Epistasis, core-genome disharmony, and adaptation in recombining bacteria.

Recombination of short DNA fragments via horizontal gene transfer (HGT) can introduce beneficial alleles, create genomic disharmony through negative epistasis, and create adaptive gene combinations through positive epistasis. For non-core (accessory) genes, the negative epistatic cost is likely to be minimal because the incoming genes have not co-evolved with the recipient genome and are frequently observed as tightly linked cassettes with major effects. By contrast, interspecific recombination in the core genome is expected to be rare because disruptive allelic replacement is likely to introduce negative epistasis. Why then is homologous recombination common in the core of bacterial genomes? To understand this enigma, we take advantage of an exceptional model system, the common enteric pathogens Campylobacter jejuni and C. coli that are known for very high magnitude interspecies gene flow in the core genome. As expected, HGT does indeed disrupt co-adapted allele pairings, indirect evidence of negative epistasis. However, multiple HGT events enable recovery of the genome's co-adaption between introgressing alleles, even in core metabolism genes (e.g., formate dehydrogenase). These findings demonstrate that, even for complex traits, genetic coalitions can be decoupled, transferred, and independently reinstated in a new genetic background-facilitating transition between fitness peaks. In this example, the two-step recombinational process is associated with C. coli that are adapted to the agricultural niche.IMPORTANCEGenetic exchange among bacteria shapes the microbial world. From the acquisition of antimicrobial resistance genes to fundamental questions about the nature of bacterial species, this powerful evolutionary force has preoccupied scientists for decades. However, the mixing of genes between species rests on a paradox: 0n one hand, promoting adaptation by conferring novel functionality; on the other, potentially introducing disharmonious gene combinations (negative epistasis) that will be selected against. Taking an interdisciplinary approach to analyze natural populations of the enteric bacteria Campylobacter, an ideal example of long-range admixture, we demonstrate that genes can independently transfer across species boundaries and rejoin in functional networks in a recipient genome. The positive impact of two-gene interactions appears to be adaptive by expanding metabolic capacity and facilitating niche shifts through interspecific hybridization. This challenges conventional ideas and highlights the possibility of multiple-step evolution of multi-gene traits by interspecific introgression.

Novel rpsK / rpsD primer-probe assay improves detection of Campylobacter jejuni and Campylobacter coli in human stool.

Campylobacter causes bacterial enteritis, dysentery, and growth faltering in children in low- and middle-income countries (LMICs). Campylobacter spp. are fastidious organisms, and their detection often relies on culture independent diagnostic technologies, especially in LMICs. Campylobacter jejuni and Campylobacter coli are most often the infectious agents and in high income settings together account for 95% of Campylobacter infections. Several other Campylobacter species have been detected in LMIC children at an increased prevalence relative to high income settings. After doing extensive whole genome sequencing of isolates of C. jejuni and C. coli in Peru, we observed heterogeneity in the binding sites for the main species-specific PCR assay (cadF) and designed an alternative rpsKD-based qPCR assay to detect both C. jejuni and C. coli. The rpsKD-based qPCR assay identified 23% more C.jejuni/ C.coli samples than the cadF assay among 47 Campylobacter genus positive cadF negative samples verified to have C. jejuni and or C. coli with shotgun metagenomics. This assay can be expected to be useful in diagnostic studies of enteric infectious diseases and be useful in revising the attribution estimates of Campylobacter in LMICs.

Genomic resistant determinants of multidrug-resistant Campylobacter spp. isolates in Peru.

OBJECTIVES: Antimicrobial resistant (AMR) Campylobacter is a global health threat; however, there is limited information on genomic determinants of resistance in low- and middle-income countries. We evaluated genomic determinants of AMR using a collection of whole genome sequenced Campylobacter jejuni and C. coli isolates from Iquitos, Peru. METHODS: Campylobacter isolates from two paediatric cohort studies enriched with isolates that demonstrated resistance to ciprofloxacin and azithromycin were sequenced and mined for AMR determinants. RESULTS: The gyrA mutation leading to the Thr86Ile amino acid change was the only gyrA mutation associated with fluoroquinolone resistance identified. The A2075G mutation in 23S rRNA was present, but three other 23S rRNA mutations previously associated with macrolide resistance were not identified. A resistant-enhancing variant of the cmeABC efflux pump genotype (RE-cmeABC) was identified in 36.1% (35/97) of C. jejuni genomes and 17.9% (12/67) of C. coli genomes. Mutations identified in the CmeR-binding site, an inverted repeat sequence in the cmeABC promoter region that increases expression of the operon, were identified in 24/97 C. jejuni and 14/67 C. coli genomes. The presence of these variants, in addition to RE-cmeABC, was noted in 18 of the 24 C. jejuni and 9 of the 14 C. coli genomes. CONCLUSIONS: Both RE-cmeABC and mutations in the CmeR-binding site were strongly associated with the MDR phenotype in C. jejuni and C. coli. This is the first report of RE-cmeABC in Peru and suggests it is a major driver of resistance to the principal therapies used to treat human campylobacteriosis in this setting.

Contrasting genes conferring short- and long-term biofilm adaptation in Listeria.

Listeria monocytogenes is an opportunistic food-borne bacterium that is capable of infecting humans with high rates of hospitalization and mortality. Natural populations are genotypically and phenotypically variable, with some lineages being responsible for most human infections. The success of L. monocytogenes is linked to its capacity to persist on food and in the environment. Biofilms are an important feature that allow these bacteria to persist and infect humans, so understanding the genetic basis of biofilm formation is key to understanding transmission. We sought to investigate the biofilm-forming ability of L. monocytogenes by identifying genetic variation that underlies biofilm formation in natural populations using genome-wide association studies (GWAS). Changes in gene expression of specific strains during biofilm formation were then investigated using RNA sequencing (RNA-seq). Genetic variation associated with enhanced biofilm formation was identified in 273 genes by GWAS and differential expression in 220 genes by RNA-seq. Statistical analyses show that the number of overlapping genes flagged by either type of experiment is less than expected by random sampling. This novel finding is consistent with an evolutionary scenario where rapid adaptation is driven by variation in gene expression of pioneer genes, and this is followed by slower adaptation driven by nucleotide changes within the core genome.

Evidence of potential Campylobacter jejuni zooanthroponosis in captive macaque populations.

Non-human primates share recent common ancestry with humans and exhibit comparable disease symptoms. Here, we explored the transmission potential of enteric bacterial pathogens in monkeys exhibiting symptoms of recurrent diarrhoea in a biomedical research facility in China. The common zoonotic bacterium Campylobacter jejuni was isolated from macaques (Macaca mulatta and Macaca fascicularis) and compared to isolates from humans and agricultural animals in Asia. Among the monkeys sampled, 5 % (44/973) tested positive for C. jejuni, 11 % (5/44) of which displayed diarrhoeal symptoms. Genomic analysis of monkey isolates, and 1254 genomes from various sources in Asia, were used to identify the most likely source of human infection. Monkey and human isolates shared high average nucleotide identity, common MLST clonal complexes and clustered together on a phylogeny. Furthermore, the profiles of putative antimicrobial resistance genes were similar between monkeys and humans. Taken together these findings suggest that housed macaques became infected with C. jejuni either directly from humans or via a common contamination source.

A high fidelity approach to assembling the complex Borrelia genome.

BACKGROUND: Bacteria of the Borrelia burgdorferi sensu lato (s.l.) complex can cause Lyme borreliosis. Different B. burgdorferi s.l. genospecies vary in their host and vector associations and human pathogenicity but the genetic basis for these adaptations is unresolved and requires completed and reliable genomes for comparative analyses. The de novo assembly of a complete Borrelia genome is challenging due to the high levels of complexity, represented by a high number of circular and linear plasmids that are dynamic, showing mosaic structure and sequence homology. Previous work demonstrated that even advanced approaches, such as a combination of short-read and long-read data, might lead to incomplete plasmid reconstruction. Here, using recently developed high-fidelity (HiFi) PacBio sequencing, we explored strategies to obtain gap-free, complete and high quality Borrelia genome assemblies. Optimizing genome assembly, quality control and refinement steps, we critically appraised existing techniques to create a workflow that lead to improved genome reconstruction. RESULTS: Despite the latest available technologies, stand-alone sequencing and assembly methods are insufficient for the generation of complete and high quality Borrelia genome assemblies. We developed a workflow pipeline for the de novo genome assembly for Borrelia using several types of sequence data and incorporating multiple assemblers to recover the complete genome including both circular and linear plasmid sequences. CONCLUSION: Our study demonstrates that, with HiFi data and an ensemble reconstruction pipeline with refinement steps, chromosomal and plasmid sequences can be fully resolved, even for complex genomes such as Borrelia. The presented pipeline may be of interest for the assembly of further complex microbial genomes.

Spatiotemporal Investigation of Antibiotic Resistance in the Urban Water Cycle Influenced by Environmental and Anthropogenic Activity.

With increasing emergence of antimicrobial resistant bacteria (ARB) and the risk this poses to public health, there are growing concerns regarding water pollution contributing to the spread of antimicrobial resistance (AMR) through inadequate amenities and the rapid rate of urbanization. In this study, the impact of different anthropogenic factors on the prevalence of AMR in the urban water cycle in Stellenbosch, South Africa (SA) was examined. Carbapenem, colistin, gentamicin and sulfamethoxazole resistant Gram-negative bacteria were recovered by selectively culturing aqueous, biofilm and sediment samples from sites impacted to varying degrees by informal settlements, residential, industrial, and agricultural activities, as well as a municipal wastewater treatment works (WWTW). A metagenomic approach determined community profiles and dominant AMR genes at various sites, while carbapenem resistant colonies were characterized using whole genome sequencing (WGS). Isolates recovered from agricultural sites exhibited relatively high levels of resistance to carbapenems and colistin, whereas sites impacted by domestic run-off had a higher prevalence of resistance to gentamicin and sulfamethoxazole, corresponding to usage data in SA. Similar microbial taxa were identified in raw sewage, sites downstream of informal settlements, and industrial areas that have limited waste removal infrastructure while WWTW were seen to reduce the prevalence of ARB in treated wastewater when operating efficiently. The results indicate the multiple complex drivers underpinning environmental dissemination of AMR and suggest that WWTW assist in removing AMR from the environment, reinforcing the necessity of adequate waste removal infrastructure and antibiotic stewardship measures to mitigate AMR transmission. IMPORTANCE The results from this study are of importance as they fill a gap in the data available on environmental AMR in South Africa to date. This study was done in parallel with co-investigators focusing on the prevalence of various antimicrobials at the same sites selected in our study, verifying that the sites that are influenced by informal settlements and WWTW influent had higher concentrations of antimicrobials and antimicrobial metabolites. The various locations of the sample sites selected, the frequency of the samples collected over a year, and the different types of samples collected at each site all contribute to informing how AMR in the environment might be affected by anthropogenic activity.

Host ecology regulates interspecies recombination in bacteria of the genus Campylobacter.

Horizontal gene transfer (HGT) can allow traits that have evolved in one bacterial species to transfer to another. This has potential to rapidly promote new adaptive trajectories such as zoonotic transfer or antimicrobial resistance. However, for this to occur requires gaps to align in barriers to recombination within a given time frame. Chief among these barriers is the physical separation of species with distinct ecologies in separate niches. Within the genus Campylobacter, there are species with divergent ecologies, from rarely isolated single-host specialists to multihost generalist species that are among the most common global causes of human bacterial gastroenteritis. Here, by characterizing these contrasting ecologies, we can quantify HGT among sympatric and allopatric species in natural populations. Analyzing recipient and donor population ancestry among genomes from 30 Campylobacter species, we show that cohabitation in the same host can lead to a six-fold increase in HGT between species. This accounts for up to 30% of all SNPs within a given species and identifies highly recombinogenic genes with functions including host adaptation and antimicrobial resistance. As described in some animal and plant species, ecological factors are a major evolutionary force for speciation in bacteria and changes to the host landscape can promote partial convergence of distinct species through HGT.

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.

Campylobacter jejuni genotypes are associated with post-infection irritable bowel syndrome in humans.

Campylobacter enterocolitis may lead to post-infection irritable bowel syndrome (PI-IBS) and while some C. jejuni strains are more likely than others to cause human disease, genomic and virulence characteristics promoting PI-IBS development remain uncharacterized. We combined pangenome-wide association studies and phenotypic assays to compare C. jejuni isolates from patients who developed PI-IBS with those who did not. We show that variation in bacterial stress response (Cj0145_phoX), adhesion protein (Cj0628_CapA), and core biosynthetic pathway genes (biotin: Cj0308_bioD; purine: Cj0514_purQ; isoprenoid: Cj0894c_ispH) were associated with PI-IBS development. In vitro assays demonstrated greater adhesion, invasion, IL-8 and TNFα secretion on colonocytes with PI-IBS compared to PI-no-IBS strains. A risk-score for PI-IBS development was generated using 22 genomic markers, four of which were from Cj1631c, a putative heme oxidase gene linked to virulence. Our finding that specific Campylobacter genotypes confer greater in vitro virulence and increased risk of PI-IBS has potential to improve understanding of the complex host-pathogen interactions underlying this condition.

Genome evolution and the emergence of pathogenicity in avian Escherichia coli.

Chickens are the most common birds on Earth and colibacillosis is among the most common diseases affecting them. This major threat to animal welfare and safe sustainable food production is difficult to combat because the etiological agent, avian pathogenic Escherichia coli (APEC), emerges from ubiquitous commensal gut bacteria, with no single virulence gene present in all disease-causing isolates. Here, we address the underlying evolutionary mechanisms of extraintestinal spread and systemic infection in poultry. Combining population scale comparative genomics and pangenome-wide association studies, we compare E. coli from commensal carriage and systemic infections. We identify phylogroup-specific and species-wide genetic elements that are enriched in APEC, including pathogenicity-associated variation in 143 genes that have diverse functions, including genes involved in metabolism, lipopolysaccharide synthesis, heat shock response, antimicrobial resistance and toxicity. We find that horizontal gene transfer spreads pathogenicity elements, allowing divergent clones to cause infection. Finally, a Random Forest model prediction of disease status (carriage vs. disease) identifies pathogenic strains in the emergent ST-117 poultry-associated lineage with 73% accuracy, demonstrating the potential for early identification of emergent APEC in healthy flocks.

A 500-year tale of co-evolution, adaptation, and virulence: Helicobacter pylori in the Americas.

Helicobacter pylori is a common component of the human stomach microbiota, possibly dating back to the speciation of Homo sapiens. A history of pathogen evolution in allopatry has led to the development of genetically distinct H. pylori subpopulations, associated with different human populations, and more recent admixture among H. pylori subpopulations can provide information about human migrations. However, little is known about the degree to which some H. pylori genes are conserved in the face of admixture, potentially indicating host adaptation, or how virulence genes spread among different populations. We analyzed H. pylori genomes from 14 countries in the Americas, strains from the Iberian Peninsula, and public genomes from Europe, Africa, and Asia, to investigate how admixture varies across different regions and gene families. Whole-genome analyses of 723 H. pylori strains from around the world showed evidence of frequent admixture in the American strains with a complex mosaic of contributions from H. pylori populations originating in the Americas as well as other continents. Despite the complex admixture, distinctive genomic fingerprints were identified for each region, revealing novel American H. pylori subpopulations. A pan-genome Fst analysis showed that variation in virulence genes had the strongest fixation in America, compared with non-American populations, and that much of the variation constituted non-synonymous substitutions in functional domains. Network analyses suggest that these virulence genes have followed unique evolutionary paths in the American populations, spreading into different genetic backgrounds, potentially contributing to the high risk of gastric cancer in the region.

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.

Genomic epidemiology of Campylobacter jejuni associated with asymptomatic pediatric infection in the Peruvian Amazon.

Campylobacter is the leading bacterial cause of gastroenteritis worldwide and its incidence is especially high in low- and middle-income countries (LMIC). Disease epidemiology in LMICs is different compared to high income countries like the USA or in Europe. Children in LMICs commonly have repeated and chronic infections even in the absence of symptoms, which can lead to deficits in early childhood development. In this study, we sequenced and characterized C. jejuni (n = 62) from a longitudinal cohort study of children under the age of 5 with and without diarrheal symptoms, and contextualized them within a global C. jejuni genome collection. Epidemiological differences in disease presentation were reflected in the genomes, specifically by the absence of some of the most common global disease-causing lineages. As in many other countries, poultry-associated strains were likely a major source of human infection but almost half of local disease cases (15 of 31) were attributable to genotypes that are rare outside of Peru. Asymptomatic infection was not limited to a single (or few) human adapted lineages but resulted from phylogenetically divergent strains suggesting an important role for host factors in the cryptic epidemiology of campylobacteriosis in LMICs.

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.

Genetic Diversity of Composite Enterotoxigenic Staphylococcus epidermidis Pathogenicity Islands.

The only known elements encoding enterotoxins in coagulase-negative staphylococci are composite Staphylococcus epidermidis pathogenicity islands (SePIs), including SePI and S. epidermidis composite insertion (SeCI) regions. We investigated 1545 Staphylococcus spp. genomes using whole-genome MLST, and queried them for genes of staphylococcal enterotoxin family and for 29 ORFs identified in prototype SePI from S. epidermidis FRI909. Enterotoxin-encoding genes were identified in 97% of Staphylococcus aureus genomes, in one Staphylococcus argenteus genome and in nine S. epidermidis genomes. All enterotoxigenic S. epidermidis strains carried composite SePI, encoding sec and sel enterotoxin genes, and were assigned to a discrete wgMLST cluster also containing genomes with incomplete islands located in the same region as complete SePI in enterotoxigenic strains. Staphylococcus epidermidis strains without SeCI and SePI genes, and strains with complete SeCI and no SePI genes were identified but no strains were found to carry only SePI and not SeCI genes. The systematic differences between SePI and SeCI regions imply a lineage-specific pattern of inheritance and support independent acquisition of the two elements in S. epidermidis. We provided evidence of reticulate evolution of mobile elements that contain elements with different putative ancestry, including composite SePI that contains genes found in other coagulase-negative staphylococci (SeCI), as well as in S. aureus (SePI-like elements). We conclude that SePI-associated elements present in nonenterotoxigenic S. epidermidis represent a scaffold associated with acquisition of virulence-associated genes. Gene exchange between S. aureus and S. epidermidis may promote emergence of new pathogenic S. epidermidis clones.

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.