- Volume 4, Issue 11, 2018

Pseudomonas aeruginosa is an extremely successful pathogen able to cause both acute and chronic infections in a range of hosts, utilizing a diverse arsenal of cell-associated and secreted virulence factors. A major cell-associated virulence factor, the Type IV pilus (T4P), is required for epithelial cell adherence and mediates a form of surface translocation termed twitching motility, which is necessary to establish a mature biofilm and actively expand these biofilms. P. aeruginosa twitching motility-mediated biofilm expansion is a coordinated, multicellular behaviour, allowing cells to rapidly colonize surfaces, including implanted medical devices. Although at least 44 proteins are known to be involved in the biogenesis, assembly and regulation of the T4P, with additional regulatory components and pathways implicated, it is unclear how these components and pathways interact to control these processes. In the current study, we used a global genomics-based random-mutagenesis technique, transposon directed insertion-site sequencing (TraDIS), coupled with a physical segregation approach, to identify all genes implicated in twitching motility-mediated biofilm expansion in P. aeruginosa. Our approach allowed identification of both known and novel genes, providing new insight into the complex molecular network that regulates this process in P. aeruginosa. Additionally, our data suggest that the flagellum-associated gene products have a differential effect on twitching motility, based on whether components are intra- or extracellular. Overall the success of our TraDIS approach supports the use of this global genomic technique for investigating virulence genes in bacterial pathogens.

Assembly of bacterial short-read whole-genome sequencing data frequently results in hundreds of contigs for which the origin, plasmid or chromosome, is unclear. Complete genomes resolved by long-read sequencing can be used to generate and label short-read contigs. These were used to train several popular machine learning methods to classify the origin of contigs from Enterococcus faecium, Klebsiella pneumoniae and Escherichia coli using pentamer frequencies. We selected support-vector machine (SVM) models as the best classifier for all three bacterial species (F1-score E. faecium=0.92, F1-score K. pneumoniae=0.90, F1-score E. coli=0.76), which outperformed other existing plasmid prediction tools using a benchmarking set of isolates. We demonstrated the scalability of our models by accurately predicting the plasmidome of a large collection of 1644 E. faecium isolates and illustrate its applicability by predicting the location of antibiotic-resistance genes in all three species. The SVM classifiers are publicly available as an R package and graphical-user interface called ‘mlplasmids’. We anticipate that this tool may significantly facilitate research on the dissemination of plasmids encoding antibiotic resistance and/or contributing to host adaptation.

Hypermutable simple sequence repeats (SSRs) are drivers of phase variation (PV) whose stochastic, high-frequency, reversible switches in gene expression are a common feature of several pathogenic bacterial species, including the human pathogen Campylobacter jejuni. Here we examine the distribution and conservation of known and putative SSR-driven phase variable genes – the phasome – in the genus Campylobacter. PhasomeIt, a new program, was specifically designed for rapid identification of SSR-mediated PV. This program detects the location, type and repeat number of every SSR. Each SSR is linked to a specific gene and its putative expression state. Other outputs include conservation of SSR-driven phase-variable genes and the ‘core phasome’ – the minimal set of PV genes in a phylogenetic grouping. Analysis of 77 complete Campylobacter genome sequences detected a ‘core phasome’ of conserved PV genes in each species and a large number of rare PV genes with few, or no, homologues in other genome sequences. Analysis of a set of partial genome sequences, with food-chain-associated metadata, detected evidence of a weak link between phasome and source host for disease-causing isolates of sequence type (ST)-828 but not the ST-21 or ST-45 complexes. Investigation of the phasomes in the genus Campylobacter provided evidence of overlapping but distinctive mechanisms of PV-mediated adaptation to specific niches. This suggests that the phasome could be involved in host adaptation and spread of campylobacters. Finally, this tool is malleable and will have utility for studying the distribution and genic effects of other repetitive elements in diverse bacterial species.

The genome of Bordetella pertussis is complex, with high G+C content and many repeats, each longer than 1000 bp. Long-read sequencing offers the opportunity to produce single-contig B. pertussis assemblies using sequencing reads which are longer than the repetitive sections, with the potential to reveal genomic features which were previously unobservable in multi-contig assemblies produced by short-read sequencing alone. We used an R9.4 MinION flow cell and barcoding to sequence five B. pertussis strains in a single sequencing run. We then trialled combinations of the many nanopore user community-built long-read analysis tools to establish the current optimal assembly pipeline for B. pertussis genome sequences. This pipeline produced closed genome sequences for four strains, allowing visualization of inter-strain genomic rearrangement. Read mapping to the Tohama I reference genome suggests that the remaining strain contains an ultra-long duplicated region (almost 200 kbp), which was not resolved by our pipeline; further investigation also revealed that a second strain that was seemingly resolved by our pipeline may contain an even longer duplication, albeit in a small subset of cells. We have therefore demonstrated the ability to resolve the structure of several B. pertussis strains per single barcoded nanopore flow cell, but the genomes with highest complexity (e.g. very large duplicated regions) remain only partially resolved using the standard library preparation and will require an alternative library preparation method. For full strain characterization, we recommend hybrid assembly of long and short reads together; for comparison of genome arrangement, assembly using long reads alone is sufficient.

Carbapenem resistance is a rapidly growing threat to our ability to treat refractory bacterial infections. To understand how carbapenem resistance is mobilized and spread between pathogens, it is important to study the genetic context of the underlying resistance mechanisms. In this study, the resistomes of six clinical carbapenem-resistant isolates of five different species – Acinetobacter baumannii, Escherichia coli, two Klebsiella pneumoniae, Proteus mirabilis and Pseudomonas aeruginosa – were characterized using whole genome sequencing. All Enterobacteriaceae isolates and the A. baumannii isolate had acquired a large number of antimicrobial resistance genes (7–18 different genes per isolate), including the following encoding carbapenemases: bla _KPC-2, bla _OXA-48, bla _OXA-72, bla _NDM-1, bla _NDM-7 and bla _VIM-1. In addition, a novel version of bla _SHV was discovered. Four new resistance plasmids were identified and their fully assembled sequences were verified using optical DNA mapping. Most of the resistance genes were co-localized on these and other plasmids, suggesting a risk for co-selection. In contrast, five out of six carbapenemase genes were present on plasmids with no or few other resistance genes. The expected level of resistance – based on acquired resistance determinants – was concordant with measured levels in most cases. There were, however, several important discrepancies for four of the six isolates concerning multiple classes of antibiotics. In conclusion, our results further elucidate the diversity of carbapenemases, their mechanisms of horizontal transfer and possible patterns of co-selection. The study also emphasizes the difficulty of using whole genome sequencing for antimicrobial susceptibility testing of pathogens with complex genotypes.