- Volume 7, Issue 6, 2021
Volume 7, Issue 6, 2021
- Outbreak Reports
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- Pathogens and Epidemiology
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Whole-genome sequencing resolves a polyclonal outbreak by extended-spectrum beta-lactam and carbapenem-resistant Klebsiella pneumoniae in a Portuguese tertiary-care hospital
Klebsiella pneumoniae has emerged as an important nosocomial pathogen, with whole-genome sequencing (WGS) significantly improving our ability to characterize associated outbreaks. Our study sought to perform a genome-wide analysis of multiclonal K. pneumoniae isolates (n=39; 23 patients) producing extended spectrum beta-lactamases and/or carbapenemases sourced between 2011 and 2016 in a Portuguese tertiary-care hospital. All isolates showed resistance to third-generation cephalosporins and six isolates (five patients) were also carbapenem resistant. Genome-wide-based phylogenetic analysis revealed a topology representing ongoing dissemination of three main sequence-type (ST) clades (ST15, ST147 and ST307) and transmission across different wards, compatible with missing links that can take the form of undetected colonized patients. Two carbapenemase-coding genes were detected: blaKPC-3 , located on a Tn4401d transposon, and blaGES-5 on a novel class 3 integron. Additionally, four genes coding for ESBLs (blaBEL-1 , blaCTX-M-8 , blaCTX-M-15 and blaCTX-M-32 ) were also detected. ESBL horizontal dissemination across five clades is highlighted by the similar genetic environments of blaCTX-M-15 gene upstream of ISEcp1 on a Tn3-like transposon. Overall, this study provides a high-resolution genome-wide perspective on the epidemiology of ESBL and carbapenemase-producing K. pneumoniae in a healthcare setting while contributing for the adoption of appropriate intervention and prevention strategies.
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- Research Articles
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- Genomic Methodologies
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rMAP: the Rapid Microbial Analysis Pipeline for ESKAPE bacterial group whole-genome sequence data
More LessThe recent re-emergence of multidrug-resistant pathogens has exacerbated their threat to worldwide public health. The evolution of the genomics era has led to the generation of huge volumes of sequencing data at an unprecedented rate due to the ever-reducing costs of whole-genome sequencing (WGS). We have developed the Rapid Microbial Analysis Pipeline (rMAP), a user-friendly pipeline capable of profiling the resistomes of ESKAPE pathogens ( Enterococcus faecium , Staphylococcus aureus , Klebsiella pneumoniae , Acinetobacter baumannii , Pseudomonas aeruginosa and Enterobacter species) using WGS data generated from Illumina’s sequencing platforms. rMAP is designed for individuals with little bioinformatics expertise, and automates the steps required for WGS analysis directly from the raw genomic sequence data, including adapter and low-quality sequence read trimming, de novo genome assembly, genome annotation, single-nucleotide polymorphism (SNP) variant calling, phylogenetic inference by maximum likelihood, antimicrobial resistance (AMR) profiling, plasmid profiling, virulence factor determination, multi-locus sequence typing (MLST), pangenome analysis and insertion sequence characterization (IS). Once the analysis is finished, rMAP generates an interactive web-like html report. rMAP installation is very simple, it can be run using very simple commands. It represents a rapid and easy way to perform comprehensive bacterial WGS analysis using a personal laptop in low-income settings where high-performance computing infrastructure is limited.
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Combination of long- and short-read sequencing fully resolves complex repeats of herpes simplex virus 2 strain MS complete genome
More LessHerpes simplex virus serotype 2 (HSV-2) is a ubiquitous human pathogen that causes recurrent genital infections and ulcerations. Many HSV-2 strains with different biological properties have been identified, but only the genomes of HSV-2 strains HG52, SD90e and 333 have been reported as complete and fully characterized sequences. We de novo assembled, annotated and manually curated the complete genome sequence of HSV-2 strain MS, a highly neurovirulent strain, originally isolated from a multiple sclerosis patient. We resolved both DNA ends, as well as the complex inverted repeats regions present in HSV genomes, usually undisclosed in previous published partial herpesvirus genomes, using long reads from Pacific Biosciences (PacBio) technology. Additionally, we identified isomeric genomes by determining the alternative relative orientation of unique fragments in the genome of the sequenced viral population. Illumina short-read sequencing was crucial to examine genetic variability, such as nucleotide polymorphisms, insertion/deletions and sequence determinants of strain-specific virulence factors. We used Illumina data to fix two disrupted open reading frames found in coding homopolymers after PacBio assembly. These results support the combination of long- and short-read sequencing technologies as a precise and effective approach for the accurate de novo assembly and curation of complex microbial genomes.
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SplitStrains, a tool to identify and separate mixed Mycobacterium tuberculosis infections from WGS data
More LessThe occurrence of multiple strains of a bacterial pathogen such as M. tuberculosis or C. difficile within a single human host, referred to as a mixed infection, has important implications for both healthcare and public health. However, methods for detecting it, and especially determining the proportion and identities of the underlying strains, from WGS (whole-genome sequencing) data, have been limited. In this paper we introduce SplitStrains, a novel method for addressing these challenges. Grounded in a rigorous statistical model, SplitStrains not only demonstrates superior performance in proportion estimation to other existing methods on both simulated as well as real M. tuberculosis data, but also successfully determines the identity of the underlying strains. We conclude that SplitStrains is a powerful addition to the existing toolkit of analytical methods for data coming from bacterial pathogens and holds the promise of enabling previously inaccessible conclusions to be drawn in the realm of public health microbiology.
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- Functional Genomics and Microbe–Niche Interactions
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Functional analysis of colonization factor antigen I positive enterotoxigenic Escherichia coli identifies genes implicated in survival in water and host colonization
Enterotoxigenic Escherichia coli (ETEC) expressing the colonization pili CFA/I are common causes of diarrhoeal infections in humans. Here, we use a combination of transposon mutagenesis and transcriptomic analysis to identify genes and pathways that contribute to ETEC persistence in water environments and colonization of a mammalian host. ETEC persisting in water exhibit a distinct RNA expression profile from those growing in richer media. Multiple pathways were identified that contribute to water survival, including lipopolysaccharide biosynthesis and stress response regulons. The analysis also indicated that ETEC growing in vivo in mice encounter a bottleneck driving down the diversity of colonizing ETEC populations.
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Insight into phenotypic and genotypic differences between vaginal Lactobacillus crispatus BC5 and Lactobacillus gasseri BC12 to unravel nutritional and stress factors influencing their metabolic activity
The vaginal microbiota, normally characterized by lactobacilli presence, is crucial for vaginal health. Members belonging to L. crispatus and L. gasseri species exert crucial protective functions against pathogens, although a total comprehension of factors that influence their dominance in healthy women is still lacking. Here we investigated the complete genome sequence and comprehensive phenotypic profile of L. crispatus strain BC5 and L. gasseri strain BC12, two vaginal strains featured by anti-bacterial and anti-viral activities. Phenotype microarray (PM) results revealed an improved capacity of BC5 to utilize different carbon sources as compared to BC12, although some specific carbon sources that can be associated to the human diet were only metabolized by BC12, i.e. uridine, amygdalin, tagatose. Additionally, the two strains were mostly distinct in the capacity to utilize the nitrogen sources under analysis. On the other hand, BC12 showed tolerance/resistance towards twice the number of stressors (i.e. antibiotics, toxic metals etc.) with respect to BC5. The divergent phenotypes observed in PM were supported by the identification in either BC5 or BC12 of specific genetic determinants that were found to be part of the core genome of each species. The PM results in combination with comparative genome data provide insights into the possible environmental factors and genetic traits supporting the predominance of either L. crispatus BC5 or L. gasseri BC12 in the vaginal niche, giving also indications for metabolic predictions at the species level.
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Mining genome traits that determine the different gut colonization potential of Lactobacillus and Bifidobacterium species
More LessAlthough the beneficial effects of probiotics are likely to be associated with their ability to colonize the gut, little is known about the characteristics of good colonizers. In a systematic analysis of the comparative genomics, we tried to elucidate the genomic contents that account for the distinct host adaptability patterns of Lactobacillus and Bifidobacterium species. The Bifidobacterium species, with species-level phylogenetic structures affected by recombination among strains, broad mucin-foraging activity, and dietary-fibre-degrading ability, represented niche conservatism and tended to be host-adapted. The Lactobacillus species stretched across three lifestyles, namely free-living, nomadic and host-adapted, as characterized by the variations of bacterial occurrence time, guanine–cytosine (GC) content and genome size, evolution event frequency, and the presence of human-adapted bacterial genes. The numbers and activity of host-adapted factors, such as bile salt hydrolase and intestinal tissue-anchored elements, were distinctly distributed among the three lifestyles. The strains of the three lifestyles could be separated with such a collection of colonization-related genomic content (genes, genome size and GC content). Thus, our work provided valuable information for rational selection and gut engraftment prediction of probiotics. Here, we have found many interesting predictive results for bacterial gut fitness, which will be validated in vitro and in vivo.
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Comparative genomics of the Pseudomonas corrugata subgroup reveals high species diversity and allows the description of Pseudomonas ogarae sp. nov.
More LessPseudomonas corrugata constitute one of the phylogenomic subgroups within the Pseudomonas fluorescens species complex and include both plant growth-promoting rhizobacteria (PGPR) and plant pathogenic bacteria. Previous studies suggest that the species diversity of this group remains largely unexplored together with frequent misclassification of strains. Using more than 1800 sequenced Pseudomonas genomes we identified 121 genomes belonging to the P. corrugata subgroup. Intergenomic distances obtained using the genome-to-genome blast distance (GBDP) algorithm and the determination of digital DNA–DNA hybridization values were further used for phylogenomic and clustering analyses, which revealed 29 putative species clusters, of which only five correspond to currently named species within the subgroup. Comparative and functional genome-scale analyses also support the species status of these clusters. The search for PGPR and plant pathogenic determinants showed that approximately half of the genomes analysed could have a pathogenic behaviour based on the presence of a pathogenicity genetic island, while all analysed genomes possess PGPR traits. Finally, this information together with the characterization of phenotypic traits, allows the reclassification proposal of Pseudomonas fluorescens F113 as Pseudomonas ogarae sp. nov., nom rev., type strain F113T (=DSM 112162T=CECT 30235T), which is substantiated by genomic, functional genomics and phenotypic differences with their closest type strains.
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Comparative genomic insights into culturable symbiotic cyanobacteria from the water fern Azolla
More LessSpecies of the floating, freshwater fern Azolla form a well-characterized symbiotic association with the non-culturable cyanobacterium Nostoc azollae, which fixes nitrogen for the plant. However, several cyanobacterial strains have over the years been isolated and cultured from Azolla from all over the world. The genomes of 10 of these strains were sequenced and compared with each other, with other symbiotic cyanobacterial strains, and with similar strains that were not isolated from a symbiotic association. The 10 strains fell into three distinct groups: six strains were nearly identical to the non-symbiotic strain, Nostoc ( Anabaena ) variabilis ATCC 29413; three were similar to the symbiotic strain, Nostoc punctiforme , and one, Nostoc sp. 2RC, was most similar to non-symbiotic strains of Nostoc linckia. However, Nostoc sp. 2RC was unusual because it has three sets of nitrogenase genes; it has complete gene clusters for two distinct Mo-nitrogenases and an alternative V-nitrogenase. Genes for Mo-nitrogenase, sugar transport, chemotaxis and pili characterized all the symbiotic strains. Several of the strains infected the liverwort Blasia, including N. variabilis ATCC 29413, which did not originate from Azolla but rather from a sewage pond. However, only Nostoc sp. 2RC, which produced highly motile hormogonia, was capable of high-frequency infection of Blasia. Thus, some of these strains, which grow readily in the laboratory, may be useful in establishing novel symbiotic associations with other plants.
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Multiple evolutionary origins reflect the importance of sialic acid transporters in the colonization potential of bacterial pathogens and commensals
More LessLocated at the tip of cell surface glycoconjugates, sialic acids are at the forefront of host–microbe interactions and, being easily liberated by sialidase enzymes, are used as metabolites by numerous bacteria, particularly by pathogens and commensals living on or near diverse mucosal surfaces. These bacteria rely on specific transporters for the acquisition of host-derived sialic acids. Here, we present the first comprehensive genomic and phylogenetic analysis of bacterial sialic acid transporters, leading to the identification of multiple new families and subfamilies. Our phylogenetic analysis suggests that sialic acid-specific transport has evolved independently at least eight times during the evolution of bacteria, from within four of the major families/superfamilies of bacterial transporters, and we propose a robust classification scheme to bring together a myriad of different nomenclatures that exist to date. The new transporters discovered occur in diverse bacteria, including Spirochaetes , Bacteroidetes , Planctomycetes and Verrucomicrobia , many of which are species that have not been previously recognized to have sialometabolic capacities. Two subfamilies of transporters stand out in being fused to the sialic acid mutarotase enzyme, NanM, and these transporter fusions are enriched in bacteria present in gut microbial communities. Our analysis supports the increasing experimental evidence that competition for host-derived sialic acid is a key phenotype for successful colonization of complex mucosal microbiomes, such that a strong evolutionary selection has occurred for the emergence of sialic acid specificity within existing transporter architectures.
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- Pathogens and Epidemiology
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Molecular characterization of respiratory syncytial viruses circulating in a paediatric cohort in Amman, Jordan
Respiratory syncytial viruses (RSVs) are an important cause of mortality worldwide and a major cause of respiratory tract infections in children, driving development of vaccine candidates. However, there are large gaps in our knowledge of the local evolutionary and transmission dynamics of RSVs, particularly in understudied regions such as the Middle East. To address this gap, we sequenced the complete genomes of 58 RSVA and 27 RSVB samples collected in a paediatric cohort in Amman, Jordan, between 2010 and 2013. RSVA and RSVB co-circulated during each winter epidemic of RSV in Amman, and each epidemic comprised multiple independent viral introductions of RSVA and RSVB. However, RSVA and RSVB alternated in dominance across years, potential evidence of immunological interactions. Children infected with RSVA tended to be older than RSVB-infected children [30 months versus 22.4 months, respectively (P value = 0.02)], and tended to developed bronchopneumonia less frequently than those with RSVB, although the difference was not statistically significant (P value = 0.06). Differences in spatial patterns were investigated, and RSVA lineages were often identified in multiple regions in Amman, whereas RSVB introductions did not spread beyond a single region of the city, although these findings were based on small sample sizes. Multiple RSVA genotypes were identified in Amman, including GA2 viruses as well as three viruses from the ON1 sub-genotype that emerged in 2009 and are now the dominant genotype circulating worldwide. As vaccine development advances, further sequencing of RSV is needed to understand viral ecology and transmission, particularly in under-studied locations.
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Enhancing genomics-based outbreak detection of endemic Salmonella enterica serovar Typhimurium using dynamic thresholds
Salmonella enterica serovar Typhimurium is the leading cause of salmonellosis in Australia, and the ability to identify outbreaks and their sources is vital to public health. Here, we examined the utility of whole-genome sequencing (WGS), including complete genome sequencing with Oxford Nanopore technologies, in examining 105 isolates from an endemic multi-locus variable number tandem repeat analysis (MLVA) type over 5 years. The MLVA type was very homogeneous, with 90 % of the isolates falling into groups with a five SNP cut-off. We developed a new two-step approach for outbreak detection using WGS. The first clustering at a zero single nucleotide polymorphism (SNP) cut-off was used to detect outbreak clusters that each occurred within a 4 week window and then a second clustering with dynamically increased SNP cut-offs were used to generate outbreak investigation clusters capable of identifying all outbreak cases. This approach offered optimal specificity and sensitivity for outbreak detection and investigation, in particular of those caused by endemic MLVA types or clones with low genetic diversity. We further showed that inclusion of complete genome sequences detected no additional mutational events for genomic outbreak surveillance. Phylogenetic analysis found that the MLVA type was likely to have been derived recently from a single source that persisted over 5 years, and seeded numerous sporadic infections and outbreaks. Our findings suggest that SNP cut-offs for outbreak cluster detection and public-health surveillance should be based on the local diversity of the relevant strains over time. These findings have general applicability to outbreak detection of bacterial pathogens.
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Genomic evolution and local epidemiology of Klebsiella pneumoniae from a major hospital in Beijing, China, over a 15 year period: dissemination of known and novel high-risk clones
Klebsiella pneumoniae is a frequent cause of nosocomial and severe community-acquired infections. Multidrug-resistant (MDR) and hypervirulent (hv) strains represent major threats, and tracking their emergence, evolution and the emerging convergence of MDR and hv traits is of major importance. We employed whole-genome sequencing (WGS) to study the evolution and epidemiology of a large longitudinal collection of clinical K. pneumoniae isolates from the H301 hospital in Beijing, China. Overall, the population was highly diverse, although some clones were predominant. Strains belonging to clonal group (CG) 258 were dominant, and represented the majority of carbapenemase-producers. While CG258 strains showed high diversity, one clone, ST11-KL47, represented the majority of isolates, and was highly associated with the KPC-2 carbapenemase and several virulence factors, including a virulence plasmid. The second dominant clone was CG23, which is the major hv clone globally. While it is usually susceptible to multiple antibiotics, we found some isolates harbouring MDR plasmids encoding for ESBLs and carbapenemases. We also reported the local emergence of a recently described high-risk clone, ST383. Conversely to strains belonging to CG258, which are usually associated to KPC-2, ST383 strains seem to readily acquire carbapenemases of different types. Moreover, we found several ST383 strains carrying the hypervirulence plasmid. Overall, we detected about 5 % of simultaneous carriage of AMR genes (ESBLs or carbapenemases) and hypervirulence genes. Tracking the emergence and evolution of such strains, causing severe infections with limited treatment options, is fundamental in order to understand their origin and evolution and to limit their spread. This article contains data hosted by Microreact.
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Phylogenetic context of Shiga toxin-producing Escherichia coli serotype O26:H11 in England
More LessThe increasing use of PCR for the detection of gastrointestinal pathogens in hospital laboratories in England has improved the detection of Shiga toxin-producing Escherichia coli (STEC), and the diagnosis of haemolytic uraemic syndrome (HUS). We aimed to analyse the microbiological characteristics and phylogenetic relationships of STEC O26:H11, clonal complex (CC) 29, in England to inform surveillance, and to assess the threat to public health. There were 502 STEC belonging to CC29 isolated between 2014 and 2019, of which 416 were from individual cases. The majority of isolates belonged to one of three major sequence types (STs), ST16 (n=37), ST21 (n=350) and ST29 (n=24). ST16 and ST29 were mainly isolated from cases reporting recent travel abroad. Within ST21, there were three main clades associated with domestic acquisition. All three domestic clades had Shiga toxin subtype gene (stx) profiles associated with causing severe clinical outcomes including STEC-HUS, specifically either stx1a, stx2a or stx1a/stx2a. Isolates from the same patient, same household or same outbreak with an established source for the most part fell within 5-SNP single linkage clusters. There were 19 5-SNP community clusters, of which six were travel-associated and one was an outbreak of 16 cases caused by the consumption of contaminated salad leaves. Of the remaining 12 clusters, 9/12 were either temporally or geographically related or both. Exposure to foodborne STEC O26:H11 ST21 capable of causing severe clinical outcomes, including STEC-HUS, is an emerging risk to public health in England. The lack of comprehensive surveillance of this STEC serotype is a concern, and there is a need to expand the implementation of methods capable of detecting STEC in local hospital settings.
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Insights into evolution and coexistence of the colibactin- and yersiniabactin secondary metabolite determinants in enterobacterial populations
The bacterial genotoxin colibactin interferes with the eukaryotic cell cycle by causing dsDNA breaks. It has been linked to bacterially induced colorectal cancer in humans. Colibactin is encoded by a 54 kb genomic region in Enterobacteriaceae . The colibactin genes commonly co-occur with the yersiniabactin biosynthetic determinant. Investigating the prevalence and sequence diversity of the colibactin determinant and its linkage to the yersiniabactin operon in prokaryotic genomes, we discovered mainly species-specific lineages of the colibactin determinant and classified three main structural settings of the colibactin–yersiniabactin genomic region in Enterobacteriaceae . The colibactin gene cluster has a similar but not identical evolutionary track to that of the yersiniabactin operon. Both determinants could have been acquired on several occasions and/or exchanged independently between enterobacteria by horizontal gene transfer. Integrative and conjugative elements play(ed) a central role in the evolution and structural diversity of the colibactin–yersiniabactin genomic region. Addition of an activating and regulating module (clbAR) to the biosynthesis and transport module (clbB-S) represents the most recent step in the evolution of the colibactin determinant. In a first attempt to correlate colibactin expression with individual lineages of colibactin determinants and different bacterial genetic backgrounds, we compared colibactin expression of selected enterobacterial isolates in vitro. Colibactin production in the tested Klebsiella species and Citrobacter koseri strains was more homogeneous and generally higher than that in most of the Escherichia coli isolates studied. Our results improve the understanding of the diversity of colibactin determinants and its expression level, and may contribute to risk assessment of colibactin-producing enterobacteria.
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Genomic epidemiology of the first epidemic wave of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Palestine
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the novel coronavirus responsible for the COVID-19 pandemic, continues to cause a significant public-health burden and disruption globally. Genomic epidemiology approaches point to most countries in the world having experienced many independent introductions of SARS-CoV-2 during the early stages of the pandemic. However, this situation may change with local lockdown policies and restrictions on travel, leading to the emergence of more geographically structured viral populations and lineages transmitting locally. Here, we report the first SARS-CoV-2 genomes from Palestine sampled from early March 2020, when the first cases were observed, through to August of 2020. SARS-CoV-2 genomes from Palestine fall across the diversity of the global phylogeny, consistent with at least nine independent introductions into the region. We identify one locally predominant lineage in circulation represented by 50 Palestinian SARS-CoV-2, grouping with genomes generated from Israel and the UK. We estimate the age of introduction of this lineage to 05/02/2020 (16/01/2020–19/02/2020), suggesting SARS-CoV-2 was already in circulation in Palestine predating its first detection in Bethlehem in early March. Our work highlights the value of ongoing genomic surveillance and monitoring to reconstruct the epidemiology of COVID-19 at both local and global scales.
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Large-scale sequencing of SARS-CoV-2 genomes from one region allows detailed epidemiology and enables local outbreak management
Andrew J. Page, Alison E. Mather, Thanh Le-Viet, Emma J. Meader, Nabil-Fareed Alikhan, Gemma L. Kay, Leonardo de Oliveira Martins, Alp Aydin, David J. Baker, Alexander J. Trotter, Steven Rudder, Ana P. Tedim, Anastasia Kolyva, Rachael Stanley, Muhammad Yasir, Maria Diaz, Will Potter, Claire Stuart, Lizzie Meadows, Andrew Bell, Ana Victoria Gutierrez, Nicholas M. Thomson, Evelien M. Adriaenssens, Tracey Swingler, Rachel A. J. Gilroy, Luke Griffith, Dheeraj K. Sethi, Dinesh Aggarwal, Colin S. Brown, Rose K. Davidson, Robert A. Kingsley, Luke Bedford, Lindsay J. Coupland, Ian G. Charles, Ngozi Elumogo, John Wain, Reenesh Prakash, Mark A. Webber, S. J. Louise Smith, Meera Chand, Samir Dervisevic, Justin O’Grady and The COVID-19 Genomics UK (COG-UK) ConsortiumThe COVID-19 pandemic has spread rapidly throughout the world. In the UK, the initial peak was in April 2020; in the county of Norfolk (UK) and surrounding areas, which has a stable, low-density population, over 3200 cases were reported between March and August 2020. As part of the activities of the national COVID-19 Genomics Consortium (COG-UK) we undertook whole genome sequencing of the SARS-CoV-2 genomes present in positive clinical samples from the Norfolk region. These samples were collected by four major hospitals, multiple minor hospitals, care facilities and community organizations within Norfolk and surrounding areas. We combined clinical metadata with the sequencing data from regional SARS-CoV-2 genomes to understand the origins, genetic variation, transmission and expansion (spread) of the virus within the region and provide context nationally. Data were fed back into the national effort for pandemic management, whilst simultaneously being used to assist local outbreak analyses. Overall, 1565 positive samples (172 per 100 000 population) from 1376 cases were evaluated; for 140 cases between two and six samples were available providing longitudinal data. This represented 42.6 % of all positive samples identified by hospital testing in the region and encompassed those with clinical need, and health and care workers and their families. In total, 1035 cases had genome sequences of sufficient quality to provide phylogenetic lineages. These genomes belonged to 26 distinct global lineages, indicating that there were multiple separate introductions into the region. Furthermore, 100 genetically distinct UK lineages were detected demonstrating local evolution, at a rate of ~2 SNPs per month, and multiple co-occurring lineages as the pandemic progressed. Our analysis: identified a discrete sublineage associated with six care facilities; found no evidence of reinfection in longitudinal samples; ruled out a nosocomial outbreak; identified 16 lineages in key workers which were not in patients, indicating infection control measures were effective; and found the D614G spike protein mutation which is linked to increased transmissibility dominates the samples and rapidly confirmed relatedness of cases in an outbreak at a food processing facility. The large-scale genome sequencing of SARS-CoV-2-positive samples has provided valuable additional data for public health epidemiology in the Norfolk region, and will continue to help identify and untangle hidden transmission chains as the pandemic evolves.
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A multisite genomic epidemiology study of Clostridioides difficile infections in the USA supports differential roles of healthcare versus community spread for two common strains
Clostridioides difficile is the leading cause of healthcare-associated infectious diarrhoea. However, it is increasingly appreciated that healthcare-associated infections derive from both community and healthcare environments, and that the primary sites of C. difficile transmission may be strain-dependent. We conducted a multisite genomic epidemiology study to assess differential genomic evidence of healthcare vs community spread for two of the most common C. difficile strains in the USA: sequence type (ST) 1 (associated with ribotype 027) and ST2 (associated with ribotype 014/020). We performed whole-genome sequencing and phylogenetic analyses on 382 ST1 and ST2 C. difficile isolates recovered from stool specimens collected during standard clinical care at 3 geographically distinct US medical centres between 2010 and 2017. ST1 and ST2 isolates both displayed some evidence of phylogenetic clustering by study site, but clustering was stronger and more apparent in ST1, consistent with our healthcare-based study more comprehensively sampling local transmission of ST1 compared to ST2 strains. Analyses of pairwise single-nucleotide variant (SNV) distance distributions were also consistent with more evidence of healthcare transmission of ST1 compared to ST2, with 44 % of ST1 isolates being within two SNVs of another isolate from the same geographical collection site compared to 5.5 % of ST2 isolates (P-value=<0.001). Conversely, ST2 isolates were more likely to have close genetic neighbours across disparate geographical sites compared to ST1 isolates, further supporting non-healthcare routes of spread for ST2 and highlighting the potential for misattributing genomic similarity among ST2 isolates to recent healthcare transmission. Finally, we estimated a lower evolutionary rate for the ST2 lineage compared to the ST1 lineage using Bayesian timed phylogenomic analyses, and hypothesize that this may contribute to observed differences in geographical concordance among closely related isolates. Together, these findings suggest that ST1 and ST2, while both common causes of C. difficile infection in hospitals, show differential reliance on community and hospital spread. This conclusion supports the need for strain-specific criteria for interpreting genomic linkages and emphasizes the importance of considering differences in the epidemiology of circulating strains when devising interventions to reduce the burden of C. difficile infections.
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Improved molecular characterization of the Klebsiella oxytoca complex reveals the prevalence of the kleboxymycin biosynthetic gene cluster
As part of the ongoing studies with clinically relevant Klebsiella spp., we characterized the genomes of three clinical GES-5-positive ST138 strains originally identified as Klebsiella oxytoca. bla OXY gene, average nucleotide identity and phylogenetic analyses showed the strains to be Klebsiella michiganensis . Affiliation of the strains to ST138 led us to demonstrate that the current multi-locus sequence typing scheme for K. oxytoca can be used to distinguish members of this genetically diverse complex of bacteria. The strains encoded the kleboxymycin biosynthetic gene cluster (BGC), previously only found in K. oxytoca strains and one strain of Klebsiella grimontii . The finding of this BGC, associated with antibiotic-associated haemorrhagic colitis, in K. michiganensis led us to carry out a wide-ranging study to determine the prevalence of this BGC in Klebsiella spp. Of 7170 publicly available Klebsiella genome sequences screened, 88 encoded the kleboxymycin BGC. All BGC-positive strains belonged to the K. oxytoca complex, with strains of four ( K. oxytoca , K. pasteurii , K. grimontii , K. michiganensis ) of the six species of complex found to encode the complete BGC. In addition to being found in K. grimontii strains isolated from preterm infants, the BGC was found in K. oxytoca and K. michiganensis metagenome-assembled genomes recovered from neonates. Detection of the kleboxymycin BGC across the K. oxytoca complex may be of clinical relevance and this cluster should be included in databases characterizing virulence factors, in addition to those characterizing BGCs.
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gbpA and chiA genes are not uniformly distributed amongst diverse Vibrio cholerae
More LessMembers of the bacterial genus Vibrio utilize chitin both as a metabolic substrate and a signal to activate natural competence. Vibrio cholerae is a bacterial enteric pathogen, sub-lineages of which can cause pandemic cholera. However, the chitin metabolic pathway in V. cholerae has been dissected using only a limited number of laboratory strains of this species. Here, we survey the complement of key chitin metabolism genes amongst 195 diverse V. cholerae . We show that the gene encoding GbpA, known to be an important colonization and virulence factor in pandemic isolates, is not ubiquitous amongst V. cholerae . We also identify a putatively novel chitinase, and present experimental evidence in support of its functionality. Our data indicate that the chitin metabolic pathway within V. cholerae is more complex than previously thought, and emphasize the importance of considering genes and functions in the context of a species in its entirety, rather than simply relying on traditional reference strains.
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Ongoing evolution of Chlamydia trachomatis lymphogranuloma venereum: exploring the genomic diversity of circulating strains
Helena M. B. Seth-Smith, Angèle Bénard, Sylvia M. Bruisten, Bart Versteeg, Björn Herrmann, Jen Kok, Ian Carter, Olivia Peuchant, Cécile Bébéar, David A. Lewis, Teresa Puerta, Darja Keše, Eszter Balla, Hana Zákoucká, Filip Rob, Servaas A. Morré, Bertille de Barbeyrac, Juan Carlos Galán, Henry J. C. de Vries, Nicholas R. Thomson, Daniel Goldenberger and Adrian EgliLymphogranuloma venereum (LGV), the invasive infection of the sexually transmissible infection (STI) Chlamydia trachomatis , is caused by strains from the LGV biovar, most commonly represented by ompA-genotypes L2b and L2. We investigated the diversity in LGV samples across an international collection over seven years using typing and genome sequencing. LGV-positive samples (n=321) from eight countries collected between 2011 and 2017 (Spain n=97, Netherlands n=67, Switzerland n=64, Australia n=53, Sweden n=37, Hungary n=31, Czechia n=30, Slovenia n=10) were genotyped for pmpH and ompA variants. All were found to contain the 9 bp insertion in the pmpH gene, previously associated with ompA-genotype L2b. However, analysis of the ompA gene shows ompA-genotype L2b (n=83), ompA-genotype L2 (n=180) and several variants of these (n=52; 12 variant types), as well as other/mixed ompA-genotypes (n=6). To elucidate the genomic diversity, whole genome sequencing (WGS) was performed from selected samples using SureSelect target enrichment, resulting in 42 genomes, covering a diversity of ompA-genotypes and representing most of the countries sampled. A phylogeny of these data clearly shows that these ompA-genotypes derive from an ompA-genotype L2b ancestor, carrying up to eight SNPs per isolate. SNPs within ompA are overrepresented among genomic changes in these samples, each of which results in an amino acid change in the variable domains of OmpA (major outer membrane protein, MOMP). A reversion to ompA-genotype L2 with the L2b genomic backbone is commonly seen. The wide diversity of ompA-genotypes found in these recent LGV samples indicates that this gene is under immunological selection. Our results suggest that the ompA-genotype L2b genomic backbone is the dominant strain circulating and evolving particularly in men who have sex with men (MSM) populations.
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- Evolution and Responses to Interventions
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Subtelomeres are fast-evolving regions of the Streptomyces linear chromosome
More LessStreptomyces possess a large linear chromosome (6–12 Mb) consisting of a conserved central region flanked by variable arms covering several megabases. In order to study the evolution of the chromosome across evolutionary times, a representative panel of Streptomyces strains and species (125) whose chromosomes are completely sequenced and assembled was selected. The pan-genome of the genus was modelled and shown to be open with a core-genome reaching 1018 genes. The evolution of Streptomyces chromosome was analysed by carrying out pairwise comparisons, and by monitoring indexes measuring the conservation of genes (presence/absence) and their synteny along the chromosome. Using the phylogenetic depth offered by the chosen panel, it was possible to infer that within the central region of the chromosome, the core-genes form a highly conserved organization, which can reveal the existence of an ancestral chromosomal skeleton. Conversely, the chromosomal arms, enriched in variable genes evolved faster than the central region under the combined effect of rearrangements and addition of new information from horizontal gene transfer. The genes hosted in these regions may be localized there because of the adaptive advantage that their rapid evolution may confer. We speculate that (i) within a bacterial population, the variability of these genes may contribute to the establishment of social characters by the production of ‘public goods’ (ii) at the evolutionary scale, this variability contributes to the diversification of the genetic pool of the bacteria.
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Evolutionary responses to codon usage of horizontally transferred genes in Pseudomonas aeruginosa: gene retention, amelioration and compensatory evolution
More LessProkaryote genome evolution is characterized by the frequent gain of genes through horizontal gene transfer (HGT). For a gene, being horizontally transferred can represent a strong change in its genomic and physiological context. If the codon usage of a transferred gene deviates from that of the receiving organism, the fitness benefits it provides can be reduced due to a mismatch with the expression machinery. Consequently, transferred genes with a deviating codon usage can be selected against or elicit evolutionary responses that enhance their integration, such as gene amelioration and compensatory evolution. Within bacterial species, the extent and relative importance of these different mechanisms has never been considered altogether. In this study, a phylogeny-based method was used to investigate the occurrence of these different evolutionary responses in Pseudomonas aeruginosa . Selection on codon usage of genes acquired through HGT was observed over evolutionary time, with the overall codon usage converging towards that of the core genome. Gene amelioration, through the accumulation of synonymous mutations after HGT, did not seem to systematically affect transferred genes. This pattern therefore seemed to be mainly driven by selective retention of transferred genes with an initial codon usage similar to that of the core genes. Additionally, variation in the copy number of tRNA genes was often associated with the acquisition of genes for which the observed variation could enhance their expression. This provides evidence that compensatory evolution might be an important mechanism for the integration of horizontally transferred genes.
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- Short Communications
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- Pathogens and Epidemiology
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Genomic contextualisation of ancient DNA molecular data from an Argentinian fifth pandemic Vibrio cholerae infection
More LessSpecific lineages of serogroup O1 Vibrio cholerae are notorious for causing cholera pandemics, of which there have been seven since the 1800s. Much is known about the sixth pandemic (1899–1923) and the ongoing seventh pandemic (1961–present), but we know very little about the bacteriology of pandemics 1 to 5. Moreover, although we are learning about the contribution of non-O1 non-pandemic V. cholerae to cholera dynamics during the current pandemic, we know almost nothing about their role in the past. A recent ancient DNA study has presented what may be the first molecular evidence of a V. cholerae infection from the fifth cholera pandemic period (1886–1887 AD) in Argentina. Here, we place the molecular evidence from that study into the genomic context of non-pandemic V. cholerae from Latin America and elsewhere, and show that a gene fragment amplified from ancient DNA is most similar to that of V. cholerae from the Americas, and from Argentina. Our results corroborate and reinforce the findings of the original study, and collectively suggest that even in the 1880s, non-pandemic V. cholerae local to the Americas may have caused sporadic infections in Argentina, just as we know this to have happened during the seventh pandemic in Latin America.
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- Research Articles
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- Functional Genomics and Microbe–Niche Interactions
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In vitro exploration of the Xanthomonas hortorum pv. vitians genome using transposon insertion sequencing and comparative genomics to discriminate between core and contextual essential genes
More LessThe essential genome of a bacterium encompasses core genes associated with basic cellular processes and conditionally essential genes dependent upon environmental conditions or the genetic context. Comprehensive knowledge of those gene sets allows for a better understanding of fundamental bacterial biology and offers new perspectives for antimicrobial drug research against detrimental bacteria such as pathogens. We investigated the essential genome of Xanthomonas hortorum pv. vitians, a gammaproteobacterial plant pathogen of lettuce (Lactuca sativa L.) which belongs to the plant-pathogen reservoir genus Xanthomonas and is affiliated to the family Xanthomonadaceae . No practical means of disease control or prevention against this pathogen is currently available, and its molecular biology is virtually unknown. To reach a comprehensive overview of the essential genome of X. hortorum pv. vitians LM16734, we developed a mixed approach combining high-quality full genome sequencing, saturated transposon insertion sequencing (Tn-Seq) in optimal growth conditions, and coupled computational analyses such as comparative genomics, synteny assessment and phylogenomics. Among the 370 essential loci identified by Tn-Seq, a majority was bound to critical cell processes conserved across bacteria. The remaining genes were either related to specific ecological features of Xanthomonas or Xanthomonadaceae species, or acquired through horizontal gene transfer of mobile genetic elements and associated with ancestral parasitic gene behaviour and bacterial defence systems. Our study sheds new light on our usual concepts about gene essentiality and is pioneering in the molecular and genomic study of X. hortorum pv. vitians.
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