A One Health Perspective on Salmonella enterica Serovar Infantis, an Emerging Human Multidrug-Resistant Pathogen.

Salmonella enterica serovar Infantis presents an ever-increasing threat to public health because of its spread throughout many countries and association with high levels of antimicrobial resistance (AMR). We analyzed whole-genome sequences of 5,284 Salmonella Infantis strains from 74 countries, isolated during 1989-2020 from a wide variety of human, animal, and food sources, to compare genetic phylogeny, AMR determinants, and plasmid presence. The global Salmonella Infantis population structure diverged into 3 clusters: a North American cluster, a European cluster, and a global cluster. The levels of AMR varied by Salmonella Infantis cluster and by isolation source; 73% of poultry isolates were multidrug resistant, compared with 35% of human isolates. This finding correlated with the presence of the pESI megaplasmid; 71% of poultry isolates contained pESI, compared with 32% of human isolates. This study provides key information for public health teams engaged in reducing the spread of this pathogen.

Geographical and temporal distribution of multidrug-resistant Salmonella Infantis in Europe and the Americas.

Recently emerged S. Infantis strains carrying resistance to several commonly used antimicrobials have been reported from different parts of the globe, causing human cases of salmonellosis and with occurrence reported predominantly in broiler chickens. Here, we performed phylogenetic and genetic clustering analyses to describe the population structure of 417 S. Infantis originating from multiple European countries and the Americas collected between 1985 and 2019. Of these, 171 were collected from 56 distinct premises located in England and Wales (E/W) between 2009 and 2019, including isolates linked to incursions of multidrug-resistant (MDR) strains from Europe associated with imported poultry meat. The analysis facilitated the comparison of isolates from different E/W sources with isolates originating from other countries. There was a high degree of congruency between the outputs of different types of population structure analyses revealing that the E/W and central European (Germany, Hungary, and Poland) isolates formed several disparate groups, which were distinct from the cluster relating to the United States (USA) and Ecuador/Peru, but that isolates from Brazil were closely related to the E/W and the central European isolates. Nearly half of the analysed strains/genomes (194/417) harboured the IncFIB(pN55391) replicon typical of the "parasitic" pESI-like megaplasmid found in diverse strains of S. Infantis. The isolates that contained the IncFIB(pN55391) replicon clustered together, despite originating from different parts of the globe. This outcome was corroborated by the time-measured phylogeny, which indicated that the initial acquisition of IncFIB(pN55391) likely occurred in Europe in the late 1980s, with a single introduction of IncFIB(pN55391)-carrying S. Infantis to the Americas several years later. Most of the antimicrobial resistance (AMR) genes were identified in isolates that harboured one or more different plasmids, but based on the short-read assemblies, only a minority of the resistance genes found in these isolates were identified as being associated with the detected plasmids, whereas the hybrid assemblies comprising the short and long reads demonstrated that the majority of the identified AMR genes were associated with IncFIB(pN55391) and other detected plasmid replicon types. This finding underlies the importance of applying appropriate methodologies to investigate associations of AMR genes with bacterial plasmids.

Development and validation of a random forest algorithm for source attribution of animal and human Salmonella Typhimurium and monophasic variants of S. Typhimurium isolates in England and Wales utilising whole genome sequencing data.

Source attribution has traditionally involved combining epidemiological data with different pathogen characterisation methods, including 7-gene multi locus sequence typing (MLST) or serotyping, however, these approaches have limited resolution. In contrast, whole genome sequencing data provide an overview of the whole genome that can be used by attribution algorithms. Here, we applied a random forest (RF) algorithm to predict the primary sources of human clinical Salmonella Typhimurium (S. Typhimurium) and monophasic variants (monophasic S. Typhimurium) isolates. To this end, we utilised single nucleotide polymorphism diversity in the core genome MLST alleles obtained from 1,061 laboratory-confirmed human and animal S. Typhimurium and monophasic S. Typhimurium isolates as inputs into a RF model. The algorithm was used for supervised learning to classify 399 animal S. Typhimurium and monophasic S. Typhimurium isolates into one of eight distinct primary source classes comprising common livestock and pet animal species: cattle, pigs, sheep, other mammals (pets: mostly dogs and horses), broilers, layers, turkeys, and game birds (pheasants, quail, and pigeons). When applied to the training set animal isolates, model accuracy was 0.929 and kappa 0.905, whereas for the test set animal isolates, for which the primary source class information was withheld from the model, the accuracy was 0.779 and kappa 0.700. Subsequently, the model was applied to assign 662 human clinical cases to the eight primary source classes. In the dataset, 60/399 (15.0%) of the animal and 141/662 (21.3%) of the human isolates were associated with a known outbreak of S. Typhimurium definitive type (DT) 104. All but two of the 141 DT104 outbreak linked human isolates were correctly attributed by the model to the primary source classes identified as the origin of the DT104 outbreak. A model that was run without the clonal DT104 animal isolates produced largely congruent outputs (training set accuracy 0.989 and kappa 0.985; test set accuracy 0.781 and kappa 0.663). Overall, our results show that RF offers considerable promise as a suitable methodology for epidemiological tracking and source attribution for foodborne pathogens.

Increased phage resistance through lysogenic conversion accompanying emergence of monophasic Salmonella Typhimurium ST34 pandemic strain.

Salmonella enterica serovar Typhimurium (S. Typhimurium) comprises a group of closely related human and animal pathogens that account for a large proportion of all Salmonella infections globally. The epidemiological record of S. Typhimurium in Europe is characterized by successive waves of dominant clones, each prevailing for approximately 10-15 years before replacement. Succession of epidemic clones may represent a moving target for interventions aimed at controlling the spread and impact of this pathogen on human and animal health. Here, we investigate the relationship of phage sensitivity and population structure of S. Typhimurium using data from the Anderson phage typing scheme. We observed greater resistance to phage predation of epidemic clones circulating in livestock over the past decades compared to variants with a restricted host range implicating increased resistance to phage in the emergence of epidemic clones of particular importance to human health. Emergence of monophasic S. Typhimurium ST34, the most recent dominant multidrug-resistant clone, was accompanied by increased resistance to phage predation during clonal expansion, in part by the acquisition of the mTmII prophage that may have contributed to the fitness of the strains that replaced ancestors lacking this prophage.

Contrasting long-term dynamics of antimicrobial resistance and virulence plasmids in Salmonella Typhimurium from animals.

Plasmids are mobile elements that can carry genes encoding traits of clinical concern, including antimicrobial resistance (AMR) and virulence. Population-level studies of Enterobacterales, including Escherichia coli, Shigella and Klebsiella, indicate that plasmids are important drivers of lineage expansions and dissemination of AMR genes. Salmonella Typhimurium is the second most common cause of salmonellosis in humans and livestock in the UK and Europe. The long-term dynamics of plasmids between S. Typhimurium were investigated using isolates collected through national surveillance of animals in England and Wales over a 25-year period. The population structure of S. Typhimurium and its virulence plasmid (where present) were inferred through phylogenetic analyses using whole-genome sequence data for 496 isolates. Antimicrobial resistance genes and plasmid markers were detected in silico. Phenotypic plasmid characterization, using the Kado and Liu method, was used to confirm the number and size of plasmids. The differences in AMR and plasmids between clades were striking, with livestock clades more likely to carry one or more AMR plasmid and be multi-drug-resistant compared to clades associated with wildlife and companion animals. Multiple small non-AMR plasmids were distributed across clades. However, all hybrid AMR-virulence plasmids and most AMR plasmids were highly clade-associated and persisted over decades, with minimal evidence of horizontal transfer between clades. This contrasts with the role of plasmids in the short-term dissemination of AMR between diverse strains in other Enterobacterales in high-antimicrobial-use settings, with implications for predicting plasmid dissemination amongst S. Typhimurium.

Development of a Multiplex Bead Assay to Detect Serological Responses to Brucella Species in Domestic Pigs and Wild Boar with the Potential to Overcome Cross-Reactivity with Yersinia enterocolitica O:9.

The aim of this study was to develop a multiplex bead assay using a Brucella rLPS antigen, a Brucella suis smooth antigen, and a Yersinia enterocolitica O:9 antigen that not only discriminates Brucella-infected from Brucella-uninfected pigs and wild boar, but also overcomes the cross reactivity with Y. enterocolitica O:9. Sera from 126 domestic pigs were tested: 29 pigs were Brucella infected, 80 were non-infected and 17 were confirmed to be false positive serological reactors (FPSR). Sera from 49 wild boar were tested: 18 were positive and 31 were negative. Using the rLPS antigen, 26/29 Brucella-infected domestic pigs and 15/18 seropositive wild boar were positive, while 75/80 non-Brucella infected domestic pigs, all FPSR, and all seronegative wild boar were negative. Using the smooth B. suis 1330 antigen, all Brucella-infected domestic pigs, 9/17 FPSR and all seropositive wild boar were positive, while all non-infected pigs and 30/31 seronegative wild boar were negative. The ratio of the readouts from the smooth B. suis antigen and Y. enterocolitica O:9 antigen enabled discriminating all Brucella infected individuals from the FPSR domestic pigs. These results demonstrate the potential of this assay for use in the surveillance of brucellosis, overcoming the cross-reactivity with Y. enterocolitica.

Dynamics of Salmonella enterica and antimicrobial resistance in the Brazilian poultry industry and global impacts on public health.

Non-typhoidal Salmonella enterica is a common cause of diarrhoeal disease; in humans, consumption of contaminated poultry meat is believed to be a major source. Brazil is the world's largest exporter of chicken meat globally, and previous studies have indicated the introduction of Salmonella serovars through imported food products from Brazil. Here we provide an in-depth genomic characterisation and evolutionary analysis to investigate the most prevalent serovars and antimicrobial resistance (AMR) in Brazilian chickens and assess the impact to public health of products contaminated with S. enterica imported into the United Kingdom from Brazil. To do so, we examine 183 Salmonella genomes from chickens in Brazil and 357 genomes from humans, domestic poultry and imported Brazilian poultry products isolated in the United Kingdom. S. enterica serovars Heidelberg and Minnesota were the most prevalent serovars in Brazil and in meat products imported from Brazil into the UK. We extended our analysis to include 1,259 publicly available Salmonella Heidelberg and Salmonella Minnesota genomes for context. The Brazil genomes form clades distinct from global isolates, with temporal analysis suggesting emergence of these Salmonella Heidelberg and Salmonella Minnesota clades in the early 2000s, around the time of the 2003 introduction of the Enteritidis vaccine in Brazilian poultry. Analysis showed genomes within the Salmonella Heidelberg and Salmonella Minnesota clades shared resistance to sulphonamides, tetracyclines and beta-lactams conferred by sul2, tetA and blaCMY-2 genes, not widely observed in other co-circulating serovars despite similar selection pressures. The sul2 and tetA genes were concomitantly carried on IncC plasmids, whereas blaCMY-2 was either co-located with the sul2 and tetA genes on IncC plasmids or independently on IncI1 plasmids. Long-term surveillance data collected in the UK showed no increase in the incidence of Salmonella Heidelberg or Salmonella Minnesota in human cases of clinical disease in the UK following the increase of these two serovars in Brazilian poultry. In addition, almost all of the small number of UK-derived genomes which cluster with the Brazilian poultry-derived sequences could either be attributed to human cases with a recent history of foreign travel or were from imported Brazilian food products. These findings indicate that even should Salmonella from imported Brazilian poultry products reach UK consumers, they are very unlikely to be causing disease. No evidence of the Brazilian strains of Salmonella Heidelberg or Salmonella Minnesota were observed in UK domestic chickens. These findings suggest that introduction of the Salmonella Enteritidis vaccine, in addition to increasing antimicrobial use, could have resulted in replacement of salmonellae in Brazilian poultry flocks with serovars that are more drug resistant, but less associated with disease in humans in the UK. The plasmids conferring resistance to beta-lactams, sulphonamides and tetracyclines likely conferred a competitive advantage to the Salmonella Minnesota and Salmonella Heidelberg serovars in this setting of high antimicrobial use, but the apparent lack of transfer to other serovars present in the same setting suggests barriers to horizontal gene transfer that could be exploited in intervention strategies to reduce AMR. The insights obtained reinforce the importance of One Health genomic surveillance.

The Spatiotemporal Dynamics and Microevolution Events That Favored the Success of the Highly Clonal Multidrug-Resistant Monophasic Salmonella Typhimurium Circulating in Europe.

The European epidemic monophasic variant of Salmonella enterica serovar Typhimurium (S. 1,4,[5],12:i:-) characterized by the multi locus sequence type ST34 and the antimicrobial resistance ASSuT profile has become one of the most common serovars in Europe (EU) and the United States (US). In this study, we reconstructed the time-scaled phylogeny and evolution of this Salmonella in Europe. The epidemic S. 1,4,[5],12:i:- ST34 emerged in the 1980s by an acquisition of the Salmonella Genomic Island (SGI)-4 at the 3' end of the phenylalanine phe tRNA locus conferring resistance to copper and arsenic toxicity. Subsequent integration of the Tn21 transposon into the fljAB locus gave resistance to mercury toxicity and several classes of antibiotics used in food-producing animals (ASSuT profile). The second step of the evolution occurred in the 1990s, with the integration of mTmV and mTmV-like prophages carrying the perC and/or sopE genes involved in the ability to reduce nitrates in intestinal contents and facilitate the disruption of the junctions of the host intestinal epithelial cells. Heavy metals are largely used as food supplements or pesticide for cultivation of seeds intended for animal feed so the expansion of the epidemic S. 1,4,[5],12:i:- ST34 was strongly related to the multiple-heavy metal resistance acquired by transposons, integrative and conjugative elements and facilitated by the escape until 2011 from the regulatory actions applied in the control of S. Typhimurium in Europe. The genomic plasticity of the epidemic S. 1,4,[5],12:i:- was demonstrated in our study by the analysis of the plasmidome. We were able to identify plasmids harboring genes mediating resistance to phenicols, colistin, and fluoroquinolone and also describe for the first time in six of the analyzed genomes the presence of two plasmids (pERR1744967-1 and pERR2174855-2) previously described only in strains of enterotoxigenic Escherichia coli and E. fergusonii.

Development of a Multiplex Bead Assay for Simultaneous Serodiagnosis of Antibodies against Mycobacterium bovis, Brucella suis, and Trichinella spiralis in Wild Boar.

The aim of this study was to evaluate the diagnostic performance of a multiplex bead assay for the simultaneous detection of antibodies against Mycobacterium bovis, Brucella suis, and Trichinella spiralis. Sera from Eurasian wild boar of known serological status for TB (64 seropositive, 106 seronegative), Brucella (30 seropositive, 39 seronegative), and Trichinella (21 seropositive, 97 seronegative) were used for the development and evaluation of the assay. Magnetic beads coated with recombinant MPB83 antigen (TB), a whole-cell B. suis 1330 antigen, and an E/S T. spiralis antigen were used for the detection of specific antibodies using Bio-Rad Bio-Plex technology. The sensitivities (Se) and specificities (Sp) of the multiplex assay were, for M. bovis, 0.98 and 0.86; for B. suis, 1.00 and 0.97; and for T. spiralis, 0.90 and 0.99 (Se and Sp, respectively). The results show the diagnostic potential of this assay for the simultaneous detection of antibodies against M. bovis, B. suis, and T. spiralis in wild boar.

Ecological niche adaptation of Salmonella Typhimurium U288 is associated with altered pathogenicity and reduced zoonotic potential.

The emergence of new bacterial pathogens is a continuing challenge for agriculture and food safety. Salmonella Typhimurium is a major cause of foodborne illness worldwide, with pigs a major zoonotic reservoir. Two phylogenetically distinct variants, U288 and ST34, emerged in UK pigs around the same time but present different risk to food safety. Here we show using genomic epidemiology that ST34 accounts for over half of all S. Typhimurium infections in people while U288 less than 2%. That the U288 clade evolved in the recent past by acquiring AMR genes, indels in the virulence plasmid pU288-1, and accumulation of loss-of-function polymorphisms in coding sequences. U288 replicates more slowly and is more sensitive to desiccation than ST34 isolates and exhibited distinct pathogenicity in the murine model of colitis and in pigs. U288 infection was more disseminated in the lymph nodes while ST34 were recovered in greater numbers in the intestinal contents. These data are consistent with the evolution of S. Typhimurium U288 adaptation to pigs that may determine their reduced zoonotic potential.

Bayesian Source Attribution of Salmonella Typhimurium Isolates From Human Patients and Farm Animals in England and Wales.

The purpose of the study was to apply a Bayesian source attribution model to England and Wales based data on Salmonella Typhimurium (ST) and monophasic variants (MST), using different subtyping approaches based on sequence data. The data consisted of laboratory confirmed human cases and mainly livestock samples collected from surveillance or monitoring schemes. Three different subtyping methods were used, 7-loci Multi-Locus Sequence Typing (MLST), Core-genome MLST, and Single Nucleotide Polymorphism distance, with the impact of varying the genetic distance over which isolates would be grouped together being varied for the latter two approaches. A Bayesian frequency matching method, known as the modified Hald method, was applied to the data from each of the subtyping approaches. Pigs were found to be the main contributor to human infection for ST/MST, with approximately 60% of human cases attributed to them, followed by other mammals (mostly horses) and cattle. It was found that the use of different clustering methods based on sequence data had minimal impact on the estimates of source attribution. However, there was an impact of genetic distance over which isolates were grouped: grouping isolates which were relatively closely related increased uncertainty but tended to have a better model fit.

Whole-genome epidemiology links phage-mediated acquisition of a virulence gene to the clonal expansion of a pandemic Salmonella enterica serovar Typhimurium clone.

Epidemic and pandemic clones of bacterial pathogens with distinct characteristics continually emerge, replacing those previously dominant through mechanisms that remain poorly characterized. Here, whole-genome-sequencing-powered epidemiology linked horizontal transfer of a virulence gene, sopE, to the emergence and clonal expansion of a new epidemic Salmonella enterica serovar Typhimurium (S. Typhimurium) clone. The sopE gene is sporadically distributed within the genus Salmonella and rare in S. enterica Typhimurium lineages, but was acquired multiple times during clonal expansion of the currently dominant pandemic monophasic S. Typhimurium sequence type (ST) 34 clone. Ancestral state reconstruction and time-scaled phylogenetic analysis indicated that sopE was not present in the common ancestor of the epidemic clade, but later acquisition resulted in increased clonal expansion of sopE-containing clones that was temporally associated with emergence of the epidemic, consistent with increased fitness. The sopE gene was mainly associated with a temperate bacteriophage mTmV, but recombination with other bacteriophage and apparent horizontal gene transfer of the sopE gene cassette resulted in distribution among at least four mobile genetic elements within the monophasic S. enterica Typhimurium ST34 epidemic clade. The mTmV prophage lysogenic transfer to other S. enterica serovars in vitro was limited, but included the common pig-associated S. enterica Derby (S. Derby). This may explain mTmV in S. Derby co-circulating on farms with monophasic S. Typhimurium ST34, highlighting the potential for further transfer of the sopE virulence gene in nature. We conclude that whole-genome epidemiology pinpoints potential drivers of evolutionary and epidemiological dynamics during pathogen emergence, and identifies targets for subsequent research in epidemiology and bacterial pathogenesis.

Evolution of Salmonella enterica serotype Typhimurium driven by anthropogenic selection and niche adaptation.

Salmonella enterica serotype Typhimurium (S. Typhimurium) is a leading cause of gastroenteritis and bacteraemia worldwide, and a model organism for the study of host-pathogen interactions. Two S. Typhimurium strains (SL1344 and ATCC14028) are widely used to study host-pathogen interactions, yet genotypic variation results in strains with diverse host range, pathogenicity and risk to food safety. The population structure of diverse strains of S. Typhimurium revealed a major phylogroup of predominantly sequence type 19 (ST19) and a minor phylogroup of ST36. The major phylogroup had a population structure with two high order clades (α and ß) and multiple subclades on extended internal branches, that exhibited distinct signatures of host adaptation and anthropogenic selection. Clade α contained a number of subclades composed of strains from well characterized epidemics in domesticated animals, while clade ß contained multiple subclades associated with wild avian species. The contrasting epidemiology of strains in clade α and ß was reflected by the distinct distribution of antimicrobial resistance (AMR) genes, accumulation of hypothetically disrupted coding sequences (HDCS), and signatures of functional diversification. These observations were consistent with elevated anthropogenic selection of clade α lineages from adaptation to circulation in populations of domesticated livestock, and the predisposition of clade ß lineages to undergo adaptation to an invasive lifestyle by a process of convergent evolution with of host adapted Salmonella serotypes. Gene flux was predominantly driven by acquisition and recombination of prophage and associated cargo genes, with only occasional loss of these elements. The acquisition of large chromosomally-encoded genetic islands was limited, but notably, a feature of two recent pandemic clones (DT104 and monophasic S. Typhimurium ST34) of clade α (SGI-1 and SGI-4).

Four European Salmonella Typhimurium datasets collected to develop WGS-based source attribution methods.

Zoonotic Salmonella causes millions of human salmonellosis infections worldwide each year. Information about the source of the bacteria guides risk managers on control and preventive strategies. Source attribution is the effort to quantify the number of sporadic human cases of a specific illness to specific sources and animal reservoirs. Source attribution methods for Salmonella have so far been based on traditional wet-lab typing methods. With the change to whole genome sequencing there is a need to develop new methods for source attribution based on sequencing data. Four European datasets collected in Denmark (DK), Germany (DE), the United Kingdom (UK) and France (FR) are presented in this descriptor. The datasets contain sequenced samples of Salmonella Typhimurium and its monophasic variants isolated from human, food, animal and the environment. The objective of the datasets was either to attribute the human salmonellosis cases to animal reservoirs or to investigate contamination of the environment by attributing the environmental isolates to different animal reservoirs.

SGI-4 in Monophasic Salmonella Typhimurium ST34 Is a Novel ICE That Enhances Resistance to Copper.

A multi drug resistant Salmonella enterica 4,[5],12:i- of sequence type 34 (monophasic S. Typhimurium ST34) is a current pandemic clone associated with livestock, particularly pigs, and numerous outbreaks in the human population. A large genomic island, termed SGI-4, is present in the monophasic Typhimurium ST34 clade and absent from other S. Typhimurium strains. SGI-4 consists of 87 open reading frames including sil and pco genes previously implicated in resistance to copper (Cu) and silver, and multiple genes predicted to be involved in mobilization and transfer by conjugation. SGI-4 was excised from the chromosome, circularized, and transferred to recipient strains of S. Typhimurium at a frequency influenced by stress induced by mitomycin C, and oxygen tension. The presence of SGI-4 was associated with increased resistance to Cu, particularly but not exclusively under anaerobic conditions. The presence of silCBA genes, predicted to encode an RND family efflux pump that transports Cu from the periplasm to the external milieu, was sufficient to impart the observed enhanced resistance to Cu, above that commonly associated with S. Typhimurium isolates. The presence of these genes resulted in the absence of Cu-dependent induction of pco genes encoding multiple proteins linked to Cu resistance, also present on SGI-4, suggesting that the system effectively limits the Cu availability in the periplasm, but did not affect SodCI-dependent macrophage survival.

Genetic Diversity of Salmonella Derby from the Poultry Sector in Europe.

Salmonella Derby (S. Derby) is emerging in Europe as a predominant serovar in fattening turkey flocks. This serovar was recorded as being predominant in the turkey sector in 2014 in the United Kingdom (UK). Only two years later, in 2016, it was also recorded in the turkey and broiler sectors in Ireland and Spain. These S. Derby isolates were characterised as members of the multilocus sequence type (MLST) profile 71 (ST71). For the first time, we characterise by whole genome sequencing (WGS) analysis a panel of 90 S. Derby ST71 genomes to understand the routes of transmission of this emerging pathogen within the poultry/turkey food trade. Selected panel included strains isolated as early as 2010 in five leading European g countries for turkey meat production. Twenty-one of the 90 genomes were extracted from a public database-Enterobase. Five of these originated from the United States (n=3), China (n=1) and Taiwan (n=1) isolated between 1986 and 2016. A phylogenomic analysis at the core-genome level revealed the presence of three groups. The largest group contained 97.5% of the European strains and included both, turkey and human isolates that were genetically related by an average of 35 ± 15 single nucleotide polymorphism substitutions (SNPs). To illustrate the diversity, the presence of antimicrobial resistance genes and phages were characteised in 30, S. Derby ST71 genomes, including 11 belonging to this study This study revealed an emergent turkey-related S. Derby ST71 clone circulating in at least five European countries (the UK, Germany, Poland, Italy, and France) since 2010 that causes human gastroenteritis. A matter of concern is the identification of a gyrA mutation involved in resistance to quinolone, present in the Italian genomes. Interestingly, the diversity of phages seems to be related to the geographic origins. These results constitute a baseline for following the spread of this emerging pathogen and identifying appropriate monitoring and prevention measures.

Antimicrobial Resistance Diversity Suggestive of Distinct Salmonella Typhimurium Sources or Selective Pressures in Food-Production Animals.

Salmonella enterica subsp. enterica serovar Typhimurium is a common cause of enterocolitis in humans globally, with multidrug resistant (MDR) strains posing an enhanced threat. S. Typhimurium is also a pathogen in food-production animals, and these populations can act as reservoirs of the bacterium. Therefore, surveillance and control measures within food-production animal populations are of importance both to animal and human health and have the potential to be enhanced though improved understanding of the epidemiology of S. Typhimurium within and between food-production animal populations. Here, data from Scotland and national surveillance England and Wales data for isolates from cattle (n = 1115), chickens (n = 248) and pigs (n = 2174) collected between 2003 and 2014 were analyzed. Ecological diversity analyses and rarefaction curves were used to compare the diversity of observed antimicrobial resistance (AMR) profiles between the host species, and within host species populations. Higher AMR profile diversity was observed in isolates from pigs compared to chickens across diversity measures and isolates from cattle for three of four diversity measures. Variation in AMR profile diversity between production sectors was noted, with higher AMR diversity of isolates from broiler compared to layer chickens, breeder compared to rearer and finisher pigs and beef compared to dairy cattle. Findings indicate variation in AMR profile diversity both within and between food-production animal host species. These observations suggest alternate sources of AMR bacteria and/or variation in selective evolutionary pressures within and between food-production animal host species populations.

Identification of Genetic Features for Attenuation of Two Salmonella Enteritidis Vaccine Strains and Differentiation of These From Wildtype Isolates Using Whole Genome Sequencing.

Salmonella Enteritidis is a major cause of salmonellosis worldwide and more than 80% of outbreaks investigated in Europe have been associated with the consumption of poorly cooked eggs or foods containing raw eggs. Vaccination has been proven to be one of the most important measures to control Salmonella Enteritidis infections in poultry farms as it can decrease colonization of the reproductive organs and intestinal tract of laying hens, thereby reducing egg contamination. Differentiation of live vaccine from field or wild type S. Enteritidis isolates in poultry is essential for monitoring of veterinary isolates and targetting control actions. Due to decreasing costs, whole genome sequencing (WGS) is becoming a key tool for characterization of Salmonella isolates, including vaccine strains. Using WGS we described the genetic changes in the live attenuated Salmovac 440 and AviPro SALMONELLA VAC E vaccine strains and developed a method for differentiation from the wildtype S. Enteritidis strains. SNP analysis confirmed that streptomycin resistance was associated with a Lys43Arg missense mutation in the rpsL gene whilst 3 missense mutations in acrB and 1 missense mutation in acrA confer erythromycin sensitivity in AviPro SALMONELLA VAC E. Further mutations Arg242His in purK and Gly236Arg in the hisB gene were related to adenine and histidine dependencies in Salmovac 440. Unique SNPs were used to construct a database of variants for differentiation of vaccine from the wildtype isolates. Two fragments from each vaccine were represented in the database to ensure high accuracy. Each of the two selected Salmovac 440 fragments differed by 6 SNPs from the wildtype and the AviPro SALMONELLA VAC E fragments differed by 4 and 6 SNPs, respectively. CD-hit software was applied to cluster similar fragments that produced the best fit output when searched with SRST2. The developed vaccine differentiation method was tested with 1,253 genome samples including field isolates of Salmovac 440 (n = 51), field isolates of AviPro SALMONELLA VAC E (n = 13), S. Gallinarum (n = 19), S. Pullorum (n = 116), S. Enteritidis (n = 244), S. Typhimurium (n = 810) and achieved 100% sensitivity and specificity.

Role of wild birds and environmental contamination in the epidemiology of Salmonella infection in an outdoor pig farm.

Foodborne outbreaks caused by Salmonella are often attributed to the pork consumption. Salmonella contamination of retail pork is directly linked to the Salmonella prevalence on farm. In UK, approximately 40% of breeding pigs are kept outdoors. Aim of this study was to investigate the role of wild birds in the epidemiology of Salmonella in one outdoor pig farm. Three sampling visits were carried out at monthly intervals to an outdoor farm consisting of two fields, one left empty of pigs for more than 2 years (field A) while the second (field B) was occupied by pigs during the first visit only. Faeces from wild bird droppings, environmental samples and pig faeces were tested for Salmonella. Salmonella spp. was isolated from environmental samples also in field A that had not been occupied by pigs more than 2 years. Interestingly, the wild bird population accessing the fields increased considerably once the pigs had left the farm and the proportion of Salmonella positive wild bird droppings increased over time with 7.4%, 15.8% and 44.3% at the first, second and third visit, respectively. The levels of Salmonella identified in some of the wild bird droppings were unusually high (105-106 CFU/g) suggesting that Salmonella was actively replicating in the gastrointestinal tract of these birds. Monophasic Salmonella Typhimurium DT193 was the predominant serotype isolated in pigs as well as in wild bird droppings and the environment, suggesting that the pigs were the original source of infection, as this serovar is typically associated with pigs.

Emergence of Klebsiella pneumoniae subspecies pneumoniae as a cause of septicaemia in pigs in England.

Between 2011 and 2014 outbreaks of septicaemia due to Klebsiella pneumoniae subspecies pneumoniae (Kpp) were diagnosed on thirteen English pig farms. The most consistent features were rapid deaths of pigs from ten-days-old to weaning, seasonal occurrence (May to September), affected farms being outdoor breeding herds and the location of all but one of the outbreaks in the East Anglia region in Eastern England. Molecular characterisation of the outbreak Kpp isolates showed that by multilocus sequencing all were sequence type 25 (ST25) of K2 capsular type with a combination of a 4.3kb plasmid (pKPMC25), three phage sequences and the rmpA virulence gene. No archived Kpp isolates of porcine origin pre-dating 2011 were identified as ST25. In 2013 there was the first detection of an outbreak Kpp isolate showing antimicrobial resistance to six antibiotics. Human infection with Kpp ST25 has not been reported in the UK.