- Volume 5, Issue 7, 2019

Clostridioides difficile is a ubiquitous, diarrhoeagenic pathogen often associated with healthcare-acquired infections that can cause a range of symptoms from mild, self-limiting disease to toxic megacolon and death. Since the early 2000s, a large proportion of C. difficile cases have been attributed to the ribotype 027 (RT027) lineage, which is associated with sequence type 1 (ST1) in the C. difficile multilocus sequence typing scheme. The spread of ST1 has been attributed, in part, to resistance to fluoroquinolones used to treat unrelated infections, which creates conditions ideal for C. difficile colonization and proliferation. In this study, we analysed 27 isolates from a healthcare network in northern Arizona, USA, and 1352 publicly available ST1 genomes to place locally sampled isolates into a global context. Whole genome, single nucleotide polymorphism analysis demonstrated that at least six separate introductions of ST1 were observed in healthcare facilities in northern Arizona over an 18-month sampling period. A reconstruction of transmission networks identified potential nosocomial transmission of isolates, which were only identified via whole genome sequence analysis. Antibiotic resistance heterogeneity was observed among ST1 genomes, including variability in resistance profiles among locally sampled ST1 isolates. To investigate why ST1 genomes are so common globally and in northern Arizona, we compared all high-quality C. difficile genomes and identified that ST1 genomes have gained and lost a number of genomic regions compared to all other C. difficile genomes; analyses of other toxigenic C. difficile sequence types demonstrate that this loss may be anomalous and could be related to niche specialization. These results suggest that a combination of antimicrobial resistance and gain and loss of specific genes may explain the prominent association of this sequence type with C. difficile infection cases worldwide. The degree of genetic variability in ST1 suggests that classifying all ST1 genomes into a quinolone-resistant hypervirulent clone category may not be appropriate. Whole genome sequencing of clinical C. difficile isolates provides a high-resolution surveillance strategy for monitoring persistence and transmission of C. difficile and for assessing the performance of infection prevention and control strategies.

The pneumococcus produces a polysaccharide capsule, encoded by the cps locus, that provides protection against phagocytosis and determines serotype. Nearly 100 serotypes have been identified with new serotypes still being discovered, especially in previously understudied regions. Here we present an analysis of the cps loci of more than 18  000 genomes from the Global Pneumococcal Sequencing (GPS) project with the aim of identifying novel cps loci with the potential to produce previously unrecognized capsule structures. Serotypes were assigned using whole genome sequence data and 66 of the approximately 100 known serotypes were included in the final dataset. Closer examination of each serotype’s sequences identified nine putative novel cps loci (9X, 11X, 16X, 18X1, 18X2, 18X3, 29X, 33X and 36X) found in ~2.6  % of the genomes. The large number and global distribution of GPS genomes provided an unprecedented opportunity to identify novel cps loci and consider their phylogenetic and geographical distribution. Nine putative novel cps loci were identified and examples of each will undergo subsequent structural and immunological analysis.

Pseudomonas aeruginosa is a highly versatile, antibiotic-resistant Gram-negative bacterium known for causing opportunistic infections and contamination of industrial products. Despite extensive genomic analysis of clinical P. aeruginosa strains, no genomes exist for preservative-tolerant industrial strains. A unique collection of 69 industrial isolates was assembled and compared to clinical and environmental strains; 16 genetically distinct industrial strains were subjected to array tube genotyping, multilocus sequence typing and whole-genome sequencing. The industrial strains possessed high preservative tolerance and were dispersed widely across P. aeruginosa as a species, but recurrence of strains from the same lineage within specific industrial products and locations was identified. The industrial P. aeruginosa genomes (mean=7.0 Mb) were significantly larger than those of previously sequenced environmental (mean=6.5 Mb; n=19) and clinical (mean=6.6 Mb; n=66) strains. Complete sequencing of the P. aeruginosa industrial strain RW109, which encoded the largest genome (7.75 Mb), revealed a multireplicon structure including a megaplasmid (555 265 bp) and large plasmid (151 612 bp). The RW109 megaplasmid represented an emerging plasmid family conserved in seven industrial and two clinical P. aeruginosa strains, and associated with extremely stress-resilient phenotypes, including antimicrobial resistance and solvent tolerance. Here, by defining the detailed phylogenomics of P. aeruginosa industrial strains, we show that they uniquely possess multireplicon, megaplasmid-bearing genomes, and significantly greater genomic content worthy of further study.

Enterococcus faecium is a gut commensal of humans and animals. In addition, it has recently emerged as an important nosocomial pathogen through the acquisition of genetic elements that confer resistance to antibiotics and virulence. We performed a whole-genome sequencing-based study on 96 multidrug-resistant E. faecium strains that asymptomatically colonized five patients with the aim of describing the genome dynamics of this species. The patients were hospitalized on multiple occasions and isolates were collected over periods ranging from 15 months to 6.5 years. Ninety-five of the sequenced isolates belonged to E. faecium clade A1, which was previously determined to be responsible for the vast majority of clinical infections. The clade A1 strains clustered into six clonal groups of highly similar isolates, three of which consisted entirely of isolates from a single patient. We also found evidence of concurrent colonization of patients by multiple distinct lineages and transfer of strains between patients during hospitalization. We estimated the evolutionary rate of two clonal groups that each colonized single patients at 12.6 and 25.2 single-nucleotide polymorphisms (SNPs)/genome/year. A detailed analysis of the accessory genome of one of the clonal groups revealed considerable variation due to gene gain and loss events, including the chromosomal acquisition of a 37 kbp prophage and the loss of an element containing carbohydrate metabolism-related genes. We determined the presence and location of 12 different insertion sequence (IS) elements, with ISEfa5 showing a unique pattern of location in 24 of the 25 isolates, suggesting widespread ISEfa5 excision and insertion into the genome during gut colonization. Our findings show that the E. faecium genome is highly dynamic during asymptomatic colonization of the human gut. We observed considerable genomic flexibility due to frequent horizontal gene transfer and recombination, which can contribute to the generation of genetic diversity within the species and, ultimately, can contribute to its success as a nosocomial pathogen.

Reference and type strains of well-known bacteria have been a cornerstone of microbiology research for decades. The sharing of well-characterized isolates among laboratories has run in parallel with research efforts and enhanced the reproducibility of experiments, leading to a wealth of knowledge about trait variation in different species and the underlying genetics. Campylobacter jejuni strain NCTC 11168, deposited at the National Collection of Type Cultures in 1977, has been adopted widely as a reference strain by researchers worldwide and was the first Campylobacter for which the complete genome was published (in 2000). In this study, we collected 23 C . jejuni NCTC 11168 reference isolates from laboratories across the UK and compared variation in simple laboratory phenotypes with genetic variation in sequenced genomes. Putatively identical isolates, identified previously to have aberrant phenotypes, varied by up to 281 SNPs (in 15 genes) compared to the most recent reference strain. Isolates also display considerable phenotype variation in motility, morphology, growth at 37 °C, invasion of chicken and human cell lines, and susceptibility to ampicillin. This study provides evidence of ongoing evolutionary change among C. jejuni isolates as they are cultured in different laboratories and highlights the need for careful consideration of genetic variation within laboratory reference strains. This article contains data hosted by Microreact.