Yersinia enterocolitica biovar 1A: An underappreciated potential pathogen in the food chain.

Yersinia enterocolitica is an underreported cause of foodborne gastroenteritis. Little is known of the diversity of Y. enterocolitica isolated from food and which food commodities contribute to human disease. In this study, Y. enterocolitica was isolated from 37/50 raw chicken, 8/10 pork, 8/10 salmon and 1/10 leafy green samples collected at retail in the UK. Up to 10 presumptive Y. enterocolitica isolates per positive sample underwent whole genome sequencing (WGS) and were compared with publicly available genomes. In total, 207 Y. enterocolitica isolates were analyzed and belonged to 38 sequence types (STs). Up to five STs of Y. enterocolitica were isolated from individual food samples and isolates belonging to the same sample and ST differed by 0-74 single nucleotide polymorphisms (SNPs). Biotype was predicted for 205 (99 %) genomes that all belonged to biotype 1A, previously described as non-pathogenic. However, around half (51 %) of food samples contained isolates belonging to the same ST as previously isolated from UK human cases. The closest human-derived isolates shared between 17 and 7978 single nucleotide polymorphisms (SNPs) with the food isolates. Extensive food surveillance is required to determine what food sources are responsible for Y. enterocolitica infections and to re-examine the role of biotype 1A as a human pathogen.

Genomic characterization of Pseudomonas spp. on food: implications for spoilage, antimicrobial resistance and human infection.

BACKGROUND: Pseudomonas species are common on food, but their contribution to the antimicrobial resistance gene (ARG) burden within food or as a source of clinical infection is unknown. Pseudomonas aeruginosa is an opportunistic pathogen responsible for a wide range of infections and is often hard to treat due to intrinsic and acquired ARGs commonly carried by this species. This study aimed to understand the potential role of Pseudomonas on food as a reservoir of ARGs and to assess the presence of potentially clinically significant Pseudomonas aeruginosa strains on food. To achieve this, we assessed the genetic relatedness (using whole genome sequencing) and virulence of food-derived isolates to those collected from humans. RESULTS: A non-specific culturing approach for Pseudomonas recovered the bacterial genus from 28 of 32 (87.5%) retail food samples, although no P. aeruginosa was identified. The Pseudomonas species recovered were not clinically relevant, contained no ARGs and are likely associated with food spoilage. A specific culture method for P. aeruginosa resulted in the recovery of P. aeruginosa from 14 of 128 (11%) retail food samples; isolates contained between four and seven ARGs each and belonged to 16 sequence types (STs), four of which have been isolated from human infections. Food P. aeruginosa isolates from these STs demonstrated high similarity to human-derived isolates, differing by 41-312 single nucleotide polymorphisms (SNPs). There were diverse P. aeruginosa collected from the same food sample with distinct STs present on some samples and isolates belonging to the same ST differing by 19-67 SNPs. The Galleria mellonella infection model showed that 15 of 16 STs isolated from food displayed virulence between a low-virulence (PAO1) and a high virulence (PA14) control. CONCLUSION: The most frequent Pseudomonas recovered from food examined in this study carried no ARGs and are more likely to play a role in food spoilage rather than infection. P. aeruginosa isolates likely to be able to cause human infections and with multidrug resistant genotypes are present on a relatively small but still substantial proportions of retail foods examined. Given the frequency of exposure, the potential contribution of food to the burden of P. aeruginosa infections in humans should be evaluated more closely.

Genomic diversity and epidemiological significance of non-typhoidal Salmonella found in retail food collected in Norfolk, UK.

Non-typhoidal Salmonella (NTS) is a major cause of bacterial gastroenteritis. Although many countries have implemented whole genome sequencing (WGS) of NTS, there is limited knowledge on NTS diversity on food and its contribution to human disease. In this study, the aim was to characterise the NTS genomes from retail foods in a particular region of the UK and assess the contribution to human NTS infections. Raw food samples were collected at retail in a repeated cross-sectional design in Norfolk, UK, including chicken (n=311), leafy green (n=311), pork (n=311), prawn (n=279) and salmon (n=157) samples. Up to eight presumptive NTS isolates per positive sample underwent WGS and were compared to publicly available NTS genomes from UK human cases. NTS was isolated from chicken (9.6 %), prawn (2.9 %) and pork (1.3 %) samples and included 14 serovars, of which Salmonella Infantis and Salmonella Enteritidis were the most common. The S. Enteritidis isolates were only isolated from imported chicken. No antimicrobial resistance determinants were found in prawn isolates, whilst 5.1 % of chicken and 0.64 % of pork samples contained multi-drug resistant NTS. The maximum number of pairwise core non-recombinant single nucleotide polymorphisms (SNPs) amongst isolates from the same sample was used to measure diversity and most samples had a median of two SNPs (range: 0-251). NTS isolates that were within five SNPs to clinical UK isolates belonged to specific serovars: S. Enteritidis and S. Infantis (chicken), and S. I 4,[5],12:i- (pork and chicken). Most NTS isolates that were closely related to human-derived isolates were obtained from imported chicken, but further epidemiological data are required to assess definitively the probable source of the human cases. Continued WGS surveillance of Salmonella on retail food involving multiple isolates from each sample is necessary to capture the diversity of Salmonella and determine the relative importance of different sources of human disease.

Repeated cross-sectional study identifies differing risk factors associated with microbial contamination in common food products in the United Kingdom.

All foods carry microbes, many of which are harmless, but foods can also carry pathogens and/or microbial indicators of contamination. Limited information exists on the co-occurrence of microbes of food safety concern and the factors associated with their presence. Here, a population-based repeated cross-sectional design was used to determine the prevalence and co-occurrence of Escherichia coli, Klebsiella spp., Salmonella spp. and Vibrio spp. in key food commodities - chicken, pork, prawns, salmon and leafy greens. Prevalence in 1,369 food samples for these four target bacterial genera/species varied, while 25.6% of all samples had at least two of the target bacteria and eight different combinations of bacteria were observed as co-occurrence profiles in raw prawns. Imported frozen chicken was 6.4 times more likely to contain Salmonella than domestic chicken, and imported salmon was 5.5 times more likely to be contaminated with E. coli. Seasonality was significantly associated with E. coli and Klebsiella spp. contamination in leafy greens, with higher detection in summer and autumn. Moreover, the odds of Klebsiella spp. contamination were higher in summer in chicken and pork samples. These results provide insight on the bacterial species present on foods at retail, and identify factors associated with the presence of individual bacteria, which are highly relevant for food safety risk assessments and the design of surveillance programmes.

Determination and quantification of microbial communities and antimicrobial resistance on food through host DNA-depleted metagenomics.

Food products carry bacteria unless specifically sterilised. These bacteria can be pathogenic, commensal or associated with food spoilage, and may also be resistant to antimicrobials. Current methods for detecting bacteria on food rely on culturing for specific bacteria, a time-consuming process, or 16S rRNA metabarcoding that can identify different taxa but not their genetic content. Directly sequencing metagenomes of food is inefficient as its own DNA vastly outnumbers the bacterial DNA present. We optimised host DNA depletion enabling efficient sequencing of food microbiota, thereby increasing the proportion of non-host DNA sequenced 13-fold (mean; range: 1.3-40-fold) compared to untreated samples. The method performed best on chicken, pork and leafy green samples which had high mean prokaryotic read proportions post-depletion (0.64, 0.74 and 0.74, respectively), with lower mean prokaryotic read proportions in salmon (0.50) and prawn samples (0.19). We show that bacterial compositions and concentrations of antimicrobial resistance (AMR) genes differed by food type, and that salmon metagenomes were influenced by the production/harvesting method. The approach described in this study is an efficient and effective method of identifying and quantifying the predominant bacteria and AMR genes on food.

Comparative genomics of Campylobacter jejuni from clinical campylobacteriosis stool specimens.

BACKGROUND: Campylobacter jejuni is a pervasive pathogen of major public health concern with a complex ecology requiring accurate and informative approaches to define pathogen diversity during outbreak investigations. Source attribution analysis may be confounded if the genetic diversity of a C. jejuni population is not adequately captured in a single specimen. The aim of this study was to determine the genomic diversity of C. jejuni within individual stool specimens from four campylobacteriosis patients. Direct plating and pre-culture filtration of one stool specimen per patient was used to culture multiple isolates per stool specimen. Whole genome sequencing and pangenome level analysis were used to investigate genomic diversity of C. jejuni within a patient. RESULTS: A total 92 C. jejuni isolates were recovered from four patients presenting with gastroenteritis. The number of isolates ranged from 13 to 30 per patient stool. Three patients yielded a single C. jejuni multilocus sequence type: ST-21 (n = 26, patient 4), ST-61 (n = 30, patient 1) and ST-2066 (n = 23, patient 2). Patient 3 was infected with two different sequence types [ST-51 (n = 12) and ST-354 (n = 1)]. Isolates belonging to the same sequence type from the same patient specimen shared 12-43 core non-recombinant SNPs and 0-20 frameshifts with each other, and the pangenomes of each sequence type consisted of 1406-1491 core genes and 231-264 accessory genes. However, neither the mutation nor the accessory genes were connected to a specific functional gene category. CONCLUSIONS: Our findings show that the C. jejuni population recovered from an individual patient's stool are genetically diverse even within the same ST and may have shared common ancestors before specimens were obtained. The population is unlikely to have evolved from a single isolate at the time point of initial patient infection, leading us to conclude that patients were likely infected with a heterogeneous C. jejuni population. The diversity of the C. jejuni population found within individual stool specimens can inform future methodological approaches to attribution and outbreak investigations.

Mobility of antimicrobial resistance across serovars and disease presentations in non-typhoidal Salmonella from animals and humans in Vietnam.

Non-typhoidal Salmonella (NTS) is a major cause of bacterial enterocolitis globally but also causes invasive bloodstream infections. Antimicrobial resistance (AMR) hampers the treatment of these infections and understanding how AMR spreads between NTS may help in developing effective strategies. We investigated NTS isolates associated with invasive disease, diarrhoeal disease and asymptomatic carriage in animals and humans from Vietnam. Isolates included multiple serovars and both common and rare phenotypic AMR profiles; long- and short-read sequencing was used to investigate the genetic mechanisms and genomic backgrounds associated with phenotypic AMR profiles. We demonstrate concordance between most AMR genotypes and phenotypes but identified large genotypic diversity in clinically relevant phenotypes and the high mobility potential of AMR genes (ARGs) in this setting. We found that 84 % of ARGs identified were located on plasmids, most commonly those containing IncHI1A_1 and IncHI1B(R27)_1_R27 replicons (33%), and those containing IncHI2_1 and IncHI2A_1 replicons (31%). The vast majority (95%) of ARGS were found within 10 kbp of IS6/IS26 elements, which provide plasmids with a mechanism to exchange ARGs between plasmids and other parts of the genome. Whole genome sequencing with targeted long-read sequencing applied in a One Health context identified a comparatively limited number of insertion sequences and plasmid replicons associated with AMR. Therefore, in the context of NTS from Vietnam and likely for other settings as well, the mechanisms by which ARGs move contribute to a more successful AMR profile than the specific ARGs, facilitating the adaptation of bacteria to different environments or selection pressures.

Extensive epigenetic modification with large-scale chromosomal and plasmid recombination characterise the Legionella longbeachae serogroup 1 genome.

Legionella longbeachae is an environmental bacterium that is the most clinically significant Legionella species in New Zealand (NZ), causing around two-thirds of all notified cases of Legionnaires' disease. Here we report the sequencing and analysis of the geo-temporal genetic diversity of 54 L. longbeachae serogroup 1 (sg1) clinical isolates, derived from cases from around NZ over a 22-year period, including one complete genome and its associated methylome. The 54 sg1 isolates belonged to two main clades that last shared a common ancestor between 95 BCE and 1694 CE. There was diversity at the genome-structural level, with large-scale arrangements occurring in some regions of the chromosome and evidence of extensive chromosomal and plasmid recombination. This includes the presence of plasmids derived from recombination and horizontal gene transfer between various Legionella species, indicating there has been both intra- and inter-species gene flow. However, because similar plasmids were found among isolates within each clade, plasmid recombination events may pre-empt the emergence of new L. longbeachae strains. Our complete NZ reference genome consisted of a 4.1 Mb chromosome and a 108 kb plasmid. The genome was highly methylated with two known epigenetic modifications, m4C and m6A, occurring in particular sequence motifs within the genome.

Tatajuba: exploring the distribution of homopolymer tracts.

Length variation of homopolymeric tracts, which induces phase variation, is known to regulate gene expression leading to phenotypic variation in a wide range of bacterial species. There is no specialized bioinformatics software which can, at scale, exhaustively explore and describe these features from sequencing data. Identifying these is non-trivial as sequencing and bioinformatics methods are prone to introducing artefacts when presented with homopolymeric tracts due to the decreased base diversity. We present tatajuba, which can automatically identify potential homopolymeric tracts and help predict their putative phenotypic impact, allowing for rapid investigation. We use it to detect all tracts in two separate datasets, one of Campylobacter jejuni and one of three Bordetella species, and to highlight those tracts that are polymorphic across samples. With this we confirm homopolymer tract variation with phenotypic impact found in previous studies and additionally find many more with potential variability. The software is written in C and is available under the open source licence GNU GPLv3.

Genomic adaptations of Campylobacter jejuni to long-term human colonization.

BACKGROUND: Campylobacter is a genus of bacteria that has been isolated from the gastrointestinal tract of humans and animals, and the environments they inhabit around the world. Campylobacter adapt to new environments by changes in their gene content and expression, but little is known about how they adapt to long-term human colonization. In this study, the genomes of 31 isolates from a New Zealand patient and 22 isolates from a United Kingdom patient belonging to Campylobacter jejuni sequence type 45 (ST45) were compared with 209 ST45 genomes from other sources to identify the mechanisms by which Campylobacter adapts to long-term human colonization. In addition, the New Zealand patient had their microbiota investigated using 16S rRNA metabarcoding, and their level of inflammation and immunosuppression analyzed using biochemical tests, to determine how Campylobacter adapts to a changing gastrointestinal tract. RESULTS: There was some evidence that long-term colonization led to genome degradation, but more evidence that Campylobacter adapted through the accumulation of non-synonymous single nucleotide polymorphisms (SNPs) and frameshifts in genes involved in cell motility, signal transduction and the major outer membrane protein (MOMP). The New Zealand patient also displayed considerable variation in their microbiome, inflammation and immunosuppression over five months, and the Campylobacter collected from this patient could be divided into two subpopulations, the proportion of which correlated with the amount of gastrointestinal inflammation. CONCLUSIONS: This study demonstrates how genomics, phylogenetics, 16S rRNA metabarcoding and biochemical markers can provide insight into how Campylobacter adapts to changing environments within human hosts. This study also demonstrates that long-term human colonization selects for changes in Campylobacter genes involved in cell motility, signal transduction and the MOMP; and that genetically distinct subpopulations of Campylobacter evolve to adapt to the changing gastrointestinal environment.

Genomic and phenotypic comparison of two Salmonella Typhimurium strains responsible for consecutive salmonellosis outbreaks in New Zealand.

Salmonella enterica serovar Typhimurium DT160 was the predominant cause of notified human salmonellosis cases in New Zealand from 2000 to 2010, before it was superseded by another S. Typhimurium strain, DT56 variant (DT56v). Whole genome sequencing and phenotypic testing were used to compare 109 DT160 isolates with eight DT56v isolates from New Zealand animal and human sources. Phylogenetic analysis provided evidence that DT160 and DT56v strains were distantly related with an estimated date of common ancestor between 1769 and 1821. The strains replicated at different rates but had similar antimicrobial susceptibility profiles. Both strains were resistant to the phage expressed from the chromosome of the other strain, which may have contributed to the emergence of DT56v. DT160 contained the pSLT virulence plasmid, and the sseJ and sseK2 genes that may have contributed to the higher reported prevalence compared to DT56v. A linear pBSSB1-family plasmid was also found in one of the DT56v isolates, but there was no evidence that this plasmid affected bacterial replication or antimicrobial susceptibility. One of the DT56v isolates was also sequenced using long-read technology and found to contain an uncommon chromosome arrangement for a Typhimurium isolate. This study demonstrates how comparative genomics and phenotypic testing can help identify strain-specific elements and factors that may have influenced the emergence and supersession of bacterial strains of public health importance.

Whole genome sequencing reveals great diversity of Vibrio spp in prawns at retail.

Consumption of prawns as a protein source has been on the rise worldwide with seafood identified as the predominant attributable source of human vibriosis. However, surveillance of non-cholera Vibrio is limited both in public health and in food. Using a population- and market share-weighted study design, 211 prawn samples were collected and cultured for Vibrio spp. Contamination was detected in 46 % of samples, and multiple diverse Vibrio isolates were obtained from 34 % of positive samples. Whole genome sequencing (WGS) and phylogenetic analysis illustrated a comprehensive view of Vibrio species diversity in prawns available at retail, with no known pathogenicity markers identified in Vibrio parahaemolyticus and V. cholerae. Antimicrobial resistance genes were found in 77 % of isolates, and 12 % carried genes conferring resistance to three or more drug classes. Resistance genes were found predominantly in V. parahaemolyticus, though multiple resistance genes were also identified in V. cholerae and V. vulnificus. This study highlights the large diversity in Vibrio derived from prawns at retail, even within a single sample. Although there was little evidence in this study that prawns are a major source of vibriosis in the UK, surveillance of non-cholera Vibrio is very limited. This study illustrates the value of expanding WGS surveillance efforts of non-cholera Vibrios in the food chain to identify critical control points for food safety through the production system and to determine the full extent of the public health impact.

Campylobacter novaezeelandiae sp. nov., isolated from birds and water in New Zealand.

Six isolates of Campylobacter with similar non-standard colonial morphologies were identified during studies isolating Campylobacter from bird faeces and rivers in New Zealand. Genomic (16S rRNA gene sequencing and whole genome analysis) and phenotypic (MALDI-TOF analysis and conventional biochemical tests) showed that the isolates form a monophyletic clade with genetic relationships to Campylobacter coli/Campylobacter jejuni and Campylobacter peloridis/Campylobacter amoricus. They may be distinguished from other Campylobacter by their MALDI-TOF spectral pattern, their florid α-haemolysis, their ability to grow anaerobically at 37 °C, and on 2 % NaCl nutrient agar, and their lack of hippuricase. This study shows that these isolates represent a novel species within the genus Campylobacter for which the name Campylobacter novaezeelandiae sp. nov. is proposed. The presence of C. novaezeelandiae in water may be a confounder for freshwater microbial risk assessment as they may not be pathogenic for humans. The type strain is B423bT (=NZRM 4741T=ATCC TSD-167T).

Whole-Genome Sequencing of Salmonella Mississippi and Typhimurium Definitive Type 160, Australia and New Zealand.

We used phylogenomic and risk factor data on isolates of Salmonella enterica serovars Mississippi and Typhimurium definitive type 160 (DT160) collected from human, animal, and environmental sources to elucidate their epidemiology and disease reservoirs in Australia and New Zealand. Sequence data suggested wild birds as a likely reservoir for DT160; animal and environmental sources varied more for Salmonella Mississippi than for Salmonella Typhimurium. Australia and New Zealand isolates sat in distinct clades for both serovars; the median single-nucleotide polymorphism distance for DT160 was 29 (range 8-66) and for Salmonella Mississippi, 619 (range 565-737). Phylogenomic data identified plausible sources of human infection from wildlife and environmental reservoirs and provided evidence supporting New Zealand-acquired DT160 in a group of travelers returning to Australia. Wider use of real-time whole-genome sequencing in new locations and for other serovars may identify sources and routes of transmission, thereby aiding prevention and control.

Investigation of the validity of two Bayesian ancestral state reconstruction models for estimating Salmonella transmission during outbreaks.

Ancestral state reconstruction models use genetic data to characterize a group of organisms' common ancestor. These models have been applied to salmonellosis outbreaks to estimate the number of transmissions between different animal species that share similar geographical locations, with animal host as the state. However, as far as we are aware, no studies have validated these models for outbreak analysis. In this study, salmonellosis outbreaks were simulated using a stochastic Susceptible-Infected-Recovered model, and the host population and transmission parameters of these simulated outbreaks were estimated using Bayesian ancestral state reconstruction models (discrete trait analysis (DTA) and structured coalescent (SC)). These models were unable to accurately estimate the number of transmissions between the host populations or the amount of time spent in each host population. The DTA model was inaccurate because it assumed the number of isolates sampled from each host population was proportional to the number of individuals infected within each host population. The SC model was inaccurate possibly because it assumed that each host population's effective population size was constant over the course of the simulated outbreaks. This study highlights the need for phylodynamic models that can take into consideration factors that influence the characteristics and behavior of outbreaks, e.g. changing effective population sizes, variation in infectious periods, intra-population transmissions, and disproportionate sampling of infected individuals.

Draft Whole-Genome Sequences of Three Isolates of a Novel Strain of a Campylobacter sp. Isolated from New Zealand Birds and Water.

Campylobacter spp. are frequently found associated with the avian intestinal tract. Most are commensals, but some can cause human campylobacteriosis. Here, we report the draft genome sequences of three strains of a novel Campylobacter sp. isolated from urban birds and a rural river in New Zealand.

Use of Genomics to Investigate Historical Importation of Shiga Toxin-Producing Escherichia coli Serogroup O26 and Nontoxigenic Variants into New Zealand.

Shiga toxin-producing Escherichia coli serogroup O26 is an important public health pathogen. Phylogenetic bacterial lineages in a country can be associated with the level and timing of international imports of live cattle, the main reservoir. We sequenced the genomes of 152 E. coli O26 isolates from New Zealand and compared them with 252 E. coli O26 genomes from 14 other countries. Gene variation among isolates from humans, animals, and food was strongly associated with country of origin and stx toxin profile but not isolation source. Time of origin estimates indicate serogroup O26 sequence type 21 was introduced at least 3 times into New Zealand from the 1920s to the 1980s, whereas nonvirulent O26 sequence type 29 strains were introduced during the early 2000s. New Zealand's remarkably fewer introductions of Shiga toxin-producing Escherichia coli O26 compared with other countries (such as Japan) might be related to patterns of trade in live cattle.

Long-term Colonization by Campylobacter jejuni Within a Human Host: Evolution, Antimicrobial Resistance, and Adaptation.

Background: Campylobacteriosis is inflammation of the gastrointestinal tract as a result of Campylobacter infection. Most campylobacteriosis cases are acute and self-limiting, with Campylobacter excretion ceasing a few weeks after symptoms cease. We identified a patient with fecal specimens positive for Campylobacter jejuni (ST45) intermittently during a 10-year period. Methods: Sixteen Campylobacter isolates were collected from the patient during 2006-2016. The isolates' genomes were sequenced to determine their relatedness, and their antimicrobial susceptibility patterns and motility were measured to determine the effects of antibiotic therapy and long-term excretion on the Campylobacter population. Results: Phylogenetic analyses estimated that the isolates shared a date of common ancestor between 1998 and 2006, coinciding with the onset of symptoms for the patient. Genomic analysis identified selection for changes in motility, and antimicrobial susceptibility testing suggested that the Campylobacter population developed resistance to several antibiotics coinciding with periods of antibiotic therapy. Conclusions: The patient was consistently colonized with organisms from a Campylobacter population that adapted to the internal environment of the patient. Genomic and phylogenetic analyses can give insight into a patient's infection history and the effect of antimicrobial treatment on Campylobacter populations in this unusual situation of long-term colonization of an individual.

Genomic Analysis of Salmonella enterica Serovar Typhimurium DT160 Associated with a 14-Year Outbreak, New Zealand, 1998-2012.

During 1998-2012, an extended outbreak of Salmonella enterica serovar Typhimurium definitive type 160 (DT160) affected >3,000 humans and killed wild birds in New Zealand. However, the relationship between DT160 within these 2 host groups and the origin of the outbreak are unknown. Whole-genome sequencing was used to compare 109 Salmonella Typhimurium DT160 isolates from sources throughout New Zealand. We provide evidence that DT160 was introduced into New Zealand around 1997 and rapidly propagated throughout the country, becoming more genetically diverse over time. The genetic heterogeneity was evenly distributed across multiple predicted functional protein groups, and we found no evidence of host group differentiation between isolates collected from human, poultry, bovid, and wild bird sources, indicating ongoing transmission between these host groups. Our findings demonstrate how a comparative genomic approach can be used to gain insight into outbreaks, disease transmission, and the evolution of a multihost pathogen after a probable point-source introduction.