Quantifying bacterial evolution in the wild: A birthday problem for Campylobacter lineages.

Measuring molecular evolution in bacteria typically requires estimation of the rate at which nucleotide changes accumulate in strains sampled at different times that share a common ancestor. This approach has been useful for dating ecological and evolutionary events that coincide with the emergence of important lineages, such as outbreak strains and obligate human pathogens. However, in multi-host (niche) transmission scenarios, where the pathogen is essentially an opportunistic environmental organism, sampling is often sporadic and rarely reflects the overall population, particularly when concentrated on clinical isolates. This means that approaches that assume recent common ancestry are not applicable. Here we present a new approach to estimate the molecular clock rate in Campylobacter that draws on the popular probability conundrum known as the 'birthday problem'. Using large genomic datasets and comparative genomic approaches, we use isolate pairs that share recent common ancestry to estimate the rate of nucleotide change for the population. Identifying synonymous and non-synonymous nucleotide changes, both within and outside of recombined regions of the genome, we quantify clock-like diversification to estimate synonymous rates of nucleotide change for the common pathogenic bacteria Campylobacter coli (2.4 x 10-6 s/s/y) and Campylobacter jejuni (3.4 x 10-6 s/s/y). Finally, using estimated total rates of nucleotide change, we infer the number of effective lineages within the sample time frame-analogous to a shared birthday-and assess the rate of turnover of lineages in our sample set over short evolutionary timescales. This provides a generalizable approach to calibrating rates in populations of environmental bacteria and shows that multiple lineages are maintained, implying that large-scale clonal sweeps may take hundreds of years or more in these species.

Genome-wide association study of signature genetic alterations among pseudomonas aeruginosa cystic fibrosis isolates.

Pseudomonas aeruginosa (PA) is an opportunistic pathogen that causes diverse human infections including chronic airway infection in patients with cystic fibrosis (CF). Comparing the genomes of CF and non-CF PA isolates has great potential to identify the genetic basis of pathogenicity. To gain a deeper understanding of PA adaptation in CF airways, we performed a genome-wide association study (GWAS) on 1,001 PA genomes. Genetic variations identified among CF isolates were categorized into (i) alterations in protein-coding regions, either large- or small-scale, and (ii) polymorphic variation in intergenic regions. We introduced each CF-associated genetic alteration into the genome of PAO1, a prototype PA strain, and validated the outcomes experimentally. Loci readily mutated among CF isolates included genes encoding a probable sulfatase, a probable TonB-dependent receptor (PA2332~PA2336), L-cystine transporter (YecS, PA0313), and a probable transcriptional regulator (PA5438). A promoter region of a heme/hemoglobin uptake outer membrane receptor (PhuR, PA4710) was also different between the CF and non-CF isolate groups. Our analysis highlights ways in which the PA genome evolves to survive and persist within the context of chronic CF infection.

Recombination-mediated dissemination of Methicillin-resistant S. aureus clonal complex 1 in the Egyptian health care settings.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) is a rapidly evolving pathogen that is frequently associated with outbreaks and sustained epidemics. This study investigated the population structure, resistome, virulome, and the correlation between antimicrobial resistance determinants with phenotypic resistance profiles of 36 representative hospital-acquired MRSA isolates recovered from hospital settings in Egypt. RESULTS: The community-acquired MRSA lineage, clonal complex 1 (CC1) was the most frequently detected clone, followed by three other globally disseminated clones, CC121, CC8, and CC22. Most isolates carried SCCmec type V and more than half of isolates demonstrated multi-drug resistant phenotypes. Resistance to linezolid, a last resort antibiotic for treating multidrug resistant MRSA, was observed in 11.11% of the isolates belonging to different genetic backgrounds. Virulome analysis indicated that most isolates harboured a large pool of virulence factors and toxins. Genes encoding aureolysin, gamma hemolysins, and serine proteases were the most frequently detected virulence encoding genes. CC1 was observed to have a high pool of AMR resistance determinants including cfr, qacA, and qacB genes, which are involved in linezolid and quaternary ammonium compounds resistance, as well as high content of virulence-related genes, including both of the PVL toxin genes. Molecular clock analysis revealed that CC1 had the greatest frequency of recombination (compared to mutation) among the four major clones, supporting the role of horizontal gene transfer in modulating AMR and hypervirulence in this clone. CONCLUSIONS: This pilot study provided evidence on the dissemination success of CA-MRSA clone CC1 among Egyptian hospitals. Co-detection of multiple AMR and virulence genes in this lineage pose a broad public health risk, with implications for successful treatment. The results of this study, together with other surveillance studies in Egypt, should be used to develop strategies for controlling MRSA infections in Egyptian health-care settings.

Agricultural intensification and the evolution of host specialism in the enteric pathogen Campylobacter jejuni.

Modern agriculture has dramatically changed the distribution of animal species on Earth. Changes to host ecology have a major impact on the microbiota, potentially increasing the risk of zoonotic pathogens being transmitted to humans, but the impact of intensive livestock production on host-associated bacteria has rarely been studied. Here, we use large isolate collections and comparative genomics techniques, linked to phenotype studies, to understand the timescale and genomic adaptations associated with the proliferation of the most common food-born bacterial pathogen (Campylobacter jejuni) in the most prolific agricultural mammal (cattle). Our findings reveal the emergence of cattle specialist C. jejuni lineages from a background of host generalist strains that coincided with the dramatic rise in cattle numbers in the 20th century. Cattle adaptation was associated with horizontal gene transfer and significant gene gain and loss. This may be related to differences in host diet, anatomy, and physiology, leading to the proliferation of globally disseminated cattle specialists of major public health importance. This work highlights how genomic plasticity can allow important zoonotic pathogens to exploit altered niches in the face of anthropogenic change and provides information for mitigating some of the risks posed by modern agricultural systems.

Signatures of selection in core and accessory genomes indicate different ecological drivers of diversification among Bacillus cereus clades.

Bacterial clades are often ecologically distinct, despite extensive horizontal gene transfer (HGT). How selection works on different parts of bacterial pan-genomes to drive and maintain the emergence of clades is unclear. Focusing on the three largest clades in the diverse and well-studied Bacillus cereus sensu lato group, we identified clade-specific core genes (present in all clade members) and then used clade-specific allelic diversity to identify genes under purifying and diversifying selection. Clade-specific accessory genes (present in a subset of strains within a clade) were characterized as being under selection using presence/absence in specific clades. Gene ontology analyses of genes under selection revealed that different gene functions were enriched in different clades. Furthermore, some gene functions were enriched only amongst clade-specific core or accessory genomes. Genes under purifying selection were often clade-specific, while genes under diversifying selection showed signs of frequent HGT. These patterns are consistent with different selection pressures acting on both the core and the accessory genomes of different clades and can lead to ecological divergence in both cases. Examining variation in allelic diversity allows us to uncover genes under clade-specific selection, allowing ready identification of strains and their ecological niche.

Stress resistance associated with multi-host transmission and enhanced biofilm formation at 42 °C among hyper-aerotolerant generalist Campylobacter jejuni.

One of the emerging conundrums of Campylobacter food-borne illness is the bacterial ability to survive stressful environmental conditions. We evaluated the heterogeneity among 90 C. jejuni and 21 C. coli isolates from different sources in Egypt with respect to biofilm formation capabilities (under microaerobic and aerobic atmosphere) and resistance to a range of stressors encountered along the food chain (aerobic stress, refrigeration, freeze-thaw, heat, peracetic acid, and osmotic stress). High prevalence (63%) of hyper-aerotolerant (HAT) isolates was observed, exhibiting also a significantly high tolerance to heat, osmotic stress, refrigeration, and freeze-thaw stress, coupled with high biofilm formation ability which was clearly enhanced under aerobic conditions, suggesting a potential link between stress adaptation and biofilm formation. Most HAT multi-stress resistant and strong biofilm producing C. jejuni isolates belonged to host generalist clonal complexes (ST-21, ST-45, ST-48 and ST-206). These findings highlight the potential role of oxidative stress response systems in providing cross-protection (resistance to other multiple stress conditions) and enhancing biofilm formation in Campylobacter and suggest that selective pressures encountered in hostile environments have shaped the epidemiology of C. jejuni in Egypt by selecting the transmission of highly adapted isolates, thus promoting the colonization of multiple host species by important disease-causing lineages.

Genome-Wide Identification of Host-Segregating Single-Nucleotide Polymorphisms for Source Attribution of Clinical Campylobacter coli Isolates.

Campylobacter is among the most common causes of gastroenteritis worldwide. Campylobacter jejuni and Campylobacter coli are the most common species causing human disease. DNA sequence-based methods for strain characterization have focused largely on C. jejuni, responsible for 80 to 90% of infections, meaning that C. coli epidemiology has lagged behind. Here, we have analyzed the genome of 450 C. coli isolates to determine genetic markers that can discriminate isolates sampled from 3 major reservoir hosts (chickens, cattle, and pigs). These markers then were applied to identify the source of infection of 147 C. coli strains from French clinical cases. Using STRUCTURE software, 259 potential host-segregating markers were revealed by probabilistic characterization of single-nucleotide polymorphism (SNP) frequency variation in strain collections from three different hosts. These SNPs were found in 41 genes or intergenic regions, mostly coding for proteins involved in motility and membrane functions. Source attribution of clinical isolates based on the differential presence of these markers confirmed chickens as the most common source of C. coli infection in France.IMPORTANCE Genome-wide and source attribution studies based on Campylobacter species have shown their importance for the understanding of foodborne infections. Although the use of multilocus sequence typing based on 7 genes from C. jejuni is a powerful method to structure populations, when applied to C. coli, results have not clearly demonstrated its robustness. Therefore, we aim to provide more accurate data based on the identification of single-nucleotide polymorphisms. Results from this study reveal an important number of host-segregating SNPs, found in proteins involved in motility, membrane functions, or DNA repair systems. These findings offer new, interesting opportunities for further study of C. coli adaptation to its environment. Additionally, the results demonstrate that poultry is potentially the main reservoir of C. coli in France.

Gene pool transmission of multidrug resistance among Campylobacter from livestock, sewage and human disease.

The use of antimicrobials in human and veterinary medicine has coincided with a rise in antimicrobial resistance (AMR) in the food-borne pathogens Campylobacter jejuni and Campylobacter coli. Faecal contamination from the main reservoir hosts (livestock, especially poultry) is the principal route of human infection but little is known about the spread of AMR among source and sink populations. In particular, questions remain about how Campylobacter resistomes interact between species and hosts, and the potential role of sewage as a conduit for the spread of AMR. Here, we investigate the genomic variation associated with AMR in 168 C. jejuni and 92 C. coli strains isolated from humans, livestock and urban effluents in Spain. AMR was tested in vitro and isolate genomes were sequenced and screened for putative AMR genes and alleles. Genes associated with resistance to multiple drug classes were observed in both species and were commonly present in multidrug-resistant genomic islands (GIs), often located on plasmids or mobile elements. In many cases, these loci had alleles that were shared among C. jejuni and C. coli consistent with horizontal transfer. Our results suggest that specific antibiotic resistance genes have spread among Campylobacter isolated from humans, animals and the environment.

Combined Analysis of Variation in Core, Accessory and Regulatory Genome Regions Provides a Super-Resolution View into the Evolution of Bacterial Populations.

The use of whole-genome phylogenetic analysis has revolutionized our understanding of the evolution and spread of many important bacterial pathogens due to the high resolution view it provides. However, the majority of such analyses do not consider the potential role of accessory genes when inferring evolutionary trajectories. Moreover, the recently discovered importance of the switching of gene regulatory elements suggests that an exhaustive analysis, combining information from core and accessory genes with regulatory elements could provide unparalleled detail of the evolution of a bacterial population. Here we demonstrate this principle by applying it to a worldwide multi-host sample of the important pathogenic E. coli lineage ST131. Our approach reveals the existence of multiple circulating subtypes of the major drug-resistant clade of ST131 and provides the first ever population level evidence of core genome substitutions in gene regulatory regions associated with the acquisition and maintenance of different accessory genome elements.

A GWAS on Helicobacter pylori strains points to genetic variants associated with gastric cancer risk.

BACKGROUND: Helicobacter pylori are stomach-dwelling bacteria that are present in about 50% of the global population. Infection is asymptomatic in most cases, but it has been associated with gastritis, gastric ulcers and gastric cancer. Epidemiological evidence shows that progression to cancer depends upon the host and pathogen factors, but questions remain about why cancer phenotypes develop in a minority of infected people. Here, we use comparative genomics approaches to understand how genetic variation amongst bacterial strains influences disease progression. RESULTS: We performed a genome-wide association study (GWAS) on 173 H. pylori isolates from the European population (hpEurope) with known disease aetiology, including 49 from individuals with gastric cancer. We identified SNPs and genes that differed in frequency between isolates from patients with gastric cancer and those with gastritis. The gastric cancer phenotype was associated with the presence of babA and genes in the cag pathogenicity island, one of the major virulence determinants of H. pylori, as well as non-synonymous variations in several less well-studied genes. We devised a simple risk score based on the risk level of associated elements present, which has the potential to identify strains that are likely to cause cancer but will require refinement and validation. CONCLUSION: There are a number of challenges to applying GWAS to bacterial infections, including the difficulty of obtaining matched controls, multiple strain colonization and the possibility that causative strains may not be present when disease is detected. Our results demonstrate that bacterial factors have a sufficiently strong influence on disease progression that even a small-scale GWAS can identify them. Therefore, H. pylori GWAS can elucidate mechanistic pathways to disease and guide clinical treatment options, including for asymptomatic carriers.

Lineage-specific plasmid acquisition and the evolution of specialized pathogens in Bacillus thuringiensis and the Bacillus cereus group.

Bacterial plasmids can vary from small selfish genetic elements to large autonomous replicons that constitute a significant proportion of total cellular DNA. By conferring novel function to the cell, plasmids may facilitate evolution but their mobility may be opposed by co-evolutionary relationships with chromosomes or encouraged via the infectious sharing of genes encoding public goods. Here, we explore these hypotheses through large-scale examination of the association between plasmids and chromosomal DNA in the phenotypically diverse Bacillus cereus group. This complex group is rich in plasmids, many of which encode essential virulence factors (Cry toxins) that are known public goods. We characterized population genomic structure, gene content and plasmid distribution to investigate the role of mobile elements in diversification. We analysed coding sequence within the core and accessory genome of 190 B. cereus group isolates, including 23 novel sequences and genes from 410 reference plasmid genomes. While cry genes were widely distributed, those with invertebrate toxicity were predominantly associated with one sequence cluster (clade 2) and phenotypically defined Bacillus thuringiensis. Cry toxin plasmids in clade 2 showed evidence of recent horizontal transfer and variable gene content, a pattern of plasmid segregation consistent with transfer during infectious cooperation. Nevertheless, comparison between clades suggests that co-evolutionary interactions may drive association between plasmids and chromosomes and limit wider transfer of key virulence traits. Proliferation of successful plasmid and chromosome combinations is a feature of specialized pathogens with characteristic niches (Bacillus anthracis, B. thuringiensis) and has occurred multiple times in the B. cereus group.

Genome-wide association of functional traits linked with Campylobacter jejuni survival from farm to fork.

Campylobacter jejuni is a major cause of bacterial gastroenteritis worldwide, primarily associated with the consumption of contaminated poultry. C. jejuni lineages vary in host range and prevalence in human infection, suggesting differences in survival throughout the poultry processing chain. From 7343 MLST-characterised isolates, we sequenced 600 C. jejuni and C. coli isolates from various stages of poultry processing and clinical cases. A genome-wide association study (GWAS) in C. jejuni ST-21 and ST-45 complexes identified genetic elements over-represented in clinical isolates that increased in frequency throughout the poultry processing chain. Disease-associated SNPs were distinct in these complexes, sometimes organised in haplotype blocks. The function of genes containing associated elements was investigated, demonstrating roles for cj1377c in formate metabolism, nuoK in aerobic survival and oxidative respiration, and cj1368-70 in nucleotide salvage. This work demonstrates the utility of GWAS for investigating transmission in natural zoonotic pathogen populations and provides evidence that major C. jejuni lineages have distinct genotypes associated with survival, within the host specific niche, from farm to fork.

Comparative Genomics Study of Staphylococcus epidermidis Isolates from Orthopedic-Device-Related Infections Correlated with Patient Outcome.

Staphylococcus epidermidis has emerged as an important opportunistic pathogen causing orthopedic-device-related infections (ODRI). This study investigated the association of genome variation and phenotypic features of the infecting S. epidermidis isolate with the clinical outcome for the infected patient. S. epidermidis isolates were collected from 104 patients with ODRI. Their clinical outcomes were evaluated, after an average of 26 months, as either "cured" or "not cured." The isolates were tested for antibiotic susceptibility and biofilm formation. Whole-genome sequencing was performed on all isolates, and genomic variation was related to features associated with "cured" and "not cured." Strong biofilm formation and aminoglycoside resistance were associated with a "not-cured" outcome (P = 0.031 and P < 0.001, respectively). Based on gene-by-gene analysis, some accessory genes were more prevalent in isolates from the "not-cured" group. These included the biofilm-associated bhp gene, the antiseptic resistance qacA gene, the cassette chromosome recombinase-encoding genes ccrA and ccrB, and the IS256-like transposase gene. This study identifies biofilm formation and antibiotic resistance as associated with poor outcome in S. epidermidis ODRI. Whole-genome sequencing identified specific genes associated with a "not-cured" outcome that should be validated in future studies. (The study has been registered at ClinicalTrials.gov with identifier NCT02640937.).

Genome-Wide Identification of Host-Segregating Epidemiological Markers for Source Attribution in Campylobacter jejuni.

Campylobacter is among the most common worldwide causes of bacterial gastroenteritis. This organism is part of the commensal microbiota of numerous host species, including livestock, and these animals constitute potential sources of human infection. Molecular typing approaches, especially multilocus sequence typing (MLST), have been used to attribute the source of human campylobacteriosis by quantifying the relative abundance of alleles at seven MLST loci among isolates from animal reservoirs and human infection, implicating chicken as a major infection source. The increasing availability of bacterial genomes provides data on allelic variation at loci across the genome, providing the potential to improve the discriminatory power of data for source attribution. Here we present a source attribution approach based on the identification of novel epidemiological markers among a reference pan-genome list of 1,810 genes identified by gene-by-gene comparison of 884 genomes of Campylobacter jejuni isolates from animal reservoirs, the environment, and clinical cases. Fifteen loci involved in metabolic activities, protein modification, signal transduction, and stress response or coding for hypothetical proteins were selected as host-segregating markers and used to attribute the source of 42 French and 281 United Kingdom clinical C. jejuni isolates. Consistent with previous studies of British campylobacteriosis, analyses performed using STRUCTURE software attributed 56.8% of British clinical cases to chicken, emphasizing the importance of this host reservoir as an infection source in the United Kingdom. However, among French clinical isolates, approximately equal proportions of isolates were attributed to chicken and ruminant reservoirs, suggesting possible differences in the relative importance of animal host reservoirs and indicating a benefit for further national-scale attribution modeling to account for differences in production, behavior, and food consumption.IMPORTANCE Accurately quantifying the relative contribution of different host reservoirs to human Campylobacter infection is an ongoing challenge. This study, based on the development of a novel source attribution approach, provides the first results of source attribution in Campylobacter jejuni in France. A systematic analysis using gene-by-gene comparison of 884 genomes of C. jejuni isolates, with a pan-genome list of genes, identified 15 novel epidemiological markers for source attribution. The different proportions of French and United Kingdom clinical isolates attributed to each host reservoir illustrate a potential role for local/national variations in C. jejuni transmission dynamics.

Local genes for local bacteria: Evidence of allopatry in the genomes of transatlantic Campylobacter populations.

The genetic structure of bacterial populations can be related to geographical locations of isolation. In some species, there is a strong correlation between geographical distance and genetic distance, which can be caused by different evolutionary mechanisms. Patterns of ancient admixture in Helicobacter pylori can be reconstructed in concordance with past human migration, whereas in Mycobacterium tuberculosis it is the lack of recombination that causes allopatric clusters. In Campylobacter, analyses of genomic data and molecular typing have been successful in determining the reservoir host species, but not geographical origin. We investigated biogeographical variation in highly recombining genes to determine the extent of clustering between genomes from geographically distinct Campylobacter populations. Whole-genome sequences from 294 Campylobacter isolates from North America and the UK were analysed. Isolates from within the same country shared more recently recombined DNA than isolates from different countries. Using 15 UK/American closely matched pairs of isolates that shared ancestors, we identify regions that have frequently and recently recombined to test their correlation with geographical origin. The seven genes that demonstrated the greatest clustering by geography were used in an attribution model to infer geographical origin which was tested using a further 383 UK clinical isolates to detect signatures of recent foreign travel. Patient records indicated that in 46 cases, travel abroad had occurred <2 weeks prior to sampling, and genomic analysis identified that 34 (74%) of these isolates were of a non-UK origin. Identification of biogeographical markers in Campylobacter genomes will contribute to improved source attribution of clinical Campylobacter infection and inform intervention strategies to reduce campylobacteriosis.

Large Sequence Diversity within the Biosynthesis Locus and Common Biochemical Features of Campylobacter coli Lipooligosaccharides.

UNLABELLED: Despite the importance of lipooligosaccharides (LOSs) in the pathogenicity of campylobacteriosis, little is known about the genetic and phenotypic diversity of LOS in Campylobacter coli In this study, we investigated the distribution of LOS locus classes among a large collection of unrelated C. coli isolates sampled from several different host species. Furthermore, we paired C. coli genomic information and LOS chemical composition for the first time to investigate possible associations between LOS locus class sequence diversity and biochemical heterogeneity. After identifying three new LOS locus classes, only 85% of the 144 isolates tested were assigned to a class, suggesting higher genetic diversity than previously thought. This genetic diversity is at the basis of a completely unexplored LOS structural heterogeneity. Mass spectrometry analysis of the LOSs of nine isolates, representing four different LOS classes, identified two features distinguishing C. coli LOS from that of Campylobacter jejuni 2-Amino-2-deoxy-d-glucose (GlcN)-GlcN disaccharides were present in the lipid A backbone, in contrast to the ß-1'-6-linked 3-diamino-2,3-dideoxy-d-glucopyranose (GlcN3N)-GlcN backbone observed in C. jejuni Moreover, despite the fact that many of the genes putatively involved in 3-acylamino-3,6-dideoxy-d-glucose (Quip3NAcyl) were apparently absent from the genomes of various isolates, this rare sugar was found in the outer core of all C. coli isolates. Therefore, regardless of the high genetic diversity of the LOS biosynthesis locus in C. coli, we identified species-specific phenotypic features of C. coli LOS that might explain differences between C. jejuni and C. coli in terms of population dynamics and host adaptation. IMPORTANCE: Despite the importance of C. coli to human health and its controversial role as a causative agent of Guillain-Barré syndrome, little is known about the genetic and phenotypic diversity of C. coli LOSs. Therefore, we paired C. coli genomic information and LOS chemical composition for the first time to address this paucity of information. We identified two species-specific phenotypic features of C. coli LOS, which might contribute to elucidating the reasons behind the differences between C. jejuni and C. coli in terms of population dynamics and host adaptation.

Genetic features of livestock-associated Staphylococcus aureus ST9 isolates from Chinese pigs that carry the lsa(E) gene for quinupristin/dalfopristin resistance.

Whole-genome sequencing (WGS) was used to investigate the genetic features of the recently identified lsa(E) gene in porcine S. aureus ST9 isolates. Three quinupristin/dalfopristin-resistant isolates harboring the lsa(E) gene (two MRSA and one MSSA) were sequenced. Phylogenetic analysis of 184S. aureus genomes showed that ST9 porcine isolates belong to a distinct sequence cluster. Further analysis showed that all isolates were deficient in the recently described type IV restriction-modification system and SCCmec type XII was identified in the two MRSA isolates, which included a rare class C2 mec gene complex. A 24kb ΨSCC fragment was found in the MRSA and MSSA isolates sharing 99% nucleotide sequence homology with the ΨSCCJCSC6690 (O-2) element of a ST9 MRSA isolate from Thailand (accession number AB705453). Comparison of these ST9 isolates with 181 publically available S. aureus genomes identified 24 genes present in all (100%) ST9 isolates, that were absent from the most closely related human isolate. Our analysis suggests that the sequenced quinupristin/dalfopristin-resistant ST9 lineage represent a reservoir of mobile genetic elements associated with resistance and virulence features.

Enhanced biofilm formation and multi-host transmission evolve from divergent genetic backgrounds in Campylobacter jejuni.

Multicellular biofilms are an ancient bacterial adaptation that offers a protective environment for survival in hostile habitats. In microaerophilic organisms such as Campylobacter, biofilms play a key role in transmission to humans as the bacteria are exposed to atmospheric oxygen concentrations when leaving the reservoir host gut. Genetic determinants of biofilm formation differ between species, but little is known about how strains of the same species achieve the biofilm phenotype with different genetic backgrounds. Our approach combines genome-wide association studies with traditional microbiology techniques to investigate the genetic basis of biofilm formation in 102 Campylobacter jejuni isolates. We quantified biofilm formation among the isolates and identified hotspots of genetic variation in homologous sequences that correspond to variation in biofilm phenotypes. Thirteen genes demonstrated a statistically robust association including those involved in adhesion, motility, glycosylation, capsule production and oxidative stress. The genes associated with biofilm formation were different in the host generalist ST-21 and ST-45 clonal complexes, which are frequently isolated from multiple host species and clinical samples. This suggests the evolution of enhanced biofilm from different genetic backgrounds and a possible role in colonization of multiple hosts and transmission to humans.

Evidence of land-sea transfer of the zoonotic pathogen Campylobacter to a wildlife marine sentinel species.

Environmental pollution often accompanies the expansion and urbanization of human populations where sewage and wastewaters commonly have an impact on the marine environments. Here, we explored the potential for faecal bacterial pathogens, of anthropic origin, to spread to marine wildlife in coastal areas. The common zoonotic bacterium Campylobacter was isolated from grey seals (Halichoerus grypus), an important sentinel species for environmental pollution, and compared to isolates from wild birds, agricultural sources and clinical samples to characterize possible transmission routes. Campylobacter jejuni was present in half of all grey seal pups sampled (24/50 dead and 46/90 live pups) in the breeding colony on the Isle of May (Scotland), where it was frequently associated with histological evidence of disease. Returning yearling animals (19/19) were negative for C. jejuni suggesting clearance of infection while away from the localized colony infection source. The genomes of 90 isolates from seals were sequenced and characterized using a whole-genome multilocus sequence typing (MLST) approach and compared to 192 published genomes from multiple sources using population genetic approaches and a probabilistic genetic attribution model to infer the source of infection from MLST data. The strong genotype-host association has enabled the application of source attribution models in epidemiological studies of human campylobacteriosis, and here assignment analyses consistently grouped seal isolates with those from human clinical samples. These findings are consistent with either a common infection source or direct transmission of human campylobacter to grey seals, raising concerns about the spread of human pathogens to wildlife marine sentinel species in coastal areas.

Assessing antimicrobial resistance profiles of Salmonella enterica in the pork production system.

Introduction. Salmonella enterica is a significant enteric pathogen affecting human and livestock health. Pork production is a common source of Salmonella contamination, with emerging multidrug resistance (MDR) posing a global health threat.Gap statement. Salmonella contamination and antimicrobial resistance (AMR) profiles in the pig production chain are underreported.Aim. To investigate the prevalence of S. enterica in the pig production chain and characterise their AMR profiles.Methodology. We collected 485 samples from pig farms, a standard pig abattoir and retail markets in Patthalung and Songkhla provinces in southern Thailand. Antimicrobial susceptibility testing was performed on these samples, and AMR profiles were determined.Results. S. enterica was detected in 68.67% of farm samples, 45.95% of abattoir samples and 50.67% of retail market samples. Analysis of 264 isolates, representing 18 serotypes, identified S. enterica serotype Rissen as the most prevalent. The predominant resistance phenotypes included ampicillin (AMP, 91.29%), tetracycline (TET, 88.26%) and streptomycin (STR, 84.47%). Over 80% of isolates showed resistance to three or more antimicrobial classes, indicating MDR. The AMP-STR-TET resistance pattern was found in nearly 70% of all MDR isolates across the production chain.Conclusions. The high prevalence of MDR is consistent with extensive antimicrobial use in the livestock sector. The presence of extensively resistant S. enterica highlights the urgent need for antimicrobial stewardship. Strengthening preventive strategies and control measures is crucial to mitigate the risk of MDR Salmonella spreading from farm to fork.