- Volume 9, Issue 6, 2023

Nucleotide sequence similarity, including k-mer plasmid composition, has been used for prediction of plasmid evolutionary host range, representing the hosts in which a plasmid has replicated at some point during its evolutionary history. However, the relationships between the bacterial taxa of experimentally identified transconjugants and the predicted evolutionary host ranges are poorly understood. Here, four different PromA group plasmids showing different k-mer compositions were used as model plasmids. Filter mating assays were performed with a donor harbouring plasmids and recipients of bacterial communities extracted from environmental samples. A broad range of transconjugants was obtained with different bacterial taxa. A calculation of the dissimilarities in k-mer compositions as Mahalanobis distance between the plasmid and its sequenced transconjugant chromosomes revealed that each plasmid and transconjugant were significantly more similar than the plasmid and other non-transconjugant chromosomes. These results indicate that plasmids with different k-mer compositions clearly have different host ranges to which the plasmid will be transferred and replicated. The similarity of the nucleotide compositions could be used for predicting not only the plasmid evolutionary host range but also future host ranges.

We delineate the evolutionary plasticity of the ecologically and biotechnologically important genus Streptomyces , by analysing the genomes of 213 species. Streptomycetes genomes demonstrate high levels of internal homology, whereas the genome of their last common ancestor was already complex. Importantly, we identify the species-specific fingerprint proteins that characterize each species. Even among closely related species, we observed high interspecies variability of chromosomal protein-coding genes, species-level core genes, accessory genes and fingerprints. Notably, secondary metabolite biosynthetic gene clusters (smBGCs), carbohydrate-active enzymes (CAZymes) and protein-coding genes bearing the rare TTA codon demonstrate high intraspecies and interspecies variability, which emphasizes the need for strain-specific genomic mining. Highly conserved genes, such as those specifying genus-level core proteins, tend to occur in the central region of the chromosome, whereas those encoding proteins with evolutionarily volatile species-level fingerprints, smBGCs, CAZymes and TTA-codon-bearing genes are often found towards the ends of the linear chromosome. Thus, the chromosomal arms emerge as the part of the genome that is mainly responsible for rapid adaptation at the species and strain level. Finally, we observed a moderate, but statistically significant, correlation between the total number of CAZymes and three categories of smBGCs (siderophores, e-Polylysin and type III lanthipeptides) that are related to competition among bacteria.

Kazachstania aerobia and Kazachstania servazzii can affect wine aroma by increasing acetate ester concentrations, most remarkably phenylethyl acetate and isoamyl acetate. The genetic basis of this is unknown, there being little to no sequence data available on the genome architecture. We report for the first time the near-complete genome sequence of the two species using long-read (PacBio) sequencing (K. aerobia 20 contigs, one scaffold; and K. servazzii 22 contigs, one scaffold). The annotated genomes of K. aerobia (12.5 Mb) and K. servazzii (12.3 Mb) were compared to Saccharomyces cerevisiae genomes (laboratory strain S288C and wine strain EC1118). Whilst a comparison of the two Kazachstania spp. genomes revealed few differences between them, divergence was evident in relation to the genes involved in ester biosynthesis, for which gene duplications or absences were apparent. The annotations of these genomes are valuable resources for future research into the evolutionary biology of Kazachstania and other yeast species (comparative genomics) as well as understanding the metabolic processes associated with alcoholic fermentation and the production of secondary ‘aromatic’ metabolites (transcriptomics, proteomics and metabolomics).

Dehalobacterium formicoaceticum is recognized for its ability to anaerobically ferment dichloromethane (DCM), and a catabolic model has recently been proposed. D. formicoaceticum is currently the only axenic representative of its class, the Dehalobacteriia, according to the Genome Taxonomy Database. However, substantial additional diversity has been revealed in this lineage through culture-independent exploration of anoxic habitats. Here we performed a comparative analysis of 10 members of the Dehalobacteriia, representing three orders, and infer that anaerobic DCM degradation appears to be a recently acquired trait only present in some members of the order Dehalobacteriales. Inferred traits common to the class include the use of amino acids as carbon and energy sources for growth, energy generation via a remarkable range of putative electron-bifurcating protein complexes and the presence of S-layers. The ability of D. formicoaceticum to grow on serine without DCM was experimentally confirmed and a high abundance of the electron-bifurcating protein complexes and S-layer proteins was noted when this organism was grown on DCM. We suggest that members of the Dehalobacteriia are low-abundance fermentative scavengers in anoxic habitats.

The gut microbiota is a reservoir for antimicrobial resistance genes (ARGs). With current sequencing methods, it is difficult to assign ARGs to their microbial hosts, particularly if these ARGs are located on plasmids. Metagenomic chromosome conformation capture approaches (meta3C and Hi-C) have recently been developed to link bacterial genes to phylogenetic markers, thus potentially allowing the assignment of ARGs to their hosts on a microbiome-wide scale. Here, we generated a meta3C dataset of a human stool sample and used previously published meta3C and Hi-C datasets to investigate bacterial hosts of ARGs in the human gut microbiome. Sequence reads mapping to repetitive elements were found to cause problematic noise in, and may importantly skew interpretation of, meta3C and Hi-C data. We provide a strategy to improve the signal-to-noise ratio by discarding reads that map to insertion sequence elements and to the end of contigs. We also show the importance of using spike-in controls to quantify whether the cross-linking step in meta3C and Hi-C protocols has been successful. After filtering to remove artefactual links, 87 ARGs were assigned to their bacterial hosts across all datasets, including 27 ARGs in the meta3C dataset we generated. We show that commensal gut bacteria are an important reservoir for ARGs, with genes coding for aminoglycoside and tetracycline resistance being widespread in anaerobic commensals of the human gut.

Campylobacter jejuni is the leading cause of bacterial gastroenteritis worldwide, but, unlike other foodborne pathogens, is not commonly reported as causing outbreaks. The population structure of the species is characterized by a high degree of genetic diversity, but the presence of stable clonally derived genotypes persisting in space and time, and potentially leading to diffuse outbreaks, has recently been identified. The spread of these recurring genotypes could be enhanced by wild birds, suspected to act as vectors for a wide range of microorganisms that can be transmissible to other animals or humans. This study assessed the genetic diversity of C. jejuni carriage in wild birds and surface waters to explore a potential link between these environments and the persistence over years of recurring lineages infecting humans in Luxembourg. These lineages corresponded to over 40 % of clinical isolates over a 4 year period from 2018 to 2021. While mainly exotic genotypes were recovered from environmental samples, 4 % of C. jejuni from wild birds corresponded to human recurring genotypes. Among them, a human clinical endemic lineage, occurring for over a decade in Luxembourg, was detected in one bird species, suggesting a possible contribution to the persistence of this clone and its multi-host feature. Whereas 27 % of wild birds were carriers of C. jejuni, confirming their role as spreader or reservoir, only three out of 59 genotypes overlapped with recurring human strains. While direct transmission of C. jejuni infection through wild birds remains questionable, they may play a key role in the environmental spreading of stable clones to livestock, and this issue merits further investigation.

Carbapenem-resistant Klebsiella pneumoniae is a major cause of hospital-acquired infections and the fastest-growing pathogen in Europe. Carbapenem resistance was detected at the Consorcio Hospital General Universitario de Valencia (CHGUV) in early 2015, and there has been a significant increase in carbapenem-resistant isolates since then. In this study, we collected carbapenem-resistant isolates from this hospital during the period of increase (from 2015 to 2019) and studied how K. pneumoniae carbapenem-resistant isolates emerged and spread in the hospital. A total of 225 isolates were subjected to whole-genome sequencing with Illumina NextSeq. We characterized the isolates by identifying lineages and antimicrobial resistance genes and plasmids, especially those related to reduced carbapenem susceptibility. Our findings show that the initial carbapenem resistance emergence and dissemination at the CHGUV occurred during a short period of 1 year. Furthermore, it was complex, involving six different lineages of types ST307, ST11, ST101 and ST437, different resistance-determinant factors, including OXA-48, NDM-1, NDM-23 and DHA-1, and different plasmids.

  Escherichia coli   is one of the most prevalent Gram-negative species associated with drug resistant infections. Strains that produce extended-spectrum beta-lactamases (ESBLs) or carbapenemases are both particularly problematic and disproportionately impact resource limited healthcare settings where last-line antimicrobials may not be available. A large number of   E. coli   genomes are now available and have allowed insights into pathogenesis and epidemiology of ESBL   E. coli   but genomes from sub-Saharan Africa (sSA) are significantly underrepresented. To reduce this gap, we investigated ESBL-producing   E. coli   colonising adults in Blantyre, Malawi to assess bacterial diversity and AMR determinants and to place these isolates in the context of the wider population structure. We performed short-read whole-genome sequencing of 473 colonising ESBL   E. coli   isolated from human stool and contextualised the genomes with a previously curated multi-country collection of 10 146   E. coli   genomes and sequence type (ST)-specific collections for our three most commonly identified STs. These were the globally successful ST131, ST410 and ST167, and the dominant ESBL genes were bla CTX-M, mirroring global trends. However, 37 % of Malawian isolates did not cluster with any isolates in the curated multicountry collection and phylogenies were consistent with locally spreading monophyletic clades, including within the globally distributed, carbapenemase-associated B4/H24RxC ST410 lineage. A single ST2083 isolate in this collection harboured a carbapenemase gene. Long read sequencing demonstrated the presence of a globally distributed ST410-associated carbapenemase carrying plasmid in this isolate, which was absent from the ST410 strains in our collection. We conclude there is a risk that carbapenem resistance in   E. coli   could proliferate rapidly in Malawi under increasing selection pressure, and that both ongoing antimicrobial stewardship and genomic surveillance are critical as local carbapenem use increases.

The type VIIb protein secretion system (T7SSb) is found in Bacillota (firmicute) bacteria and has been shown to mediate interbacterial competition. EssC is a membrane-bound ATPase that is a critical component of the T7SSb and plays a key role in substrate recognition. Prior analysis of available genome sequences of the foodborne bacterial pathogen Listeria monocytogenes has shown that although the T7SSb was encoded as part of the core genome, EssC could be found as one of seven different sequence variants. While each sequence variant was associated with a specific suite of candidate substrate proteins encoded immediately downstream of essC, many LXG-domain proteins were encoded across multiple essC sequence variants. Here, we have extended this analysis using a diverse collection of 37 930 L . monocytogenes genomes. We have identified a rare eighth variant of EssC present in ten L. monocytogenes lineage III genomes. These genomes also encode a large toxin of the rearrangement hotspot (Rhs) repeat family adjacent to essC8, along with a probable immunity protein and three small accessory proteins. We have further identified nine novel LXG-domain proteins, and four additional chromosomal hotspots across L. monocytogenes genomes where LXG proteins can be encoded. The eight L. monocytogenes EssC variants were also found in other Listeria species, with additional novel EssC types also identified. Across the genus, species frequently encoded multiple EssC types, indicating that T7SSb diversity is a primary feature of the genus Listeria .

Vibrio parahaemolyticus has been identified as an emerging human pathogen worldwide with cases undergoing a global expansion over recent decades in phase with climate change. New Zealand had remained free of outbreaks until 2019, but different outbreaks have been reported consecutively since then. To provide new insights into the recent emergence of cases associated with outbreak clones over recent years, a comparative genomic study was carried out using a selection of clinical (mostly outbreak) and environmental isolates of V. parahaemolyticus obtained in New Zealand between 1973 and 2021. Among 151 isolates of clinical (n=60) and environmental (n=91) origin, 47 sequence types (STs) were identified, including 31 novel STs. The population of environmental isolates generated 30 novel STs, whereas only 1 novel ST (ST2658) was identified among the population of clinical isolates. The novel clinical ST was a single-locus variant of the pandemic ST36 strain, indicating further evolution of this pandemic strain. The environmental isolates exhibited a significant genetic heterogeneity compared to the clinical isolates. The whole-genome phylogeny separated the population of clinical isolates from their environmental counterparts, clearly indicating their distant genetic relatedness. In addition to differences in ancestral profiles and genetic relatedness, these two groups of isolates exhibited a profound difference in their virulence profiles. While the entire population of clinical isolates harboured the thermostable direct haemolysin (tdh) and/or the thermostable-related haemolysin (trh), only a few isolates of environmental origin possessed the same virulence genes. In contrast to tdh and trh, adhesin-encoding genes, vpadF and MSHA, showed a significantly (P<0.001) greater association with the environmental isolates compared to the clinical isolates. The effectors, VopQ, VPA0450 and VopS, which belong to T3SS1, were ubiquitous, being present in each isolate regardless of its origin. The effectors VopC and VopA, which belong to T3SS2, were rarely detected in any of the examined isolates. Our data indicate that the clinical and environmental isolates of V. parahaemolyticus from New Zealand differ in their population structures, ancestral profiles, genetic relatedness and virulence profiles. In addition, we identified numerous unique non-synonymous single-nucleotide polymorphisms (nsSNPs) in adhesins and effectors, exclusively associated with the clinical isolates tested, which may suggest a possible role of these mutations in the overall virulence of the clinical isolates.

The outer core locus (OCL) that includes genes for the synthesis of the variable outer core region of the lipooligosaccharide (LOS) is one of the key epidemiological markers used for tracing the spread of Acinetobacter baumannii , a bacterial pathogen of global concern. In this study, we screened 12 476 publicly available A. baumannii genome assemblies for novel OCL sequences, detecting six new OCL types that were designated OCL17–OCL22. These were compiled with previously characterized OCL sequences to create an updated version of the A. baumannii OCL reference database, providing a total of 22 OCL reference sequences for use with the bioinformatics tool Kaptive. Use of this database against the 12 476 downloaded assemblies found OCL1 to be the most common locus, present in 73.6 % of sequenced genomes assigned by Kaptive with a match confidence score of good or above. OCL1 was most common amongst isolates belonging to sequence types (STs) ST1, ST2, ST3 and ST78, all of which are over-represented clonal lineages. The highest level of diversity in OCL types was found in ST2, with eight different OCLs identified. The updated OCL reference database is available for download from GitHub (https://github.com/klebgenomics/Kaptive; under version v. 2.0.5), and has been integrated for use on Kaptive-Web (https://kaptive-web.erc.monash.edu/) and PathogenWatch (https://pathogen.watch/), enhancing current methods for A. baumannii strain identification, classification and surveillance.

The National Institute for Communicable Diseases in South Africa participates in national laboratory-based surveillance for human isolates of Salmonella species. Laboratory analysis includes whole-genome sequencing (WGS) of isolates. We report on WGS-based surveillance of Salmonella enterica serovar Typhi ( Salmonella Typhi) in South Africa from 2020 through 2021. We describe how WGS analysis identified clusters of enteric fever in the Western Cape Province of South Africa and describe the epidemiological investigations associated with these clusters. A total of 206 Salmonella Typhi isolates were received for analysis. Genomic DNA was isolated from bacteria and WGS was performed using Illumina NextSeq technology. WGS data were investigated using multiple bioinformatics tools, including those available at the Centre for Genomic Epidemiology, EnteroBase and Pathogenwatch. Core-genome multilocus sequence typing was used to investigate the phylogeny of isolates and identify clusters. Three major clusters of enteric fever were identified in the Western Cape Province; cluster one (n=11 isolates), cluster two (n=13 isolates), and cluster three (n=14 isolates). To date, no likely source has been identified for any of the clusters. All isolates associated with the clusters, showed the same genotype (4.3.1.1.EA1) and resistome (antimicrobial resistance genes: bla TEM-1B, catA1, sul1, sul2, dfrA7). The implementation of genomic surveillance of Salmonella Typhi in South Africa has enabled rapid detection of clusters indicative of possible outbreaks. Cluster identification allows for targeted epidemiological investigations and a timely, coordinated public health response.

Although infections caused by Clostridioides difficile have historically been attributed to hospital acquisition, growing evidence supports the role of community acquisition in C. difficile infection (CDI). Symptoms of CDI can range from mild, self-resolving diarrhoea to toxic megacolon, pseudomembranous colitis, and death. In this study, we sampled C. difficile from clinical, environmental, and canine reservoirs in Flagstaff, Arizona, USA, to understand the distribution and transmission of the pathogen in a One Health framework; Flagstaff is a medium-sized, geographically isolated city with a single hospital system, making it an ideal site to characterize genomic overlap between sequenced C. difficile isolates across reservoirs. An analysis of 562 genomes from Flagstaff isolates identified 65 sequence types (STs), with eight STs being found across all three reservoirs and another nine found across two reservoirs. A screen of toxin genes in the pathogenicity locus identified nine STs where all isolates lost the toxin genes needed for CDI manifestation (tcdB, tcdA), demonstrating the widespread distribution of non-toxigenic C. difficile (NTCD) isolates in all three reservoirs; 15 NTCD genomes were sequenced from symptomatic, clinical samples, including two from mixed infections that contained both tcdB+ and tcdB- isolates. A comparative single nucleotide polymorphism (SNP) analysis of clinically derived isolates identified 78 genomes falling within clusters separated by ≤2 SNPs, indicating that ~19 % of clinical isolates are associated with potential healthcare-associated transmission clusters; only symptomatic cases were sampled in this study, and we did not sample asymptomatic transmission. Using this same SNP threshold, we identified genomic overlap between canine and soil isolates, as well as putative transmission between environmental and human reservoirs. The core genome of isolates sequenced in this study plus a representative set of public C. difficile genomes (n=136), was 2690 coding region sequences, which constitutes ~70 % of an individual C. difficile genome; this number is significantly higher than has been published in some other studies, suggesting that genome data quality is important in understanding the minimal number of genes needed by C. difficile . This study demonstrates the close genomic overlap among isolates sampled across reservoirs, which was facilitated by maximizing the genomic search space used for comprehensive identification of potential transmission events. Understanding the distribution of toxigenic and non-toxigenic C. difficile across reservoirs has implications for surveillance sampling strategies, characterizing routes of infections, and implementing mitigation measures to limit human infection.

The genomic diversity of microbes is commonly parameterized as SNPs relative to a reference genome of a well-characterized, but arbitrary, isolate. However, any reference genome contains only a fraction of the microbial pangenome, the total set of genes observed in a given species. Reference-based approaches are thus blind to the dynamics of the accessory genome, as well as variation within gene order and copy number. With the widespread usage of long-read sequencing, the number of high-quality, complete genome assemblies has increased dramatically. In addition to pangenomic approaches that focus on the variation in the sets of genes present in different genomes, complete assemblies allow investigations of the evolution of genome structure and gene order. This latter problem, however, is computationally demanding with few tools available that shed light on these dynamics. Here, we present PanGraph, a Julia-based library and command line interface for aligning whole genomes into a graph. Each genome is represented as a path along vertices, which in turn encapsulate homologous multiple sequence alignments. The resultant data structure succinctly summarizes population-level nucleotide and structural polymorphisms and can be exported into several common formats for either downstream analysis or immediate visualization.

Acidithiobacillus ferrooxidans serves as a model chemolithoautotrophic organism in extremely acidic environments, which has attracted much attention due to its unique metabolism and strong adaptability. However, little was known about the divergences along the evolutionary process based on whole genomes. Herein, we isolated six strains of A. ferrooxidans from mining areas in China and Zambia, and used comparative genomics to investigate the intra-species divergences. The results indicated that A. ferrooxidans diverged into three groups from a common ancestor, and the pan-genome is ‘open’. The ancestral reconstruction of A. ferrooxidans indicated that genome sizes experienced a trend of increase in the very earliest days before a decreasing tendency during the evolutionary process, suggesting that both gene gain and gene loss played crucial roles in A. ferrooxidans genome flexibility. Meanwhile, 23 single-copy orthologous groups (OGs) were under positive selection. The differences of rusticyanin (Rus) sequences (the key protein in the iron oxidation pathway) and type IV secretion system (T4SS) composition in the A. ferrooxidans were both related to their group divergences, which contributed to their intraspecific diversity. This study improved our understanding of the divergent evolution and environmental adaptation of A. ferrooxidans at the genome level in extreme conditions, which provided theoretical support for the survival mechanism of living creatures at the extreme.