The mycotoxin deoxynivalenol potentiates intestinal inflammation by Salmonella typhimurium in porcine ileal loops

Background and Aims

Both deoxynivalenol (DON) and nontyphoidal salmonellosis are emerging threats with possible hazardous effects on both human and animal health. The objective of this study was to examine whether DON at low but relevant concentrations interacts with the intestinal inflammation induced by Salmonella Typhimurium.

Methodology

By using a porcine intestinal ileal loop model, we investigated whether intake of low concentrations of DON interacts with the early intestinal inflammatory response induced by Salmonella Typhimurium.

Results

A significant higher expression of IL-12 and TNFα and a clear potentiation of the expression of IL-1β, IL-8, MCP-1 and IL-6 was seen in loops co-exposed to 1 µg/mL of DON and Salmonella Typhimurium compared to loops exposed to Salmonella Typhimurium alone. This potentiation coincided with a significantly enhanced Salmonella invasion in and translocation over the intestinal epithelial IPEC-J2 cells, exposed to non-cytotoxic concentrations of DON for 24 h. Exposure of Salmonella Typhimurium to 0.250 µg/mL of DON affected the bacterial gene expression level of a limited number of genes, however none of these expression changes seemed to give an explanation for the increased invasion and translocation of Salmonella Typhimurium and the potentiated inflammatory response in combination with DON.

Conclusion

These data imply that the intake of low and relevant concentrations of DON renders the intestinal epithelium more susceptible to Salmonella Typhimurium with a subsequent potentiation of the inflammatory response in the gut.     

Prevalence and Characterization of Monophasic Salmonella Serovar 1,4,[5],12:i:- of Food Origin in China

Salmonella enterica subsp. enterica serovar 1,4,[5],12:i:- is a monophasic variant of Salmonella Typhimurium, which has recently been recognized as an emerging cause of infection worldwide. This bacterium has also ranked among the four most frequent serovars causing human salmonellosis in China. However, there are no reports on its contamination in Chinese food. Serotyping, polymerase chain reaction, antibiotic resistance, virulotyping, and multilocus sequence typing (MLST) assays were used to investigate the prevalence of this serological variant in food products in China, and to determine phenotypic and genotypic difference of monophasic isolates and Salmonella Typhimurium isolated over the same period. Salmonella 1,4,[5],12:i:- was prevalent in various food sources, including beef, pork, chicken, and pigeon. The study also confirmed the high prevalence (53.8%) of resistance to ampicillin, streptomycin, sulfonamides, and tetracycline in Salmonella 1,4,[5],12:i:-, which was higher than that in Salmonella Typhimurium. Moreover, Salmonella 1,4,[5],12:i:- isolates in our study were different from Salmonella Typhimurium isolates by the absence of three plasmid-borne genes (spvC, pefA, and rck) and the presence of gipA in all isolates. All Salmonella 1,4,[5],12:i:- isolates demonstrated MLST pattern ST34. Genomic deletions within the fljBA operon and surrounding genes were only found in Salmonella 1,4,[5],12:i:- isolates, with all isolates containing a deletion of fljB. However, hin and iroB were identified in all Salmonella 1,4,[5],12:i:- isolates. Three different deletion profiles were observed and two of them were different from the reported Salmonella 1,4,[5],12:i:- clones from Spain, America, and Italy, which provided some new evidence on the independent evolution of the multiple successful monophasic clones from Salmonella Typhimurium ancestors. This study is the first report of Salmonella 1,4,[5],12:i:- in food products from China. The data are more comprehensive and representative, providing valuable information for epidemiological studies, risk management, and public health strategies.     

Virulence Gene Profiling and Pathogenicity Characterization of Non-Typhoidal Salmonella Accounted for Invasive Disease in Humans

Human infection with non-typhoidal Salmonella serovars (NTS) infrequently causes invasive systemic disease and bacteremia. To understand better the nature of invasive NTS (iNTS), we studied the gene content and the pathogenicity of bacteremic strains from twelve serovars (Typhimurium, Enteritidis, Choleraesuis, Dublin, Virchow, Newport, Bredeney, Heidelberg, Montevideo, Schwarzengrund, 9,12:l,v:- and Hadar). Comparative genomic hybridization using a Salmonella enterica microarray revealed a core of 3233 genes present in all of the iNTS strains, which include the Salmonella pathogenicity islands 1–5, 9, 13, 14; five fimbrial operons (bcf, csg, stb, sth, sti); three colonization factors (misL, bapA, sinH); and the invasion gene, pagN. In the iNTS variable genome, we identified 16 novel genomic islets; various NTS virulence factors; and six typhoid-associated virulence genes (tcfA, cdtB, hlyE, taiA, STY1413, STY1360), displaying a wider distribution among NTS than was previously known. Characterization of the bacteremic strains in C3H/HeN mice showed clear differences in disease manifestation. Previously unreported characterization of serovars Schwarzengrund, 9,12:l,v:-, Bredeney and Virchow in the mouse model showed low ability to elicit systemic disease, but a profound and elongated shedding of serovars Schwarzengrund and 9,12:l,v:- (as well as Enteritidis and Heidelberg) due to chronic infection of the mouse. Phenotypic comparison in macrophages and epithelial cell lines demonstrated a remarkable intra-serovar variation, but also showed that S. Typhimurium bacteremic strains tend to present lower intracellular growth than gastroenteritis isolates. Collectively, our data demonstrated a common core of virulence genes, which might be required for invasive salmonellosis, but also an impressive degree of genetic and phenotypic heterogeneity, highlighting that bacteremia is a complex phenotype, which cannot be attributed merely to an enhanced invasion or intracellular growth of a particular strain.     

Antimicrobial Resistance in Invasive Non-typhoid Salmonella from the Democratic Republic of the Congo: Emergence of Decreased Fluoroquinolone Susceptibility and Extended-spectrum Beta Lactamases

Background

Co-resistance against the first-line antibiotics ampicillin, chloramphenicol and trimethoprim/sulphamethoxazole or multidrug resistance (MDR) is common in non typhoid Salmonella (NTS). Use of alternative antibiotics, such as fluoroquinolones or third generation cephalosporins is threatened by increasing resistance, but remains poorly documented in Central-Africa.

Methodology/Principal findings

As part of a microbiological surveillance study in DR Congo, blood cultures were collected between 2007 and 2011. Isolated NTS were assessed for serotype and antimicrobial resistance including decreased ciprofloxacin susceptibility and extended-spectrum beta-lactamase (ESBL) production. In total, 233 NTS isolates (representing 23.6% of clinically significant organisms) were collected, mainly consisting of Salmonella Typhimurium (79%) and Salmonella Enteritidis (18%). The majority of NTS were isolated in the rainy season, and recovered from children ≤2 years old. MDR, decreased ciprofloxacin susceptibility, azithromycin and cefotaxime resistance were 80.7%, 4.3%, 3.0% and 2.1% respectively. ESBL production was noted in three (1.3%) isolates. Decreased ciprofloxacin susceptibility was associated with mutations in codon 87 of the gyrA gene, while ESBLs all belonged to the SHV-2a type.

Conclusions/Significance

Presence of almost full MDR among NTS isolates from blood cultures in Central Africa was confirmed. Resistance to fluoroquinolones, azithromycin and third generation cephalosporins is still low, but emerging. Increased microbiological surveillance in DR Congo is crucial for adapted antibiotic therapy and the development of treatment guidelines.     

Genomic Comparison between Salmonella Gallinarum and Pullorum: Differential Pseudogene Formation under Common Host Restriction

Background

Salmonella serovars Enteritidis and Gallinarum are closely related, but their host ranges are very different: the former is host-promiscuous and the latter can infect poultry only. Comparison of their genomic sequences reveals that Gallinarum has undergone much more extensive degradation than Enteritidis. This phenomenon has also been observed in other host restricted Salmonella serovars, such as Typhi and Paratyphi A. The serovar Gallinarum can be further split into two biovars: Gallinarum and Pullorum, which take poultry as their common host but cause distinct diseases, with the former eliciting typhoid and the latter being a dysentery agent. Genomic comparison of the two pathogens, with a focus on pseudogenes, would provide insights into the evolutionary processes that might have facilitated the formation of host-restricted Salmonella pathogens.

Methodologies/Principal Findings

We sequenced the complete genome of Pullorum strains and made comparison with Gallinarum and other Salmonella lineages. The gene contents of Gallinarum and Pullorum were highly similar, but their pseudogene compositions differed considerably. About one fourth of pseudogenes had the same inactivation mutations in Gallinarum and Pullorum but these genes remained intact in Enteritidis, suggesting that the ancestral Gallinarum may have already been restricted to poultry. On the other hand, the remaining pseudogenes were either in the same genes but with different inactivation sites or unique to Gallinarum or Pullorum, reflecting unnecessary functions in infecting poultry.

Conclusions

Our results support the hypothesis that the divergence between Gallinarum and Pullorum was initiated and facilitated by host restriction. Formation of pseudogenes instead of gene deletion is the major form of genomic degradation. Given the short divergence history of Gallinarum and Pullorum, the effect of host restriction on genomic degradation is huge and rapid, and such effect seems to be continuing to work. The pseudogenes may reflect the unnecessary functions for Salmonella within the poultry host.     

An incomplete TCA cycle increases survival of Salmonella Typhimurium during infection of resting and activated murine macrophages

Background

In comparison to the comprehensive analyses performed on virulence gene expression, regulation and action, the intracellular metabolism of Salmonella during infection is a relatively under-studied area. We investigated the role of the tricarboxylic acid (TCA) cycle in the intracellular replication of Salmonella Typhimurium in resting and activated macrophages, epithelial cells, and during infection of mice.

Methodology/Principal Findings

We constructed deletion mutations of 5 TCA cycle genes in S. Typhimurium including gltA, mdh, sdhCDAB, sucAB, and sucCD. We found that the mutants exhibited increased net intracellular replication in resting and activated murine macrophages compared to the wild-type. In contrast, an epithelial cell infection model showed that the S. Typhimurium ΔsucCD and ΔgltA strains had reduced net intracellular replication compared to the wild-type. The glyoxylate shunt was not responsible for the net increased replication of the TCA cycle mutants within resting macrophages. We also confirmed that, in a murine infection model, the S. Typhimurium ΔsucAB and ΔsucCD strains are attenuated for virulence.

Conclusions/Significance

Our results suggest that disruption of the TCA cycle increases the ability of S. Typhimurium to survive within resting and activated murine macrophages. In contrast, epithelial cells are non-phagocytic cells and unlike macrophages cannot mount an oxidative and nitrosative defence response against pathogens; our results show that in HeLa cells the S. Typhimurium TCA cycle mutant strains show reduced or no change in intracellular levels compared to the wild-type [1]. The attenuation of the S. Typhimurium ΔsucAB and ΔsucCD mutants in mice, compared to their increased net intracellular replication in resting and activated macrophages suggest that Salmonella may encounter environments within the host where a complete TCA cycle is advantageous.     

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.     

Salmonella paratyphi C: Genetic Divergence from Salmonella choleraesuis and Pathogenic Convergence with Salmonella typhi

Background

Although over 1400 Salmonella serovars cause usually self-limited gastroenteritis in humans, a few, e.g., Salmonella typhi and S. paratyphi C, cause typhoid, a potentially fatal systemic infection. It is not known whether the typhoid agents have evolved from a common ancestor (by divergent processes) or acquired similar pathogenic traits independently (by convergent processes). Comparison of different typhoid agents with non-typhoidal Salmonella lineages will provide excellent models for studies on how similar pathogens might have evolved.

Methodologies/Principal Findings

We sequenced a strain of S. paratyphi C, RKS4594, and compared it with previously sequenced Salmonella strains. RKS4594 contains a chromosome of 4,833,080 bp and a plasmid of 55,414 bp. We predicted 4,640 intact coding sequences (4,578 in the chromosome and 62 in the plasmid) and 152 pseudogenes (149 in the chromosome and 3 in the plasmid). RKS4594 shares as many as 4346 of the 4,640 genes with a strain of S. choleraesuis, which is primarily a swine pathogen, but only 4008 genes with another human-adapted typhoid agent, S. typhi. Comparison of 3691 genes shared by all six sequenced Salmonella strains placed S. paratyphi C and S. choleraesuis together at one end, and S. typhi at the opposite end, of the phylogenetic tree, demonstrating separate ancestries of the human-adapted typhoid agents. S. paratyphi C seemed to have suffered enormous selection pressures during its adaptation to man as suggested by the differential nucleotide substitutions and different sets of pseudogenes, between S. paratyphi C and S. choleraesuis.

Conclusions

S. paratyphi C does not share a common ancestor with other human-adapted typhoid agents, supporting the convergent evolution model of the typhoid agents. S. paratyphi C has diverged from a common ancestor with S. choleraesuis by accumulating genomic novelty during adaptation to man.     

Antimicrobial Resistance,Class 1 Integrons,and Genomic Island 1 in Salmonella Isolates from Vietnam

Background

The objective was to investigate the phenotypic and genotypic resistance and the horizontal transfer of resistance determinants from Salmonella isolates from humans and animals in Vietnam.

Methodology/Principal Findings

The susceptibility of 297 epidemiologically unrelated non-typhoid Salmonella isolates was investigated by disk diffusion assay. The isolates were screened for the presence of class 1 integrons and Salmonella genomic island 1 by PCR. The potential for the transfer of resistance determinants was investigated by conjugation experiments. Resistance to gentamicin, kanamycin, chloramphenicol, streptomycin, trimethoprim, ampicillin, nalidixic acid, sulphonamides, and tetracycline was found in 13 to 50% of the isolates. Nine distinct integron types were detected in 28% of the isolates belonging to 11 Salmonella serovars including S. Tallahassee. Gene cassettes identified were aadA1, aadA2, aadA5, bla _PSE-1, bla _OXA-30, dfrA1, dfrA12, dfrA17, and sat, as well as open reading frames with unknown functions. Most integrons were located on conjugative plasmids, which can transfer their antimicrobial resistance determinants to Escherichia coli or Salmonella Enteritidis, or with Salmonella Genomic Island 1 or its variants. The resistance gene cluster in serovar Emek identified by PCR mapping and nucleotide sequencing contained SGI1-J3 which is integrated in SGI1 at another position than the majority of SGI1. This is the second report on the insertion of SGI1 at this position. High-level resistance to fluoroquinolones was found in 3 multiresistant S. Typhimurium isolates and was associated with mutations in the gyrA gene leading to the amino acid changes Ser83Phe and Asp87Asn.

Conclusions

Resistance was common among Vietnamese Salmonella isolates from different sources. Legislation to enforce a more prudent use of antibiotics in both human and veterinary medicine should be implemented by the authorities in Vietnam.     

Development of a real-time multiplex PCR assay for the detection of multiple <Emphasis Type="Italic">Salmonella</Emphasis> serotypes in chicken samples

Background  

A real-time multiplex PCR assay was developed for the detection of multiple Salmonella serotypes in chicken samples. Poultry-associated serotypes detected in the assay include Enteritidis, Gallinarum, Typhimurium, Kentucky and Dublin. The traditional cultural method according to EN ISO 6579:2002 for the detection of Salmonella in food was performed in parallel. The real-time PCR based method comprised a pre-enrichment step in Buffered Peptone Water (BPW) overnight, followed by a shortened selective enrichment in Rappaport Vasilliadis Soya Broth (RVS) for 6 hours and subsequent DNA extraction.     

Next generation genome sequencing reveals phylogenetic clades with different level of virulence among Salmonella Typhimurium clinical human isolates in Hong Kong

Background

Salmonella Typhimurium is frequently isolated from foodborne infection cases in Hong Kong, but the lack of genome sequences has hindered in-depth epidemiological and phylogenetic studies. In this study, we sought to reconstruct the phylogenetic relationship and investigate the distribution and mutation patterns of virulence determinants among local S. Typhimurium clinical isolates using their genome sequences.

Results

We obtained genome sequences of 20 S. Typhimurium clinical isolates from a local hospital cluster using a 454 GS FLX Titanium sequencing platform. Phylogenetic analysis was performed based on single nucleotide polymorphism positions of the core genome against the reference strain LT2. Antimicrobial susceptibility was determined using minimal inhibitory concentration for five antimicrobial agents and analyses of virulence determinants were performed through referencing to various databases. Through phylogenetic analysis, we revealed two distinct clades of S. Typhimurium isolates and three outliers in Hong Kong, which differ remarkably in antimicrobial susceptibility and presentation and mutations of virulence determinants. The local isolates were not closely related to many of the previously sequenced S. Typhimurium isolates, except LT2. As the isolates in the two clades spanned over 10 years of isolation, they probably represent endemic strains. The outliers are possibly introduced from outside of Hong Kong. The close relatedness of members in one of the clades to LT2 and the Japanese stool isolate T000240 suggests the potential reemergence of LT2 progeny in regions nearby.

Conclusions

Our study demonstrated the utility of next-generation sequencing coupled to traditional microbiological testing method in a retrospective epidemiological study involving multiple clinical isolates. The evolution of multidrug- and ciprofloxacin-resistant strains among the more virulent clade is also an increasing concern.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1900-y) contains supplementary material, which is available to authorized users.     

HLA-B27 modulates intracellular growth of Salmonella pathogenicity island 2 mutants and production of cytokines in infected monocytic U937 cells

Background

Salmonella enterica serovar Enteritidis PT4 KS8822/88 replicates rapidly in HLA-B27-transfected human monocytic U937 cells. In this process, Salmonella pathogenicity island 2 (SPI-2) genes play a crucial role. Our previous study indicated that 118 Salmonella genes, including 8 SPI-2 genes were affected by HLA-B27 antigen during Salmonella infection of U937 cells.

Methods/Principal Findings

To further investigate Salmonella replication in HLA-B27-positive U937 monocytic cells, two SPI-2 genes, ssaS and sscA up-regulated most during Salmonella infection of HLA-B27-transfected U937 cells, were mutated by using one-step gene disruption method. Intracellular survival and replication of the mutants in the U937 cells was compared to that of the wild type strain. Surprisingly, the two mutated strains replicated significantly more than the wild type bacteria in HLA-B27-transfected cells. Secretion of tumor necrosis factor alpha (TNF-α) and interleukin 10 (IL-10) was significantly induced during the infection of HLA-B27-transfected U937 cells with the mutants. The results indicated that the certain SPI-2 genes in wild type bacteria suppress Salmonella intracellular growth and production of cytokines in infected HLA-B27-transfected cells. HLA-B27-associated modulation of Salmonella SPI-2 genes and cytokine production may have importance in the persistent infection of the bacteria and the pathogenesis of reactive arthritis.

Conclusions

The study provides evidence that certain virulence factors of pathogens can reduce the intracellular growth in the host cells. We suggest that the limiting intracellular growth might be a strategy for persistence of bacteria in host cells, keeping a balance between pathogenic growth and pathogenesis.     

Microbial-resistant Salmonella enteritidis isolated from poultry samples

Background:

Multidrug resistance in Salmonella enteritidis isolates is a public health problem worldwide; the present study, therefore, was designed for antimicrobial-resistance determination in this strain.

Methods:

Salmonella strains isolated from poultry samples by biochemical positive and negative tests were subjected to PCR and identified as Salmonella enteritidis. For detection and identification of Salmonella enteritidis isolates, sdfI gene-specific primers were used.

Results:

We found that 100% of isolates were resistant to ampicillin, 90% were resistant to cephalothin and streptomycin, 70% were resistant to cefotaxime, and 60% were resistant to kanamycin and gentamicin.

Conclusion:

Salmonella enteritidis isolates had antimicrobial resistance to mentioned antibiotics. Key Words: Antibiotic Resistance, PCR, Poultry, Salmonella enteritidis     

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.     

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.     

Receptor Diversity and Host Interaction of Bacteriophages Infecting Salmonella enterica Serovar Typhimurium

Background

Salmonella enterica subspecies enterica serovar Typhimurium is a Gram-negative pathogen causing salmonellosis. Salmonella Typhimurium-targeting bacteriophages have been proposed as an alternative biocontrol agent to antibiotics. To further understand infection and interaction mechanisms between the host strains and the bacteriophages, the receptor diversity of these phages needs to be elucidated.

Methodology/Principal Findings

Twenty-five Salmonella phages were isolated and their receptors were identified by screening a Tn5 random mutant library of S. Typhimurium SL1344. Among them, three types of receptors were identified flagella (11 phages), vitamin B₁₂ uptake outer membrane protein, BtuB (7 phages) and lipopolysaccharide-related O-antigen (7 phages). TEM observation revealed that the phages using flagella (group F) or BtuB (group B) as a receptor belong to Siphoviridae family, and the phages using O-antigen of LPS as a receptor (group L) belong to Podoviridae family. Interestingly, while some of group F phages (F-I) target FliC host receptor, others (F-II) target both FliC and FljB receptors, suggesting that two subgroups are present in group F phages. Cross-resistance assay of group B and L revealed that group L phages could not infect group B phage-resistant strains and reversely group B phages could not infect group L SPN9TCW-resistant strain.

Conclusions/Significance

In this report, three receptor groups of 25 newly isolated S. Typhimurium-targeting phages were determined. Among them, two subgroups of group F phages interact with their host receptors in different manner. In addition, the host receptors of group B or group L SPN9TCW phages hinder other group phage infection, probably due to interaction between receptors of their groups. This study provides novel insights into phage-host receptor interaction for Salmonella phages and will inform development of optimal phage therapy for protection against Salmonella.     

Interaction of Saccharomyces boulardii with Salmonella enterica Serovar Typhimurium Protects Mice and Modifies T84 Cell Response to the Infection

Background

Salmonella pathogenesis engages host cells in two-way biochemical interactions: phagocytosis of bacteria by recruitment of cellular small GTP-binding proteins induced by the bacteria, and by triggering a pro-inflammatory response through activation of MAPKs and nuclear translocation of NF-κB. Worldwide interest in the use of functional foods containing probiotic bacteria for health promotion and disease prevention has increased significantly. Saccharomyces boulardii is a non-pathogenic yeast used as a probiotic in infectious diarrhea.

Methodology/Principal Findings

In this study, we reported that S. boulardii (Sb) protected mice from Salmonella enterica serovar Typhimurium (ST)-induced death and prevented bacterial translocation to the liver. At a molecular level, using T84 human colorectal cancer cells, we demonstrate that incubation with Sb before infection totally abolished Salmonella invasion. This correlates with a decrease of activation of Rac1. Sb preserved T84 barrier function and decreased ST-induced IL-8 synthesis. This anti-inflammatory effect was correlated with an inhibitory effect of Sb on ST-induced activation of the MAPKs ERK1/2, p38 and JNK as well as on activation of NF-κB. Electron and confocal microscopy experiments showed an adhesion of bacteria to yeast cells, which could represent one of the mechanisms by which Sb exerts its protective effects.

Conclusions

Sb shows modulating effects on permeability, inflammation, and signal transduction pathway in T84 cells infected by ST and an in vivo protective effect against ST infection. The present results also demonstrate that Sb modifies invasive properties of Salmonella.