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Methodologies for source attribution (SA) of foodborne illnesses comprise a rapidly expanding suite of techniques for estimating the most important source or sources of human infection. Recently, the increasing availability of whole genome sequencing (WGS) data for a wide range of bacterial strains has led to the development of novel SA methods. These techniques utilize the unique features of bacterial genomes adapted to different host types and hence offer increased resolution of the outputs. Comparative studies of different SA techniques reliant on WGS data are currently lacking. Here, we critically assessed and compared the outputs of three SA methods: a supervised classification random forest machine learning algorithm (RandomForest), an Accessory genes-Based Source Attribution method (AB_SA), and a Bayesian frequency matching method (Bayesian). Each technique was applied to the WGS data of a panel of 902 reservoir host and human monophasic and biphasic Salmonella enterica subsp. enterica serovar Typhimurium isolates sampled in the British Isles (BI) and Denmark from 2012 to 2016. Additionally, for RandomForest and Bayesian, we explored whether utilization of accessory genome features as model inputs improved attribution accuracy of these methods over using the core genome derived features only. Results indicated that this was the case for RandomForest, but for Bayesian the overall attribution estimates varied little regardless of the inclusion or not of the accessory genome features. All three methods attributed the vast majority of human isolates to the Pigs primary source class, which was expected given the known high relative prevalence rates in pigs, and hence routes of infection into the human population, of monophasic and biphasic S. Typhimurium in the BI and Denmark. The accuracy of AB_SA was lower than of RandomForest when attributing the primary source classes to the 120 animal test set isolates with known primary sources. A major advantage of both AB_SA and Bayesian was a much faster execution time as compared to RandomForest. Overall, the SA method comparison presented in this study describes the strengths and weaknesses of each of the three methods applied to attributing potential monophasic and biphasic S. Typhimurium animal sources to human infections that could be valuable when deciding which SA methodology would be the most applicable to foodborne disease outbreak scenarios involving monophasic and biphasic S. Typhimurium.

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.

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.

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.

Mark Kirkwood et al. take an epidemiological, genomic, phylogenetic, and experimental approach to assess the evolutionary origins and virulence of the U288 and ST34 isolates of Salmonella enterica in affecting livestock and potentially humans. Their results indicate that the molecular evolution of these isolates is associated with altered pathogenicity in pigs and reduced zoonotic potential of U288.

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. Author summary 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. Here we determine that S. enterica serovars Heidelberg and Minnesota were the most prevalent serovars in Brazilian poultry and in poultry products imported from Brazil into the UK. However, long-term surveillance data collected by the UK Health Security Agency 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. 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. 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.

Abstract Background UK public health organisations perform routine antimicrobial susceptibility tests (ASTs) to characterise the potential for antimicrobial resistance in Salmonella enterica serovars. Genetic determinants of these resistance mechanisms are detectable by whole genome sequencing (WGS), however the viability of WGS-based genotyping as an alternative resistance screening tool remains uncertain. We compared WGS-based genotyping, disk diffusion and agar dilution to the broth microdilution reference AST for 102 Salmonella enterica serovar Typhimurium (S. Typhimurium) isolates across 11 antimicrobial compounds. Results Genotyping concordance, interpreted using epidemiological cut-offs (ECOFFs), was 89.8% (1007/1122) with 0.83 sensitivity and 0.96 specificity. For seven antimicrobials interpreted using Salmonella clinical breakpoints, genotyping produced 0.84 sensitivity and 0.88 specificity. Although less accurate than disk diffusion (0.94 sensitivity, 0.93 specificity) and agar dilution (0.83 sensitivity, 0.98 specificity), genotyping performance improved to 0.89 sensitivity and 0.97 specificity when two antimicrobials with relatively high very major error rates were excluded (streptomycin and sulfamethoxazole). Conclusions An 89.8% concordance from WGS-based AST predictions using ECOFF interpretations suggest that WGS would serve as an effective screening tool for the tracking of antimicrobial resistance mechanisms in S. Typhimurium. For use as a standalone clinical diagnostic screen, further work is required to reduce the error rates for specific antimicrobials.

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.

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.

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.

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 β) 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 β contained multiple subclades associated with wild avian species. The contrasting epidemiology of strains in clade α and β 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 β 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).

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.

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.

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.

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.

Microevolution associated with emergence and expansion of new epidemic clones of bacterial pathogens holds the key to epidemiologic success. To determine microevolution associated with monophasic Salmonella Typhimurium during an epidemic, we performed comparative whole-genome sequencing and phylogenomic analysis of isolates from the United Kingdom and Italy during 2005–2012. These isolates formed a single clade distinct from recent monophasic epidemic clones previously described from North America and Spain. The UK monophasic epidemic clones showed a novel genomic island encoding resistance to heavy metals and a composite transposon encoding antimicrobial drug resistance genes not present in other Salmonella Typhimurium isolates, which may have contributed to epidemiologic success. A remarkable amount of genotypic variation accumulated during clonal expansion that occurred during the epidemic, including multiple independent acquisitions of a novel prophage carrying the sopE gene and multiple deletion events affecting the phase II flagellin locus. This high level of microevolution may affect antigenicity, pathogenicity, and transmission.

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.

The term “spotty liver disease” (SLD) has been used since the late 1990s for a condition seen in the UK and Australia that primarily affects free range laying hens around peak lay, causing acute mortality and a fall in egg production. A novel thermophilic SLD-associated Campylobacter was reported in the United Kingdom (UK) in 2015. Subsequently, similar isolates occurring in Australia were formally described as a new species, Campylobacter hepaticus. We describe the comparative genomics of 10 C. hepaticus isolates recovered from 5 geographically distinct poultry holdings in the UK between 2010 and 2012. Hierarchical gene-by-gene analyses of the study isolates and representatives of 24 known Campylobacter species indicated that C. hepaticus is most closely related to the major pathogens Campylobacter jejuni and Campylobacter coli. We observed low levels of within-farm variation, even between isolates collected over almost 3 years. With respect to C. hepaticus genome features, we noted that the study isolates had a ~140 Kb reduction in genome size, ~144 fewer genes, and a lower GC content compared to C. jejuni. The most notable reduction was in the subsystem containing genes for iron acquisition and metabolism, supported by reduced growth of C. hepaticus in an iron depletion assay. Genome reduction is common among many pathogens and in C. hepaticus has likely been driven at least in part by specialization following the occupation of a new niche, the chicken liver.

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.

The ability of Shiga toxin-producing Escherichia coli (STEC) to cause severe illness in humans is determined by multiple host factors and bacterial characteristics, including Shiga toxin (Stx) subtype. Given the link between Stx2a subtype and disease severity, we sought to identify the stx subtypes present in whole genome sequences (WGS) of 444 isolates of STEC O157. Difficulties in assembling the stx genes in some strains were overcome by using two complementary bioinformatics methods: mapping and de novo assembly. We compared the WGS analysis with the results obtained using a PCR approach and investigated the diversity within and between the subtypes. All strains of STEC O157 in this study had stx1a, stx2a or stx2c or a combination of these three genes. There was over 99% (442/444) concordance between PCR and WGS. When common source strains were excluded, 236/349 strains of STEC O157 had multiple copies of different Stx subtypes and 54 had multiple copies of the same Stx subtype. Of those strains harbouring multiple copies of the same Stx subtype, 33 had variants between the alleles while 21 had identical copies. Strains harbouring Stx2a only were most commonly found to have multiple alleles of the same subtype (42%). Both the PCR and WGS approach to stx subtyping provided a good level of sensitivity and specificity. In addition, the WGS data also showed there were a significant proportion of strains harbouring multiple alleles of the same Stx subtype associated with clinical disease in England.

Salmonella enterica serovars Derby and Mbandaka are isolated from different groups of livestock species in the UK. S. Derby is predominantly isolated from pigs and turkeys and S. Mbandaka is predominantly isolated from cattle and chickens. Alignment of the genome sequences of two isolates of each serovar led to the discovery of a new putative Salmonella pathogenicity island, SPI-23, in the chromosome sequence of S. Derby isolates. SPI-23 is 37 kb in length and contains 42 ORFs, ten of which are putative type III effector proteins. In this study we use porcine jejunum derived cell line IPEC-J2 and in vitro organ culture of porcine jejunum and colon, to characterise the association and invasion rates of S. Derby and S. Mbandaka, and tissue tropism of S. Derby respectively. We show that S. Derby invades and associates to an IPEC-J2 monolayer in significantly greater numbers than S. Mbandaka, and that S. Derby preferentially attaches to porcine jejunum over colon explants. We also show that nine genes across SPI-23 are up-regulated to a greater degree in the jejunum compared to the colon explants. Furthermore, we constructed a mutant of the highly up-regulated, pilV-like gene, potR, and find that it produces an excess of surface pili compared to the parent strain which form a strong agglutinating phenotype interfering with association and invasion of IPEC-J2 monolayers. We suggest that potR may play a role in tissue tropism.

West Nile Virus (WNV) is the causative agent of a vector-borne, zoonotic disease with a worldwide distribution. Recent expansion and introduction of WNV into new areas, including southern Europe, has been associated with severe disease in humans and equids, and has increased concerns regarding the need to prevent and control future WNV outbreaks. Since 2010, 524 confirmed human cases of the disease have been reported in Greece with greater than 10% mortality. Infected mosquitoes, wild birds, equids, and chickens have been detected and associated with human disease. The aim of our study was to establish a monitoring system with wild birds and reported human cases data using Geographical Information System (GIS). Potential distribution of WNV was modelled by combining wild bird serological surveillance data with environmental factors (e.g. elevation, slope, land use, vegetation density, temperature, precipitation indices, and population density). Local factors including areas of low altitude and proximity to water were important predictors of appearance of both human and wild bird cases (Odds Ratio = 1,001 95%CI = 0,723-1,386). Using GIS analysis, the identified risk factors were applied across Greece identifying the northern part of Greece (Macedonia, Thrace) western Greece and a number of Greek islands as being at highest risk of future outbreaks. The results of the analysis were evaluated and confirmed using the 161 reported human cases of the 2012 outbreak predicting correctly (Odds = 130/31 = 4,194 95%CI = 2,841-6,189) and more areas were identified for potential dispersion in the following years. Our approach verified that WNV risk can be modelled in a fast cost-effective way indicating high risk areas where prevention measures should be implemented in order to reduce the disease incidence.

Campylobacter jejuni is an important cause of human foodborne gastroenteritis; strategies to prevent infection are hampered by a poor understanding of the complex interactions between host and pathogen. Previous work showed that C. jejuni could bind human histo-blood group antigens (BgAgs) in vitro and that BgAgs could inhibit the binding of C. jejuni to human intestinal mucosa ex vivo. Here, the major flagella subunit protein (FlaA) and the major outer membrane protein (MOMP) were identified as BgAg-binding adhesins in C. jejuni NCTC11168. Significantly, the MOMP was shown to be O-glycosylated at Thr268; previously only flagellin proteins were known to be O-glycosylated in C. jejuni. Substitution of MOMP Thr268 led to significantly reduced binding to BgAgs. The O-glycan moiety was characterized as Gal(β1–3)-GalNAc(β1–4)-GalNAc(β1–4)-GalNAcα1-Thr268; modelling suggested that O-glycosylation has a notable effect on the conformation of MOMP and this modulates BgAg-binding capacity. Glycosylation of MOMP at Thr268 promoted cell-to-cell binding, biofilm formation and adhesion to Caco-2 cells, and was required for the optimal colonization of chickens by C. jejuni, confirming the significance of this O-glycosylation in pathogenesis.

A study was conducted in order to determine the occurrence of European Brown Hare Syndrome virus (EBHSV) in Denmark and possible relation between disease pathogenesis and Major Histocompatibility Complex (MHC) host genotype. Liver samples were examined from 170 brown hares (hunted, found sick or dead), collected between 2004 and 2009. Macroscopical and histopathological findings consistent with EBHS were detected in 24 (14.1%) hares; 35 (20.6%) had liver lesions not typical of the syndrome, 50 (29.4%) had lesions in other tissues and 61 (35.9%) had no lesions. Sixty five (38.2%) of 170 samples were found to be EBHSV-positive (RT-PCR, VP60 gene). In order to investigate associations between viral pathogenesis and host genotype, variation within the exon 2 DQA gene of MHC was assessed. DQA exon 2 analysis revealed the occurrence of seven different alleles in Denmark. Consistent with other populations examined so far in Europe, observed heterozygosity of DQA (H o = 0.1180) was lower than expected (H e = 0.5835). The overall variation for both nucleotide and amino acid differences (2.9% and 14.9%, respectively) were lower in Denmark than those assessed in other European countries (8.3% and 16.9%, respectively). Within the peptide binding region codons the number of nonsynonymous substitutions (dN) was much higher than synonymous substitutions (dS), which would be expected for MHC alleles under balancing selection. Allele frequencies did not significantly differ between EBHSV-positive and -negative hares. However, allele Leeu-DQA*30 was detected in significantly higher (P = 0.000006) frequency among the positive hares found dead with severe histopathological lesions than among those found sick or apparently healthy. In contrast, the latter group was characterized by a higher frequency of the allele Leeu-DQA*14 as well as the proportion of heterozygous individuals (P = 0.000006 and P = 0.027). These data reveal a polarisation between EBHSV pathogenesis and MHC class II genotype within the European brown hare in Denmark.