Microbial Genomics - Online First

Next-generation sequencing (NGS) is now widely used in microbiology to explore genome evolution and the structure of pathogen outbreaks. Bioinformatics pipelines readily detect single-nucleotide polymorphisms or short indels. However, bacterial genomes also evolve through the action of small transposable elements called insertion sequences (ISs), which are difficult to detect due to their short length and multiple repetitions throughout the genome. We designed panISa software for the ab initio detection of IS insertions in the genomes of prokaryotes. PanISa has been released as open source software (GPL3) available from https://github.com/bvalot/panISa. In this study, we assessed the utility of this software for evolutionary studies, by reanalysing five published datasets for outbreaks of human major pathogens in which ISs had not been specifically investigated. We reanalysed the raw data from each study, by aligning the reads against reference genomes and running panISa on the alignments. Each hit was automatically curated and IS-related events were validated on the basis of nucleotide sequence similarity, by comparison with the ISFinder database. In Acinetobacter baumannii , the panISa pipeline identified ISAba1 or ISAba125 upstream from the ampC gene, which encodes a cephalosporinase in all third-generation cephalosporin-resistant isolates. In the genomes of Vibrio cholerae isolates, we found that early Haitian isolates had the same ISs as Nepalese isolates, confirming the inferred history of the contamination of this island. In Enterococcus faecalis , panISa identified regions of high plasticity, including a pathogenicity island enriched in IS-related events. The overall distribution of ISs deduced with panISa was consistent with SNP-based phylogenic trees, for all species considered. The role of ISs in pathogen evolution has probably been underestimated due to difficulties detecting these transposable elements. We show here that panISa is a useful addition to the bioinformatics toolbox for analyses of the evolution of bacterial genomes. PanISa will facilitate explorations of the functional impact of ISs and improve our understanding of prokaryote evolution.

Acinetobacter baumannii (Aba) is an emerging opportunistic pathogen associated to nosocomial infections. The rapid increase in multidrug resistance (MDR) among Aba strains underscores the urgency of understanding how this pathogen evolves in the clinical environment. We conducted here a whole-genome sequence comparative analysis of three phylogenetically and epidemiologically related MDR Aba strains from Argentinean hospitals, assigned to the CC104O/CC15P clonal complex. While the Ab244 strain was carbapenem-susceptible, Ab242 and Ab825, isolated after the introduction of carbapenem therapy, displayed resistance to these last resource β-lactams. We found a high chromosomal synteny among the three strains, but significant differences at their accessory genomes. Most importantly, carbapenem resistance in Ab242 and Ab825 was attributed to the acquisition of a Rep_3 family plasmid carrying a bla OXA-58 gene. Other differences involved a genomic island carrying resistance to toxic compounds and a Tn10 element exclusive to Ab244 and Ab825, respectively. Also remarkably, 44 insertion sequences (ISs) were uncovered in Ab825, in contrast with the 14 and 11 detected in Ab242 and Ab244, respectively. Moreover, Ab825 showed a higher killing capacity as compared to the other two strains in the Galleria mellonella infection model. A search for virulence and persistence determinants indicated the loss or IS-mediated interruption of genes encoding many surface-exposed macromolecules in Ab825, suggesting that these events are responsible for its higher relative virulence. The comparative genomic analyses of the CC104O/CC15P strains conducted here revealed the contribution of acquired mobile genetic elements such as ISs and plasmids to the adaptation of A. baumannii to the clinical setting.

Clostridium difficile B1/NAP1/RT027/ST01 has been responsible for outbreaks of antibiotic-associated diarrhoea in clinical settings worldwide and is associated with severe disease presentations and increased mortality rates. Two fluoroquinolone-resistant (FQR) lineages of the epidemic B1/NAP1/RT027/ST01 strain emerged in the USA in the early 1990s and disseminated trans continentally (FQR1 and FQR2). However, it is unclear when and from where they entered Latin America (LA) and whether isolates from LA exhibit unique genomic features when compared to B1/NAP1/RT027/ST01 isolates from other regions of the world. To answer the first issue we compared whole-genome sequences (WGS) of 25 clinical isolates typed as NAP1, RT027 or ST01 in Costa Rica (n=16), Chile (n=5), Honduras (n=3) and Mexico (n=1) to WGS of 129 global isolates from the same genotype using Bayesian phylogenomics. The second question was addressed through a detailed analysis of the number and type of mutations of the LA isolates and their mobile resistome. All but two B1/NAP1/RT027/ST01 isolates from LA belong to the FQR2 lineage (n=23, 92 %), confirming its widespread distribution. As indicated by analysis of a dataset composed of 154 WGS, the B1/NAP1/RT027/ST01 strain was introduced into the four LA countries analysed between 1998 and 2005 from North America (twice) and Europe (at least four times). These events occurred soon after the emergence of the FQR lineages and more than one decade before the first report of the detection of the B1/NAP1/RT027/ST01 in LA. A total of 552 SNPs were identified across all genomes examined (3.8–4.3 Mb) in pairwise comparisons to the R20291 reference genome. Moreover, pairwise SNP distances were among the smallest distances determined in this species so far (0 to 55). Despite this high level of genomic conservation, 39 unique SNPs (7 %) in genes that play roles in the infection process (i.e. slpA) or antibiotic resistance (i.e. rpoB, fusA) distinguished the LA isolates. In addition, isolates from Chile, Honduras and Mexico had twice as many antibiotic resistance genes (ARGs, n=4) than related isolates from other regions. Their unique set of ARGs includes a cfr-like gene and tetM, which were found as part of putative mobile genetic elements whose sequences resemble undescribed integrative and conjugative elements. These results show multiple, independent introductions of B1/NAP1/RT027/ST01 isolates from the FQR1 and FQR2 lineages from different geographical sources into LA and a rather rapid accumulation of distinct mutations and acquired ARG by the LA isolates.

Blood stream invasion by Escherichia coli is the commonest cause of bacteremia in the UK and elsewhere with an attributable mortality of about 15–20 %; antibiotic resistance to multiple agents is common in this microbe and is associated with worse outcomes. Genes conferring antimicrobial resistance, and their frequent location on horizontally transferred genetic elements is well-recognised, but the origin of these determinants, and their ability to be maintained and spread within clinically-relevant bacterial populations is unclear. Here, we set out to examine the distribution of antimicrobial resistance genes in chromosomes and plasmids of 16 bloodstream isolates of E. coli from patients within Scotland, and how these genes are maintained and spread. Using a combination of short and long-read whole genome sequencing methods, we were able to assemble complete sequences of 44 plasmids, with 16 Inc group F and 20 col plasmids; antibiotic resistance genes located almost exclusively within the F group. bla CTX-M15 genes had re-arranged in some strains into the chromosome alone (five strains), while others contained plasmid copies alone (two strains). Integrons containing multiple antibiotic genes were widespread in plasmids, notably many with a dfrA7 gene encoding resistance to trimethoprim, thus linking trimethoprim resistance to the other antibiotic resistance genes within the plasmids. This will allow even narrow spectrum antibiotics such as trimethoprim to act as a selective agent for plasmids containing antibiotic resistance genes mediating much broader resistance, including blaCTX-M15. To our knowledge, this is the first analysis to provide complete sequence data of chromosomes and plasmids in a collection of pathogenic human bloodstream isolates of E. coli . Our findings reveal the interplay between plasmids and integrative and conjugative elements in the maintenance and spread of antibiotic resistance genes within pathogenic E. coli .

Linezolid-resistant Enterococcus faecalis (LREfs) carrying optrA are increasingly reported globally from multiple sources, but we lack a comprehensive analysis of human and animal optrA-LREfs strains. To assess if optrA is dispersed in isolates with varied genetic backgrounds or with common genetic features, we investigated the phylogenetic structure, genetic content [antimicrobial resistance (AMR), virulence, prophages, plasmidome] and optrA-containing platforms of 27 publicly available optrA-positive E. faecalis genomes from different hosts in seven countries. At the genome-level analysis, an in-house database with 64 virulence genes was tested for the first time. Our analysis showed a diversity of clones and adaptive gene sequences related to a wide range of genera from Firmicutes . Phylogenies of core and accessory genomes were not congruent, and at least PAI-associated and prophage genes contribute to such differences. Epidemiologically unrelated clones (ST21, ST476-like and ST489) obtained from human clinical and animal hosts in different continents over eight years (2010–2017) could be phylogenetically related (3–126 SNPs difference). optrA was located on the chromosome within a Tn6674-like element (n=10) or on medium-size plasmids (30–60 kb; n=14) belonging to main plasmid families (RepA_N/Inc18/Rep_3). In most cases, the immediate gene vicinity of optrA was generally identical in chromosomal (Tn6674) or plasmid (impB-fexA-optrA) backbones. Tn6674 was always inserted into the same ∆radC integration site and embedded in a 32 kb chromosomal platform common to strains from different origins (patients, healthy humans, and animals) in Europe, Africa, and Asia during 2012–2017. This platform is conserved among hundreds of E. faecalis genomes and proposed as a chromosomal hotspot for optrA integration. The finding of optrA in strains sharing common adaptive features and genetic backgrounds across different hosts and countries suggests the occurrence of common and independent genetic events occurring in distant regions and might explain the easy de novo generation of optrA-positive strains. It also anticipates a dramatic increase of optrA carriage and spread with a serious impact on the efficacy of linezolid for the treatment of Gram-positive infections.

  Vibrio parahaemolyticus   is an important cause of foodborne gastroenteritis globally. Thermostable direct haemolysin (TDH) and the TDH-related haemolysin are the two key virulence factors in V. parahaemolyticus. Vibrio pathogenicity islands harbour the genes encoding these two haemolysins. The serotyping of   V. parahaemolyticus   is based on the combination of O and K antigens. Frequent recombination has been observed in   V. parahaemolyticus  , including in the genomic regions encoding the O and K antigens.   V. parahaemolyticus   serotype O4:K12 has caused gastroenteritis outbreaks in the USA and Spain. Recently, outbreaks caused by this serotype of   V. parahaemolyticus   have been reported in China. However, the relationships among this serotype of   V. parahaemolyticus   strains isolated in different regions have not been addressed. Here, we investigated the genome variation of the   V. parahaemolyticus   serotype O4:K12 using the whole-genome sequences of 29 isolates. We determined five distinct lineages in this strain collection. We observed frequent recombination among different lineages. In contrast, little recombination was observed within each individual lineage. We showed that the lineage of this serotype of   V. parahaemolyticus   isolated in America was different from those isolated in Asia and identified genes that exclusively existed in the strains isolated in America. Pan-genome analysis showed that strain-specific and cluster-specific genes were mostly located in the genomic islands. Pan-genome analysis also showed that the vast majority of the accessory genes in the O4:K12 serotype of   V. parahaemolyticus   were acquired from within the genus   Vibrio  . Hence, we have shown that multiple distinct lineages exist in   V. parahaemolyticus   serotype O4:K12 and have provided more evidence about the gene segregation found in   V. parahaemolyticus   isolated in different continents.

Pseudomonas aeruginosa is a highly adaptive opportunistic pathogen that can have serious health consequences in patients with lung disorders. Taxonomic outliers of P. aeruginosa of environmental origin have recently emerged as infectious for humans. Here, we present the first genome-wide analysis of an isolate that caused fatal haemorrhagic pneumonia. In two clones, CLJ1 and CLJ3, sequentially recovered from a patient with chronic pulmonary disease, insertion of a mobile genetic element into the P. aeruginosa chromosome affected major virulence-associated phenotypes and led to increased resistance to the antibiotics used to combat the infection. Comparative genome, proteome and transcriptome analyses revealed that this ISL3-family insertion sequence disrupted the genes for flagellar components, type IV pili, O-specific antigens, translesion polymerase and enzymes producing hydrogen cyanide. Seven-fold more insertions were detected in the later isolate, CLJ3, than in CLJ1, some of which modified strain susceptibility to antibiotics by disrupting the genes for the outer-membrane porin OprD and the regulator of β-lactamase expression AmpD. In the Galleria mellonella larvae model, the two strains displayed different levels of virulence, with CLJ1 being highly pathogenic. This study revealed insertion sequences to be major players in enhancing the pathogenic potential of a P. aeruginosa taxonomic outlier by modulating both its virulence and its resistance to antimicrobials, and explains how this bacterium adapts from the environment to a human host.