Salmonella Serovars in the Herpetofauna of Indiana County, Pennsylvania

Herpetofaunal Salmonella enterica serovars have not been fully examined in any U.S. region. Thirty-three Salmonella serovars were isolated from 156 samples from 34 species, all within Indiana County, Pennsylvania. Results suggest that herpetofaunas could potentially pose a threat to humans. Further understanding of Salmonella in herpetofaunas may prevent future human cases.     

Identification of Salmonella enterica subspecies I, Salmonella enterica serovars Typhimurium, Enteritidis and Typhi using multiplex PCR

This study was designed to develop a multiplex PCR method with five specific primer pairs for the detection of Salmonella spp., Salmonella subspecies I, Salmonella enterica serovars Typhimurium, Typhi and Enteritidis. A multiplex PCR was constructed with five primer pairs for the detection of Salmonella and pathogenic Salmonella serovars, including a specific primer pair for Salmonella Typhi, based on the sequence comparison between genomic DNA sequences of 12 Salmonella strains. Each primer pair was specifically targeted to Salmonella spp., Salmonella subspecies I, Salmonella Typhimurium, Typhi and Enteritidis. This multiplex PCR was evaluated with various DNAs of Salmonella serovars that yielded high specificity for amplifying the expected PCR products of Salmonella serovars. Using this primer pair, a set of multiplex PCR was performed for the rapid identification of salmonellae and major pathogenic Salmonella serovars. Although this multiplex PCR method will need to be evaluated for a wide range of Salmonella serovars among multilaboratories, it should be useful for identifying clinically significant strains of Salmonella serovars rapidly and accurately without the need for serological testing.     

Characterization of Salmonella enterica subspecies I genovars by use of microarrays

Subspecies 1 of Salmonella enterica is responsible for almost all Salmonella infections of warm-blooded animals. Within subspecies 1 there are over 2,300 known serovars that differ in their prevalence and the diseases that they cause in different hosts. Only a few of these serovars are responsible for most Salmonella infections in humans and domestic animals. The gene contents of 79 strains from the most prevalent serovars were profiled by microarray analysis. Strains within the same serovar often differed by the presence and absence of hundreds of genes. Gene contents sometimes differed more within a serovar than between serovars. Groups of strains that share a distinct profile of gene content can be referred to as "genovars" to distinguish them from serovars. Several misassignments within the Salmonella reference B collection were detected by genovar typing and were subsequently confirmed serologically. Just as serology has proved useful for understanding the host range and pathogenic manifestations of Salmonella, genovars are likely to further define previously unrecognized specific features of Salmonella infections.     

Prevalence and serovars of Salmonella in the feces of free-ranging white-tailed deer (Odocoileus virginianus) in Nebraska

To determine the prevalence and serovars of Salmonella in free-ranging deer, we cultured feces from white-tailed deer (Odocoileus virginianus) harvested by hunters during a regular firearm season in southeastern Nebraska (USA). We recovered Salmonella from 5 (1%; 95% confidence interval: 0.37-2.20%) of 500 samples and identified four different Salmonella enterica serovars [Litchfield (1), Dessau (1), Infantis (2), and Enteritidis (1)]. Although the prevalence of Salmonella in free-ranging deer appears to be low, the serovars recovered are known to be pathogenic to humans and animals.     

Distribution and genotypic characterization of Salmonella serovars isolated from tropical seafood of Cochin, India

Aims:  To determine the distribution of Salmonella serovars in seafood and to examine the intraserovar genetic variations in Salmonella enterica subsp. enterica serovar Rissen and Salmonella Weltevreden by polymerase chain reaction (PCR)-ribotyping and enterobacterial repetitive intergenic consensus (ERIC)-PCR methods.
Method and Results:  A total of 417 seafood samples collected over 2003–2006 from fishing harbours and fish markets of Cochin (India) was studied for presence of Salmonella serovars. Seafood samples were analysed for the presence of Salmonella by Bacteriological Analytical Manual (BAM), U.S. Food & Drug Administration (USFDA) method. The study indicated that 23·2% of the seafood samples were positive for Salmonella and a total of 241 Salmonella isolates comprising of 27 different serovars were isolated from seafood. S. Weltevreden, Salmonella Rissen, Salmonella Typhimurium and Salmonella Derby were found to be the most predominant serovars in seafood. PCR-ribotypes and ERIC-PCR profiles showed multiple genotypic profiles for S. Rissen and S. Weltevreden in seafood and the level of discrimination indices obtained was at 0·974 for S. Rissen and 0·988 for S. Weltevreden, respectively.
Conclusion:  The study highlighted the major Salmonella serovars in the seafood of Cochin (India) and molecular fingerprinting pattern revealed genetic variation among S. Rissen and S. Weltevreden.
Significance and Impact of the Study:  Widespread occurrence of Salmonella contamination in seafood and multiple clones of S. Rissen and S. Weltevreden detected in seafood, thus, indicated the diverse routes of Salmonella contamination in seafood.     

Comparison of commercially available kits with standard methods for detection of Salmonella strains in foods.

Six commercial kits were compared with the U.S. Food and Drug Administration (USFDA) method and the Japanese standard method for Salmonella isolation in foods. When only Salmonella serovars were tested, many of the methods performed well; however, when foods were artificially inoculated, only the USFDA method and immunomagnetic separation coupled with the xylose-lysine-brilliant green agar method (MS-XLBG) could positively detect Salmonella serovars. All seven wild-type Salmonella serovars were detected by the USFDA method, and the MS-XLBG method detected salmonellae from six samples.     

Genomic rearrangements at rrn operons in Salmonella

Most Salmonella serovars are general pathogens that infect a variety of hosts. These "generalist" serovars cause disease in many animals from reptiles to mammals. In contrast, a few serovars cause disease only in a specific host. Host-specific serovars can cause a systemic, often fatal disease in one species yet remain avirulent in other species. Host-specific Salmonella frequently have large genomic rearrangements due to recombination at the ribosomal RNA (rrn) operons while the generalists consistently have a conserved chromosomal arrangement. To determine whether this is the result of an intrinsic difference in recombination frequency or a consequence of lifestyle difference between generalist and host-specific Salmonella, we determined the frequency of rearrangements in vitro. Using lacZ genes as portable regions of homology for inversion analysis, we found that both generalist and host-specific serovars of Salmonella have similar tolerances to chromosomal rearrangements in vitro. Using PCR and genetic selection, we found that generalist and host-specific serovars also undergo rearrangements at rrn operons at similar frequencies in vitro. These observations indicate that the observed difference in genomic stability between generalist and host-specific serovars is a consequence of their distinct lifestyles, not intrinsic differences in recombination frequencies.     

Comparison of Salmonella enterica serovar Typhimurium LT2 and non-LT2 salmonella genomic sequences, and genotyping of salmonellae by using PCR

Genes of Salmonella enterica serovar Typhimurium LT2 expected to be specifically present in Salmonella were selected using the Basic Local Alignment Search Tool (BLAST) program. The 152 selected genes were compared with 11 genomic sequences of Salmonella serovars, including Salmonella enterica subsp. I and IIIb and Salmonella bongori (V), and were clustered into 17 groups by their comparison patterns. A total of 38 primer pairs were constructed to represent each of the 17 groups, and PCR was performed with various Salmonella subspecies including Salmonella enterica subsp. I, II, IIIa, IIIb, IV, VI, and V to evaluate a comprehensive DNA-based scheme for identification of Salmonella subspecies and the major disease-causing Salmonella serovars. Analysis of PCR results showed that Salmonella enterica subsp. I was critically divided from other subspecies, and Salmonella strains belonging to S. enterica subsp. I were clustered based on their serovars. In addition, genotypic relationships within S. enterica subsp. I by PCR results were investigated. Also, Salmonella signature genes, Salmonella enterica serovar Typhimurium signature genes, and Salmonella enterica subsp. I signature genes were demonstrated based on their PCR results. The described PCR method suggests a rapid and convenient method for identification of Salmonella serovars that can be used by nonspecialized laboratories. Genome sequence comparison can be a useful tool in epidemiologic and taxonomic studies of Salmonella.     

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.     

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.     

Features of Salmonella serovars among food handlers in Kyushu, Japan

Salmonella were isolated from 106 (0.032%) of 331,644 fecal samples from food handlers, and from 144 of 11,478 fecal samples from symptomatic patients in Japan to determine the incidence and features of Salmonella serovars among food handlers. S. enterica subspecies enterica serovar Infantis (S. serovar Infantis) was the dominant serovar (accounting for 48.1%), followed by S. serovar Corvallis, which showed poor genetic diversity, and S. serovar Enteritidis among food handlers. The former two serovars were not dominant among symptomatic patients. The present study demonstrates the need for education on the sanitary handling of chicken eggs and chicken meat, which are possible infectious sources of these Salmonella serovars.     

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.     

Genomic comparison of Salmonella enterica serovars and Salmonella bongori by use of an S. enterica serovar typhimurium DNA microarray

The genus Salmonella consists of over 2,200 serovars that differ in their host range and ability to cause disease despite their close genetic relatedness. The genetic factors that influence each serovar's level of host adaptation, how they evolved or were acquired, their influence on the evolution of each serovar, and the phylogenic relationships between the serovars are of great interest as they provide insight into the mechanisms behind these differences in host range and disease progression. We have used an Salmonella enterica serovar Typhimurium spotted DNA microarray to perform genomic hybridizations of various serovars and strains of both S. enterica (subspecies I and IIIa) and Salmonella bongori to gain insight into the genetic organization of the serovars. Our results are generally consistent with previously published DNA association and multilocus enzyme electrophoresis data. Our findings also reveal novel information. We observe a more distant relationship of serovar Arizona (subspecies IIIa) from the subspecies I serovars than previously measured. We also observe variability in the Arizona SPI-2 pathogenicity island, indicating that it has evolved in a manner distinct from the other serovars. In addition, we identify shared genetic features of S. enterica serovars Typhi, Paratyphi A, and Sendai that parallel their unique ability to cause enteric fever in humans. Therefore, whereas the taxonomic organization of Salmonella into serogroups provides a good first approximation of genetic relatedness, we show that it does not account for genomic changes that contribute to a serovar's degree of host adaptation.     

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.     

Horizontal gene transfer of a ColV plasmid has resulted in a dominant avian clonal type of Salmonella enterica serovar Kentucky

Salmonella enterica continues to be a significant cause of foodborne gastrointestinal illness in humans. A wide variety of Salmonella serovars have been isolated from production birds and from retail poultry meat. Recently, though, S. enterica subsp. enterica serovar Kentucky has emerged as one of the prominent Salmonella serovars isolated from broiler chickens. Recent work suggests that its emergence apparently coincides with its acquisition of a ColV virulence plasmid. In the present study, we examined 902 Salmonella isolates belonging to 59 different serovars for the presence of this plasmid. Of the serovars examined, the ColV plasmid was found only among isolates belonging to the serovars Kentucky (72.9%), Typhimurium (15.0%) and Heidelberg (1.7%). We demonstrated that a single PFGE clonal type of S. Kentucky harbors this plasmid, and acquisition of this plasmid by S. Kentucky significantly increased its ability to colonize the chicken cecum and cause extraintestinal disease. Comparison of the completed sequences of three ColV plasmids from S. Kentucky isolated from different geographical locales, timepoints and sources revealed a nearly identical genetic structure with few single nucleotide changes or insertions/deletions. Overall, it appears that the ColV plasmid was recently acquired by a single clonal type S. Kentucky and confers to its host enhanced colonization and fitness capabilities. Thus, the potential for horizontal gene transfer of virulence and fitness factors to Salmonella from other enteric bacteria exists in poultry, representing a potential human health hazard.     

Comparison of Salmonella enterica serovar distribution and antibiotic resistance patterns in wastewater at municipal water treatment plants in two California cities

AIM: To determine Salmonella enterica serovars and antibiotic resistance (ABR) in the human waste stream. METHODS AND RESULTS: Sampling of influent wastewater at municipal treatment plants in two California cities was performed by collecting composite samples, over a 24-h period, from the treatment plants on five to six occasions. Serial water quantities were filtered and cultured with a Salmonella selective method and an oxytetracycline-supplemented Salmonella selective method. Antibiotic susceptibilities to 12 antibiotics were determined and the isolates were grouped based on ABR patterns. From 983 S. enterica isolated, 102 represented unique sampling-serovar-ABR patterns. Thirty-five different serovars were identified to be distributed over 17 different ABR patterns. The serovar distribution differed between the sampling sites, whereas there was no significant trend in levels of multiple ABR. CONCLUSIONS: Salmonella enterica was recovered with ease from small sample volumes of wastewater received by municipal water treatment plants. A large variety of serovars and ABR profiles were represented in the recovered Salmonella. SIGNIFICANCE AND IMPACT OF THE STUDY: The ease of sampling and recovery of Salmonella from municipal wastewater from treatment plants makes it a valuable sampling approach for monitoring the presence of Salmonella in the human population.     

Etiological structure of salmonellosis and the serotype passage of salmonellae isolated in separate regions of the USSR in 1980-1982

At the period of 1980-1982 the isolation of salmonellae belonging to 394 serovars was registered in the USSR. Of these, 116 Salmonella serovars were registered in the USSR for the first time. 12 dominating serovars constituted 83.1% of salmonellae isolated from humans, 99% of salmonellae isolated from animals and 70% of all salmonellae isolated from different environmental objects. S. typhimurium was the predominant serovar, found to determine 50% of cases of Salmonella infection. The isolation rate of S. infantis and S. virchow was shown to increase. The existence of definite ecological relationships between infective agents isolated from different sources was established.     

Rapid identification of Salmonella typhimurium, S. enteritidis and S. virchow isolates by polymerase chain reaction based fingerprinting methods.

In this study we used and evaluated three rapid molecular typing methods for the identification of three frequent, clinically significant Salmonella serovars on the basis of the ease, simplicity and reproducibility of the chosen methods. We determined the genetic diversity among several isolates of Salmonella enteritidis, S. typhimiurium and S. virchow, and compared them with other enterobacteria by using the repetitive extragenic palindromic (REP) sequences, the enterobacterial repetitive intergenic consensus (ERIC) sequences, and the 16S-23S rDNA intergenic spacer region (ITS 1). The objective was to evaluate their potential application to discriminate among members of the species Salmonella enterica subspecies enterica using the genetic diversity of the group found by genomic fingerprinting. The three different serovars of Salmonella studied gave reproducible and distinguishable profiles using whichever of the above mentioned polymerase chain reaction (PCR) methods assayed. The conserved patterns in each serovar allowed for easy differentiation from other serovars of Salmonella.     

Identification and characterization of novel salmonella mobile elements involved in the dissemination of genes linked to virulence and transmission

The genetic diversity represented by >2,500 different Salmonella serovars provides a yet largely uncharacterized reservoir of mobile elements that can contribute to the frequent emergence of new pathogenic strains of this important zoonotic pathogen. Currently, our understanding of Salmonella mobile elements is skewed by the fact that most studies have focused on highly virulent or common serovars. To gain a more global picture of mobile elements in Salmonella, we used prediction algorithms to screen for mobile elements in 16 sequenced Salmonella genomes representing serovars for which no prior genome scale mobile element data were available. From these results, selected mobile elements underwent further analyses in the form of validation studies, comparative analyses, and PCR-based population screens. Through this analysis we identified a novel plasmid that has two cointegrated replicons (IncI1-IncFIB); this plasmid type was found in four genomes representing different Salmonella serovars and contained a virulence gene array that had not been previously identified. A Salmonella Montevideo isolate contained an IncHI and an IncN2 plasmid, which both encoded antimicrobial resistance genes. We also identified two novel genomic islands (SGI2 and SGI3), and 42 prophages with mosaic architecture, seven of them harboring known virulence genes. Finally, we identified a novel integrative conjugative element (ICE) encoding a type IVb pilus operon in three non-typhoidal Salmonella serovars. Our analyses not only identified a considerable number of mobile elements that have not been previously reported in Salmonella, but also found evidence that these elements facilitate transfer of genes that were previously thought to be limited in their distribution among Salmonella serovars. The abundance of mobile elements encoding pathogenic properties may facilitate the emergence of strains with novel combinations of pathogenic traits.     

Recombination of Salmonella phase 1 flagellin genes generates new serovars.

To determine the evolutionary mechanisms generating serotypic diversity in Salmonella strains, we sequenced the central, antigen-determining part of the phase 1 flagellin gene (fliC) in strains of several serovars for which estimates of chromosomal genomic relatedness had been obtained by multilocus enzyme electrophoresis. The nucleotide sequence of this region was identical in several chromosomally divergent strains of Salmonella heidelberg (phase 1 antigen r) but differed by 19% from the corresponding and similarly invariant sequence in strains of the closely related serovar Salmonella typhimurium (phase 1 antigen i). Mutational drift of the sequence present in the common ancestor is unlikely to have generated the difference between the phase 1 flagellins of these two serovars, which we attribute instead to a recombination event. This interpretation is supported by evidence that Salmonella strains of very diverse chromosomal backgrounds but similar phase 1 antigens may have closely similar nucleotide sequences for this highly polymorphic region. We suggest that lateral transfer and recombination of phase 1 flagellin genes is a major evolutionary mechanism generating new Salmonella serovars.