- Volume 6, Issue 2, 2020

To establish the prevalence of mobile colistin resistance (mcr) genes amongst Salmonella enterica isolates obtained through public health surveillance in England (April 2014 to September 2017), 33 205 S . enterica genome sequences obtained from human, food, animal and environmental isolates were screened for the presence of mcr variants 1 to 8. The mcr-positive genomes were assembled, annotated and characterized according to plasmid type. Nanopore sequencing was performed on six selected isolates with putative novel plasmids, and phylogenetic analysis was used to provide an evolutionary context for the most commonly isolated clones. Fifty-two mcr-positive isolates were identified, of which 32 were positive for mcr-1, 19 for mcr-3 and 1 for mcr-5. The combination of Illumina and Nanopore sequencing identified three novel mcr-3 plasmids and one novel mcr-5 plasmid, as well as the presence of chromosomally integrated mcr-1 and mcr-3. Monophasic S. enterica serovar Typhimurium accounted for 27/52 (52 %) of the mcr-positive isolates, with the majority clustering in clades associated with travel to Southeast Asia. Isolates in these clades were associated with a specific plasmid range and an additional extended-spectrum beta-lactamase genotype. Routine whole-genome sequencing for public health surveillance provides an effective screen for novel and emerging antimicrobial determinants, including mcr. Complementary long-read technologies elucidated the genomic context of resistance determinants, offering insights into plasmid dissemination and linkage to other resistance genes.

Here, we report comprehensive transcriptomic profiles from Fusobacterium nucleatum under conditions that mimic the first stages of bacterial infection in a highly differentiated adenocarcinoma epithelial cell line. Our transcriptomic in vitro adenocarcinoma approach allows us to measure the expression dynamics and regulation of bacterial virulence and response factors in real time, and is a novel strategy for clarifying the role of F. nucleatum infection in colorectal cancer (CRC) progression. Our data show that: (i) infection alters metabolic and functional pathways in F. nucleatum , allowing the bacterium to adapt to the host-imposed milieu; (ii) infection also stimulates the expression of genes required to help induce and promote a hypoxic and inflammatory microenvironment in the host; and (iii) F. nucleatum invasion occurs by a haematogenous route of infection. Our study identifies novel gene targets from F. nucleatum that are activated during invasion and which may aid in determining how this species invades and promotes disease within the human gastrointestinal tract. These invasion-specific genes may be useful as biomarkers for CRC progression in a host and could also assist in the development of new diagnostic tools and treatments (such as vaccines or small molecule drug targets), which will be able to combat infection and inflammation in the host while circumventing the potential problem of F. nucleatum tolerization.

Edwardsiella ictaluri and Edwardsiella piscicida are important fish pathogens affecting cultured and wild fish worldwide. To investigate the genome-level differences and similarities between catfish-adapted strains in these two species, the complete E. ictaluri 93-146 and E. piscicida  C07-087 genomes were evaluated by applying comparative genomics analysis. All available complete (10) and non-complete (19) genomes from five Edwardsiella species were also included in a systematic analysis. Average nucleotide identity and core-genome phylogenetic tree analyses indicated that the five Edwardsiella species were separated from each other. Pan-/core-genome analyses for the 29 strains from the five species showed that genus Edwardsiella members have 9474 genes in their pan genome, while the core genome consists of 1421 genes. Orthology cluster analysis showed that E. ictaluri and E. piscicida  genomes have the greatest number of shared clusters. However, E. ictaluri and E. piscicida  also have unique features; for example, the E. ictaluri genome encodes urease enzymes and cytochrome o ubiquinol oxidase subunits, whereas E. piscicida  genomes encode tetrathionate reductase operons, capsular polysaccharide synthesis enzymes and vibrioferrin-related genes. Additionally, we report for what is believed to be the first time that E. ictaluri 93-146 and three other E. ictaluri genomes encode a type IV secretion system (T4SS), whereas none of the E. piscicida  genomes encode this system. Additionally, the E. piscicida  C07-087 genome encodes two different type VI secretion systems. E. ictaluri genomes tend to encode more insertion elements, phage regions and genomic islands than E. piscicida . We speculate that the T4SS could contribute to the increased number of mobilome elements in E. ictaluri compared to E. piscicida . Two of the E. piscicida  genomes encode full CRISPR-Cas regions, whereas none of the E. ictaluri genomes encode Cas proteins. Overall, comparison of the E. ictaluri and E. piscicida  genomes reveals unique features and provides new insights on pathogenicity that may reflect the host adaptation of the two species.

Prophages are integrated phage elements that are a pervasive feature of bacterial genomes. The fitness of bacteria is enhanced by prophages that confer beneficial functions such as virulence, stress tolerance or phage resistance, and these functions are encoded by ‘accessory’ or ‘moron’ loci. Whilst the majority of phage-encoded genes are repressed during lysogeny, accessory loci are often highly expressed. However, it is challenging to identify novel prophage accessory loci from DNA sequence data alone. Here, we use bacterial RNA-seq data to examine the transcriptional landscapes of five Salmonella prophages. We show that transcriptomic data can be used to heuristically enrich for prophage features that are highly expressed within bacterial cells and represent functionally important accessory loci. Using this approach, we identify a novel antisense RNA species in prophage BTP1, STnc6030, which mediates superinfection exclusion of phage BTP1. Bacterial transcriptomic datasets are a powerful tool to explore the molecular biology of temperate phages.

Antimicrobial resistance (AMR) poses a threat to public health. Clinical microbiology laboratories typically rely on culturing bacteria for antimicrobial-susceptibility testing (AST). As the implementation costs and technical barriers fall, whole-genome sequencing (WGS) has emerged as a ‘one-stop’ test for epidemiological and predictive AST results. Few published comparisons exist for the myriad analytical pipelines used for predicting AMR. To address this, we performed an inter-laboratory study providing sets of participating researchers with identical short-read WGS data from clinical isolates, allowing us to assess the reproducibility of the bioinformatic prediction of AMR between participants, and identify problem cases and factors that lead to discordant results. We produced ten WGS datasets of varying quality from cultured carbapenem-resistant organisms obtained from clinical samples sequenced on either an Illumina NextSeq or HiSeq instrument. Nine participating teams (‘participants’) were provided these sequence data without any other contextual information. Each participant used their choice of pipeline to determine the species, the presence of resistance-associated genes, and to predict susceptibility or resistance to amikacin, gentamicin, ciprofloxacin and cefotaxime. We found participants predicted different numbers of AMR-associated genes and different gene variants from the same clinical samples. The quality of the sequence data, choice of bioinformatic pipeline and interpretation of the results all contributed to discordance between participants. Although much of the inaccurate gene variant annotation did not affect genotypic resistance predictions, we observed low specificity when compared to phenotypic AST results, but this improved in samples with higher read depths. Had the results been used to predict AST and guide treatment, a different antibiotic would have been recommended for each isolate by at least one participant. These challenges, at the final analytical stage of using WGS to predict AMR, suggest the need for refinements when using this technology in clinical settings. Comprehensive public resistance sequence databases, full recommendations on sequence data quality and standardization in the comparisons between genotype and resistance phenotypes will all play a fundamental role in the successful implementation of AST prediction using WGS in clinical microbiology laboratories.

Proteins interacting with DNA are fundamental for mediating processes such as gene expression, DNA replication and maintenance of genome integrity. Accumulating evidence suggests that the chromatin of apicomplexan parasites, such as Plasmodium falciparum, is highly organized, and this structure provides an epigenetic mechanism for transcriptional regulation. To investigate how parasite chromatin structure is being regulated, we undertook comparative genomics analysis using 12 distinct eukaryotic genomes. We identified conserved and parasite-specific chromatin-associated domains (CADs) and proteins (CAPs). We then used the chromatin enrichment for proteomics (ChEP) approach to experimentally capture CAPs in P. falciparum. A topological scoring analysis of the proteomics dataset revealed stage-specific enrichments of CADs and CAPs. Finally, we characterized, two candidate CAPs: a conserved homologue of the structural maintenance of chromosome 3 protein and a homologue of the crowded-like nuclei protein, a plant-like protein functionally analogous to animal nuclear lamina proteins. Collectively, our results provide a comprehensive overview of CAPs in apicomplexans, and contribute to our understanding of the complex molecular components regulating chromatin structure and genome architecture in these deadly parasites.