Presence of optrA-mediated linezolid resistance in multiple lineages and plasmids of Enterococcus faecalis revealed by long read sequencing

Funding: This work was supported by the Chief Scientist Office (Scotland) through the Scottish Healthcare Associated Infection Prevention Institute (Reference SIRN/10). Bioinformatics and Computational Biology analyses were supported by the University of St Andrews Bioinformatics Unit, which is funded by a Wellcome Trust ISSF award [grant 105621/Z/14/Z].Transferable linezolid resistance due to optrA, poxtA, cfr and cfr-like genes is increasingly detected in enterococci associated with animals and humans globally. We aimed to characterize the genetic environment of optrA in linezolid-resistant Enterococcus faecalis isolates from Scotland. Six linezolid-resistant E. faecalis isolated from urogenital samples were confirmed to carry the optrA gene by PCR. Short read (Illumina) sequencing showed the isolates were genetically distinct (>13900 core SNPs) and belonged to different MLST sequence types. Plasmid contents were examined using hybrid assembly of short and long read (Oxford Nanopore MinION) sequencing technologies. The optrA gene was located on distinct plasmids in each isolate, suggesting that transfer of a single plasmid did not contribute to optrA dissemination in this collection. pTM6294-2, BX5936-1 and pWE0438-1 were similar to optrA-positive plasmids from China and Japan, while the remaining three plasmids had limited similarity to other published examples. We identified the novel Tn6993 transposon in pWE0254-1 carrying linezolid (optrA), macrolide (ermB) and spectinomycin [ANT(9)-Ia] resistance genes. OptrA amino acid sequences differed by 0–20 residues. We report multiple variants of optrA on distinct plasmids in diverse strains of E. faecalis . It is important to identify the selection pressures driving the emergence and maintenance of resistance against linezolid to retain the clinical utility of this antibiotic.Publisher PDFPeer reviewe

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Emergence of optrA-mediated linezolid resistance in multiple lineages and plasmids of Enterococcus faecalis revealed by long read sequencing

Objectives To characterise the genetic environment of optrA in linezolid-resistant Enterococcus faecalis isolates from Scotland.Methods Linezolid-resistant E. faecalis were identified in three Scottish Health Boards and confirmed to carry the optrA gene at the national reference laboratory. WGS was performed with short read (Illumina MiSeq) and long read (Oxford Nanopore MinION) technologies to generate complete genome assemblies. Illumina reads for 94 E. faecalis bloodstream isolates were used to place the optrA-positive isolates in a larger UK phylogeny.Results Six optrA-positive linezolid-resistant E. faecalis were isolated from urogenital samples in three Scottish Health Boards (2014-2017). No epidemiological links were identified between the patients, four were community-based, and only one had recent linezolid exposure. Reference-based mapping confirmed the isolates were genetically distinct (&gt;13,900 core SNPs). optrA was located on a plasmid in each isolate and these plasmids showed limited nucleotide similarity. There was variable presence of transposable elements surrounding optrA, (including IS1216, IS3, and Tn3) and not always as a recognisable gene cassette. OptrA amino acid sequences were also divergent, resulting in four protein variants differing in 1-20 residues. One isolate belonged to ST16 and clustered with three other isolates in the UK collection (76-182 SNPs), otherwise the optrA-positive isolates were genetically distinct from the bloodstream isolates (&gt;6,000 SNPs).Conclusions We report multiple variants of the linezolid resistance gene optrA in diverse E. faecalis strain and plasmid backgrounds, suggesting multiple introductions of the gene into the E. faecalis population and selection driving recent emergence.<br/

Presence of optrA-mediated linezolid resistance in multiple lineages and plasmids of Enterococcus faecalis revealed by long read sequencing

Transferable linezolid resistance due to optrA, poxtA, cfr and cfr-like genes is increasingly detected in enterococci associated with animals and humans globally. We aimed to characterize the genetic environment of optrA in linezolid-resistant Enterococcus faecalis isolates from Scotland. Six linezolid-resistant E. faecalis isolated from urogenital samples were confirmed to carry the optrA gene by PCR. Short read (Illumina) sequencing showed the isolates were genetically distinct (>13900 core SNPs) and belonged to different MLST sequence types. Plasmid contents were examined using hybrid assembly of short and long read (Oxford Nanopore MinION) sequencing technologies. The optrA gene was located on distinct plasmids in each isolate, suggesting that transfer of a single plasmid did not contribute to optrA dissemination in this collection. pTM6294-2, BX5936-1 and pWE0438-1 were similar to optrA-positive plasmids from China and Japan, while the remaining three plasmids had limited similarity to other published examples. We identified the novel Tn6993 transposon in pWE0254-1 carrying linezolid (optrA), macrolide (ermB) and spectinomycin [ANT(9)-Ia] resistance genes. OptrA amino acid sequences differed by 0-20 residues. We report multiple variants of optrA on distinct plasmids in diverse strains of E. faecalis. It is important to identify the selection pressures driving the emergence and maintenance of resistance against linezolid to retain the clinical utility of this antibiotic

Mating sequence, dominance and paternity success in captive male tammar wallabies

The tammar wallaby (Macropus eugenii) is a small, promiscuous, macropodid marsupial. Females usually produce a single young each year and there is a clear dominance hierarchy between adult males. The dominant male usually mates first and then guards the female to prevent access to her by other males. In this study, agonistic encounters and mating behaviour were observed to determine male dominance hierarchies in six groups of captive tammars consisting of a total of 23 males and 50 females. Mating behaviour was observed immediately post-partum when females were in oestrus and was correlated with plasma testosterone concentrations. Male mating sequences were recorded, and the paternity of offspring was determined by using seven macropodid marsupial microsatellites. Rates of sexual checking and aggression by males housed with females in oestrus in the non-breeding season were lower than in the breeding season. These males also had lower concentrations of testosterone, but were still able to sire young. High testosterone concentrations neither ensured dominance nor appeared to control directly the level of sexual activity. Females usually mated with more than one male. The dominant male most often secured the initial copulation (60%), but the first-mating male did not always secure parentage, with second and third matings resulting in as many young as first matings. Using these data, we were unable to discount first sire, last sire or equal chance models of paternity in this species. Half the young (50%) were sired by the dominant α male, but of the remaining progeny, the β male sired more (35%) than γ and δ males (15%). Dominance therefore is only a moderately effective predictor of paternity in the tammar. Although the dominant males gained most first matings and individually sired half of the offspring, the subdominant males still contributed significantly to the population, at least in captivity