- Volume 6, Issue 4, 2020

Escherichia coli sequence type 131 (ST131) is a pandemic clone that is evolving rapidly with increasing levels of antimicrobial resistance. Here, we investigated an outbreak of E. coli ST131 producing extended spectrum β-lactamases (ESBLs) in a long-term care facility (LTCF) in Ireland by combining data from this LTCF (n=69) with other Irish (n=35) and global (n=690) ST131 genomes to reconstruct the evolutionary history and understand changes in population structure and genome architecture over time. This required a combination of short- and long-read genome sequencing, de novo assembly, read mapping, ESBL gene screening, plasmid alignment and temporal phylogenetics. We found that Clade C was the most prevalent (686 out of 794 isolates, 86 %) of the three major ST131 clades circulating worldwide (A with fimH41, B with fimH22, C with fimH30), and was associated with the presence of different ESBL alleles, diverse plasmids and transposable elements. Clade C was estimated to have emerged in c. 1985 and subsequently acquired different ESBL gene variants (bla CTX-M-14 vs bla CTX-M-15 ). An ISEcp1-mediated transposition of the bla CTX-M-15 gene further increased the diversity within Clade C. We discovered a local clonal expansion of a rare C2 lineage (C2_8) with a chromosomal insertion of bla CTX-M-15 at the mppA gene. This was acquired from an IncFIA plasmid. The C2_8 lineage clonally expanded in the Irish LTCF from 2006, displacing the existing C1 strain (C1_10), highlighting the potential for novel ESBL-producing ST131 with a distinct genetic profile to cause outbreaks strongly associated with specific healthcare environments.

Lawsonia intracellularis is a Gram-negative obligate intracellular bacterium that is the aetiological agent of proliferative enteropathy (PE), a common intestinal disease of major economic importance in pigs and other animal species. To date, progress in understanding the biology of L. intracellularis for improved disease control has been hampered by the inability to culture the organism in vitro. In particular, our understanding of the genomic diversity and population structure of clinical L. intercellularis is very limited. Here, we utilized a metagenomic shotgun approach to directly sequence and assemble 21 L . intracellularis genomes from faecal and ileum samples of infected pigs and horses across three continents. Phylogenetic analysis revealed a genetically monomorphic clonal lineage responsible for infections in pigs, with distinct subtypes associated with infections in horses. The genome was highly conserved, with 94 % of genes shared by all isolates and a very small accessory genome made up of only 84 genes across all sequenced strains. In part, the accessory genome was represented by regions with a high density of SNPs, indicative of recombination events importing novel gene alleles. In summary, our analysis provides the first view of the population structure for L. intracellularis , revealing a single major lineage associated with disease of pigs. The limited diversity and broad geographical distribution suggest the recent emergence and clonal expansion of an important livestock pathogen.

This study presents the nationwide epidemiology of Neisseria gonorrhoeae , using whole-genome sequencing of all culture-positive cases, which comprise roughly 40 % of all cases of gonorrhea reported in Norway from 2016 to 2017. Isolates were assigned to sequence types and Bayesian analysis clusters and variation in genes coding for antibiotic resistance was linked to phenotypic resistance data. The study also included isolates taken from the same patients from different anatomical sites at one or more time points. Comparing these isolates allows for observation of patterns of infections, i.e. multiple reinfections of genetically related clones vs. reinfections of genetically distant clones, and quantification of the genomic variation of closely related isolates from samples taken from a patient within the same day. Demographically, the patients in the study could be split into two groups; one group of patients from the capital with a high proportion of men who have sex with men (MSM), and another consisting of young adults with transmission primarily between males and females from outside the capital. Some clusters of N. gonorrhoeae were restricted to one of these two demographic groups. Pairwise comparison of multiple isolates from the same patients revealed that most were reinfected with different clones. Observations of frequent reinfections in patients is a concern and should be taken into account in the development of improved information and treatment guidelines.

Bacteria can evade cohabiting phages through mutations in phage receptors, but these mutations may come at a cost if they disrupt the receptor’s native cellular function. To investigate the relationship between these two conflicting activities, we generated sequence–function maps of Escherichia coli LamB with respect to sensitivity to phage λ and transport of maltodextrin. By comparing 413 missense mutations whose effect on both traits could be analysed, we find that these two phenotypes were correlated, implying that most mutations affect these phenotypes through a common mechanism such as loss of protein stability. However, individual mutations could be found that specifically disrupt λ-sensitivity without affecting maltodextrin transport. We identify and individually assay nine such mutations, whose spatial positions implicate loop L6 of LamB in λ binding. Although missense mutations that lead to λ-resistance are rare, they were approximately as likely to be maltodextrin-utilizing (Mal+) as not (Mal-), implying that E. coli can adapt to λ while conserving the receptor’s native function. We propose that in order for E. coli and λ to stably cohabitate, selection for λ-resistance and maltose transport must be spatially or temporally separated.

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 .

Understanding host and pathogen factors that influence tuberculosis (TB) transmission can inform strategies to eliminate the spread of Mycobacterium tuberculosis (Mtb). Determining transmission links between cases of TB is complicated by a long and variable latency period and undiagnosed cases, although methods are improving through the application of probabilistic modelling and whole-genome sequence analysis. Using a large dataset of 1857 whole-genome sequences and comprehensive metadata from Karonga District, Malawi, over 19 years, we reconstructed Mtb transmission networks using a two-step Bayesian approach that identified likely infector and recipient cases, whilst robustly allowing for incomplete case sampling. We investigated demographic and pathogen genomic variation associated with transmission and clustering in our networks. We found that whilst there was a significant decrease in the proportion of infectors over time, we found higher transmissibility and large transmission clusters for lineage 2 (Beijing) strains. By performing evolutionary convergence testing (phyC) and genome-wide association analysis (GWAS) on transmitting versus non-transmitting cases, we identified six loci, PPE54, accD2, PE_PGRS62, rplI, Rv3751 and Rv2077c, that were associated with transmission. This study provides a framework for reconstructing large-scale Mtb transmission networks. We have highlighted potential host and pathogen characteristics that were linked to increased transmission in a high-burden setting and identified genomic variants that, with validation, could inform further studies into transmissibility and TB eradication.

The complete genome sequence of Rhodococcus sp. WAY2 (WAY2) consists of a circular chromosome, three linear replicons and a small circular plasmid. The linear replicons contain typical actinobacterial invertron-type telomeres with the central CGTXCGC motif. Comparative phylogenetic analysis of the 16S rRNA gene along with phylogenomic analysis based on the genome-to-genome blast distance phylogeny (GBDP) algorithm and digital DNA–DNA hybridization (dDDH) with other Rhodococcus type strains resulted in a clear differentiation of WAY2, which is likely a new species. The genome of WAY2 contains five distinct clusters of bph, etb and nah genes, putatively involved in the degradation of several aromatic compounds. These clusters are distributed throughout the linear plasmids. The high sequence homology of the ring-hydroxylating subunits of these systems with other known enzymes has allowed us to model the range of aromatic substrates they could degrade. Further functional characterization revealed that WAY2 was able to grow with biphenyl, naphthalene and xylene as sole carbon and energy sources, and could oxidize multiple aromatic compounds, including ethylbenzene, phenanthrene, dibenzofuran and toluene. In addition, WAY2 was able to co-metabolize 23 polychlorinated biphenyl congeners, consistent with the five different ring-hydroxylating systems encoded by its genome. WAY2 could also use n-alkanes of various chain-lengths as a sole carbon source, probably due to the presence of alkB and ladA gene copies, which are only found in its chromosome. These results show that WAY2 has a potential to be used for the biodegradation of multiple organic compounds.

Bovine viral diarrhoea (BVD) is an important disease of cattle, with significant impacts on animal health and welfare. The wide host range of the causative pestiviruses may lead to formation of virus reservoirs in other ruminant or wildlife species, presenting a concern for the long-term success of BVD eradication campaigns. It is likely that the quasispecies nature of these RNA viruses contributes to their interspecies transmission by providing genetic plasticity. Understanding the spectrum of sequence variants present in persistently infected (PI) animals is, therefore, essential for studies of virus transmission. To analyse quasispecies diversity without amplification bias, we extracted viral RNA from the serum of a PI cow, and from cell culture fluid after three passages of the same virus in culture, to produce cDNA without amplification. Sequencing of this material using Illumina 250 bp paired-read technology produced full-length virus consensus sequences from both sources and demonstrated the quasispecies diversity of this pestivirus A genotype 1a field strain within serum and after culture. We report the distribution and diversity of over 800 SNPs and provide evidence for a loss of diversity after only three passages in cell culture, implying that cultured viruses cannot be used to understand quasispecies diversity and may not provide reliable molecular markers for source tracing or transmission studies. Additionally, both serum and cultured viruses could be sequenced as a set of 25 overlapping PCR amplicons that demonstrated the same consensus sequences and the presence of many of the same quasispecies variants. The observation that aspects of the quasispecies structure revealed by massively parallel sequencing are also detected after PCR and Sanger sequencing suggests that this approach may be useful for small or difficult to analyse samples.

Rhizobia supply legumes with fixed nitrogen using a set of symbiosis genes. These can cross rhizobium species boundaries, but it is unclear how many other genes show similar mobility. Here, we investigate inter-species introgression using de novo assembly of 196   Rhizobium leguminosarum   sv. trifolii genomes. The 196 strains constituted a five-species complex, and we calculated introgression scores based on gene-tree traversal to identify 171 genes that frequently cross species boundaries. Rather than relying on the gene order of a single reference strain, we clustered the introgressing genes into four blocks based on population structure-corrected linkage disequilibrium patterns. The two largest blocks comprised 125 genes and included the symbiosis genes, a smaller block contained 43 mainly chromosomal genes, and the last block consisted of three genes with variable genomic location. All introgression events were likely mediated by conjugation, but only the genes in the symbiosis linkage blocks displayed overrepresentation of distinct, high-frequency haplotypes. The three genes in the last block were core genes essential for symbiosis that had, in some cases, been mobilized on symbiosis plasmids. Inter-species introgression is thus not limited to symbiosis genes and plasmids, but other cases are infrequent and show distinct selection signatures.

The availability of next-generation sequencing techniques provides an unprecedented opportunity for the assignment of gene function. Streptococcus equi subspecies equi is the causative agent of strangles in horses, one of the most prevalent and important diseases of equids worldwide. However, the live attenuated vaccines that are utilized to control this disease cause adverse reactions in some animals. Here, we employ transposon-directed insertion-site sequencing (TraDIS) to identify genes that are required for the fitness of S. equi in whole equine blood or in the presence of H2O2 to model selective pressures exerted by the equine immune response during infection. We report the fitness values of 1503 and 1471 genes, representing 94.5 and 92.5 % of non-essential genes in S. equi , following incubation in whole blood and in the presence of H2O2, respectively. Of these genes, 36 and 15 were identified as being important to the fitness of S. equi in whole blood or H2O2, respectively, with 14 genes being important in both conditions. Allelic replacement mutants were generated to validate the fitness results. Our data identify genes that are important for S. equi to resist aspects of the immune response in vitro, which can be exploited for the development of safer live attenuated vaccines to prevent strangles.