On the Evolutionary History,Population Genetics and Diversity among Isolates of Salmonella Enteritidis PFGE Pattern JEGX01.0004

Facile laboratory tools are needed to augment identification in contamination events to trace the contamination back to the source (traceback) of Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis). Understanding the evolution and diversity within and among outbreak strains is the first step towards this goal. To this end, we collected 106 new S. Enteriditis isolates within S. Enteriditis Pulsed-Field Gel Electrophoresis (PFGE) pattern JEGX01.0004 and close relatives, and determined their genome sequences. Sources for these isolates spanned food, clinical and environmental farm sources collected during the 2010 S. Enteritidis shell egg outbreak in the United States along with closely related serovars, S. Dublin, S. Gallinarum biovar Pullorum and S. Gallinarum. Despite the highly homogeneous structure of this population, S. Enteritidis isolates examined in this study revealed thousands of SNP differences and numerous variable genes (n = 366). Twenty-one of these genes from the lineages leading to outbreak-associated samples had nonsynonymous (causing amino acid changes) changes and five genes are putatively involved in known Salmonella virulence pathways. While chromosome synteny and genome organization appeared to be stable among these isolates, genome size differences were observed due to variation in the presence or absence of several phages and plasmids, including phage RE-2010, phage P125109, plasmid pSEEE3072_19 (similar to pSENV), plasmid pOU1114 and two newly observed mobile plasmid elements pSEEE1729_15 and pSEEE0956_35. These differences produced modifications to the assembled bases for these draft genomes in the size range of approximately 4.6 to 4.8 mbp, with S. Dublin being larger (∼4.9 mbp) and S. Gallinarum smaller (4.55 mbp) when compared to S. Enteritidis. Finally, we identified variable S. Enteritidis genes associated with virulence pathways that may be useful markers for the development of rapid surveillance and typing methods, potentially aiding in traceback efforts during future outbreaks involving S. Enteritidis PFGE pattern JEGX01.0004.     

Salmonella enterica Serovar Enteritidis Genes Induced during Oviduct Colonization and Egg Contamination in Laying Hens

Salmonella enterica serovar Enteritidis is the predominant serovar associated with salmonellosis worldwide, which is in part due to its ability to contaminate the internal contents of the hen's egg. It has been shown that S. enterica serovar Enteritidis has an unusual tropism for the avian reproductive tract and an ability to persist in the oviduct and ovary. Factors allowing S. enterica serovar Enteritidis strains to contaminate eggs could be a specific interaction with the oviduct tissue, leading to persisting oviduct colonization. In vivo expression technology, a promoter-trap strategy, was used to identify genes expressed during oviduct colonization and egg contamination with S. enterica serovar Enteritidis. A total of 25 clones with in vivo-induced promoters were isolated from the oviduct tissue and from laid eggs. Among the 25 clones, 7 were isolated from both the oviducts and the eggs. DNA sequencing of the cloned promoters revealed that genes involved in amino acid and nucleic acid metabolism, motility, cell wall integrity, and stress responses were highly expressed in the reproductive tract tissues of laying hens.     

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.     

Comparison of Genotypes of Salmonella enterica Serovar Enteritidis Phage Type 30 and 9c Strains Isolated during Three Outbreaks Associated with Raw Almonds

In 2000 to 2001, 2003 to 2004, and 2005 to 2006, three outbreaks of Salmonella enterica serovar Enteritidis were linked with the consumption of raw almonds. The S. Enteritidis strains from these outbreaks had rare phage types (PT), PT30 and PT9c. Clinical and environmental S. Enteritidis strains were subjected to pulsed-field gel electrophoresis (PFGE), multilocus variable-number tandem repeat analysis (MLVA), and DNA microarray-based comparative genomic indexing (CGI) to evaluate their genetic relatedness. All three methods differentiated these S. Enteritidis strains in a manner that correlated with PT. The CGI analysis confirmed that the majority of the differences between the S. Enteritidis PT9c and PT30 strains corresponded to bacteriophage-related genes present in the sequenced genomes of S. Enteritidis PT4 and S. enterica serovar Typhimurium LT2. However, PFGE, MLVA, and CGI failed to discriminate between S. Enteritidis PT30 strains related to outbreaks from unrelated clinical strains or between strains separated by up to 5 years. However, metabolic fingerprinting demonstrated that S. Enteritidis PT4, PT8, PT13a, and clinical PT30 strains metabolized l-aspartic acid, l-glutamic acid, l-proline, l-alanine, and d-alanine amino acids more efficiently than S. Enteritidis PT30 strains isolated from orchards. These data indicate that S. Enteritidis PT9c and 30 strains are highly related genetically and that PT30 orchard strains differ from clinical PT30 strains metabolically, possibly due to fitness adaptations.Salmonella enterica is one of the major causes of bacterial food-borne illness worldwide. Many serovars of S. enterica serovar Enteritidis emerged as serious problems in the human food supply during the 1980s, and these cases were associated mostly with undercooked eggs and poultry (26). The phage typing of S. Enteritidis strains associated with egg-associated outbreaks had indicated that phage types 8 (PT8) and PT13a were the most common PTs in the United States (12), and PT4 was the most common in Europe (22). Through education and quality improvements, the incidence of S. Enteritidis due to egg products has decreased in the United States (18). However, several recent outbreaks have identified new sources for S. Enteritidis, specifically mung bean sprouts, tomatoes, and raw whole almonds (3, 13, 31).At the time of the 2001 outbreak, almonds and other low-moisture foods were considered an unlikely source of food-borne illness. Almonds are California''s major tree nut crop and have ranked first in California agricultural exports for many years, accounting for 60% of world production in 2000 (14) and 80% in 2008 (http://www.almondboard.com/AboutTheAlmondBoard/Documents/2008-Almond-Board-Almanac.pdf). However, no outbreaks associated with almonds had been reported before 2001. In the spring of 2001, Canadian health officials identified a link between illnesses caused by S. Enteritidis and the consumption of raw almonds (6). Outbreak-related cases were identified from November 2001 to July 2001 in several provinces across Canada and in several regions in the United States (13). During the traceback investigation, almond retailers, processors, and growers were identified, and S. Enteritidis PT30 was cultured from almond samples, a huller/sheller facility, and environmental samples from the orchards (30). The ability to identify the contaminated food source for this outbreak was aided significantly by the previously rare occurrence of S. Enteritidis PT30. S. Enteritidis PT30 continued to be isolated from one of the outbreak-associated orchards during a 5-year period, suggesting that this organism was highly fit for persistence in this environment (30).In 2004, another rare S. Enteritidis PT (PT9c) was linked to a second outbreak associated with raw almonds. Similarly to the first outbreak, both phage typing and pulsed-field gel electrophoresis (PFGE) aided the identification of related cases caused by S. Enteritidis PT9c that occurred over a large geographical region of the United States and Canada (3). A third S. Enteritidis PT30 outbreak associated with raw almonds was reported in Sweden in 2005 to 2006 (15).We have characterized, by molecular methods, S. Enteritidis strains recovered from clinical, almond, and orchard samples related to these three outbreaks to determine whether they were related genotypically. Additional S. Enteritidis strains representing some common phage types also were examined for comparison. Strains were genotyped by PFGE profiling, multilocus variable-number tandem repeat analysis (MLVA), and comparative genomic indexing (CGI) with a S. enterica serovar Typhimurium LT2/Enteritidis PT4 microarray to determine relatedness and whether an association with the source could be determined.     

Multi Locus Variable-Number Tandem Repeat (MLVA) Typing Tools Improved the Surveillance of Salmonella Enteritidis: A 6 Years Retrospective Study

Surveillance of Salmonella enterica subsp. enterica serovar Enteritidis is generally considered to benefit from molecular techniques like multiple-locus variable-number of tandem repeats analysis (MLVA), which allow early detection and confinement of outbreaks. Here, a surveillance study, including phage typing, antimicrobial susceptibility testing and MLVA on 1,535 S. Enteritidis isolates collected between 2007 and 2012, was used to evaluate the added value of MLVA for public health surveillance in Belgium. Phage types PT4, PT8, PT21, PT1, PT6, PT14b, PT28 and PT13 dominate the Belgian S. Enteritidis population. The isolates of S. Enteritidis were most frequently susceptible to all antibiotics tested. 172 different MLVA profiles were detected, of which 9 frequent profiles included 67.2% of the S. Enteritidis population. During a serial passage experiment on selected isolates to investigate the in vitro stability of the 5 MLVA loci, no variations over time were observed indicating that the MLVA profiles were stable. The MLVA profile of isolates originating from different outbreaks in the Democratic Republic of the Congo (DRC) between 2010 and 2011 were distinct from any of the MLVA profiles found in Belgian isolates throughout the six year observational period and demonstrates that MLVA improves public health surveillance of S. Enteritidis. However, MLVA should be complemented with other subtyping methods when investigating outbreaks is caused by the most common MLVA profile.     

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.     

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.     

Limited genetic diversity in <Emphasis Type="Italic">Salmonella enterica</Emphasis> Serovar Enteritidis PT13

Background  

Salmonella enterica serovar Enteritidis has emerged as a significant foodborne pathogen throughout the world and is commonly characterized by phage typing. In Canada phage types (PT) 4, 8 and 13 predominate and in 2005 a large foodborne PT13 outbreak occurred in the province of Ontario. The ability to link strains during this outbreak was difficult due to the apparent clonality of PT13 isolates in Canada, as there was a single dominant pulsed-field gel electrophoresis (PFGE) profile amongst epidemiologically linked human and food isolates as well as concurrent sporadic strains. The aim of this study was to perform comparative genomic hybridization (CGH), DNA sequence-based typing (SBT) genomic analyses, plasmid analyses, and automated repetitive sequence-based PCR (rep-PCR) to identify epidemiologically significant traits capable of subtyping S. Enteritidis PT13.     

Public Health Assessment of Salmonella enterica Serovar Enteritidis Inactivated-Vaccine Treatment in Layer Flocks

Although there have been several reports on the efficacy assessment of a Salmonella enterica serovar Enteritidis vaccine against intestinal and parenchymatous organ diseases of laying hens, no public health risk characterization of its long-term effect on eggs has been reported. In this study, we attempted to assess the public health effect of an inactivated S. enterica serovar Enteritidis vaccine against serovar Enteritidis contamination of chicken eggs. We analyzed serovar Enteritidis isolation test results from four windowless farms in which inactivated-vaccine administration was initiated based on the sanitary monitoring program of a farm. When flocks with and without S. enterica serovar Enteritidis vaccine treatments were mixed, the application of an inactivated serovar Enteritidis vaccine decreased the most probable number (MPN) of bacteria by at least 100-fold in broken (liquid) egg samples positive for serovar Enteritidis, although a statistical difference between those MPNs could not be obtained. The isolation frequency after the vaccine application was less than 1/10 (P < 0.01). No S. enterica serovar Enteritidis bacteria were isolated approximately 1 year after all of the chickens had received the inactivated serovar Enteritidis vaccine. It was suggested that an adequate administration of an inactivated serovar Enteritidis vaccine reduced the contamination risk of eggs (the number of isolated serovar Enteritidis cells and detection frequency) compared to the contamination risk of eggs laid by nonvaccinated hens.     

Subpopulation Characteristics of Egg-Contaminating Salmonella enterica serovar Enteritidis as Defined by the Lipopolysaccharide O Chain

Characterization of Salmonella enterica serovar Enteritidis was refined by incorporating new data from isolates obtained from avian sources, from the spleens of naturally infected mice, and from the United Kingdom into an existing lipopolysaccharide (LPS) O-chain compositional database. From least to greatest, the probability of avian isolates producing high-molecular-mass LPS O chain ranked as follows: pooled kidney, liver, and spleen; intestine; cecum; ovary and oviduct; albumen; yolk; and whole egg. Mouse isolates were most like avian intestinal samples, whereas United Kingdom isolates were most like those from the avian reproductive tract and egg. Non-reproductive tract organ isolates had significant loss of O chain. Isogenic isolates that varied in ability to make biofilm and to be orally invasive produced different O-chain structures at 25°C but not at 37°C. Hens infected at a 91:9 biofilm-positive/-negative colony phenotype ratio yielded only the negative phenotype from eggs. These results indicate that the environment within the hen applies stringent selection pressure on subpopulations of S. enterica serovar Enteritidis at certain points in the infection pathway that ends in egg contamination. The avian cecum, rather than the intestines, is the early interface between the environment and the host that supports emergence of subpopulation diversity. These results suggest that diet and other factors that alter cecal physiology should be investigated as a means to reduce egg contamination.     

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.     

Exposure to a dog elicits different cardiovascular and behavioral effects in pregnant and lactating goats

Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) was responsible for a worldwide pandemic during the 1980s and 1990s; however, changes in the dominant lineage before and after this event remain unknown. This study determined S. Enteritidis lineages before and after this pandemic event in Japan using multilocus sequence typing (MLST). Thirty S. Enteritidis strains were collected in Japan between 1973 and 2004, consisting of 27 human strains from individual episodes, a bovine strain, a liquid egg strain and an eggshell strain. Strains showed nine phage types and 17 pulsed-field profiles with pulsed-field gel electrophoresis. All strains had homologous type 11 sequences without any nucleotide differences in seven housekeeping genes. These MLST results suggest that S. Enteritidis with the diversities revealed by phage typing and pulsed-field profiling has a highly clonal population. Although type 11 S. Enteritidis may exhibit both pleiotropic surface structure and pulsed-field type variation, it is likely to be a stable lineage derived from an ancestor before the 1980s and/or 1990s pandemic in Japan.     

Conjugal Transfer of the Pathogenicity Island ROD21 in Salmonella enterica serovar Enteritidis Depends on Environmental Conditions

Unstable pathogenicity islands are chromosomal elements that can be transferred from one bacterium to another. Salmonella enterica serovar Enteritidis (S. Enteritidis) is a pathogenic bacterium containing such unstable pathogenicity islands. One of them, denominated ROD21, is 26.5 kb in size and capable of excising from the chromosome in certain culture conditions, as well as during bacterial infection of phagocytic cells. In this study we have evaluated whether ROD21 can be effectively transferred from one bacterium to another. We generated a donor and several recipient strains of S. Enteritidis to carry out transfer assays in liquid LB medium. These assays showed that ROD21 is effectively transferred from donor to recipient strains of S. Enteritidis and S. Typhimurium. When Escherichia coli was used as the recipient strain, ROD21 transfer failed to be observed. Subsequently, we showed that a conjugative process was required for the transfer of the island and that changes in temperature and pH increased the transfer frequency between Salmonella strains. Our data indicate that ROD21 is an unstable pathogenicity island that can be transferred by conjugation in a species-specific manner between Salmonellae. Further, ROD21 transfer frequency increases in response to environmental changes, such as pH and temperature.     

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.     

Immunogenicity of Salmonella enterica serovar Enteritidis virulence protein,InvH, and cross-reactivity of its antisera with Salmonella strains

Acellular vaccines containing bacterial immunodominant components such as surface proteins may be potent alternatives to live attenuated vaccines in order to reduce salmonellosis risk to human health. invH gene, an important part of needle complex in type three secretion system (TTSS) plays important role in efficient bacterial adherence and entry into epithelial cells. In this work we hypothesize that use of a 15 kDa recombinant InvH as Salmonella enterica serovar Enteritidis surface protein could provoke antibody production in mouse and would help us study feasibility of its potential for diagnosis and/or a recombinant vaccine. The purified InvH provoked significant rise of IgG in mice. Active protection induced by immunization with InvH against variable doses of S. enterica serovar Enteritidis, indicated that the immunized mice were completely protected against challenge with 10⁴ LD₅₀. The immunoreaction of sera from immunized mice with other Salmonella strains or cross reaction with sera of Salmonella strains inoculated mice is indicative of possessing by Salmonella strains of the surface protein, InvH, that can be employed in both prophylactic and diagnostic measures against S. enterica. Bacteria free spleen and ileum of the immunized mice in this study indicate that the invH gene affects bacterial invasion. Efficacy of the virulence protein, InvH, in shuttling into host cells in injectisome of S. enterica serovar Enteritidis and inhibition of this phenomenon by active immunization was shown in this study. In conclusion immunization with InvH protein can develop protection against S. enterica serovar Enteritidis infections. InvH in Salmonella strains can be exploited in protective measures as well as a diagnostic tool in Salmonella infections.     

Genomic Comparison of the Closely-Related Salmonella enterica Serovars Enteritidis,Dublin and Gallinarum

The Salmonella enterica serovars Enteritidis, Dublin, and Gallinarum are closely related but differ in virulence and host range. To identify the genetic elements responsible for these differences and to better understand how these serovars are evolving, we sequenced the genomes of Enteritidis strain LK5 and Dublin strain SARB12 and compared these genomes to the publicly available Enteritidis P125109, Dublin CT 02021853 and Dublin SD3246 genome sequences. We also compared the publicly available Gallinarum genome sequences from biotype Gallinarum 287/91 and Pullorum RKS5078. Using bioinformatic approaches, we identified single nucleotide polymorphisms, insertions, deletions, and differences in prophage and pseudogene content between strains belonging to the same serovar. Through our analysis we also identified several prophage cargo genes and pseudogenes that affect virulence and may contribute to a host-specific, systemic lifestyle. These results strongly argue that the Enteritidis, Dublin and Gallinarum serovars of Salmonella enterica evolve by acquiring new genes through horizontal gene transfer, followed by the formation of pseudogenes. The loss of genes necessary for a gastrointestinal lifestyle ultimately leads to a systemic lifestyle and niche exclusion in the host-specific serovars.     

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.     

Pathogenic Role of SEF14, SEF17, and SEF21 Fimbriae in Salmonella enterica Serovar Enteritidis Infection of Chickens

Very little is known about the contribution of surface appendages of Salmonella enterica serovar Enteritidis to pathogenesis in chickens. This study was designed to clarify the role of SEF14, SEF17, and SEF21 fimbriae in serovar Enteritidis pathogenesis. Stable, single, defined sefA (SEF14), agfA (SEF17), and fimA (SEF21) insertionally inactivated fimbrial gene mutants of serovar Enteritidis were constructed. All mutant strains invaded Caco-2 and HT-29 enterocytes at levels similar to that of the wild type. Both mutant and wild-type strains were ingested equally well by chicken macrophage cell lines HD11 and MQ-NCSU. There were no significant differences in the abilities of these strains to colonize chicken ceca. The SEF14⁻ strain was isolated in lower numbers from the livers of infected chickens and was cleared from the spleens faster than other strains. No significant differences in fecal shedding of these strains were observed.     

Genome analysis and CRISPR typing of Salmonella enterica serovar Virchow

Background

Salmonella enterica subsp. enterica serovar Virchow has been recognized as a significant health burden in Asia, Australia and Europe. In addition to its global distribution, S. Virchow is clinically significant due to the frequency at which it causes invasive infections and its association with outbreaks arising from food-borne transmission. Here, we examine the genome of an invasive isolate of S. Virchow SVQ1 (phage type 8) from an outbreak in southeast Queensland, Australia. In addition to identifying new potential genotyping targets that could be used for discriminating between S. Virchow strains in outbreak scenarios, we also aimed to carry out a comprehensive comparative analysis of the S. Virchow genomes.

Results

Genome comparisons between S. Virchow SVQ1 and S. Virchow SL491, a previously published strain, identified a high degree of genomic similarity between the two strains with fewer than 200 single nucleotide differences. Clustered Regularly Interspaced Palindromic Repeats (CRISPR) regions were identified as a highly variable region that could be used to discriminate between S. Virchow isolates. We amplified and sequenced the CRISPR regions of fifteen S. Virchow isolates collected from seven different outbreaks across Australia. We observed three allelic types of the CRISPR region from these isolates based on the presence/absence of the spacers and were able to discriminate S. Virchow phage type 8 isolates originating from different outbreaks. A comparison with 27 published Salmonella genomes found that the S. Virchow SVQ1 genome encodes 11 previously described Salmonella Pathogenicity Islands (SPI), as well as additional genomic islands including a remnant integrative conjugative element that is distinct from SPI-7. In addition, the S. Virchow genome possesses a novel prophage that encodes the Type III secretion system effector protein SopE, a key Salmonella virulence factor. The prophage shares very little similarity to the SopE prophages found in other Salmonella serovars suggesting an independent acquisition of sopE.

Conclusions

The availability of this genome will serve as a genome template and facilitate further studies on understanding the virulence and global distribution of the S. Virchow serovar, as well as the development of genotyping methods for outbreak investigations.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-389) contains supplementary material, which is available to authorized users.