We investigated the molecular properties of SGI1-carrying Salmonella enterica serovars selected from a European strain collection. A total of 38 strains belonging to S. enterica serovar Agona, S. enterica serovar Albany, S. enterica serovar Derby, S. enterica serovar Kentucky, S. enterica serovar Newport, S. enterica serovar Paratyphi B dT+, and S. enterica serovar Typhimurium, isolated between 2002 and 2006 in eight European countries from humans, animals, and food, were subjected to antimicrobial susceptibility testing, molecular typing methods (XbaI pulsed-field gel electrophoresis [PFGE], plasmid analysis, and multilocus variable-number tandem-repeat analysis [MLVA]), as well as detection of resistance and virulence determinants (PCR/sequencing and DNA microarray analysis). Typing experiments revealed wide heterogeneity inside the strain collection and even within serovars. PFGE analysis distinguished a total of 26 different patterns. In contrast, the characterization of the phenotypic and genotypic antimicrobial resistance revealed serovar-specific features. Apart from the classical SGI1 organization found in 61% of the strains, seven different variants were identified with antimicrobial resistance properties associated with SGI1-A (S. Derby), SGI1-C (S. Derby), SGI1-F (S. Albany), SGI1-L (S. Newport), SGI1-K (S. Kentucky), SGI1-M (S. Typhimurium), and, eventually, a novel variant similar to SGI1-C with additional gentamicin resistance encoded by aadB. Only minor serovar-specific differences among virulence patterns were detected. In conclusion, the SGI1 carriers exhibited pathogenetic backgrounds comparable to the ones published for susceptible isolates. However, because of their multidrug resistance, they may be more relevant in clinical settings.     

Molecular analyses of Salmonella enterica isolates from fish feed factories and fish feed ingredients

Isolates of the most commonly observed salmonella serovars in Norwegian fish feed factories from 1998 to 2000 (Salmonella enterica serovar Agona, S. enterica serovar Montevideo, S. enterica serovar Senftenberg, and S. enterica serovar Kentucky) were studied by pulsed-field gel electrophoresis (PFGE) and plasmid profile analysis and compared to isolates of the same serovars from fish feed ingredients, humans, and other sources (a total of 112 isolates). Within each serovar, a variety of distinct PFGE types (with similarity levels less than 90%) were observed in the feed ingredients and other sources, while only two distinct types of each serovar were identified in the factories. The combined results of PFGE and plasmid analyses showed that each factory harbored only a few S. enterica clones. Some of these clones persisted for at least 3 years in the factories, indicating that there was long-lasting contamination probably due to inadequate decontamination procedures.     

Population dynamics of Salmonella enterica serotypes in commercial egg and poultry production

Fresh and processed poultry have been frequently implicated in cases of human salmonellosis. Furthermore, increased consumption of meat and poultry has increased the potential for exposure to Salmonella enterica. While advances have been made in reducing the prevalence and frequency of Salmonella contamination in processed poultry, there is mounting pressure on commercial growers to prevent and/or eliminate these human pathogens in preharvest production facilities. Several factors contribute to Salmonella colonization in commercial poultry, including the serovar and the infectious dose. In the early 1900s, Salmonella enterica serovars Pullorum and Gallinarum caused widespread diseases in poultry, but vaccination and other voluntary programs helped eradicate pullorum disease and fowl typhoid from commercial flocks. However, the niche created by the eradication of these serovars was likely filled by S. Enteritidis, which proliferated in the bird populations. While this pathogen remains a significant problem in commercial egg and poultry production, its prevalence among poultry has been declining since the 1990s. Coinciding with the decrease of S. Enteritidis, S. Heidelberg and S. Kentucky have emerged as the predominant serovars in commercial broilers. In this review, we have highlighted bacterial genetic and host-related factors that may contribute to such shifts in Salmonella populations in commercial poultry and intervention strategies that could limit their colonization.     

Uptake and replication of Salmonella enterica in Acanthamoeba rhysodes

The ability of salmonellae to become internalized and to survive and replicate in amoebae was evaluated by using three separate serovars of Salmonella enterica and five different isolates of axenic Acanthamoeba spp. In gentamicin protection assays, Salmonella enterica serovar Dublin was internalized more efficiently than Salmonella enterica serovar Enteritidis or Salmonella enterica serovar Typhimurium in all of the amoeba isolates tested. The bacteria appeared to be most efficiently internalized by Acanthamoeba rhysodes. Variations in bacterial growth conditions affected internalization efficiency, but this effect was not altered by inactivation of hilA, a key regulator in the expression of the invasion-associated Salmonella pathogenicity island 1. Microscopy of infected A. rhysodes revealed that S. enterica resided within vacuoles. Prolonged incubation resulted in a loss of intracellular bacteria associated with morphological changes and loss of amoebae. In part, these alterations were associated with hilA and the Salmonella virulence plasmid. The data show that Acanthamoeba spp. can differentiate between different serovars of salmonellae and that internalization is associated with cytotoxic effects mediated by defined Salmonella virulence loci.     

Salmonella virulence plasmid. Modular acquisition of the spv virulence region by an F-plasmid in Salmonella enterica subspecies I and insertion into the chromosome of subspecies II,IIIa, IV and VII isolates.

The spv operon is common to all Salmonella virulence plasmids. DNA hybridization analysis indicates that the spv region is limited in distribution to serovars of Salmonella enterica subspecies I, II, IIIa, IV, and VII and is absent from Salmonella bongori isolates. Among strains of subspecies II, IIIa, and VII, all isolates examined contained sequences that hybridized with the spv region. However, among isolates of subspecies I, DNA sequences capable of hybridizing with the spv region were found in some isolates of certain serovars. Furthermore, in isolates of subspecies I, the virulence plasmid was found in the same set of isolates as an F-related plasmid, as determined by the presence of the spv region of the virulence plasmid and the finO, traD, and repA sequences of the F-plasmid. The concordance of the virulence plasmid and all three F-plasmid sequences in subspecies I serovar Choleraesuis, Paratyphi, and Typhimurium is most easily explained if the spv region is carried in an F-related plasmid in these isolates. In contrast, among S. enterica subspecies II, IIIa, IV, and VII, the isolates that contain spv sequences did not hybridize with an F-related plasmid or any other identifiable plasmid. With the use of pulse-field gel electrophoresis, the spv region in subspecies II, IIIa, and VII was found to be encoded on the chromosome. Analysis of the phylogenetic distribution of spv among Salmonella isolates and comparative nucleotide sequence analysis of spvA and spvC suggests that the spv region was acquired very recently, after speciation of the salmonellae.     

Evolution and population structure of Salmonella enterica serovar Newport

Salmonellosis caused by Salmonella enterica serovar Newport is a major global public health concern, particularly because S. Newport isolates that are resistant to multiple drugs (MDR), including third-generation cephalosporins (MDR-AmpC phenotype), have been commonly isolated from food animals. We analyzed 384 S. Newport isolates from various sources by a multilocus sequence typing (MLST) scheme to study the evolution and population structure of the serovar. These were compared to the population structure of S. enterica serovars Enteritidis, Kentucky, Paratyphi B, and Typhimurium. Our S. Newport collection fell into three lineages, Newport-I, Newport-II, and Newport-III, each of which contained multiple sequence types (STs). Newport-I has only a few STs, unlike Newport-II or Newport-III, and has possibly emerged recently. Newport-I is more prevalent among humans in Europe than in North America, whereas Newport-II is preferentially associated with animals. Two STs of Newport-II encompassed all MDR-AmpC isolates, suggesting recent global spread after the acquisition of the bla(CMY-2) gene. In contrast, most Newport-III isolates were from humans in North America and were pansusceptible to antibiotics. Newport was intermediate in population structure to the other serovars, which varied from a single monophyletic lineage in S. Enteritidis or S. Typhimurium to four discrete lineages within S. Paratyphi B. Both mutation and homologous recombination are responsible for diversification within each of these lineages, but the relative frequencies differed with the lineage. We conclude that serovars of S. enterica provide a variety of different population structures.     

Haemolysins of Salmonella, their role in pathogenesis and subtyping of Salmonella serovars

Haemolysin patterns of 175 strains of different Salmonella enterica subspecies enterica serovars isolated from different animal sources and places were determined using 11 different blood agar media made with either non-washed horse/sheep erythrocytes or with washed erythrocytes of cattle, sheep, horse, goat, rabbit, guinea pig, and human A, O and B blood groups. Study on 47 strains belonging to 10 serovars of Salmonella from buffalo meat (buffen), 42 strains of 11 serovars from goat meat (chevon): 16 strains of Salmonella enterica serovar Paratyphi B and 25 of S. enterica serovar Paratyphi B var Java from fish, meat, meat products and clinical cases; 45 isolates of S. Abortusequi from aborted mares (18), fetal contents (21), aborted donkey mares (2) and 4 reference strains, revealed that all host restricted Salmonella namely, S. enterica serovar Gallinarum, S. enterica serovar Anatum, S. enterica serovar Abortusequi and S. enterica serovar Paratyphi B could be divided into different haemolysin types based on their inability to produce haemolysis on one or more types of blood agar, while strains of all zoonotic Salmonella serovars induced haemolysis on all the 9 types of blood agar made of washed erythrocytes. None of 175 Salmonella could produce hemolytic colonies on blood agar made of non-washed horse/ sheep erythrocytes. Haemolysin type I (lysing all types of washed erythrocytes) was the commonest one among all serovars except S. Abortusequi, none of which lysed horse erythrocytes. Salmonella enterica serovar Abortusequi having hemolytic activity against sheep erythrocytes were more invasive but had lesser ability to survive in sheep mononuclear cells than non-hemolytic strains. Multiplicity of haemolysins appeared significant epidemiological tool.     

The virulence plasmids of Salmonella.

Certain Salmonella serovars belonging to subspecies I carry a large, low-copy-number plasmid that contains virulence genes. Virulence plasmids are required to trigger systemic disease; their involvement in the enteric stage of the infection is unclear. Salmonella virulence plasmids are heterogeneous in size (50-90 kb), but all share a 7.8 kb region, spv, required for bacterial multiplication in the reticuloendothelial system. Other loci of the plasmid, such as the fimbrial operon pef, the conjugal transfer gene traT and the enigmatic rck and rsk loci, may play a role in other stages of the infection process. The virulence plasmid of Salmonella typhimurium LT2 is self-transmissible; virulence plasmids from other serovars, such as Salmonella enteritidis and Salmonella choleraesuis, carry incomplete tra operons. The presence of virulence plasmids in host-adapted serovars suggests that virulence plasmid acquisition may have expanded the host range of Salmonella.     

Epidemiology of Salmonella sofia in Australia.

In recent years, the incidence of isolation of Salmonella sofia in Australia has risen from 33% of all poultry isolates in 1982 to a peak of 49% of isolates in 1988. A parallel rise has not been seen in S. sofia isolated from humans. In Israel, however, S. sofia has been commonly isolated from both humans and poultry. We investigated the possibility that the Israeli strains may belong to a different clonal group and express virulence determinants not seen in the Australian isolates, accounting for the apparent differences in the virulence seen within this species. A number of S. sofia isolates from Australian chickens and humans, as well as from Israeli humans and chickens, were compared by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of outer membrane proteins, plasmid profiles, and restriction fragment length polymorphism analysis. No reproducible differences could be detected by analysis of outer membrane proteins. A small 6.4-kb plasmid, pIMVS2, was detected in all Australian isolates from chickens but not in the Israeli isolates. Restriction fragment length polymorphism studies with cosmid clones as probes provided the most discrimination among isolates, allowing us to divide them into seven groups. This technique revealed that significant differences exist between Australian and Israeli isolates and provided additional insights into the epidemiology of these Salmonella isolates.     

Epidemiology of Salmonella sofia in Australia.

In recent years, the incidence of isolation of Salmonella sofia in Australia has risen from 33% of all poultry isolates in 1982 to a peak of 49% of isolates in 1988. A parallel rise has not been seen in S. sofia isolated from humans. In Israel, however, S. sofia has been commonly isolated from both humans and poultry. We investigated the possibility that the Israeli strains may belong to a different clonal group and express virulence determinants not seen in the Australian isolates, accounting for the apparent differences in the virulence seen within this species. A number of S. sofia isolates from Australian chickens and humans, as well as from Israeli humans and chickens, were compared by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of outer membrane proteins, plasmid profiles, and restriction fragment length polymorphism analysis. No reproducible differences could be detected by analysis of outer membrane proteins. A small 6.4-kb plasmid, pIMVS2, was detected in all Australian isolates from chickens but not in the Israeli isolates. Restriction fragment length polymorphism studies with cosmid clones as probes provided the most discrimination among isolates, allowing us to divide them into seven groups. This technique revealed that significant differences exist between Australian and Israeli isolates and provided additional insights into the epidemiology of these Salmonella isolates.     

Molecular Characterization of Motile Serovars of Salmonella enterica from Breeder and Commercial Broiler Poultry Farms in Bangladesh

Contaminated poultry and poultry products are a major source of motile Salmonellae for human salmonellosis worldwide. Local circulation of any motile Salmonella serovar in poultry has a wider public health impact beyond its source of origin for being dispersed elsewhere through poultry trades or human travels. To investigate the status of motile Salmonella serovars in breeder farms in Bangladesh, multiple flocks of two breeder farms were observed for a period of six months. In addition, a cross-sectional survey was carried out to determine the prevalence and serovar distribution of motile Salmonella by randomly selecting 100 commercial broiler poultry farms. Five pooled faecal samples representing an entire housed flock of breeders or broilers were screened for presence of motile Salmonella following conventional bacteriological procedures. The Salmonella isolates obtained were subsequently serotyped, and characterized by plasmid profiling and pulsed-field gel electrophoresis (PFGE). The results revealed that both the breeder farms were positive with three Salmonella serovars: S. Virchow, S. Paratyphi B var Java (S. Java) and S. Enteritidis. Eleven of the 100 broiler farms investigated were positive for motile Salmonella, giving a farm-level prevalence of 11% (95% confidence interval 5–17%). S. Virchow and S. Kentucky were the two predominant serovars isolated from the broiler farms. The PFGE genotyping demonstrated that the isolates belonging to the same serovars were closely related due to variation in only 1–4 bands. All the S. Virchow and S. Java isolates, irrespective of breeder or broiler farm origin, were plasmid-free, except for one S. Virchow isolate from a broiler farm that harboured a 9.7 kb-sized plasmid. The S. Kentucky isolates belonged to three plasmid profiles having plasmids of four different sizes, ranging from 2.7 to 109 kb. This is the first report of any motile Salmonella serovars from breeder and commercial broiler poultry farms in Bangladesh.     

Occurrence of sopE gene and its phenotypic expression among different serovars of Salmonella enterica isolated from man and animals

Salmonella pathogenesis is a complex phenomenon and a Type III secretion system plays a central role in the development of Salmonella-induced enteritis. One such Type III secretion protein is Salmonella outer protein E (SopE). Prevalence of sopE gene and its phenotypic expression (SopE protein) among different serovars of Salmonella enterica isolated from man and animals were investigated. Of 305 strains of S. enterica belonging to 11 serovars tested for the presence of sopE, 130 strains belonging to three serovars viz., Enteritidis, Gallinarum and Virchow were found to carry sopE gene irrespective of their source of isolation when tested by PCR amplification technique using its specific primers. Of these 130 strains, 112 strains were found to express SopE protein phenotypically as detected by Dot-ELISA using SopE antibody. Among the different serovars tested only serovars Gallinarum, Enteritidis and Virchow expressed SopE protein phenotypically in vitro. Role of SopE protein in pathogenesis of salmonellosis has been discussed.     

Detection of a Salmonella enterica serovar California strain spreading in spanish feed mills and genetic characterization with DNA microarrays

We performed an epidemiological study on Salmonella isolated from raw plant-based feed in Spanish mills. Overall, 32 different Salmonella serovars were detected. Despite its rare occurrence in humans and animals, Salmonella enterica serovar California was found to be the predominant serovar in Spanish feed mills. Different typing techniques showed that isolates of this serovar were genetically closely related, and comparative genomic hybridization using microarray technology revealed 23 S. enterica serovar Typhimurium LT2 gene clusters that are absent from serovar California.     

Novel virulence gene and clustered regularly interspaced short palindromic repeat (CRISPR) multilocus sequence typing scheme for subtyping of the major serovars of Salmonella enterica subsp. enterica

Salmonella enterica subsp. enterica is the leading cause of bacterial food-borne disease in the United States. Molecular subtyping methods are powerful tools for tracking the farm-to-fork spread of food-borne pathogens during outbreaks. In order to develop a novel multilocus sequence typing (MLST) scheme for subtyping the major serovars of S. enterica subsp. enterica, the virulence genes sseL and fimH and clustered regularly interspaced short palindromic repeat (CRISPR) loci were sequenced from 171 clinical isolates from nine Salmonella serovars, Salmonella serovars Typhimurium, Enteritidis, Newport, Heidelberg, Javiana, I 4,[5],12:i:-, Montevideo, Muenchen, and Saintpaul. The MLST scheme using only virulence genes was congruent with serotyping and identified epidemic clones but could not differentiate outbreaks. The addition of CRISPR sequences dramatically improved discriminatory power by differentiating individual outbreak strains/clones. Of particular note, the present MLST scheme provided better discrimination of Salmonella serovar Enteritidis strains than pulsed-field gel electrophoresis (PFGE). This method showed high epidemiologic concordance for all serovars screened except for Salmonella serovar Muenchen. In conclusion, the novel MLST scheme described in the present study accurately differentiated outbreak strains/clones of the major serovars of Salmonella, and therefore, it shows promise for subtyping this important food-borne pathogen during investigations of outbreaks.     

Prevalence of the virulence plasmids of nontyphoid Salmonella in the serovars isolated from humans and their association with bacteremia.

To determine if the virulence plasmid is one of the elements contributing to Salmonella bacteremia in humans, 436 clinical Salmonella isolates of different serovars were examined by a specific multiplex polymerase chain reaction assay for the presence of a virulence plasmid. These serovars showed differences in their ability to produce particular disease syndrome in humans. In the serovars usually causing bacteremia without concomitant gastroenteritis (primary bacteremia), i.e., S. choleraesuis, S. dublin, and S. enteritidis in this study, the rate of virulence plasmid carriage was 100%, while among those occasionally generating bacteremia following an episode of gastroenteritis (secondary bacteremia), the majority were plasmidless. Only a portion of S. typhimurium strains harbored a virulence plasmid; however, the rates of virulence plasmid carriage in S. typhimurium were not statistically different between non-fecal and fecal isolates (90% vs. 85%, 0.1 < P < 0.9). These results indicate that the virulence plasmids may be important for primary bacteremia, but not secondary bacteremia, to occur.     

Genome sequence of the invasive Salmonella enterica subsp. enterica serotype enteritidis strain LA5

Salmonella enterica subsp. enterica serotype Enteritidis is one of the major causes of gastroenteritis in humans due to consumption of poultry derivatives. Here we report the whole-genome sequence and annotation, including the virulence plasmid, of S. Enteritidis LA5, which is a chicken isolate used by numerous laboratories in virulence studies.     

Characterization of the RpoS status of clinical isolates of Salmonella enterica

The stationary-phase-inducible sigma factor, sigma(S) (RpoS), is the master regulator of the general stress response in Salmonella and is required for virulence in mice. rpoS mutants can frequently be isolated from highly passaged laboratory strains of Salmonella: We examined the rpoS status of 116 human clinical isolates of Salmonella, including 41 Salmonella enterica serotype Typhi strains isolated from blood, 38 S. enterica serotype Typhimurium strains isolated from blood, and 37 Salmonella serotype Typhimurium strains isolated from feces. We examined the abilities of these strains to produce the sigma(S) protein, to express RpoS-dependent catalase activity, and to resist to oxidative stress in the stationary phase of growth. We also carried out complementation experiments with a cloned wild-type rpoS gene. Our results showed that 15 of the 41 Salmonella serotype Typhi isolates were defective in RpoS. We sequenced the rpoS allele of 12 strains. This led to identification of small insertions, deletions, and point mutations resulting in premature stop codons or affecting regions 1 and 2 of sigma(S), showing that the rpoS mutations are not clonal. Thus, mutant rpoS alleles can be found in freshly isolated clinical strains of Salmonella serotype Typhi, and they may affect virulence properties. Interestingly however, no rpoS mutants were found among the 75 Salmonella serotype Typhimurium isolates. Strains that differed in catalase activity and resistance to hydrogen peroxide were found, but the differences were not linked to the rpoS status. This suggests that Salmonella serotype Typhimurium rpoS mutants are counterselected because rpoS plays a role in the pathogenesis of Salmonella serotype Typhimurium in humans or in the transmission cycle of the disease.