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.     

Identification of Novel Salmonella enterica Serovar Typhimurium DT104-Specific Prophage and Nonprophage Chromosomal Sequences among Serovar Typhimurium Isolates by Genomic Subtractive Hybridization

Genomic subtractive hybridization was performed between Salmonella enterica serovar Typhimurium LT2 and DT104 to search for novel Salmonella serovar Typhimurium DT104-specific sequences. The subtraction resulted mainly in the isolation of DNA fragments with sequence similarity to phages. Two fragments identified were associated with possible virulence factors. One fragment was identical to irsA of Salmonella serovar Typhimurium ATCC 14028, which is suggested to be involved in macrophage survival. The other fragment was homologous to HldD, an Escherichia coli O157:H7 lipopolysaccharide assembly-related protein. Five selected DNA fragments—irsA, the HldD homologue, and three fragments with sequence similarity to prophages—were tested for their presence in 17 Salmonella serovar Typhimurium DT104 isolates and 27 non-DT104 isolates by PCR. All five selected DNA fragments were Salmonella serovar Typhimurium DT104 specific among the serovar Typhimurium isolates tested. These DNA fragments can be useful for better detection and typing of Salmonella serovar Typhimurium DT104.     

1株表现为鞭毛抗原3相的韦太夫雷登沙门氏菌的鉴定和基因组序列分析

利用高通量测序获取1株抗原式为3,10∶a,r,z6的沙门氏菌(Salmonella)GX150603的全基因组序列.根据鞭毛抗原序列、致病性和抗性基因预测GX150603的血清型,利用分子生物学软件分析基因组岛和前噬菌体,并与其他菌株进行全基因组系统发育分析.经鉴定GX150603为韦太夫雷登沙门氏菌(Salmone...     

Neutral Genomic Microevolution of a Recently Emerged Pathogen,Salmonella enterica Serovar Agona

Salmonella enterica serovar Agona has caused multiple food-borne outbreaks of gastroenteritis since it was first isolated in 1952. We analyzed the genomes of 73 isolates from global sources, comparing five distinct outbreaks with sporadic infections as well as food contamination and the environment. Agona consists of three lineages with minimal mutational diversity: only 846 single nucleotide polymorphisms (SNPs) have accumulated in the non-repetitive, core genome since Agona evolved in 1932 and subsequently underwent a major population expansion in the 1960s. Homologous recombination with other serovars of S. enterica imported 42 recombinational tracts (360 kb) in 5/143 nodes within the genealogy, which resulted in 3,164 additional SNPs. In contrast to this paucity of genetic diversity, Agona is highly diverse according to pulsed-field gel electrophoresis (PFGE), which is used to assign isolates to outbreaks. PFGE diversity reflects a highly dynamic accessory genome associated with the gain or loss (indels) of 51 bacteriophages, 10 plasmids, and 6 integrative conjugational elements (ICE/IMEs), but did not correlate uniquely with outbreaks. Unlike the core genome, indels occurred repeatedly in independent nodes (homoplasies), resulting in inaccurate PFGE genealogies. The accessory genome contained only few cargo genes relevant to infection, other than antibiotic resistance. Thus, most of the genetic diversity within this recently emerged pathogen reflects changes in the accessory genome, or is due to recombination, but these changes seemed to reflect neutral processes rather than Darwinian selection. Each outbreak was caused by an independent clade, without universal, outbreak-associated genomic features, and none of the variable genes in the pan-genome seemed to be associated with an ability to cause outbreaks.     

Genetic and Phenotypic Variability among Salmonella enterica Serovar Typhimurium Isolates from California Dairy Cattle and Humans

Fifty-six human and 24 adult dairy cattle isolates of Salmonella enterica serovar Typhimurium from a single county in California were compared using ribotyping, insertion sequence typing (IS200), pulsed-field gel electrophoresis, plasmid typing, phage typing, and antimicrobial resistance testing. The majority of the isolates fell into one of two groups which were phage types DT104 and DT193. Combining the information from all typing methods, a total of 45 different “clusters” were defined, with 35 of those including only a single isolate. The library of isolates had a high degree of variability, but antibiotic resistance and plasmid typing each defined single clusters in which human or bovine isolates predominated (χ², P < 0.05).     

Clinical and Veterinary Isolates of Salmonella enterica Serovar Enteritidis Defective in Lipopolysaccharide O-Chain Polymerization

Twelve human and chicken isolates of Salmonella enterica serovar Enteritidis belonging to phage types 4, 8, 13a, and 23 were characterized for variability in lipopolysaccharide (LPS) composition. Isolates were differentiated into two groups, i.e., those that lacked immunoreactive O-chain, termed rough isolates, and those that had immunoreactive O-chain, termed smooth isolates. Isolates within these groups could be further differentiated by LPS compositional differences as detected by gel electrophoresis and gas liquid chromatography of samples extracted with water, which yielded significantly more LPS in comparison to phenol-chloroform extraction. The rough isolates were of two types, the O-antigen synthesis mutants and the O-antigen polymerization (wzy) mutants. Smooth isolates were also of two types, one producing low-molecular-weight (LMW) LPS and the other producing high-molecular-weight (HMW) LPS. To determine the genetic basis for the O-chain variability of the smooth isolates, we analyzed the effects of a null mutation in the O-chain length determinant gene, wzz (cld) of serovar Typhimurium. This mutation results in a loss of HMW LPS; however, the LMW LPS of this mutant was longer and more glucosylated than that from clinical isolates of serovar Enteritidis. Cluster analysis of these data and of those from two previously characterized isogenic strains of serovar Enteritidis that had different virulence attributes indicated that glucosylation of HMW LPS (via oafR function) is variable and results in two types of HMW structures, one that is highly glucosylated and one that is minimally glucosylated. These results strongly indicate that naturally occurring variability in wzy, wzz, and oafR function can be used to subtype isolates of serovar Enteritidis during epidemiological investigations.     

伤寒沙门菌基因组DNA芯片的制备与基因表达谱分析应用

伤寒沙门菌是一种具有鞭毛的革兰阴性人类肠道致病菌,也是一种重要的原核生物研究用模式菌．基因组芯片能够系统、全面且高效地观察生物的基因表达及进行基因组结构比较．利用伤寒沙门菌现有的全基因组序列,以Ty2菌株的基因组为基准,选取CT18菌株和z66阳性菌株的特异性蛋白编码基因,设计特异性引物,经PCR有效扩增出4 201个基因,产物纯化后点样于多聚赖氨酸玻片制备伤寒沙门菌基因组DNA芯片,并验证了芯片样点位次与效果．通过对基因表达谱分析的各种条件进行优化,建立相应的表达谱分析方法,并用于比较伤寒沙门菌野生株在高渗、低渗条件下的基因表达差异,结果与以前的报道基本一致．结果表明,成功建立了伤寒沙门菌基因组DNA芯片及表达谱分析方法,可为有关伤寒沙门菌基因表达调控及致病性机理、进化和基因多样性等方面的深入研究提供有效的技术支持．     

Antimicrobial Resistance in Salmonella enterica Serovar Heidelberg Isolates from Retail Meats, Including Poultry, from 2002 to 2006

Salmonella enterica serovar Heidelberg frequently causes food-borne illness in humans. There are few data on the prevalence, antimicrobial susceptibility, and genetic diversity of Salmonella serovar Heidelberg isolates in retail meats. We compared the prevalences of Salmonella serovar Heidelberg in a sampling of 20,295 meats, including chicken breast (n = 5,075), ground turkey (n = 5,044), ground beef (n = 5,100), and pork chops (n = 5,076), collected during 2002 to 2006. Isolates were analyzed for antimicrobial susceptibility and compared genetically using pulsed-field gel electrophoresis (PFGE) and PCR for the bla_CMY gene. A total of 298 Salmonella serovar Heidelberg isolates were recovered, representing 21.6% of all Salmonella serovars from retail meats. One hundred seventy-eight (59.7%) were from ground turkey, 110 (36.9%) were from chicken breast, and 10 (3.4%) were from pork chops; none was found in ground beef. One hundred ninety-eight isolates (66.4%) were resistant to at least one compound, and 49 (16.4%) were resistant to at least five compounds. Six isolates (2.0%), all from ground turkey, were resistant to at least nine antimicrobials. The highest resistance in poultry isolates was to tetracycline (39.9%), followed by streptomycin (37.8%), sulfamethoxazole (27.7%), gentamicin (25.7%), kanamycin (21.5%), ampicillin (19.8%), amoxicillin-clavulanic acid (10.4%), and ceftiofur (9.0%). All isolates were susceptible to ceftriaxone and ciprofloxacin. All ceftiofur-resistant strains carried bla_CMY. PFGE using XbaI and BlnI showed that certain clones were widely dispersed in different types of meats and meat brands from different store chains in all five sampling years. These data indicate that Salmonella serovar Heidelberg is a common serovar in retail poultry meats and includes widespread clones of multidrug-resistant strains.     

Genomic Evidence Reveals Numerous Salmonella enterica Serovar Newport Reintroduction Events in Suwannee Watershed Irrigation Ponds

Our previous work indicated a predominance (56.8%) of Salmonella enterica serovar Newport among isolates recovered from irrigation ponds used in produce farms over a 2-year period (B. Li et al., Appl Environ Microbiol 80:6355–6365, http://dx.doi.org/10.1128/AEM.02063-14). This observation provided a valuable set of metrics to explore an underaddressed issue of environmental survival of Salmonella by DNA microarray. Microarray analysis correctly identified all the isolates (n = 53) and differentiated the S. Newport isolates into two phylogenetic lineages (S. Newport II and S. Newport III). Serovar distribution analysis showed no instances where the same serovar was recovered from a pond for more than a month. Furthermore, during the study, numerous isolates with an indistinguishable genotype were recovered from different ponds as far as 180 km apart for time intervals as long as 2 years. Although isolates within either lineage were phylogenetically related as determined by microarray analysis, subtle genotypic differences were detected within the lineages, suggesting that isolates in either lineage could have come from several unique hosts. For example, strains in four different subgroups (A, B, C, and D) possessed an indistinguishable genotype within their subgroups as measured by gene differences, suggesting that strains in each subgroup shared a common host. Based on this comparative genomic evidence and the spatial and temporal factors, we speculated that the presence of Salmonella in the ponds was likely due to numerous punctuated reintroduction events associated with several different but common hosts in the environment. These findings may have implications for the development of strategies for efficient and safe irrigation to minimize the risk of Salmonella outbreaks associated with fresh produce.     

Survival and Filamentation of Salmonella enterica Serovar Enteritidis PT4 and Salmonella enterica Serovar Typhimurium DT104 at Low Water Activity

In this study we investigated the long-term survival of and morphological changes in Salmonella strains at low water activity (a_w). Salmonella enterica serovar Enteritidis PT4 and Salmonella enterica serovar Typhimurium DT104 survived at low a_w for long periods, but minimum humectant concentrations of 8% NaCl (a_w, 0.95), 96% sucrose (a_w, 0.94), and 32% glycerol (a_w, 0.92) were bactericidal under most conditions. Salmonella rpoS mutants were usually more sensitive to bactericidal levels of NaCl, sucrose, and glycerol. At a lethal a_w, incubation at 37°C resulted in more rapid loss of viability than incubation at 21°C. At a_w values of 0.93 to 0.98, strains of S. enterica serovar Enteritidis and S. enterica serovar Typhimurium formed filaments, some of which were at least 200 μm long. Filamentation was independent of rpoS expression. When the preparations were returned to high-a_w conditions, the filaments formed septa, and division was complete within approximately 2 to 3 h. The variable survival of Salmonella strains at low a_w highlights the importance of strain choice when researchers produce modelling data to simulate worst-case scenarios or conduct risk assessments based on laboratory data. The continued increase in Salmonella biomass at low a_w (without a concomitant increase in microbial count) would not have been detected by traditional microbiological enumeration tests if the tests had been performed immediately after low-a_w storage. If Salmonella strains form filaments in food products that have low a_w values (0.92 to 0.98), there are significant implications for public health and for designing methods for microbiological monitoring.     

Isolation of Salmonella enterica Serovar Enteritidis from Houseflies (Musca domestica) Found in Rooms Containing Salmonella Serovar Enteritidis-Challenged Hens

Houseflies (Musca domestica) released into rooms containing hens challenged with Salmonella enterica serovar Enteritidis (Salmonella serovar Enteritidis) rapidly became contaminated with Salmonella serovar Enteritidis. Forty to 50% of the flies were contaminated at 48 h, and the percentage increased to 50 to 70% at 4 and 7 days postexposure and then decreased to 30% at day 15. Initial attempts at recovering surface organisms for culture using an aqueous rinse were largely unsuccessful, while cultures of internal contents readily recovered Salmonella serovar Enteritidis. However, when 0.5% detergent was incorporated into the rinse, high recovery levels of bacteria were observed from both external and internal culture regimens, indicating equal distribution of the organism on and in the fly and a tighter interaction of the organism with the host than previously thought. Salmonella serovar Enteritidis was isolated routinely from the fly gut, on rare occasions from the crop, and never from the salivary gland. Feeding contaminated flies to hens resulted in gut colonization of a third of the birds, but release of contaminated flies in a room containing previously unchallenged hens failed to result in colonization of any of the subject birds. These results indicate that flies exposed to an environment containing Salmonella serovar Enteritidis can become colonized with the organism and might serve as a source for transmission of Salmonella serovar Enteritidis within a flock situation.     

Antimicrobial Resistance-Conferring Plasmids with Similarity to Virulence Plasmids from Avian Pathogenic Escherichia coli Strains in Salmonella enterica Serovar Kentucky Isolates from Poultry

Salmonella enterica, a leading cause of food-borne gastroenteritis worldwide, may be found in any raw food of animal, vegetable, or fruit origin. Salmonella serovars differ in distribution, virulence, and host specificity. Salmonella enterica serovar Kentucky, though often found in the food supply, is less commonly isolated from ill humans. The multidrug-resistant isolate S. Kentucky {"type":"entrez-protein","attrs":{"text":"CVM29188","term_id":"985925135","term_text":"CVM29188"}}CVM29188, isolated from a chicken breast sample in 2003, contains three plasmids (146,811 bp, 101,461 bp, and 46,121 bp), two of which carry resistance determinants (pCVM29188_146 [strAB and tetRA] and pCVM29188_101 [bla_CMY-2 and sugE]). Both resistance plasmids were transferable by conjugation, alone or in combination, to S. Kentucky, Salmonella enterica serovar Newport, and Escherichia coli recipients. pCVM29188_146 shares a highly conserved plasmid backbone of 106 kb (>90% nucleotide identity) with two virulence plasmids from avian pathogenic Escherichia coli strains (pAPEC-O1-ColBM and pAPEC-O2-ColV). Shared avian pathogenic E. coli (APEC) virulence factors include iutA iucABCD, sitABCD, etsABC, iss, and iroBCDEN. PCR analyses of recent (1997 to 2005) S. Kentucky isolates from food animal, retail meat, and human sources revealed that 172 (60%) contained similar APEC-like plasmid backbones. Notably, though rare in human- and cattle-derived isolates, this plasmid backbone was found at a high frequency (50 to 100%) among S. Kentucky isolates from chickens within the same time span. Ninety-four percent of the APEC-positive isolates showed resistance to tetracycline and streptomycin. Together, our findings of a resistance-conferring APEC virulence plasmid in a poultry-derived S. Kentucky isolate and of similar resistance/virulence plasmids in most recent S. Kentucky isolates from chickens and, to lesser degree, from humans and cattle highlight the need for additional research in order to examine the prevalence and spread of combined virulence and resistance plasmids in bacteria in agricultural, environmental, and clinical settings.Nontyphoidal Salmonella enterica infections are one of the leading causes of bacterial food-borne gastroenteritis worldwide and an important public health problem in the United States, causing an estimated 1.4 million cases of infection, 15,000 hospitalizations, and more than 400 deaths annually in the United States alone (41). Although Salmonella infection usually presents with self-limiting diarrhea, in some patient populations, such as the immunocompromised, it can lead to life-threatening systemic infections that require effective and immediate antimicrobial therapy (21). The global emergence of multidrug resistance in S. enterica isolates from agricultural and clinical settings has therefore raised concerns and resulted in the establishment of several national resistance surveillance programs, such as the European Antimicrobial Resistance Surveillance System and the National Antimicrobial Monitoring System (NARMS) in the United States.In the United States, antimicrobial compounds are widely used in agriculture, not only to treat and prevent disease in plants, fruits, vegetables, and animals but also to promote growth in poultry and other livestock (25, 38). As a consequence, multidrug resistance is commonly detected in enterobacteria isolated from veterinary sources, including nontyphoidal Salmonella and other food-borne pathogens (40). Several studies have indicated the possibility that resistance reservoirs in animals can promote the transmission of resistance determinants from agricultural to clinical settings via food contaminants (1, 2, 45). Whether antimicrobial use in agriculture enhances the distribution and spectrum of antimicrobial resistance phenotypes in clinical settings has been the focus of vigorous debate within the public health and research communities (15, 19, 24, 27, 43). The extent of multidrug resistance in food-borne pathogens (10, 40), however, remains a concern. In 2006, only 17.7%, 25.0%, 38.8%, and 73.7% of the nontyphoidal Salmonella isolates from ground turkey, pork chop, chicken breast, and ground beef samples, respectively, showed susceptibility to all 16 antimicrobial compounds tested as part of the NARMS program (10). On the other hand, the overall prevalence of antimicrobial resistance phenotypes in nontyphoidal Salmonella isolates from human sources has slightly decreased, from 33.8% of all 876 isolates tested in 1996 to 19.4% of all 1,654 isolates tested in 2005 displaying a detectable resistance phenotype to at least one out of five antimicrobial subclasses as defined by the Clinical and Laboratory Standards Institute. However, in the same interval, increases from 0.4% to 2.4% and 0.2% to 2.9% in resistance to the clinically important subclasses of quinolones (nalidixic acid [Nal]) and cephalosporins (ceftiofur), respectively, were observed for the same set of human Salmonella isolates (6). Altogether, these reports demonstrate the need for further investigations on the influence of antimicrobial selection on the evolution, distribution, and transmission of resistance and virulence phenotypes among enteric bacteria derived from agricultural and clinical settings in order to prevent or at least limit the future spread of resistant zoonotic pathogens between these environments.Salmonella enterica subsp. enterica serovar Kentucky is widespread in poultry meats but is relatively uncommon in human cases of salmonellosis (7). S. Kentucky did not rank among the 20 most frequent Salmonella serotypes isolated from human sources in 2006 (7). In food-related sources, however, it is often found in animal samples and has been the most common serotype isolated from chickens (48.8%) (40) and chicken meat (38.8%) (10). At lower proportions, it is also present in turkey and cattle (2.6% and 3.6% of all nontyphoidal Salmonella isolates, respectively) (40). In the past decade, the fraction of S. Kentucky isolates from chickens, compared with other serotypes, has been increasing steadily, from 25% in 1997 to almost 50% in 2006 (40). Interestingly, while on average only 63 (0.174%) S. Kentucky isolates were reported between 1996 and 2004 among all nontyphoidal salmonellae from human samples, this number increased in 2005 (81 isolates [0.224%]) and 2006 (123 isolates [0.302%]) (7). Antimicrobial resistance phenotypes in S. Kentucky isolates from chicken meat are overrepresented compared to those in other serovars, with resistance to tetracycline (72.9%) and streptomycin (69.5%) being most commonly found (10). Although a causal connection between the increase of S. Kentucky in chickens and the number of human infections caused by the same serovar has not been demonstrated, further investigation is warranted, particularly in light of the high prevalence of antimicrobial resistance phenotypes in S. Kentucky isolates from chickens and the increasing resistance to beta-lactam compounds.Here, we describe the plasmid component of the genome of S. Kentucky {"type":"entrez-protein","attrs":{"text":"CVM29188","term_id":"985925135","term_text":"CVM29188"}}CVM29188, a multidrug-resistant strain that was isolated in 2003 from a chicken meat sample with resistance to streptomycin, tetracycline, ampicillin, and ceftiofur. Using a combination of in silico and in vivo approaches, including comparative plasmid sequence analysis, conjugative plasmid transfer, and PCR-based plasmid screenings, we present new insights into the genetic basis for multidrug resistance phenotypes of this isolate that provide new clues about virulence evolution and host adaptation in this Salmonella serovar.