Preliminary Characterization of Phage-like Particles from the Male-Killing Mollicute Spiroplasma poulsonii (an Endosymbiont of Drosophila)

Viruses are vastly abundant and influential in all ecosystems, and are generally regarded as pathogens. Viruses of prokaryotes (themselves highly diverse and abundant) are known as bacteriophages or phages. Phages engage in diverse associations with their hosts, and contribute to regulation of biogeochemical processes, horizontal movement of genes, and control of bacterial populations. Recent studies have revealed the influential role of phage in the association of arthropods and their heritable endosymbiotic bacteria (e.g. the Proteobacteria genera Wolbachia and Hamiltonella). Despite prior studies (~30 years ago) documenting presence of phage in the mollicute Spiroplasma infecting Drosophila, genomic sequences of such phage are lacking, and their effects on the Spiroplasma-Drosophila interaction have not been comprehensively characterized. The present work isolated phage-like particles from the male-killing Spiroplasma poulsonii (strains NSRO and MSRO-Br) harbored by Drosophila melanogaster. Isolated particles were subjected to DNA sequencing, assembly, and annotation. Our results recovered three ~19 kb phage-like contigs (two in NSRO and one in MSRO-Br), and two smaller non-phage-like contigs encoding a known Spiroplasma toxin and an insertion element. Whole or parts of the particle-derived contigs were found in the genome assemblies of members of the Spiroplasma poulsonii clade. Although our results do not allow us to distinguish whether the contigs obtained represent infective phage-like particles capable of transmitting their DNA to new hosts, their encoding of several typical phage genes suggests that they are at least remnants of functional phage. We discuss potential implications of our findings and suggest future directions.

Bacterial LomR Induces the Vibriophage VP882 VqmA-Directed Quorum-Sensing Lysogeny-Lysis Transition

The bacterial cell-cell communication process called quorum sensing enables groups of bacteria to synchronously alter behavior in response to changes in cell population density. Quorum sensing relies on the production, release, accumulation, and detection of extracellular signal molecules called autoinducers. Here, we investigate a mechanism employed by a vibriophage to surveil host quorum sensing and tune its lysogeny-lysis decision to host cell density. The phage possesses a gene called vqmAPhage encoding a quorum-sensing receptor homologous to vibrio VqmA. Both VqmA receptors can detect the host bacteria-produced autoinducer called DPO. DPO-bound VqmAPhage launches the phage lysis process. We discover that the bacterial host produces an inducer of the VqmAPhage-directed quorum-sensing lysogeny-lysis transition. Production of the inducer appears to be widespread among bacteria. A screen of the Escherichia coli Keio collection for mutants impaired for inducer production revealed lomR, located in a prophage, and encoding a poorly understood protein. In the E. coli screening strain, lomR is interrupted by DNA encoding an insertion element. The 3’ domain of this LomR protein is sufficient to induce VqmAPhage-directed lysis. Alanine-scanning mutagenesis showed that substitution at either of two key residues abrogates inducer activity. Full-length LomR is similar to the outer membrane porin OmpX in E. coli and Vibrio parahaemolyticus O3:K6, and OmpT in Vibrio cholerae C6706, and indeed, OmpX and OmpT can induce VqmAPhage-directed activity. Possibly, development of the LomR, OmpX, or OmpT proteins as tools to direct phage lysis of host cells could be used to control bacteria in medical or industrial settings.

Diagnostic test accuracy study for the detection of bovine genital campylobacteriosis of preputial scrapings from beef bulls, in Uruguay: A Bayesian latent class model approach

The sensitivity (Se) and specificity (Sp) of three diagnostic tests for the detection of Campylobacter fetus venerealis (Cfv) using field samples were estimated using a Bayesian latent class model (BLCM), accounting for the absence of a gold standard. The tests were direct immunofluorescence antibody test (IFAT), polymerase chain reaction (PCR), and real-time PCR (RT-PCR). Twelve farms from two different populations were selected and bull prepuce samples were collected. The IFAT was performed according to the OIE Manual. The conventional PCR was performed as multiplex, targeting the gene nahE for C. fetus species identification and insertion element ISCfe1 for Cfv identification. The RT-PCR was performed as uniplex one targeting the gene nahE for C. fetus and the other targeting the insertion ISCfe1 (ISC2) for Cfv. Results from the BLCM showed a median Se of 11.7% (Bayesian credibility interval (BCI): 1.93% − 29.79%), 53.7% (BCI: 23.1% − 95.0%) and 36.1% (BCI: 14.5% − 71.7%) for IFAT, PCR and RT-PCR respectively. The Sp were 94.5% (BCI: 90.1% -97.9%), 97.0% (BCI: 92.9% − 99.3%) and 98.4% (BCI: 95.3% − 99.7%) for IFAT, PCR and RT-PCR respectively. The correlation between PCR and RT-PCR was positive and low in samples from both sampled population (0.63% vs 8.47%). These results suggest that diagnostic sensitivity of the studied tests is lower using field samples than using pure Cfv strains.

Genomic evaluation of Bordetella spp. originating from Australia

Bordetella pertussis is the primary causative agent of pertussis, a highly infectious respiratory disease associated with prolonged coughing episodes. Pertussis infections are typically mild in adults, however in neonates, infections can be fatal. Despite successful vaccine uptake, the disease is re-emerging across the globe, therefore it is critical to determine the mechanism by which B. pertussis is escaping vaccination control. Studies have suggested that significant changes have occurred in B. pertussis genomes in response to whole cell and acellular vaccines. Continued molecular monitoring is therefore crucial for public health surveillance.High-resolution molecular surveillance of B. pertussis can be achieved through the sequencing of the whole genome. In public health laboratories, whole genome sequencing is primarily performed by short-read sequencing technologies as they are most cost-effective. However short read sequencing does not resolve the extensive genomic rearrangement evident in Bordetella genomes. This is because repeat regions present in Bordetella genomes are collapsed by downstream analysis. For example, the B. pertussis genome contains more than 200 copies of the IS481 insertion element, hence assemblies generally consist of >200 contigs. Advancements in long-read technologies however increase the potential to circularise and close genomes by bridging the locations of the IS481 insertion element.In this study, we aimed to contextualise the Bordetella spp. circulating in NSW, Australia and assess their relationship with global isolates utilising core genome, SNP and structural clustering analysis using long read technology. We report five closed genomes of Bordetella spp. isolated from Australian patients. Two of the three B. pertussis closed isolates, were unique with their own genomic structure, while the other structurally clustered with global isolates. We found that Australian B. holmesii and B. parapertussis strains cluster with global isolates and do not appear to be unique to Australia. Australian draft B. holmesii SNP analysis showed that between 1999 and 2007, isolates were relatively similar, however post-2012, isolates were distinct from each other. The closed isolates can also be used as high-quality reference sequences for both surveillance and other investigations into pertussis spread.

Emergence of vancomycin- and teicoplanin-resistant Enterococcus faecium via vanD5-harbouring large genomic island

Background Treatment of VRE is of clinical concern. While certain numbers of vanD-type VRE have been isolated, only two vanD5-harbouring Enterococcus faecium isolates have been reported in Canada and Japan. Methods We report the isolation of vanD5-type E. faecium and the first ever determination of the whole-genome sequence to investigate the possible mechanisms of the acquisition of the vanD5 gene cluster in E. faecium. Results Two vanD5-harbouring vancomycin-resistant E. faecium were isolated from the skin (SMVRE19) and faeces (SMVRE20) of a patient with a skin ulcer in Japan. The isolates exhibited vancomycin and teicoplanin MIC values of 128 mg/L, whilst the previous isolates of vanD5-harbouring E. faecium were only resistant to vancomycin. SMVRE19 and SMVRE20 were clones related to ST18, which is also seen in vanA- and vanB-type VRE. These isolates harboured an insertion element, ISEfm1, in the ddl gene, similar to a previously described teicoplanin-resistant vanD3-type E. faecium. The vanD5 gene cluster was integrated into the SMVRE20 chromosome as a part of a large genomic island (approximately 127 kb), similar to other recently spreading vanD variants in the Netherlands. The genomic island shared the greatest similarity with a part of the Blautia coccoides genome sequence, except for the region surrounding the vanD gene cluster. Conclusions This study reports that emergence of vancomycin- and teicoplanin-resistant vanD5-type E. faecium occurred via acquisition of the vanD5 cluster and ISEfm1 insertion into ddl. Considering the genetic similarity between the various VRE strains, the current study should serve as a warning against the spread of vanD5-type VRE.

Genetic characterisation of a large antibiotic resistant environmental ST131 E. coli plasmid using a long read hybrid assembly approach

Escherichia coli Strain Type 131 are a globally disseminated environmental E. coli that has been linked to the capture and spread of plasmid mediated blaCTX-M type extended spectrum beta-lactamase (ESBLs). Accurately identifying such resistance genes in their wider genetic context provides a greater understanding of the mechanisms of selection and persistence in the environment. In this study we use a novel DNA extraction and enrichment method in combination with a custom long-read scaffold hybrid-assembly and polishing pipe line to identify the genetic context of the plasmid borne blaCTX-M gene previously identified in an ST131 environmental E. coli isolate. This has allowed us to discern the complete structure of a ~100kb environmental plasmid and further resolve the blaCTX-M variant to the group 9 blaCTX-M-27 gene. The upstream IS26 insertion element associated with the global capture and dissemination of blaCTX-M-15 was also identified in proximity to blaCTX-M-27. Furthermore, the lack of conjugative machinery identified on this plasmid, in combination with a toxin-antitoxin and plasmid partitioning system, indicates a mechanism of vertical transmission to maintain persistence in a population.

High-Resolution, Multidimensional Phylogenetic Metrics Identify Class I Aminoacyl-tRNA Synthetase Evolutionary Mosaicity and Inter-modular ‘Coupling

The provenance of the aminoacyl-tRNA synthetases (aaRS) poses unusually challenging questions because of their role in the emergence and evolution of genetic coding. We investigate evidence about their ancestry from highly curated structure-based multiple sequence alignments of a small “scaffold” that is structurally invariant in all 10 canonical Class I aaRS. Statistically different values of two uncorrelated phylogenetic metrics—residue by residue conservation derived from Clustal and row-by-row cladistic congruence derived from BEAST2—suggest that the Class I scaffold is a mosaic assembled from distinct, successive genetic sources. These data are especially significant in light of: (i) experimental fragmentations of the Class I scaffold into three partitions that retain catalytic activities in proportion to their length; and (ii) multiple sources of evidence that two of these partitions arose from an ancestral Class I aaRS gene encoding a Class II ancestor in frame on the opposite strand. Two additional metrics output by BEAST2 vary in accordance with the presumed functionality endowed by the various modules. The new evidence supplements previous aaRS phylogenies. It identifies a previously characterized 46-residue Class I “protozyme” as preceding the adaptive radiation of the superfamily containing variations of the Rossmann dinucleotide binding fold related to amino acid discrimination, and thus as root of that molecular tree. Such a rooting is consistent with near simultaneous emergence of genetic coding and the origin of the proteome, resolving a conundrum posed by previous inferences that Class I aaRS evolved long after the genetic code had been implemented in an RNA world. Further, it establishes a timeline for the growth of coding from a binary amino acid alphabet by pinpointing discontinuous enhancements of aaRS fidelity.Author SummaryPhylogenetic analysis uncovers evolutionary connections between different protein superfamily members. We describe complementary, uncorrelated, phylogenetic metrics that support multiple evolutionary histories for different segments within members of the Class I aminoacyl-tRNA synthetase superfamily. Using a carefully curated 3D crystal structure superposition as the primary source of the multiple sequence alignment substantially reduced dependence of these metrics on empirical amino acid substitution matrices. Two metrics are derived from the amino acid distribution observed in each successive position. A third depends on how individual sequences distribute into phylogenetic tree branches for each of the ten amino acids activated by the superfamily. All metrics confirm that a segment previously identified as an inserted element is, indeed, a more recent acquisition, despite its structural conservation. The residue-by-residue conservation metrics reveal significant co-variation of mutational frequencies between a core segment that forms the amino acid binding site and a neighboring segment derived from the more recent insertion element. We attribute that covariation to the differentiation of superfamily members as evolutionary divergence enhanced amino acid specificity. Finally, evidence that the insertion element is a recent acquisition implies a new branching order for much of the proteome.

Applying Nanopore sequencing to a One-Health scenario for colistin resistance transmission among pigs, cows and the farmer

One-Health studies applying massive-parallel and single-molecule sequencing are a suitable approximation to try to understand how antibiotic resistances flow between the human-animal-environment scenario. Colistin has been withdrawn in human medicine due to its toxicity, limiting its usage as a last-resort treatment option for multidrug-resistant Gram-negative bacteria. However, it is still used orally to treat Enterobacteriaceae infections in veterinary medicine. Since 2015, colistin resistance appeared to be located in mobile genetic elements, raising the concern of the likelihood of transmission by horizontal gene transfer between animals and humans. In this study, 202 faecal samples were collected in a mixed farm from pigs, calves, and the farmer. PCR for the mcr-1 gene was positive for 18 of the isolates, and Nanopore sequencing allowed us to determine the location of the gene, either on the chromosome or in plasmids. Three types of replicons were found within the positive isolates harbouring the mcr-1: IncX4, IncI2, and IncHI2. Four different genetic contexts probably indicate different stages of gene stabilization, either in the chromosome or plasmid, with ISApl1 as the main insertion element flanking the gene. Moreover, 43 other resistance genes were found in our samples, related to more than six different antibiotic families (e.g. aminoglycosides, lincosamides, beta-lactams, macrolides, trimethoprim, phenicols, and sulphonamides). We found resistance genes against colistin and that six antibiotic families together in at least one of the isolates from human, swine, and bovine. Isolate 15B-22 harboured one plasmid with seven resistance genes related to four families of antibiotics other than polymyxins, meaning that there are more chances to maintain colistin resistance even with the withdrawn of colistin. Nanopore long reads allowed us to assemble the DNA elements within the isolates easily and determine the genetic context of the mcr-1 gene. Furthermore, they allowed us to describe and locate more antimicrobial resistance genes to other antibiotic families and antiseptic compounds.