Genomic Comparison of Non-Typhoidal Salmonella enterica Serovars Typhimurium,Enteritidis, Heidelberg,Hadar and Kentucky Isolates from Broiler Chickens

BackgroundNon-typhoidal Salmonella enterica serovars, associated with different foods including poultry products, are important causes of bacterial gastroenteritis worldwide. The colonization of the chicken gut by S. enterica could result in the contamination of the environment and food chain. The aim of this study was to compare the genomes of 25 S. enterica serovars isolated from broiler chicken farms to assess their intra- and inter-genetic variability, with a focus on virulence and antibiotic resistance characteristics.Conclusions/SignificanceThis study showed that the predominant Salmonella serovars in broiler chickens harbor genes encoding adhesins, flagellar proteins, T3SS, iron acquisition systems, and antibiotic and metal resistance genes that may explain their pathogenicity, colonization ability and persistence in chicken. The existence of mobile genetic elements indicates that isolates from a given serovar could acquire and transfer genetic material. Conserved genes in the T3SS and T4SS that we have identified are promising candidates for identification of diagnostic, antimicrobial or vaccine targets for the control of Salmonella in broiler chickens.     

Differences in gene content between Salmonella enterica serovar Enteritidis isolates and comparison to closely related serovars Gallinarum and Dublin

Salmonella enterica serovar Enteritidis is often transmitted into the human food supply through eggs of hens that appear healthy. This pathogen became far more prevalent in poultry following eradication of the fowl pathogen S. enterica serovar Gallinarum in the mid-20th century. To investigate whether changes in serovar Enteritidis gene content contributed to this increased prevalence, and to evaluate genetic heterogeneity within the serovar, comparative genomic hybridization was performed on eight 60-year-old and nineteen 10- to 20-year-old serovar Enteritidis strains from various hosts, using a Salmonella-specific microarray. Overall, almost all the serovar Enteritidis genomes were very similar to each other. Excluding two rare strains classified as serovar Enteritidis in the Salmonella reference collection B, only eleven regions of the serovar Enteritidis phage type 4 (PT4) chromosome (sequenced at the Sanger Center) were absent or divergent in any of the other serovar Enteritidis strains tested. The more recent isolates did not have consistent differences from 60-year-old field isolates, suggesting that no large genomic additions on a whole-gene scale were needed for serovar Enteritidis to become more prevalent in domestic fowl. Cross-hybridization of phage genes on the array with related genes in the examined genomes grouped the serovar Enteritidis isolates into two major lineages. Microarray comparisons of the sequenced serovar Enteritidis PT4 to isolates of the closely related serovars Dublin and Gallinarum (biovars Gallinarum and Pullorum) revealed several genomic areas that distinguished them from serovar Enteritidis and from each other. These differences in gene content could be useful in DNA-based typing and in understanding the different phenotypes of these related serovars.     

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.     

Novel haemolysins of Salmonella enterica spp. enterica serovar Gallinarum

Haemolysins of Salmonella are important due to their probable role in pathogenesis of systemic salmonellosis and use in sub-serovar level typing. The present study was undertaken to determine haemolytic potential of Salmonella Gallinarum strains through phenotypic and genotypic methods. Amplification of haemolysin gene (clyA) and cytolysin gene (slyA) was attempted in order to determine their role in haemolysin production. Study on 94 strains of S. Gallinarum revealed the production of two types of haemolysis viz., beneath the colony haemolysis (BCH) or contact haemolysis and clear zone haemolysis (CZH). Haemolysis was observed on blood agar prepared with blood of cattle, buffalo, sheep, goat, horse, rabbit, guinea pig, fowl, and human blood group A, B, AB and O. Although, haemolysis was also observed on blood agar prepared with whole blood, clarity of zone was more evident on blood agar made from washed erythrocytes. Clear zone haemolysis was best observed on blood agar prepared with washed erythrocytes of goat and a total of 12% (11 of 94) S. Gallinarum strains under study produced CZH on it. The clyA gene could not be detected in any of the 94 strains under study, while slyA gene could be amplified uniformly irrespective of haemolytic potential (CZH) and haemolytic pattern (BCH) of the strains. The study suggested that the two types of haemolysis (CZH and BCH) observed among S. Gallinarum strains may not be due to either slyA or clyA gene products and thus there may be some other gene responsible for haemolytic trait in Gallinarum serovar. Different haemolytic patterns of strains under study indicated multiplicity of haemolysins in S. Gallinarum.     

Genome-Wide Methylation Patterns in Salmonella enterica Subsp. enterica Serovars

The methylation of DNA bases plays an important role in numerous biological processes including development, gene expression, and DNA replication. Salmonella is an important foodborne pathogen, and methylation in Salmonella is implicated in virulence. Using single molecule real-time (SMRT) DNA-sequencing, we sequenced and assembled the complete genomes of eleven Salmonella enterica isolates from nine different serovars, and analysed the whole-genome methylation patterns of each genome. We describe 16 distinct N⁶-methyladenine (m6A) methylated motifs, one N⁴-methylcytosine (m4C) motif, and one combined m6A-m4C motif. Eight of these motifs are novel, i.e., they have not been previously described. We also identified the methyltransferases (MTases) associated with 13 of the motifs. Some motifs are conserved across all Salmonella serovars tested, while others were found only in a subset of serovars. Eight of the nine serovars contained a unique methylated motif that was not found in any other serovar (most of these motifs were part of Type I restriction modification systems), indicating the high diversity of methylation patterns present in Salmonella.     

Genome sequences of Salmonella enterica serovar typhimurium, Choleraesuis, Dublin, and Gallinarum strains of well- defined virulence in food-producing animals

Salmonella enterica is an animal and zoonotic pathogen of worldwide importance and may be classified into serovars differing in virulence and host range. We sequenced and annotated the genomes of serovar Typhimurium, Choleraesuis, Dublin, and Gallinarum strains of defined virulence in each of three food-producing animal hosts. This provides valuable measures of intraserovar diversity and opportunities to formally link genotypes to phenotypes in target animals.     

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.     

Contribution of the Type VI Secretion System Encoded in SPI-19 to Chicken Colonization by Salmonella enterica Serotypes Gallinarum and Enteritidis

Salmonella Gallinarum is a pathogen with a host range specific to poultry, while Salmonella Enteritidis is a broad host range pathogen that colonizes poultry sub-clinically but is a leading cause of gastrointestinal salmonellosis in humans and many other species. Despite recent advances in our understanding of the complex interplay between Salmonella and their hosts, the molecular basis of host range restriction and unique pathobiology of Gallinarum remain largely unknown. Type VI Secretion System (T6SS) represents a new paradigm of protein secretion that is critical for the pathogenesis of many Gram-negative bacteria. We recently identified a putative T6SS in the Salmonella Pathogenicity Island 19 (SPI-19) of Gallinarum. In Enteritidis, SPI-19 is a degenerate element that has lost most of the T6SS functions encoded in the island. In this work, we studied the contribution of SPI-19 to the colonization of Salmonella Gallinarum strain 287/91 in chickens. Non-polar deletion mutants of SPI-19 and the clpV gene, an essential T6SS component, colonized the ileum, ceca, liver and spleen of White Leghorn chicks poorly compared to the wild-type strain after oral inoculation. Return of SPI-19 to the ΔSPI-19 mutant, using VEX-Capture, complemented this colonization defect. In contrast, transfer of SPI-19 from Gallinarum to Enteritidis resulted in transient increase in the colonization of the ileum, liver and spleen at day 1 post-infection, but at days 3 and 5 post-infection a strong colonization defect of the gut and internal organs of the experimentally infected chickens was observed. Our data indicate that SPI-19 and the T6SS encoded in this region contribute to the colonization of the gastrointestinal tract and internal organs of chickens by Salmonella Gallinarum and suggest that degradation of SPI-19 T6SS in Salmonella Enteritidis conferred an advantage in colonization of the avian host.     

Poultry Body Temperature Contributes to Invasion Control through Reduced Expression of Salmonella Pathogenicity Island 1 Genes in Salmonella enterica Serovars Typhimurium and Enteritidis

Salmonella enterica serovars Typhimurium (S. Typhimurium) and Enteritidis (S. Enteritidis) are foodborne pathogens, and outbreaks are often associated with poultry products. Chickens are typically asymptomatic when colonized by these serovars; however, the factors contributing to this observation are uncharacterized. Whereas symptomatic mammals have a body temperature between 37°C and 39°C, chickens have a body temperature of 41°C to 42°C. Here, in vivo experiments using chicks demonstrated that numbers of viable S. Typhimurium or S. Enteritidis bacteria within the liver and spleen organ sites were ≥4 orders of magnitude lower than those within the ceca. When similar doses of S. Typhimurium or S. Enteritidis were given to C3H/HeN mice, the ratio of the intestinal concentration to the liver/spleen concentration was 1:1. In the avian host, this suggested poor survival within these tissues or a reduced capacity to traverse the host epithelial layer and reach liver/spleen sites or both. Salmonella pathogenicity island 1 (SPI-1) promotes localization to liver/spleen tissues through invasion of the epithelial cell layer. Following in vitro growth at 42°C, SPI-1 genes sipC, invF, and hilA and the SPI-1 rtsA activator were downregulated compared to expression at 37°C. Overexpression of the hilA activators fur, fliZ, and hilD was capable of inducing hilA-lacZ at 37°C but not at 42°C despite the presence of similar levels of protein at the two temperatures. In contrast, overexpression of either hilC or rtsA was capable of inducing hilA and sipC at 42°C. These data indicate that physiological parameters of the poultry host, such as body temperature, have a role in modulating expression of virulence.     

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.     

Circulation dynamics of Salmonella enterica subsp. enterica ser. Gallinarum biovar Gallinarum in a poultry farm infested by Dermanyssus gallinae

Dermanyssus gallinae (Mesostigmata: Dermanyssidae, De Geer, 1778) is an ectoparasite of poultry, suspected to play a role as a vector of Salmonella enterica subsp. enterica ser. Gallinarum. Despite an association between them being reported, the actual dynamics in field remain unclear. Therefore, the present study aimed to confirm the interactions among mites, pathogen and chickens. The study was carried out in an industrial poultry farm infested by D. gallinae, during an outbreak of fowl typhoid. The presence of S. Gallinarum in mites was assessed and quantified by a semi‐nested polymerase chain reaction (PCR) and real‐time PCR, respectively, in mites collected during two subsequent productive cycles and the sanitary break. The anti‐group D Salmonella antibodies were quantified by an enzyme‐linked immunosorbent assay. During the outbreak and the sanitary break, S. Gallinarum was constantly present in mites. In the second cycle, scattered positivity was observed, although hens did not exhibit signs of fowl typhoid, as a result of the vaccination with BIO‐VAC SGP695 (Fatro, Ozzano Emilia Bo, Italy). The data strongly suggest that D. gallinae acts as reservoir of S. Gallinarum, thus allowing the pathogen to persist in farms. Furthermore, the present study has highlighted the interactions among D. gallinae, S. Gallinarum and hens with respect to enhancing the mite‐mediated circulation of S. Gallinarum in an infested poultry farm.     

Salmonella enterica subsp. enterica serovar Enteritidis harbours ColE1, ColE2, and rolling-circle-like replicating plasmids

Using DNA hybridization, at least three distinct groups of low molecular mass plasmids were identified in Salmonella enterica subsp. enterica serovar Enteritidis. After sequencing representative plasmids from each group, we concluded that they belonged to ColE1, ColE2, and rolling-circle-like replicating plasmids. Plasmid pK (4245 bp) is a representative of widely distributed ColE1 plasmids. Plasmid pP (4301 bp) is homologous to ColE2 plasmids and was present predominantly in single-stranded DNA form. The smallest plasmids pJ (2096 bp) and pB (1983 bp) were classified as rolling-circle-like replicating plasmids. Both encoded only a single protein essential for their own replication, and they must have existed in an unusual molecular structure, as (i) they were capable of hybridization without denaturation, (ii) their DNA could be linearized with S1 nuclease, and (iii) even after such treatment, the ability to hybridize without denaturation did not disappear.     

wksl3, a New Biocontrol Agent for Salmonella enterica Serovars Enteritidis and Typhimurium in Foods: Characterization,Application, Sequence Analysis,and Oral Acute Toxicity Study

Of the Salmonella enterica serovars, S. Enteritidis and S. Typhimurium are responsible for most of the Salmonella outbreaks implicated in the consumption of contaminated foods in the Republic of Korea. Because of the widespread occurrence of antimicrobial-resistant Salmonella in foods and food processing environments, bacteriophages have recently surfaced as an alternative biocontrol tool. In this study, we isolated a virulent bacteriophage (wksl3) that could specifically infect S. Enteritidis, S. Typhimurium, and several additional serovars. Transmission electron microscopy revealed that phage wksl3 belongs to the family Siphoviridae. Complete genome sequence analysis and bioinformatic analysis revealed that the DNA of phage wksl3 is composed of 42,766 bp with 64 open reading frames. Since it does not encode any phage lysogeny factors, toxins, pathogen-related genes, or food-borne allergens, phage wksl3 may be considered a virulent phage with no side effects. Analysis of genetic similarities between phage wksl3 and four of its relatives (SS3e, vB_SenS-Ent1, SE2, and SETP3) allowed wksl3 to be categorized as a SETP3-like phage. A single-dose test of oral toxicity with BALB/c mice resulted in no abnormal clinical observations. Moreover, phage application to chicken skin at 8°C resulted in an about 2.5-log reduction in the number of Salmonella bacteria during the test period. The strong, stable lytic activity, the significant reduction of the number of S. Enteritidis bacteria after application to food, and the lack of clinical symptoms of this phage suggest that wksl3 may be a useful agent for the protection of foods against S. Enteritidis and S. Typhimurium contamination.