Population Genetic Structure of 4,12:a:? Salmonella enterica Strains from Harbor Porpoises

According to pulsed-field gel electrophoresis (PFGE) typing, 4,12:a:− Salmonella enterica isolates from harbor porpoises are highly diverse. However, porpoise isolates belong to only two multilocus sequence types within the eBurst group 18 (eBG18) genetic cluster, which also includes S. enterica serovars Bispebjerg and Abortusequi. Isolates of other, serologically similar serovars belong to unrelated eBGs. These assignments to eBGs were supported by eBG-specific sequences of the flagellar gene fliC.     

Comparison of a semi-automated rep-PCR system and multilocus sequence typing for differentiation of Salmonella enterica isolates

The accurate sub-typing of Salmonella enterica isolates is essential for epidemiological investigations and surveillance of Salmonella infections. Salmonella isolates are currently identified using the Kauffman-White serotyping scheme. Multilocus sequence typing (MLST) schemes have been developed for the major bacterial pathogens including Salmonella and have assisted in understanding the molecular epidemiology and population biology of these organisms. Recently, the DiversiLab rep-PCR system has been developed using micro-fluidic chips to provide standardized, semi-automated fingerprinting for pathogens including S. enterica. In the current study, 71 isolates of S. enterica, representing 21 serovars, were analyzed using MLST and the DiversiLab rep-PCR system. MLST was able to identify 31 sequence types (STs), while the DiversiLab system revealed 38 DiversiLab types (DTs). The rep-PCR distinguished isolates of different serovars and showed greater discriminatory power (0.95) than MLST typing (0.89). Rep-PCR exhibited 92% concordance with MLST and 90% with serotyping, while the concordance level of MLST typing with serotyping was 96%, representing a strong association. Comparison of rep-PCR profiles with those held in an online library database led to the accurate prediction of serovar in 63% of cases and resulted in inaccurate predictions for 10% of profiles. MLST and the rep-PCR system may provide useful additional informative techniques for the molecular identification of S. enterica. We conclude that the DiversiLab rep-PCR system may provide a rapid (less than 4 h) and standardized method for sub-typing isolates of S. enterica.     

Population Structure of Salmonella enterica Serovar 4,[5],12:b:? Strains and Likely Sources of Human Infection

Salmonella enterica serovar 4,[5],12:b:− is a monophasic serovar not able to express the second-phase flagellar antigen (H2 antigen). In Germany, the serovar is occasionally isolated from poultry, reptiles, fish, food, and humans. In this study, a selection of 67 epidemiologically unrelated Salmonella enterica serovar 4,[5],12:b:− strains isolated in Germany between 2000 and 2011 from the environment, animal, food, and humans was investigated by phenotypic and genotypic methods to better understand the population structure and to identify potential sources of human infections. Strains of this monophasic serovar were highly diverse. Within the 67 strains analyzed, we identified 52 different pulsed-field gel electrophoresis XbaI profiles, 12 different multilocus sequence types (STs), and 18 different pathogenicity array types. The relatedness of strains based on the pathogenicity gene repertoire (102 markers tested) was in good agreement with grouping by MLST. S. enterica serovar 4,[5],12:b:− is distributed across multiple unrelated eBurst groups and consequently is highly polyphyletic. Two sequence types (ST88 and ST127) were linked to S. enterica serovar Paratyphi B (d-tartrate positive), two single-locus variants of ST1583 were linked to S. enterica serovar Abony, and one sequence type (ST1484) was associated with S. enterica serovar Mygdal, a recently defined, new serovar. From the characterization of clinical isolates and those of nonhuman origin, it can be concluded that the potential sources of sporadic human infections with S. enterica serovar 4,[5],12:b:− most likely are mushrooms, shellfish/fish, and poultry.     

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.     

Rapid identification of Salmonella enterica serovars, Typhimurium, Choleraesuis, Infantis, Hadar, Enteritidis, Dublin and Gallinarum, by multiplex PCR

Salmonella enterica subsp. enterica poses a threat to both human and animal health, with more than 2500 serovars having been reported to date. Salmonella serovars are identified by slide and tube agglutination tests using O and H antigen-specific anti-sera, although this procedure is both labor intensive and time consuming. Establishment of a method for rapid screening of the major Salmonella serovars is therefore required. We have established multiplex polymerase chain reaction (m-PCR) assays for identification of seven serovars of Salmonella, i.e., Typhimurium, Choleraesuis, Infantis, Hadar, Enteritidis, Dublin and Gallinarum. Three serovar-specific genomic regions (SSGRs) of each serovar were selected using an approach in comparative genomics. The Salmonella-specific invA gene was used to confirm the genetic background of the organisms. The isolates tested were identified as a target serovar when the three selected SSGRs and invA were all positive for amplification. The specificity of each m-PCR assay was investigated using 118 serovars of Salmonella and 12 species of non-Salmonella strains. Although a small number of false-positive results were observed in the m-PCR assays used to identify Typhimurium, Choleraesuis, Enteritidis and Dublin for closely related serovars, false-negative results were not observed in any assays. These assays had sufficient specificity to identify the seven Salmonella serovars, and therefore, have the potential for use as rapid screening methods.     

Salmonella enterica Diversity in Central Californian Coastal Waterways

Salmonella enterica is one of the most important bacterial enteric pathogens worldwide. However, little is known about its distribution and diversity in the environment. The present study explored the diversity of 104 strains of Salmonella enterica isolated over 2 years from 12 coastal waterways in central California. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing were used to probe species diversity. Seventy-four PFGE patterns and 38 sequence types (STs) were found, including 18 newly described STs. Nineteen of 25 PFGE patterns were indistinguishable from those of clinical isolates in PulseNet. The most common ST was consistent with S. enterica serovar Typhimurium, and other frequently detected STs were associated with the serovars Heidelberg and Enteritidis; all of these serovars are important etiologies of salmonellosis. An investigation into S. enterica biogeography was conducted at the level of ST and subspecies. At the ST and subspecies level, we found a taxon-time relationship but no taxon-area or taxon-environmental distance relationships. STs collected during wet versus dry conditions tended to be more similar; however, STs collected from waterways adjacent to watersheds with similar land covers did not tend to be similar. The results suggest that the lack of dispersal limitation may be an important factor affecting the diversity of S. enterica in the region.     

Clonal Expansion May Account for High Levels of Quinolone Resistance in Salmonella enterica Serovar Enteritidis

We have observed a high incidence of isolated nalidixic acid resistance in Salmonella enterica serovar Enteritidis isolates in Ireland, particularly isolates of phage type 1 (PT1). A group of nalidixic acid-resistant (n = 22) and nalidixic acid-susceptible (n = 28) isolates of serovar Enteritidis from multiple sites in Ireland were selected. Isolates were typed by pulsed-field gel electrophoresis (PFGE) with XbaI, and the MICs for nalidixic acid and ciprofloxacin were determined. Mutations associated with nalidixic acid resistance in clinical isolates and laboratory mutants of serovar Enteritidis and 32 nalidixic acid-resistant isolates of 15 other salmonella serovars were identified. PFGE had limited discriminatory power. A specific point mutation (G246T) associated with amino acid substitution Asp87Tyr in the quinolone resistance determining region of the gyrA gene accounted for 95% of all mutations in serovar Enteritidis and for all mutations in PT1 isolates. Greater diversity of mutations was observed among all non-Enteritidis salmonella serovars studied. Rates of nalidixic acid resistance in serovar Enteritidis may predominantly reflect clonal expansion after infrequent mutation or selection events.     

An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium

Current commercial PCRs tests for identifying Salmonella target genes unique to this genus. However, there are two species, six subspecies, and over 2,500 different Salmonella serovars, and not all are equal in their significance to public health. For example, finding S. enterica subspecies IIIa Arizona on a table egg layer farm is insignificant compared to the isolation of S. enterica subspecies I serovar Enteritidis, the leading cause of salmonellosis linked to the consumption of table eggs. Serovars are identified based on antigenic differences in lipopolysaccharide (LPS)(O antigen) and flagellin (H1 and H2 antigens). These antigenic differences are the outward appearance of the diversity of genes and gene alleles associated with this phenotype.We have developed an allelotyping, multiplex PCR that keys on genetic differences between four major S. enterica subspecies I serovars found in poultry and associated with significant human disease in the US. The PCR primer pairs were targeted to key genes or sequences unique to a specific Salmonella serovar and designed to produce an amplicon with size specific for that gene or allele. Salmonella serovar is assigned to an isolate based on the combination of PCR test results for specific LPS and flagellin gene alleles. The multiplex PCRs described in this article are specific for the detection of S. enterica subspecies I serovars Enteritidis, Hadar, Heidelberg, and Typhimurium.Here we demonstrate how to use the multiplex PCRs to identify serovar for a Salmonella isolate.     

Identification of Genes Associated with Survival of Salmonella enterica Serovar Enteritidis in Chicken Egg Albumen

Salmonella enterica consists of over 2,000 serovars that are major causes of morbidity and mortality associated with contaminated food. Despite similarities among serovars of Salmonella enterica, many demonstrate unique host specificities, epidemiological characteristics, and clinical manifestations. One of the unique epidemiological characteristics of the serovar Enteritidis is that it is the only bacterium routinely transmitted to humans through intact chicken eggs. Therefore, Salmonella enterica serovar Enteritidis must be able to persist inside chicken eggs to be transmitted to humans, and its survival in egg is important for its transmission to the human population. The ability of Salmonella enterica serovar Enteritidis to survive in and transmit through eggs may have contributed to its drastically increased prevalence in the 1980s and 1990s. In the present study, using transposon-mediated mutagenesis, we have identified genes important for the association of Salmonella enterica serovar Enteritidis with chicken eggs. Our results indicate that genes involved in cell wall structural and functional integrity, and nucleic acid and amino acid metabolism are important for Salmonella enterica serovar Enteritidis to persist in egg albumen. Two regions unique to Salmonella enterica serovar Enteritidis were also identified, one of which enhanced the survival of a Salmonella enterica serovar Typhimurium isolate in egg albumen. The implication of our results to the serovar specificity of Salmonella enterica is also explored in the present study.     

Identification by Subtractive Hybridization of Sequences Specific for Salmonella enterica Serovar Enteritidis

Salmonella enterica serovar Enteritidis, a major cause of food poisoning, can be transmitted to humans through intact chicken eggs when the contents have not been thoroughly cooked. Infection in chickens is asymptomatic; therefore, simple, sensitive, and specific detection methods are crucial for efforts to limit human exposure. Suppression subtractive hybridization was used to isolate DNA restriction fragments present in Salmonella serovar Enteritidis but absent in other bacteria found in poultry environments. Oligonucleotide primers to candidate regions were used in polymerase chain reactions to test 73 non-Enteritidis S. enterica isolates comprising 34 different serovars, including Dublin and Pullorum, two very close relatives of Enteritidis. A primer pair to one Salmonella difference fragment (termed Sdf I) clearly distinguished serovar Enteritidis from all other serovars tested, while two other primer pairs only identified a few non-Enteritidis strains. These primer pairs were also useful for the detection of a diverse collection of clinical and environmental Salmonella serovar Enteritidis isolates. In addition, five bacterial genera commonly found with Salmonella serovar Enteritidis were not detected. By treating total DNA with an exonuclease that degrades sheared chromosomal DNA but not intact circular plasmid DNA, it was shown that Sdf I is located on the chromosome. The Sdf I primers were used to screen a Salmonella serovar Enteritidis genomic library and a unique 4,060-bp region was defined. These results provide a basis for developing a rapid, sensitive, and highly specific detection system for Salmonella serovar Enteritidis and provide sequence information that may be relevant to the unique characteristics of this serovar.     

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.     

Human Infections Attributable to the d-Tartrate-Fermenting Variant of Salmonella enterica Serovar Paratyphi B in Germany Originate in Reptiles and,on Rare Occasions,Poultry

In this study, the population structure, incidence, and potential sources of human infection caused by the d-tartrate-fermenting variant of Salmonella enterica serovar Paratyphi B [S. Paratyphi B (dT+)] was investigated. In Germany, the serovar is frequently isolated from broilers. Therefore, a selection of 108 epidemiologically unrelated S. enterica serovar Paratyphi B (dT+) strains isolated in Germany between 2002 and 2010 especially from humans, poultry/poultry meat, and reptiles was investigated by phenotypic and genotypic methods. Strains isolated from poultry and products thereof were strongly associated with multilocus sequence type ST28 and showed antimicrobial multiresistance profiles. Pulsed-field gel electrophoresis XbaI profiles were highly homogeneous, with only a few minor XbaI profile variants. All strains isolated from reptiles, except one, were strongly associated with ST88, another distantly related type. Most of the strains were susceptible to antimicrobial agents, and XbaI profiles were heterogeneous. Strains isolated from humans yielded seven sequence types (STs) clustering in three distantly related lineages. The first lineage, comprising five STs, represented mainly strains belonging to ST43 and ST149. The other two lineages were represented only by one ST each, ST28 and ST88. The relatedness of strains based on the pathogenicity gene repertoire (102 markers tested) was mostly in agreement with the multilocus sequence type. Because ST28 was frequently isolated from poultry but rarely in humans over the 9-year period investigated, overall, this study indicates that in Germany S. enterica serovar Paratyphi B (dT+) poses a health risk preferentially by contact with reptiles and, to a less extent, by exposure to poultry or poultry meat.     

Pork Contaminated with Salmonella enterica Serovar 4,[5],12:i:?, an Emerging Health Risk for Humans

Salmonella enterica subsp. enterica serovar 4,[5],12:i:− is a monophasic variant of S. enterica serovar Typhimurium (antigenic formula 4,[5],12:i:1,2). Worldwide, especially in several European countries and the United States, it has been reported among the 10 most frequently isolated serovars in pigs and humans. In the study reported here, 148 strains of the monophasic serovar isolated from pigs, pork, and humans in 2006 and 2007 in Germany were characterized by various phenotypic and genotypic methods. This characterization was done in order to investigate their clonality, the prevalence of identical subtypes in pigs, pork, and humans, and the genetic relatedness to other S. enterica serovar Typhimurium subtypes in respect to the pathogenic and resistance gene repertoire. Two major clonal lineages of the monophasic serovar were detected which can be differentiated by their phage types and pulsed-field gel electrophoresis (PFGE) profiles. Seventy percent of the strains tested belonged to definite phage type DT193, and those strains were mainly assigned to PFGE cluster B. Nineteen percent of the strains were typed to phage type DT120 and of these 86% belonged to PFGE cluster A. Sixty-five percent of the isolates of both lineages carried core multiresistance to ampicillin, streptomycin, tetracycline, and sulfamethoxazole encoded by the genes bla_TEM1-like, strA-strB, tet(B), and sul2. No correlation to the source of isolation was observed in either lineage. Microarray analysis of 61 S. enterica serovar 4,[5],12:i:− and 20 S. enterica serovar Typhimurium isolates tested determining the presence or absence of 102 representative pathogenicity genes in Salmonella revealed no differences except minor variations in single strains within and between the serovars, e.g., by presence of the virulence plasmid in four strains. Overall the study indicates that in Germany S. enterica serovar 4,[5],12:i:− strains isolated from pig, pork, and human are highly related, showing their transmission along the food chain. Since the pathogenicity gene repertoire is highly similar to that of S. enterica serovar Typhimurium, it is essential that interventions are introduced at the farm level in order to limit human infection.Salmonella enterica subsp. enterica serovar Typhimurium is a ubiquitous serovar that usually induces gastroenteritis in a broad range of unrelated host species. Following the White-Kauffmann-Le Minor scheme, the seroformula for S. enterica serovar Typhimurium is 4,[5],12:i:1,2 (14). Salmonella serotyping is based on antigenic variability of lipopolysaccharides (O antigen) and flagellar proteins (H1 and H2 antigens).In the mid-1990s a monophasic S. enterica serovar with the seroformula 4,[5],12:i:− started to emerge in Europe (10). Initial characterization of isolates from pig samples in Spain in 1997 demonstrated that this serovar in comparison with S. enterica serovar Typhimurium (4,[5],12:i:1,2) lacked the fljB gene encoding the structural subunit of the phase two flagellar (H2) antigen (11). The predominant phage type was U302. Another DNA microarray-based typing study indicated that the monophasic serovar had a gene repertoire highly similar to that of S. enterica serovar Typhimurium, indicating a close genetic relatedness between the serovars (13). Similarly, multi-locus sequence typing showed that S. enterica serovar 4,[5],12:i:− and S. enterica serovar Typhimurium represent a highly clonal group (23).Within the last years S. enterica serovar 4,[5],12:i:− has increasingly been implicated in human disease worldwide (1, 10, 24, 25). Recently, larger outbreaks caused by this serovar have been reported from Luxembourg and the United States (5, 19). A European Union (EU) baseline survey on the prevalence of Salmonella in slaughter-age pigs in 2006 to 2007 revealed that the monophasic serovar was isolated from pigs in 9 of 25 participating member states (12). At the EU level, S. enterica serovar 4,[5],12:i:− was the fourth most prevalent serovar in slaughter-age pigs. In Germany it was the second most prevalent serovar after S. enterica serovar Typhimurium (12). Between 1999 and 2008 the proportion of S. enterica serovar 4,[5],12:i:− isolates among all S. enterica isolates received by the German National Reference Laboratory for Salmonella increased from 0.1% to 8.3% (305 isolates in 2008), with the most remarkable increase between 2006 and 2007. Most of these strains (48% on average between 2006 and 2008) were isolated from pigs, followed by cattle (13%), poultry (5%), and other isolates sporadically found in the environment, wildlife, and reptiles. Remarkably, the annual proportion of the monophasic serovar among all S. enterica serovar 4,[5],12:i:− and S. enterica serovar Typhimurium isolates increased from 0.3% to 32.7% in the same decade. Interestingly, the number of S. enterica serovar 4,[5],12:i:− strains isolated from humans and sent on voluntary basis to the National Reference Centre for Salmonella and other Enterics increased from 0.1% in 1999 to 14.0% (456 isolates) in 2008. Likewise, the proportion of the monophasic serovar among all S. enterica serovar 4,[5],12:i:− and S. enterica serovar Typhimurium isolates increased from 0.3% to 42.8% in the same time because of declining numbers of S. enterica serovar Typhimurium isolates.In the present study a collection of S. enterica serovar 4,[5],12:i:− strains isolated from pigs, pork, and humans in Germany during the years 2006 and 2007 was examined using phenotypic and molecular methods. The aim of the analyses was to gain a better understanding of the clonality of the serovar and of the ability of its subtypes to be transmitted to humans via pigs and pork. Additionally, the genetic relatedness as well as the pathogenicity and antimicrobial resistance gene repertoire of S. enterica serovar 4,[5],12:i:− was compared with selected S. enterica serovar Typhimurium strains representing corresponding phage types in order to estimate the potential health risk for humans.     

Prevalence and Fimbrial Genotype Distribution of Poultry Salmonella Isolates in China (2006 to 2012)

In this study, a total of 323 Salmonella enterica strains were isolated from 3,566 rectal swab samples of 51 poultry farms in seven regions of 12 provinces of China between 2006 and 2012. The prevalences of Salmonella sp. carriage were 12.4% in geese (66 positive/533 samples), 10.4% in turkeys (32/309), 9.8% in chickens (167/1,706), 6.8% in ducks (41/601), and 4.1% in pigeons (17/417), respectively. These isolates belonged to 20 serovars, in which the most frequent serovars were S. enterica serovar Gallinarum biovar Pullorum (herein, S. Pullorum) (55 isolates, 17.0%), S. enterica serovar Typhimurium (50 isolates, 15.5%), and S. enterica serovar Enteritidis (39 isolates, 12.1%). Overall, S. Typhimurium was the most commonly detected serovar; among the individual species, S. Pullorum was most commonly isolated from chickens, S. Enteritidis was most common in ducks, S. Typhimurium was most common in geese and pigeons, and S. enterica serovar Saintpaul was most common in turkeys. PCR determination of 20 fimbrial genes demonstrated the presence of bcfD, csgA, fimA, stdB, and sthE genes and the absence of staA and stgA genes in these isolates, and other loci were variably distributed, with frequency values ranging from 11.8 to 99.1%. These 323 Salmonella isolates were subdivided into 41 different fimbrial genotypes, and of these isolate, 285 strains (88.2%) had 12 to 14 fimbrial genes. Our findings indicated that the Salmonella isolates from different poultry species were phenotypically and genetically diverse and that some fimbrial genes are more frequently associated with serovars or serogroups.     

Detection of hilA gene sequences in serovars of Salmonella enterica subspecies enterica

hilA gene promoter, component of the Salmonella Pathogenicity Island 1, has been found in Salmonella serovar Typhimurium, being important for the regulation of type III secretion apparatus genes. We detected hilA gene sequences in Salmonella serovars Typhi, Enteritidis, Choleraesuis, Paratyphi A and B, and Pullorum, by polymerase chain reaction (PCR) and hybridization techniques. The primers to carry out PCR were designed according to hilA sequence. A low stringency hybridization with the probe pVV441 (hilA open-reading-frame plasmid) was carried out. To find hilA gene sequences in other Salmonella sp. suggest that these serovars could have similar sequences of this kind of virulence genes.     

Multilocus Sequence Typing Supports the Hypothesis that Cow- and Human-Associated Salmonella Isolates Represent Distinct and Overlapping Populations

A collection of 179 human and 156 bovine clinical Salmonella isolates obtained from across New York state over the course of 1 year was characterized using serotyping and a multilocus sequence typing (MLST) scheme based on the sequencing of three genes (fimA, manB, and mdh). The 335 isolates were differentiated into 52 serotypes and 72 sequence types (STs). Analyses of bovine isolates collected on different farms over time indicated that specific subtypes can persist over time on a given farm; in particular, a number of farms showed evidence for the persistence of a specific Salmonella enterica serotype Newport sequence type. Serotypes and STs were not randomly distributed among human and bovine isolates, and selected serotypes and STs were associated exclusively with either human or bovine sources. A number of common STs were geographically widespread. For example, ST6, which includes isolates representing serotype Typhimurium as well as the emerging serotype 4,5,12:i:-, was found among human and bovine isolates in a number of counties in New York state. Phylogenetic analyses supported the possibility that serotype 4,5,12:i:- is closely related to Salmonella serotype Typhimurium. Salmonella serotype Newport was found to represent two distinct evolutionary lineages that differ in their frequencies among human and bovine isolates. A number of Salmonella isolates carried two copies of manB (33 isolates) or showed small deletion events in fimA (nine isolates); these duplication and deletion events may provide mechanisms for the rapid diversification of Salmonella surface molecules. We conclude that the combined use of an economical three-gene MLST scheme and serotyping can provide considerable new insights into the evolution and transmission of Salmonella.     

Draft Genome Sequences of 21 Salmonella enterica Serovar Enteritidis Strains

Salmonella enterica subsp. enterica serovar Enteritidis is a common food-borne pathogen, often associated with shell eggs and poultry. Here, we report draft genomes of 21 S. Enteritidis strains associated with or related to the U.S.-wide 2010 shell egg recall. Eleven of these genomes were from environmental isolates associated with the egg outbreak, and 10 were reference isolates from previous years, unrelated to the outbreak. The whole-genome sequence data for these 21 human pathogen strains are being released in conjunction with the newly formed 100K Genome Project.     

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.     

Diversity and Antimicrobial Resistance of Salmonella enterica Isolates from Surface Water in Southeastern United States

A study of prevalence, diversity, and antimicrobial resistance of Salmonella enterica in surface water in the southeastern United States was conducted. A new scheme was developed for recovery of Salmonella from irrigation pond water and compared with the FDA''s Bacteriological Analytical Manual (8th ed., 2014) (BAM) method. Fifty-one isolates were recovered from 10 irrigation ponds in produce farms over a 2-year period; nine Salmonella serovars were identified by pulsed-field gel electrophoresis analysis, and the major serovar was Salmonella enterica serovar Newport (S. Newport, n = 29), followed by S. enterica serovar Enteritidis (n = 6), S. enterica serovar Muenchen (n = 4), S. enterica serovar Javiana (n = 3), S. enterica serovar Thompson (n = 2), and other serovars. It is noteworthy that the PulseNet patterns of some of the isolates were identical to those of the strains that were associated with the S. Thompson outbreaks in 2010, 2012, and 2013, S. Enteritidis outbreaks in 2011 and 2013, and an S. Javiana outbreak in 2012. Antimicrobial susceptibility testing confirmed 16 S. Newport isolates of the multidrug resistant-AmpC (MDR-AmpC) phenotype, which exhibited resistance to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline (ACSSuT), and to the 1st, 2nd, and 3rd generations of cephalosporins (cephalothin, amoxicillin-clavulanic acid, and ceftriaxone). Moreover, the S. Newport MDR-AmpC isolates had a PFGE pattern indistinguishable from the patterns of the isolates from clinical settings. These findings suggest that the irrigation water may be a potential source of contamination of Salmonella in fresh produce. The new Salmonella isolation scheme significantly increased recovery efficiency from 21.2 (36/170) to 29.4% (50/170) (P = 0.0002) and streamlined the turnaround time from 5 to 9 days with the BAM method to 4 days and thus may facilitate microbiological analysis of environmental water.