Genomic Diversity of Campylobacter lari Group Isolates from Europe and Australia in a One Health Context.

Members of the Campylobacter lari group are causative agents of human gastroenteritis and are frequently found in shellfish, marine waters, shorebirds, and marine mammals. Within a One Health context, we used comparative genomics to characterize isolates from a diverse range of sources and geographical locations within Europe and Australia and assess possible transmission of food, animal, and environmental isolates to the human host. A total of 158 C. lari isolates from Australia, Denmark, France, and Germany, which included 82 isolates from human stool and blood, 12 from food, 14 from domestic animal, 19 from waterbirds, and 31 from the environment were analyzed. Genome-wide analysis of the genetic diversity, virulence, and antimicrobial resistance (AMR) traits was carried-out. Most of the isolates belonged to C. lari subsp. lari (Cll; 98, 62.0%), while C. lari subsp. concheus and C. lari urease-positive thermotolerant Campylobacter (UPTC) were represented by 12 (7.6%) and 15 (9.5%) isolates, respectively. Furthermore, 33 (20.9%) isolates were not assigned a subspecies and were thus attributed to distant Campylobacter spp. clades. Whole-genome sequence-derived multilocus sequence typing (MLST) and core-genome MLST (cgMLST) analyses revealed a high genetic diversity with 97 sequence types (STs), including 60 novel STs and 14 cgMLST clusters (≤10 allele differences), respectively. The most prevalent STs were ST-21, ST-70, ST-24, and ST-58 (accounting for 13.3%, 4.4%, 3.8%, and 3.2% of isolates, respectively). A high prevalence of the 125 examined virulence-related loci (from 76.8 to 98.4% per isolate) was observed, especially in Cll isolates, suggesting a probable human pathogenicity of these strains. IMPORTANCE Currently, relatedness between bacterial isolates impacting human health is easily monitored by molecular typing methods. These approaches rely on discrete loci or whole-genome sequence (WGS) analyses. Campylobacter lari is an emergent human pathogen isolated from diverse ecological niches, including fecal material from humans and animals, aquatic environments, and seafood. The presence of C. lari in such diverse sources underlines the importance of adopting an integrated One Health approach in studying C. lari population structure for conducting epidemiological risk assessment. This retrospective study presents a comparative genomics analysis of C. lari isolates retrieved from two different continents (Europe and Australia) and from different sources (human, domestic animals, waterbirds, food, and environment). It was designed to improve knowledge regarding C. lari ecology and pathogenicity, important for developing effective surveillance and disease prevention strategies.

Emergence and global spread of Listeria monocytogenes main clinical clonal complex.

The bacterial foodborne pathogen Listeria monocytogenes clonal complex 1 (Lm-CC1) is the most prevalent clonal group associated with human listeriosis and is strongly associated with cattle and dairy products. Here, we analyze 2021 isolates collected from 40 countries, covering Lm-CC1 first isolation to present days, to define its evolutionary history and population dynamics. We show that Lm-CC1 spread worldwide from North America following the Industrial Revolution through two waves of expansion, coinciding with the transatlantic livestock trade in the second half of the 19th century and the rapid growth of cattle farming and food industrialization in the 20th century. In sharp contrast to its global spread over the past century, transmission chains are now mostly local, with limited inter- and intra-country spread. This study provides an unprecedented insight into L. monocytogenes phylogeography and population dynamics and highlights the importance of genome analyses for a better control of pathogen transmission.

Identification of Novel Phage Resistance Mechanisms in Campylobacter jejuni by Comparative Genomics.

Phages infecting Campylobacter jejuni are considered a promising intervention strategy at broiler farms, yet phage sensitivity of naturally occurring poultry isolates is not well studied. Here, we investigated phage sensitivity and identified resistance mechanisms of C. jejuni strains originating from Danish broilers belonging to the most prevalent MLST (ST) types. Determining plaque formation of 51 phages belonging to Fletchervirus or Firehammervirus showed that 21 out of 31 C. jejuni strains were susceptible to at least one phage. While C. jejuni ST-21 strains encoded the common phase variable O-methyl phosphoramidate (MeOPN) receptor of the Fletchervirus and were only infected by these phages, ST-45 strains did not encode this receptor and were exclusively infected by Firehammervirus phages. To identify internal phage resistance mechanism in ST-21 strains, we performed comparative genomics of two strains, CAMSA2002 sensitive to almost all Fletchervirus phages and CAMSA2038, resistant to all 51 phages. The strains encoded diverse clustered regularly interspaced short palindromic repeats (CRISPR) spacers but none matched the tested phages. Sequence divergence was also observed in a predicted SspE homolog and putative restriction modification systems including a methyl-specific McrBC endonuclease. Furthermore, when mcrB was deleted, CAMSA2038 became sensitive to 17 out of 43 phages, three being Firehammervirus phages that otherwise did not infect any ST-21 strains. Yet, 16 phages demonstrated significantly lower efficiencies of plating on the mcrB mutant suggesting additional resistance mechanism still restricting phage propagation in CAMSA2038. Thus, our work demonstrates that C. jejuni isolates originating from broilers may have acquired several resistance mechanisms to successfully prevent phage infection in their natural habitat.

Whole-Genome Sequencing to Detect Numerous Campylobacter jejuni Outbreaks and Match Patient Isolates to Sources, Denmark, 2015-2017.

In industrialized countries, the leading cause of bacterial gastroenteritis is Campylobacter jejuni. However, outbreaks are rarely reported, which may reflect limitations of surveillance, for which molecular typing is not routinely performed. To determine the frequency of genetic clusters among patients and to find links to concurrent isolates from poultry meat, broiler chickens, cattle, pigs, and dogs, we performed whole-genome sequencing on 1,509 C. jejuni isolates from 774 patients and 735 food or animal sources in Denmark during 2015-2017. We found numerous clusters; 366/774 (47.3%) clinical isolates formed 104 clusters of >2 isolates. A total of 41 patient clusters representing 199/366 (54%) patients matched a potential source, primarily domestic chickens/broilers. This study revealed serial outbreaks and numerous matches to concurrent food and animal isolates and highlighted the potential of whole-genome sequencing for improving routine surveillance of C. jejuni by enhancing outbreak detection, source tracing, and potentially prevention of human infections.

WGS based study of the population structure of Salmonella enterica serovar Infantis.

BACKGROUND: Salmonella Infantis (S. Infantis) is one of the most frequent Salmonella serovars isolated from human cases of salmonellosis and the most detected serovar from animal and food sources in Europe. The serovar is commonly associated with poultry and there is increasing concern over multidrug resistant clones spreading worldwide, as the dominating clones are characterized by presence of large plasmids carrying multiple resistance genes. Increasing the knowledge of the S. Infantis population and evolution is important for understanding and preventing further spread. In this study, we analysed a collection of strains representing different decades, sources and geographic locations. We analysed the population structure and the accessory genome, in particular we identified prophages with a view to understand the role of prophages in relation to the evolution of this serovar. RESULTS: We sequenced a global collection of 100 S. Infantis strains. A core-genome SNP analysis separated five strains in e-Burst Group (eBG) 297 with a long branch. The remaining strains, all in eBG31, were divided into three lineages that were estimated to have separated approximately 150 years ago. One lineage contained the vast majority of strains. In five of six clusters, no obvious correlation with source or geographical locations was seen. However, one cluster contained mostly strains from human and avian sources, indicating a clone with preference for these sources. The majority of strains within this cluster harboured a pESI-like plasmid with multiple resistance genes. Another lineage contained three genetic clusters with more rarely isolated strains of mainly animal origin, possibly less sampled or less infectious clones. Conserved prophages were identified in all strains, likely representing bacteriophages which integrated into the chromosome of a common ancestor to S. Infantis. We also saw that some prophages were specific to clusters and were probably introduced when the clusters were formed. CONCLUSIONS: This study analysed a global S. Infantis population and described its genetic structure. We hypothesize that the population has evolved in three separate lineages, with one more successfully emerging lineage. We furthermore detected conserved prophages present in the entire population and cluster specific prophages, which probably shaped the population structure.

Cross-border outbreak of listeriosis caused by cold-smoked salmon, revealed by integrated surveillance and whole genome sequencing (WGS), Denmark and France, 2015 to 2017.

In August 2017, an outbreak of six listeriosis cases in Denmark was traced to cold-smoked salmon, using epidemiological investigations and whole-genome sequencing (WGS) analyses. Exchange of genome sequences allowed identification in France of a food isolate from a salmon-derived product and a human isolate from 2016 within the same cgMLST cluster as the Danish isolates (L2-SL8-ST8-CT771). The salmon product came from a third European Union country. WGS can rapidly link human cases and food isolates across Europe.

Plasmid-borne colistin resistance gene mcr-3 in Salmonella isolates from human infections, Denmark, 2009-17.

This report describes one Salmonella isolate harbouring both mcr-1 and mcr-3. We also found nine other Salmonella isolates positive for the plasmid-borne colistin resistance gene, mcr-3. The strains were isolated from patients in Denmark between 2009 and 2017 and five of the patients had travelled to Asia. In addition to mcr-3, all strains were found positive for blaTEM-1, strA, strB, sul2 and tet(A) or tet(B), and most strains were positive for blaCTX-M-55 and qnrS.

Whole genome-based population biology and epidemiological surveillance of Listeria monocytogenes.

Listeria monocytogenes (Lm) is a major human foodborne pathogen. Numerous Lm outbreaks have been reported worldwide and associated with a high case fatality rate, reinforcing the need for strongly coordinated surveillance and outbreak control. We developed a universally applicable genome-wide strain genotyping approach and investigated the population diversity of Lm using 1,696 isolates from diverse sources and geographical locations. We define, with unprecedented precision, the population structure of Lm, demonstrate the occurrence of international circulation of strains and reveal the extent of heterogeneity in virulence and stress resistance genomic features among clinical and food isolates. Using historical isolates, we show that the evolutionary rate of Lm from lineage I and lineage II is low (∼2.5 × 10-7 substitutions per site per year, as inferred from the core genome) and that major sublineages (corresponding to so-called 'epidemic clones') are estimated to be at least 50-150 years old. This work demonstrates the urgent need to monitor Lm strains at the global level and provides the unified approach needed for global harmonization of Lm genome-based typing and population biology.

Application of Molecular Typing Results in Source Attribution Models: The Case of Multiple Locus Variable Number Tandem Repeat Analysis (MLVA) of Salmonella Isolates Obtained from Integrated Surveillance in Denmark.

Salmonella is an important cause of bacterial foodborne infections in Denmark. To identify the main animal-food sources of human salmonellosis, risk managers have relied on a routine application of a microbial subtyping-based source attribution model since 1995. In 2013, multiple locus variable number tandem repeat analysis (MLVA) substituted phage typing as the subtyping method for surveillance of S. Enteritidis and S. Typhimurium isolated from animals, food, and humans in Denmark. The purpose of this study was to develop a modeling approach applying a combination of serovars, MLVA types, and antibiotic resistance profiles for the Salmonella source attribution, and assess the utility of the results for the food safety decisionmakers. Full and simplified MLVA schemes from surveillance data were tested, and model fit and consistency of results were assessed using statistical measures. We conclude that loci schemes STTR5/STTR10/STTR3 for S. Typhimurium and SE9/SE5/SE2/SE1/SE3 for S. Enteritidis can be used in microbial subtyping-based source attribution models. Based on the results, we discuss that an adjustment of the discriminatory level of the subtyping method applied often will be required to fit the purpose of the study and the available data. The issues discussed are also considered highly relevant when applying, e.g., extended multi-locus sequence typing or next-generation sequencing techniques.

Real-time whole-genome sequencing for routine typing, surveillance, and outbreak detection of verotoxigenic Escherichia coli.

Fast and accurate identification and typing of pathogens are essential for effective surveillance and outbreak detection. The current routine procedure is based on a variety of techniques, making the procedure laborious, time-consuming, and expensive. With whole-genome sequencing (WGS) becoming cheaper, it has huge potential in both diagnostics and routine surveillance. The aim of this study was to perform a real-time evaluation of WGS for routine typing and surveillance of verocytotoxin-producing Escherichia coli (VTEC). In Denmark, the Statens Serum Institut (SSI) routinely receives all suspected VTEC isolates. During a 7-week period in the fall of 2012, all incoming isolates were concurrently subjected to WGS using IonTorrent PGM. Real-time bioinformatics analysis was performed using web-tools (www.genomicepidemiology.org) for species determination, multilocus sequence type (MLST) typing, and determination of phylogenetic relationship, and a specific VirulenceFinder for detection of E. coli virulence genes was developed as part of this study. In total, 46 suspected VTEC isolates were characterized in parallel during the study. VirulenceFinder proved successful in detecting virulence genes included in routine typing, explicitly verocytotoxin 1 (vtx1), verocytotoxin 2 (vtx2), and intimin (eae), and also detected additional virulence genes. VirulenceFinder is also a robust method for assigning verocytotoxin (vtx) subtypes. A real-time clustering of isolates in agreement with the epidemiology was established from WGS, enabling discrimination between sporadic and outbreak isolates. Overall, WGS typing produced results faster and at a lower cost than the current routine. Therefore, WGS typing is a superior alternative to conventional typing strategies. This approach may also be applied to typing and surveillance of other pathogens.

Evaluation of whole genome sequencing for outbreak detection of Salmonella enterica.

Salmonella enterica is a common cause of minor and large food borne outbreaks. To achieve successful and nearly 'real-time' monitoring and identification of outbreaks, reliable sub-typing is essential. Whole genome sequencing (WGS) shows great promises for using as a routine epidemiological typing tool. Here we evaluate WGS for typing of S. Typhimurium including different approaches for analyzing and comparing the data. A collection of 34 S. Typhimurium isolates was sequenced. This consisted of 18 isolates from six outbreaks and 16 epidemiologically unrelated background strains. In addition, 8 S. Enteritidis and 5 S. Derby were also sequenced and used for comparison. A number of different bioinformatics approaches were applied on the data; including pan-genome tree, k-mer tree, nucleotide difference tree and SNP tree. The outcome of each approach was evaluated in relation to the association of the isolates to specific outbreaks. The pan-genome tree clustered 65% of the S. Typhimurium isolates according to the pre-defined epidemiology, the k-mer tree 88%, the nucleotide difference tree 100% and the SNP tree 100% of the strains within S. Typhimurium. The resulting outcome of the four phylogenetic analyses were also compared to PFGE revealing that WGS typing achieved the greater performance than the traditional method. In conclusion, for S. Typhimurium, SNP analysis and nucleotide difference approach of WGS data seem to be the superior methods for epidemiological typing compared to other phylogenetic analytic approaches that may be used on WGS. These approaches were also superior to the more classical typing method, PFGE. Our study also indicates that WGS alone is insufficient to determine whether strains are related or un-related to outbreaks. This still requires the combination of epidemiological data and whole genome sequencing results.

Characterization of water and wildlife strains as a subgroup of Campylobacter jejuni using DNA microarrays.

Campylobacter jejuni is the leading cause of human bacterial gastroenteritis worldwide, but source attribution of the organism is difficult. Previously, DNA microarrays were used to investigate isolate source, which suggested a non-livestock source of infection. In this study we analysed the genome content of 162 clinical, livestock and water and wildlife (WW) associated isolates combined with the previous study. Isolates were grouped by genotypes into nine clusters (C1 to C9). Multilocus sequence typing (MLST) data demonstrated that livestock associated clonal complexes dominated clusters C1-C6. The majority of WW isolates were present in the C9 cluster. Analysis of previously reported genomic variable regions demonstrated that these regions were linked to specific clusters. Two novel variable regions were identified. A six gene multiplex PCR (mPCR) assay, designed to effectively differentiated strains into clusters, was validated with 30 isolates. A further five WW isolates were tested by mPCR and were assigned to the C7-C9 group of clusters. The predictive mPCR test could be used to indicate if a clinical case has come from domesticated or WW sources. Our findings provide further evidence that WW C. jejuni subtypes show niche adaptation and may be important in causing human infection.

Standardization and international multicenter validation of a PulseNet pulsed-field gel electrophoresis protocol for subtyping Shigella flexneri isolates.

Shigella flexneri is one of the agents most frequently linked to diarrheal illness in developing countries and often causes outbreaks in settings with poor hygiene or sanitary conditions. Travel is one of the means by which S. flexneri can be imported into developed countries, where this pathogen is not commonly seen. A robust and discriminatory subtyping method is needed for the surveillance of S. flexneri locally and regionally, and to aid in the detection and investigation of outbreaks. The PulseNet International network utilizes standardized pulsed-field gel electrophoresis (PFGE) protocols to carry out laboratory-based surveillance of foodborne pathogens in combination with epidemiologic data. A multicenter validation was carried out in nine PulseNet laboratories located in North and South America, Europe, and Asia, and it demonstrated that a new protocol is highly robust and reproducible for subtyping of S. flexneri. This protocol, already approved for PulseNet laboratories, applies NotI and XbaI as primary and secondary restriction enzymes, respectively, under electrophoresis conditions of initial switch time of 5 s to final switch time of 35 s, at 6 volts/cm.

Molecular characterization of Salmonella Typhimurium highly successful outbreak strains.

Three large clusters of Salmonella Typhimurium infections in Denmark in 2008 and 2009 were defined by multilocus variable number of tandem repeat analysis (MLVA). One of these proved to be the hereto largest Danish cluster of salmonellosis with 1446 cases. Two smaller clusters with a total of 197 and 89 cases, respectively, were seen concurrently. These clusters shared epidemiological characteristics such as age distribution, geography, and time. To investigate the possible genetic relationship between the cluster strains, these were further characterized by phage typing, pulsed-field gel electrophoresis, and Optical Mapping. Although the MLVA method proved robust and well-performing in detecting and defining clusters, the employment of a second typing method detected an additional fourth cluster among the isolates. The cluster strains were stable throughout the almost 2-year period, even though we detected changes in three of five MLVA loci in a small fraction of isolates. These changes were mainly due to the gain or loss of single repeats. Optical Mapping of the large cluster strain indicated no increased content of virulence genes; however, Optical Mapping did reveal a large insert, a probable prophage, in the main cluster. This probable prophage may give the cluster strain a competitive advantage. The molecular methods employed suggested that the four clusters represented four distinct strains, although they seemed to be epidemiologically linked and shared genotypic characteristics.

Association between phylogeny, virulence potential and serovars of Salmonella enterica.

Salmonella enterica subsp. enterica is one of the leading causes of zoonotic food-borne disease worldwide. The consequence of these infections is a serious impact on economics of the society in the form of lost productivity and expenses for medical care. The objective of this study was to analyze the difference in genomic content between selected serovars, especially the content of pathogenicity genes and this was done with a DNA microarray. Furthermore, we investigated the phylogenetic relationship between serovars using multilocus sequence typing (MLST). We chose serovars Typhimurium and Enteritidis as they are responsible for 75% of human infections in Europe. Additionally, we included serovars Derby, Dublin, Saintpaul, 4,5,12:i:-, Java and 4,5,12:b:- which are suspected to have different degrees of virulence to humans. MLST analysis clustered strains according to serovar with the exception of Java and Derby. DNA microarray clustered strains according to serovar and serogroup except for serovar 4,5,12:b:-. Differences in content of pathogenicity related genes between serovars with various host preferences and virulence towards humans were not observed. However, our strains from the supposedly less virulent serovar Derby lacked a combination of genes important for virulence. It might be speculated that other serovars can sustain their pathogenicity lacking one or two of these genes, whereas lack of many virulence genes will result in reduced virulence. A partial lack of concordance between MLST and microarray was found and this can be explained by the underlying data. On one hand, microarray data include highly variable regions which are known to be involved in horizontal gene transfer. On the other hand, MLST data is restricted to seven sequences and disregards contribution of horizontally acquired genes when evaluating evolution. The DNA microarray and MLST analysis complement each other giving a clearer image of evolution of these serovars and, furthermore, a visualization of the horizontally acquired genes.

DNA microarray analysis of Salmonella serotype Typhimurium strains causing different symptoms of disease.

BACKGROUND: Salmonella enterica subsp. enterica is one of the leading food-borne pathogens in the USA and European countries. Outcome of human Salmonella serotype Typhimurium infections ranges from mild self-limiting diarrhoea to severe diarrhoea that requires hospitalization. Increased knowledge of the mechanisms that are responsible for causing infection and especially the severity of infection is of high interest. RESULTS: Strains were selected from patients with mild infections (n = 9) and patients with severe infections (n = 9) and clinical data allowed us to correct for known underlying diseases. Additionally, outbreak isolates (n = 3) were selected. Strains were analyzed on a DNA-DNA microarray for presence or absence of 281 genes covering marker groups of genes related to pathogenicity, phages, antimicrobial resistance, fimbriae, mobility, serotype and metabolism. Strains showed highly similar profiles when comparing virulence associated genes, but differences between strains were detected in the prophage marker group. The Salmonella virulence plasmid was present in 72% of the strains, but presence or absence of the virulence plasmid did not correspond to disease symptoms. A dendrogram clustered strains into four groups. Clustering confirmed DT104 as being a clonal phagetype. Clustering of the remaining strains was mainly correlated to presence or absence of the virulence plasmid and mobile elements such as transposons. Each of the four clusters in the tree represented an almost equal amount of strains causing severe or mild symptoms of infection. CONCLUSIONS: We investigated clinical significance of known virulence factors of Salmonella serotype Typhimurium strains causing different disease symptoms, and conclude that the few detected differences in Salmonella serotype Typhimurium do not affect outcome of human disease.

A phylogenetic group of Escherichia coli associated with active left-sided inflammatory bowel disease.

BACKGROUND: Escherichia coli have been found in increased numbers in tissues from patients with Inflammatory Bowel Disease (IBD) and adherent-invasive E. coli have been found in resected ileum from patients with Crohn's disease. This study aimed to characterize possible differences in phylogenetic group (triplex PCR), extraintestinal pathogenic E. coli (ExPEC) genes and multilocus sequence type (MLST) between E. coli strains isolated from IBD patients with past or present involvement of the left side of the colon and from controls. RESULTS: Fecal samples were collected from 18 patients and from 10 healthy controls. Disease activity was evaluated by sigmoidoscopy. Interestingly, E. coli strains of the phylogenetic group B2 were cultured from 60% of patients with IBD compared to 11% of healthy controls (p < 0.05). Furthermore, when comparing the number of E. coli B2 strains with at least one positive ExPEC gene among different groups, 86% were found positive among active IBD patients, significantly more than 13% among inactive IBD patients (p < 0.05), and 11% among healthy controls (p < 0.05). The B2 phylogenetic group was found in a specific cluster based on MLST, but no further separation between E. coli strains associated with active compared to inactive IBD was achieved. CONCLUSION: In conclusion, E. coli of the phylogenetic group B2 were isolated more frequently from IBD patients with past or present involvement of the left side of the colon compared to healthy controls, and B2 strains with ExPEC genes were found more frequently among IBD patients with active disease compared to patients with inactive disease.

Packed with Salmonella--investigation of an international outbreak of Salmonella Senftenberg infection linked to contamination of prepacked basil in 2007.

Salmonella Senftenberg is uncommon in the United Kingdom. In January-June 2007, the Health Protection Agency reported on 55 primary human cases of Salmonella Senftenberg in England and Wales. In May 2007, fresh basil sold in the United Kingdom was found to be contaminated with Salmonella Senftenberg. We launched an investigation to elucidate the cause of this outbreak. Isolates were examined using plasmid profiling and pulsed-field gel electrophoresis, and the outbreak strain (SSFTXB.0014) was identified. We enquired via Enter-net whether other countries had isolated the outbreak strain, analyzed samples of fresh herbs from U.K. retailers, and interviewed patients on food history. Thirty-two patient-cases were referred to this outbreak in England and Wales. Onsets of illness occurred between 5 March and 6 June 2007. Fifty-six percent of patient-cases were females and 90% adults (>20 years old); three were admitted to hospital as a result of Salmonella infection. Scotland, Denmark, the Netherlands, and the United States reported on 19 cases of Salmonella Senftenberg infection presenting with the outbreak strain since January 2007. Eight samples of prepacked fresh basil imported from Israel tested positive with the same strain. A minority of patients could recall the consumption of basil before illness, and some reported consumption of products where basil was a likely ingredient. Environmental investigations in Israel did not identify the contamination source. Microbiological evidence suggested an association between contamination of fresh basil and the cases of Salmonella Senftenberg infection, leading to withdrawal of basil from all potentially affected batches from the U.K. market.