Five-year VIM-producing Pseudomonas aeruginosa outbreak in four Belgian ICUs, an investigation report (2019-2023).

BACKGROUND: Verona integron-encoded metallo-ß-lactamase-producing Pseudomonas aeruginosa (VIM-PA) outbreaks are frequently linked to contaminated sink-drains in the intensive care unit (ICU). OBJECTIVES: This study aims to investigate a VIM-PA outbreak occurring at four ICUs in a Belgian university center. METHODS: Between the 1st January 2019 and the 30th July 2023, data were retrospectively retrieved. Whole genome sequencing of VIM-PA was carried out for available clinical and sink-drains isolates and the core genome multilocus sequencing typing (cgMLST) was used to confirm clonality. New case incidence was estimated by analyzing the weekly data of at-risk and VIM-PA colonized patients, fitting a gamma regression with a log link. RESULTS: Fifty-one patients were colonized, among them 32 (63%) were infected by VIM-PA, which contributed to 7 deaths. The outbreak investigation showed that 19 (47%) of the examined sink-drains grew at least once a VIM-PA. Two major clusters were observed by cgMLST: ST 111 (59 clones with 40 clinical isolates), and ST17 (8 clones with 6 clinical isolates). The estimated incidence rate of new cases was significantly higher in one ICU. CONCLUSION: A five-year prolonged outbreak at the ICU of the UZ Brussel was caused by only two VIM-PA clones, both linked to sink-drains, with minimal mutations occurring throughout the years. Statistical modelling found different incidence rates between units. Tailored interventions were hence prioritized.

Evidence for the occurrence of Acinetobacter faecalis in cattle feces and its emended description.

This study provides an emended description of Acinetobacter faecalis, a species previously described based on a single isolate (YIM 103518T) from elephant feces in China. Our emended description is based on 15 novel isolates conspecific with the A. faecalis type strain, obtained from eight cattle farms in the Czech Republic. The A. faecalis strains have relatively small genomes (≈2.5-2.7 Mbp), with a GC content of 36.3-36.7 mol%. Core genome-based phylogenetic analysis showed that the 15 strains, together with the type strain of A. faecalis, form a distinct and internally coherent phylogroup within the genus. Pairwise genomic ANIb values for the 16 A. faecalis strains were 97.32-99.04 %, while ANIb values between the genomes of the 16 strains and those of the other Acinetobacter spp. were ≤ 86.2 %. Analysis of whole-cell MALDI-TOF mass spectra supported the distinctness and cohesiveness of the taxon. The A. faecalis strains could be differentiated from the other validly named Acinetobacter spp. by the absence of hemolytic activity along with their ability to grow at 37 °C and on L-aspartate, ethanol, and L-glutamate but not at 41 °C or on adipate or 2,3-butanediol. Reduced susceptibility to sulfamethoxazole, trimethoprim and/or streptomycin was shown in eight strains, along with the presence of corresponding antibiotic resistance genes. In conclusion, this study provides a comprehensive description of A. faecalis and demonstrates its occurrence in cattle feces. Though the ecological role of A. faecalis remains unknown, our results show its ability to acquire antibiotic resistance genes, likely as an adaptation to antibiotic selection pressure in livestock farms.

High prevalence of the recently identified clonal lineage ST1299/CT3109 vanA among vancomycin-resistant Enterococcus faecium strains isolated from municipal wastewater.

Previously, we demonstrated that the majority of vancomycin-resistant Enterococcus faecium (VREfm) strains from in-patients of the University Hospital Erlangen, Germany, belonged to only three clonal lineages, namely ST117/CT71 vanB and two novel ST1299 vanA lineages classified as CT3109 and CT1903. The goal of the current study was (i) to investigate whether VREfm is also detectable in wastewater of the city of Erlangen, (ii) to identify their molecular features, and (iii) to clarify whether VREfm could arise from the community of the city of Erlangen or can be (directly) connected to nosocomial infections in the hospital setting. From April to May 2023, a total of 244 VREfm strains from raw wastewater of the city of Erlangen were analyzed by core genome multilocus sequence typing (cgMLST). Moreover, 20 of them were further investigated for single nucleotide polymorphisms (SNPs). The molecular characterization of the wastewater VREfm strains revealed a high prevalence (27.9%) of the recently identified clonal lineage ST1299/CT3109 vanA, which is mainly characterized by the presence of the tetracycline-resistance determinant tet(M) and the virulence genes pilA and prpA. The SNPs analysis revealed the presence of two major clusters, namely cluster I (≤65 SNPs), which included well-known hospital-adapted vanB clonal lineages such as ST117/CT71 and ST80/CT1065 and cluster II (≤70 SNPs), which were mainly characterized by the lineage ST1299/CT3109 vanA. Based on the concomitant resistance to vancomycin and tetracycline, we propose that ST1299/CT3109 vanA primarily originated and spread outside of hospital settings.IMPORTANCEThis study provides a detailed genomic analysis of vancomycin-resistant Enterococcus faecium (VREfm) strains isolated from municipal wastewater with a particular focus on clonal lineages, antimicrobial resistance, and the presence of virulence genes. The high wastewater prevalence of the recently identified clonal lineage ST1299/CT3109 vanA, which has been previously detected in hospitals, suggests an enormous potential for future spread in Germany.

Development and implementation of a core genome multilocus sequence typing scheme for Haemophilus influenzae.

Haemophilus influenzae is part of the human nasopharyngeal microbiota and a pathogen causing invasive disease. The extensive genetic diversity observed in H. influenzae necessitates discriminatory analytical approaches to evaluate its population structure. This study developed a core genome multilocus sequence typing (cgMLST) scheme for H. influenzae using pangenome analysis tools and validated the cgMLST scheme using datasets consisting of complete reference genomes (N = 14) and high-quality draft H. influenzae genomes (N = 2297). The draft genome dataset was divided into a development dataset (N = 921) and a validation dataset (N = 1376). The development dataset was used to identify potential core genes, and the validation dataset was used to refine the final core gene list to ensure the reliability of the proposed cgMLST scheme. Functional classifications were made for all the resulting core genes. Phylogenetic analyses were performed using both allelic profiles and nucleotide sequence alignments of the core genome to test congruence, as assessed by Spearman's correlation and ordinary least square linear regression tests. Preliminary analyses using the development dataset identified 1067 core genes, which were refined to 1037 with the validation dataset. More than 70% of core genes were predicted to encode proteins essential for metabolism or genetic information processing. Phylogenetic and statistical analyses indicated that the core genome allelic profile accurately represented phylogenetic relatedness among the isolates (R 2 = 0.945). We used this cgMLST scheme to define a high-resolution population structure for H. influenzae, which enhances the genomic analysis of this clinically relevant human pathogen.

A universal and constant rate of gene content change traces pangenome flux to LUCA.

Prokaryotic genomes constantly undergo gene flux via lateral gene transfer, generating a pangenome structure consisting of a conserved core genome surrounded by a more variable accessory genome shell. Over time, flux generates change in genome content. Here we measure and compare the rate of genome flux for 5 655 prokaryotic genomes as a function of amino acid sequence divergence in 36 universally distributed proteins of the informational core (IC). We find a clock of gene content change. The long-term average rate of gene content flux is remarkably constant across all higher prokaryotic taxa sampled, whereby the size of the accessory genome-the proportion of the genome harboring gene content difference for genome pairs-varies across taxa. The proportion of species-level accessory genes per genome, varies from 0% (Chlamydia) to 30-33% (Alphaproteobacteria, Gammaproteobacteria, Clostridia). A clock-like rate of gene content change across all prokaryotic taxa sampled suggest that pangenome structure is a general feature of prokaryotic genomes and that it has been in existence since the divergence of bacteria and archaea.

Emergence and Comparative Genome Analysis of Salmonella Ohio Strains from Brown Rats, Poultry, and Swine in Hungary.

Rats are particularly important from an epidemiological point of view, because they are regarded as reservoirs for diverse zoonotic pathogens including enteric bacteria. This study is the first to report the emergence of Salmonella serovar Ohio in brown rats (Rattus norvegicus) and food-producing animals in Hungary. We first reveal the genomic diversity of the strains and their phylogenomic relationships in the context of the international collection of S. Ohio genomes. This pathogen was detected in 4.3% (4/92) of rats, captured from multiple sites in Hungary. A whole-genome-based genotype comparison of S. Ohio, Infantis, Enteritidis, and Typhimurium strains showed that 76.4% (117/153) of the virulence and antimicrobial resistance genes were conserved among these serovars, and none of the genes were specific to S. Ohio. All S. Ohio strains lacked virulence and resistance plasmids. The cgMLST phylogenomic comparison highlighted a close genetic relationship between rat and poultry strains of S. Ohio from Hungary. These strains clustered together with the international S. Ohio genomes from aquatic environments. Overall, this study contributes to our understanding of the epidemiology of Salmonella spp. in brown rats and highlights the importance of monitoring to minimize the public health risk of rodent populations. However, further research is needed to understand the route of infection and evolution of this serovar.

Evolution and Competitive Struggles of Lactiplantibacillus plantarum under Different Oxygen Contents.

Lactiplantibacillus (Lb.) plantarum is known as a benign bacterium found in various habitats, including the intestines of animals and fermented foods. Since animal intestines lack oxygen, while fermented foods provide a limited or more oxygen environment, this study aimed to investigate whether there were genetic differences in the growth of Lb. plantarum under aerobic vs. anaerobic conditions. Genomic analysis of Lb. plantarum obtained from five sources-animals, dairy products, fermented meat, fermented vegetables, and humans-was conducted. The analysis included not only an examination of oxygen-utilizing genes but also a comparative pan-genomic analysis to investigate evolutionary relationships between genomes. The ancestral gene analysis of the evolutionary pathway classified Lb. plantarum into groups A and B, with group A further subdivided into A1 and A2. It was confirmed that group A1 does not possess the narGHIJ operon, which is necessary for energy production under limited oxygen conditions. Additionally, it was found that group A1 has experienced more gene acquisition and loss compared to groups A2 and B. Despite an initial assumption that there would be genetic distinctions based on the origin (aerobic or anaerobic conditions), it was observed that such differentiation could not be attributed to the origin. However, the evolutionary process indicated that the loss of genes related to nitrate metabolism was essential in anaerobic or limited oxygen conditions, contrary to the initial hypothesis.

Comparative genomics of quinolone-resistant Escherichia coli from broilers and humans in Norway.

BACKGROUND: The usage of fluoroquinolones in Norwegian livestock production is very low, including in broiler production. Historically, quinolone-resistant Escherichia coli (QREC) isolated from Norwegian production animals rarely occur. However, with the introduction of a selective screening method for QREC in the Norwegian monitoring programme for antimicrobial resistance in the veterinary sector in 2014; 89.5% of broiler caecal samples and 70.7% of broiler meat samples were positive. This triggered the concern if there could be possible links between broiler and human reservoirs of QREC. We are addressing this by characterizing genomes of QREC from humans (healthy carriers and patients) and broiler isolates (meat and caecum). RESULTS: The most frequent mechanism for quinolone resistance in both broiler and human E. coli isolates were mutations in the chromosomally located gyrA and parC genes, although plasmid mediated quinolone resistance (PMQR) was also identified. There was some relatedness of the isolates within human and broiler groups, but little between these two groups. Further, some overlap was seen for isolates with the same sequence type isolated from broiler and humans, but overall, the SNP distance was high. CONCLUSION: Based on data from this study, QREC from broiler makes a limited contribution to the incidence of QREC in humans in Norway.

Liquorilactobacillus: A Context of the Evolutionary History and Metabolic Adaptation of a Bacterial Genus from Fermentation Liquid Environments.

In the present work, we carried out a comparative genomic analysis to trace the evolutionary trajectory of the bacterial species that make up the Liquorilactobacillus genus, from the identification of genes and speciation/adaptation mechanisms in their unique characteristics to the identification of the pattern grouping these species. We present phylogenetic relationships between Liquorilactobacillus and related taxa such as Bacillus, basal lactobacilli and Ligilactobacillus, highlighting evolutionary divergences and lifestyle transitions across different taxa. The species of this genus share a core genome of 1023 genes, distributed in all COGs, which made it possible to characterize it as Liquorilactobacillus sensu lato: few amino acid auxotrophy, low genes number for resistance to antibiotics and general and specific cellular reprogramming mechanisms for environmental responses. These species were divided into four clades, with diversity being enhanced mainly by the diversity of genes involved in sugar metabolism. Clade 1 presented lower (< 70%) average amino acid identity with the other clades, with exclusive or absent genes, and greater distance in the genome compared to clades 2, 3 and 4. The data pointed to an ancestor of clades 2, 3 and 4 as being the origin of the genus Ligilactobacillus, while the species of clade 1 being closer to the ancestral Bacillus. All these traits indicated that the species of clade 1 could be soon separated in a distinct genus.

A novel variant of Chlamydia psittaci causing human psittacosis in China.

From January 2022 to November 2022, sporadic psittacosis occurred in Lishui city, China. The patients were presented with fever, cough, and pulmonary infiltration. Their clinical symptoms were not relieved after receiving cephalosporin, penicillin, beta-lactamase inhibitors, and quinolones. Metagenomic next-generation sequencing of bronchoalveolar lavage fluid samples from the patients revealed Chlamydia psittaci infection. Then, three C. psittaci strains were isolated from the patients. Their whole genome sequences (WGSs) were obtained, and a core genome multilocus sequence typing (cgMLST) method was developed to study the population structure of C. psittaci. Using the constructed cgMLST method, 72 WGSs were divided into four related groups and ten sub-clusters. The Lishui strains formed a unique population of C. psittaci, which might represent a new variant of C. psittaci. In vitro antimicrobial susceptibility testing suggested that the Lishui strains were sensitive to tetracycline, macrolides, quinolones, and no drug-resistance was observed.

Whole-Genome Sequencing of Human and Porcine Escherichia coli Isolates on a Commercial Pig Farm in South Africa.

Escherichia coli is an indicator micro-organism in One Health antibiotic resistance surveillance programs. The purpose of the study was to describe and compare E. coli isolates obtained from pigs and human contacts from a commercial farm in South Africa using conventional methods and whole-genome sequencing (WGS). Porcine E. coli isolates were proportionally more resistant phenotypically and harbored a richer diversity of antibiotic resistance genes as compared to human E. coli isolates. Different pathovars, namely ExPEC (12.43%, 21/169), ETEC (4.14%, 7/169), EPEC (2.96%, 5/169), EAEC (2.96%, 5/169) and STEC (1.18%, 2/169), were detected at low frequencies. Sequence type complex (STc) 10 was the most prevalent (85.51%, 59/169) among human and porcine isolates. Six STcs (STc10, STc86, STc168, STc206, STc278 and STc469) were shared at the human-livestock interface according to multilocus sequence typing (MLST). Core-genome MLST and hierarchical clustering (HC) showed that human and porcine isolates were overall genetically diverse, but some clustering at HC2-HC200 was observed. In conclusion, even though the isolates shared a spatiotemporal relationship, there were still differences in the virulence potential, antibiotic resistance profiles and cgMLST and HC according to the source of isolation.

Characterization of Acinetobacter baumannii at a tertiary hospital in Guangzhou: a genomic and clinical study.

Acinetobacter baumannii (AB) infections have become a global public health concern due to the continued increase in the incidence of infection and the rate of resistance to carbapenems. This study aimed to investigate the genomic features of AB strains recovered from a tertiary hospital and assess the clinical implications of the findings. A total of 217 AB strains were collected between 2016 and 2018 at a tertiary hospital in Guangzhou, with 183 (84.33%) being carbapenem-resistant AB (CRAB), with the main mechanism being the carriage of the blaOXA-23 gene. The overall mortality rate of patients caused by such strains was 15.21% (n = 33). Artificial lung ventilation and the use of meropenem were mortality risk factors in AB-infected patients, while KL2 AB infection was negatively associated. Core genome multilocus sequence typing and clustering analysis were performed on the integrated AB genome collection from the NCBI database and this study to illustrate the population structure among China. The results revealed diverse core genome profiles (n = 17) among AB strains from China, and strains from this single hospital exhibited most of the core genome profiles (n = 13), suggesting genetic variability within the hospital and transmission across the country. These findings show that the high transmission potential of the CRAB strains and meropenem usage that confers a selective advantage of CRAB clinically are two major factors that pose significant challenges to the effective clinical management of AB infections. Understanding the genetic features and transmission patterns of clinical AB strains is crucial for the effective control of infections caused by this pathogen.

Antimicrobial resistance and genetic relatedness among Campylobacter coli and Campylobacter jejuni from humans and retail chicken meat in Taiwan.

OBJECTIVES: Campylobacter is a significant zoonotic pathogen primarily transmitted through poultry. Our study aimed to assess antimicrobial resistance and genetic relationships among Campylobacter isolates from retail chicken meat and humans in Taiwan. METHODS: Campylobacter isolates were analysed using whole-genome sequencing to investigate their antimicrobial resistance, genetic determinants of resistance, and genotypes. RESULTS: Campylobacter coli and Campylobacter jejuni accounted for 44.9% and 55.1% of chicken meat isolates, and 11.4% and 88.6% of human isolates, respectively. C. coli displayed significantly higher resistance levels. Furthermore, isolates from chicken meat exhibited higher levels of resistance to most tested antimicrobials compared to isolates from humans. Multidrug resistance was observed in 96.3% of C. coli and 43.3% of C. jejuni isolates from chicken meat and 80.6% of C. coli and 15.8% of C. jejuni isolates from humans. Macrolide resistance was observed in 85.5% of C. coli isolates, primarily attributed to the erm(B) rather than the A2075G mutation in 23S rRNA. Among the 511 genomes, we identified 133 conventional MLST sequence types, indicating significant diversity among Campylobacter strains. Notably, hierarchical Core-genome multilocus sequence typing clustering, including HC0, HC5, and HC10, revealed a significant proportion of closely related isolates from chicken meat and humans. CONCLUSIONS: Our research highlights significant associations in antimicrobial resistance and genetic relatedness between Campylobacter isolates from chicken meat and humans in Taiwan. The genetic analysis data suggest that campylobacteriosis outbreaks may occur more frequently in Taiwan than previously assumed. Our study emphasizes the need for strategies to control multidrug-resistant strains and enhance outbreak prevention.

Implications of ad-hoc molecular typing for infection control measures in a multi-cluster, multi-phenotypic Serratia marcescens outbreak in a neonatal intensive care unit.

BACKGROUND: Serratia marcescens is known to cause outbreaks in neonatal intensive care units (NICUs). Traditionally epidemiological data, antimicrobial resistance patterns and epidemiological typing have been used to guide infection prevention methods. Whole-genome sequencing (WGS) applications such as core-genome multi-locus sequence typing (cgMLST) applied during an outbreak would potentially yield more information. AIM: To use cgMLST to acquire detailed information on the source and spread of bacteria, enabling more efficient control measures during an S. marcescens outbreak at a NICU. METHODS: Neonates admitted to the NICU of the Leiden University Medical Center (LUMC) during an outbreak between September 2023 and January 2024, with S. marcescens being cultured, were included. Environmental samples were taken to search for a common source, antibiotic susceptibility testing was performed, and antimicrobial resistance genes were analysed. FINDINGS: S. marcescens strains from 17 of the 20 positive patients were available for molecular typing. The cgMLST scheme revealed five different complex types consisting of four separate clusters. Multiple clusters made an unidentified persistent environmental source as cause of the outbreak less likely, leading to a quick downscaling of infection prevention measures. Differences were shown in aminoglycoside resistance patterns of isolates within the same complex types and patients. CONCLUSION: The use of ad-hoc cgMLST provided timely data for rational decision-making during an S. marcescens outbreak at the NICU. Antibiotic phenotyping alone was found not to be suitable for studying clonal spread during this outbreak with S. marcescens.

Aspergillus flavus pangenome (AflaPan) uncovers novel aflatoxin and secondary metabolite associated gene clusters.

BACKGROUND: Aspergillus flavus is an important agricultural and food safety threat due to its production of carcinogenic aflatoxins. It has high level of genetic diversity that is adapted to various environments. Recently, we reported two reference genomes of A. flavus isolates, AF13 (MAT1-2 and highly aflatoxigenic isolate) and NRRL3357 (MAT1-1 and moderate aflatoxin producer). Where, an insertion of 310 kb in AF13 included an aflatoxin producing gene bZIP transcription factor, named atfC. Observations of significant genomic variants between these isolates of contrasting phenotypes prompted an investigation into variation among other agricultural isolates of A. flavus with the goal of discovering novel genes potentially associated with aflatoxin production regulation. Present study was designed with three main objectives: (1) collection of large number of A. flavus isolates from diverse sources including maize plants and field soils; (2) whole genome sequencing of collected isolates and development of a pangenome; and (3) pangenome-wide association study (Pan-GWAS) to identify novel secondary metabolite cluster genes. RESULTS: Pangenome analysis of 346 A. flavus isolates identified a total of 17,855 unique orthologous gene clusters, with mere 41% (7,315) core genes and 59% (10,540) accessory genes indicating accumulation of high genomic diversity during domestication. 5,994 orthologous gene clusters in accessory genome not annotated in either the A. flavus AF13 or NRRL3357 reference genomes. Pan-genome wide association analysis of the genomic variations identified 391 significant associated pan-genes associated with aflatoxin production. Interestingly, most of the significantly associated pan-genes (94%; 369 associations) belonged to accessory genome indicating that genome expansion has resulted in the incorporation of new genes associated with aflatoxin and other secondary metabolites. CONCLUSION: In summary, this study provides complete pangenome framework for the species of Aspergillus flavus along with associated genes for pathogen survival and aflatoxin production. The large accessory genome indicated large genome diversity in the species A. flavus, however AflaPan is a closed pangenome represents optimum diversity of species A. flavus. Most importantly, the newly identified aflatoxin producing gene clusters will be a new source for seeking aflatoxin mitigation strategies and needs new attention in research.

Methods for Pangenomic Core Detection.

Computational pangenomics deals with the joint analysis of all genomic sequences of a species. It has already been successfully applied to various tasks in many research areas. Further advances in DNA sequencing technologies constantly let more and more genomic sequences become available for many species, leading to an increasing attractiveness of pangenomic studies. At the same time, larger datasets also pose new challenges for data structures and algorithms that are needed to handle the data. Efficient methods oftentimes make use of the concept of k-mers.Core detection is a common way of analyzing a pangenome. The pangenome's core is defined as the subset of genomic information shared among all individual members. Classically, it is not only determined on the abstract level of genes but can also be described on the sequence level.In this chapter, we provide an overview of k-mer-based methods in the context of pangenomics studies. We first revisit existing software solutions for k-mer counting and k-mer set representation. Afterward, we describe the usage of two k-mer-based approaches, Pangrowth and Corer, for pangenomic core detection.

Temporal variations in the serogroup distribution of invasive meningococcal disease in Quebec, Canada, due to emerging unique clade of serogroup Y strain belonging to the Sequence Type-23 clonal complex.

OBJECTIVE: To identify recent trends in invasive meningococcal diseases (IMD) in Quebec, Canada, with a focus on MenY cases and MenY strains. METHODS: IMD cases and MenY strains from January 1, 2015 to August 11, 2023 were analyzed for clonal analysis and prediction of susceptibility to MenB vaccines. MenY strains of ST-23 CC from Quebec were analyzed with global MenY strains by core-genomic multi-locus sequence typing (cg-MLST). RESULTS: Since 2015 the serogroup distribution of IMD in Quebec has shifted from predominantly MenB to mainly MenY, with most (80.9 %) of the latter belonging to ST-23 CC. The median age of MenY cases due to ST-23 CC were statistically younger than MenY cases due to non-ST-23 CC. MenY of ST-23 CC showed genetic diversity and the major genetic cluster were similar to the Swedish Y1 strain. The increase in invasive MenY disease in Quebec was due to a sub-clade of Lineage 23.1 which caused an elevated proportion of severe disease in young adults. CONCLUSION: The increase in invasive MenY disease in Quebec, Canada was driven by the expansion of a sub-clade of Lineage 23.1 in young adults. Currently available quadrivalent A,C,W,Y-conjugate meningococcal vaccines were predicted to provide protection against these strains.

Pan-genome analysis of Streptococcus suis serotype 2 highlights genes associated with virulence and antibiotic resistance.

Streptococcus suis serotype 2 (SS2) is a Gram-positive bacterium. It is a common and significant pathogen in pigs and a common cause of zoonotic meningitis in humans. It can lead to sepsis, endocarditis, arthritis, and pneumonia. If not diagnosed and treated promptly, it has a high mortality rate. The pan-genome of SS2 is open, and with an increasing number of genes, the core genome and accessory genome may exhibit more pronounced differences. Due to the diversity of SS2, the genes related to its virulence and resistance are still unclear. In this study, a strain of SS2 was isolated from a pig farm in Sichuan Province, China, and subjected to whole-genome sequencing and characterization. Subsequently, we conducted a Pan-Genome-Wide Association Study (Pan-GWAS) on 230 strains of SS2. Our analysis indicates that the core genome is composed of 1,458 genes related to the basic life processes of the bacterium. The accessory genome, consisting of 4,337 genes, is highly variable and a major contributor to the genetic diversity of SS2. Furthermore, we identified important virulence and resistance genes in SS2 through pan-GWAS. The virulence genes of SS2 are mainly associated with bacterial adhesion. In addition, resistance genes in the core genome may confer natural resistance of SS2 to fluoroquinolone and glycopeptide antibiotics. This study lays the foundation for further research on the virulence and resistance of SS2, providing potential new drug and vaccine targets against SS2.

A core genome multi-locus sequence typing scheme for Streptococcus uberis: an evolution in typing a genetically diverse pathogen.

Streptococcus uberis is a globally endemic and poorly controlled cause of bovine mastitis impacting the sustainability of the modern dairy industry. A core genome was derived from 579 newly sequenced S. uberis isolates, along with 305 publicly available genome sequences of S. uberis isolated from 11 countries around the world and used to develop a core genome multi-locus sequence typing (cgMLST) scheme. The S. uberis core genome comprised 1475 genes, and these were used to identify 1447 curated loci that were indexed into the cgMLST scheme. This was able to type 1012 of 1037 (>97  %) isolates used and differentiated the associated sequences into 932 discrete core genome sequence types (cgSTs). Analysis of the phylogenetic relationships of cgSTs revealed no clear clustering of isolates based on metadata such as disease status or year of isolation. Geographical clustering of cgSTs was limited to identification of a UK-centric clade, but cgSTs from UK isolates were also dispersed with those originating from other geographical regions across the entire phylogenetic topology. The cgMLST scheme offers a new tool for the detailed analysis of this globally important pathogen of dairy cattle. Initial analysis has re-emphasized and exemplified the genetically diverse nature of the global population of this opportunistic pathogen.

Uncovering the boundaries of Campylobacter species through large-scale phylogenetic and nucleotide identity analyses.

Campylobacter species are typically helical shaped, Gram-negative, and non-spore-forming bacteria. Species in this genus include established foodborne and animal pathogens as well as emerging pathogens. The accumulation of genomic data from the Campylobacter genus has increased exponentially in recent years, accompanied by the discovery of putative new species. At present, the lack of a standardized species boundary complicates distinguishing established and novel species. We defined the Campylobacter genus core genome (500 loci) using publicly available Campylobacter complete genomes (n = 498) and constructed a core genome phylogeny using 2,193 publicly available Campylobacter genomes to examine inter-species diversity and species boundaries. Utilizing 8,440 Campylobacter genomes representing 33 species and 8 subspecies, we found species delineation based on an average nucleotide identity (ANI) cutoff of 94.2% is consistent with the core genome phylogeny. We identified 60 ANI genomic species that delineated Campylobacter species in concordance with previous comparative genetic studies. All pairwise ANI genomic species pairs had in silico DNA-DNA hybridization scores of less than 70%, supporting their delineation as separate species. We provide the tool Campylobacter Genomic Species typer (CampyGStyper) that assigns ANI genomic species to query genomes based on ANI similarities to medoid genomes from each ANI genomic species with an accuracy of 99.96%. The ANI genomic species definitions proposed here allow consistent species definition in the Campylobacter genus and will facilitate the detection of novel species in the future.IMPORTANCEIn recent years, Campylobacter has gained recognition as the leading cause of bacterial gastroenteritis worldwide, leading to a substantial rise in the collection of genomic data of the Campylobacter genus in public databases. Currently, a standardized Campylobacter species boundary at the genomic level is absent, leading to challenges in detecting emerging pathogens and defining putative novel species within this genus. We used a comprehensive representation of genomes of the Campylobacter genus to construct a core genome phylogenetic tree. Furthermore, we found an average nucleotide identity (ANI) of 94.2% as the optimal cutoff to define the Campylobacter species. Using this cutoff, we identified 60 ANI genomic species which provided a standardized species definition and nomenclature. Importantly, we have developed Campylobacter Genomic Species typer (CampyGStyper), which can robustly and accurately assign these ANI genomic species to Campylobacter genomes, thereby aiding pathogen surveillance and facilitating evolutionary and epidemiological studies of existing and emerging pathogens in the genus Campylobacter.