Staphylococcal phages and pathogenicity islands drive plasmid evolution

Conjugation has classically been considered the main mechanism driving plasmid transfer in nature. Yet bacteria frequently carry so-called non-transmissible plasmids, raising questions about how these plasmids spread. Interestingly, the size of many mobilisable and non-transmissible plasmids coincides with the average size of phages (~40 kb) or that of a family of pathogenicity islands, the phage-inducible chromosomal islands (PICIs, ~11 kb). Here, we show that phages and PICIs from Staphylococcus aureus can mediate intra- and inter-species plasmid transfer via generalised transduction, potentially contributing to non-transmissible plasmid spread in nature. Further, staphylococcal PICIs enhance plasmid packaging efficiency, and phages and PICIs exert selective pressures on plasmids via the physical capacity of their capsids, explaining the bimodal size distribution observed for non-conjugative plasmids. Our results highlight that transducing agents (phages, PICIs) have important roles in bacterial plasmid evolution and, potentially, in antimicrobial resistance transmission.

Identification of the conjugative and mobilizable plasmid fragments in the plasmidome using sequence signatures

Plasmids are the key element in horizontal gene transfer in the microbial community. Recently, a large number of experimental and computational methods have been developed to obtain the plasmidomes of microbial communities. Distinguishing transmissible plasmid sequences, which are derived from conjugative or at least mobilizable plasmids, from non-transmissible plasmid sequences in the plasmidome is essential for understanding the diversity of plasmids and how they regulate the microbial community. Unfortunately, due to the highly fragmented characteristics of DNA sequences in the plasmidome, effective identification methods are lacking. In this work, we used information entropy from information theory to assess the randomness of synonymous codon usage over 4424 plasmid genomes. The results showed that for all amino acids, the choice of a synonymous codon in conjugative and mobilizable plasmids is more random than that in non-transmissible plasmids, indicating that transmissible plasmids have different sequence signatures from non-transmissible plasmids. Inspired by this phenomenon, we further developed a novel algorithm named PlasTrans. PlasTrans takes the triplet code sequences and base sequences of plasmid DNA fragments as input and uses the convolutional neural network of the deep learning technique to further extract the more complex signatures of the plasmid sequences and identify the conjugative and mobilizable DNA fragments. Tests showed that PlasTrans could achieve an AUC of as high as 84–91%, even though the fragments only contained hundreds of base pairs. To the best of our knowledge, this is the first quantitative analysis of the difference in sequence signatures between transmissible and non-transmissible plasmids, and we developed the first tool to perform transferability annotation for DNA fragments in the plasmidome. We expect that PlasTrans will be a useful tool for researchers who analyse the properties of novel plasmids in the microbial community and horizontal gene transfer, especially the spread of resistance genes and virulence factors associated with plasmids. PlasTrans is freely available via https://github.com/zhenchengfang/PlasTrans

A Pandrug-Resistant Providencia Carrying Two blaIMP Carbapenemase-Encoding Genes Including blaIMP-69, a New blaIMP Variant, on a Newly Identified Worldwide-Distributed IncC Plasmid

Imipenemase (IMP) is a metallo-β-lactamase that confers resistance to almost all β-lactams. Identification of IMP genes is essential for understanding and combatting antibiotic resistance. In this study, we report a pandrug-resistant Providencia strain from a human rectal swab. This strain carried 2 blaIMP carbapenemase genes, blaIMP-69 and blaIMP-4. IMP-69 is a novel IMP variant with an amino acid substitution at A21T compared with IMP-8. blaIMP-69 was found in a blaIMP-69-aacA4 array of an integron on a 165-kilobase (kb) IncC self-transmissible plasmid, whereas blaIMP-4 was located in a blaIMP-4-qacG-aacA4-catB3 array of an integron on a 19-kb nonself-transmissible plasmid. Such coexistence has the potential to allow the generation of new, hybrid blaIMP variants by homologous recombination. The blaIMP-69-carrying IncC plasmid belonged to the core-genome plasmid multilocus sequence typing (cgPMLST) 3.5 type. We found that cgPMLST 3.5 IncC plasmids have been circulating worldwide for decades and may represent a common vehicle mediating the spread of antimicrobial resistance.

A Cluster of Colistin- and Carbapenem-Resistant Klebsiella pneumoniae Carrying blaNDM-1 and mcr-8.2

Background Klebsiella pneumoniae resistant to both carbapenems and colistin imposes severe challenges for management. In this study, we report a cluster of 5 carbapenem-resistant K pneumoniae clinical strains belonging to ST1 and K57 types, 4 of which were also resistant to colistin, from 2 hospitals. Methods The 5 strains were subjected to whole-genome sequencing (WGS) using the short-read Illumina HiSeq platform, and 2 strains were also selected for long-read WGS using MinION. Clonal relatedness of the 5 strains was determined based on single-nucleotide polymorphisms (SNPs). Conjugation experiments were performed to obtain self-transmissible plasmids. Results All 5 strains carried the carbapenemase-encoding gene blaNDM-1, whereas the 4 colistin-resistant strains also harbored a new variant of the mcr-8 colistin resistance gene, namely, mcr-8.2. MCR-8.2 differs from MCR-8.1 by four amino acid substitutions (A51V, A232S, N365Y, and N480K). mcr-8.2 was located on a large, hybrid, nonself-transmissible plasmid containing IncQ, IncR, and IncFII replicons, whereas blaNDM-1 was carried by self-transmissible IncX3 plasmids. Phylogenetic analysis based on SNPs revealed that the 5 strains were likely to have a common origin. Conclusions Both the intra- and interhospital transfer of strains carrying mcr-8 and blaNDM-1 were identified, which represents an emerging threat for clinical management and infection control.

Survival of the unfittest: Metabolic trade-offs expose unforeseen benefits of plasmid carriage

Population genetics theory defines fitness as reproductive success: Mutants reproducing faster than their wild-type counterpart are favoured by selection. Otherwise, the mutations are lost. Here I show that unfit mutants can thrive when selection favours non-reproductive traits if they engage in a trade-off with fitness. I co-maintained two constructs of Escherichia coli, with and without a non-transmissible plasmid, for more than 80 generations in competition assays that favoured yield. Plasmid carriage prompted a known metabolic trade-off in the bacterium between growth rate per capita—reproductive success—and yield. Importantly the plasmid carries a tetracycline-resistance gene, tet(36). By favouring yield, cells harbouring the plasmid preserved it without exposure to the antibiotic. Unsurprisingly, these cells outgrew their fitter plasmid-free competitor with trace low tetracycline concentrations. Fitness competition assays are widely used, but experimental validation of their underlying principle is rare. These assays are the ‘gold-standard’ in genetics, but my work suggests their reliability may be lower than previously thought.

A large self-transmissible plasmid from Nigeria confers resistance to multiple antibacterials without a carrying cost

Antimicrobial resistance is rapidly expanding, in a large part due to mobile genetic elements. We screened 94 fecal fluoroquinolone-resistantEscherichia coliisolates from Nigeria for six plasmid-mediated quinolone resistance (PMQR) genes. Sixteen isolates harbored at least one of the PMQR genes and four were positive foraac-6-Ib-cr. In one strain,aac-6-Ib-crwas mapped to a 125 Kb self-transmissible IncFII plasmid, pMB2, which also bearsblaCTX-M-15, seven other functional resistance genes and multiple resistance pseudogenes. We hypothesized that pMB2 had been selected by antimicrobials and that its large size would confer a growth disadvantage. However, laboratory strains carrying pMB2 grew at least as fast as isogenic strains lacking the plasmid in both rich and minimal media. We excised a 32 Kb fragment containing thesitABCDand another putative transporter,pefB, apapBhomolog, and several open-reading frames of unknown function. The resulting 93 Kb mini-plasmid conferred slower growth rates and lower fitness than wildtype pMB2. Trans-complementing the deletion with the clonedsitABCDgenes confirmed that they accounted for the growth advantage conferred by pMB2 in iron-depleted media. The mini-plasmid additionally conferred autoaggregation and was less transmissible and both phenotypes could be complemented with apefBclone. pMB2 is a large plasmid with a flexible resistance region that contains multiple loci that can account for evolutionary success in the absence of antimicrobials. Ancillary functions conferred by resistance plasmids can mediate their retention and transmissibility, worsening the trajectory for antimicrobial resistance and potentially circumventing efforts to contain resistance through restricted use.

1201. A Prolonged Multispecies Outbreak of Carbapenemase-Producing Enterobacteriaceae Due to Transmissible Plasmid With Carbapenemase Gene

Background In 2010, a multispecies outbreak of IMP type carbapenemase-producing Enterobacteriaceae (IMP-CPE) occurred at a large acute care hospital in Japan. The outbreak continued for years involving more than 100 patients mainly in surgical wards. Methods Because of the long period of the outbreak, investigation were focused on hospitalized patients whose clinical samples were positive for IMP-CPE between July 2013 and March 2014. A case–control study was conducted for cases who underwent abdominal surgery with controls from whom meropenem-susceptible Enterobacteriaceae were isolated. Pulsed-field gel electrophoresis (PFGE) was used for molecular typing. To evaluate genetic relationship among IMP-CPE isolates of different species, plasmid analysis using S1 nuclease to separate plasmid and chromosomal DNA followed by plasmid DNA extraction and whole-genome sequencing (WGS) was conducted. Results During the study period, 22 cases were identified and 22 IMP-CPE isolates which consisted of eight Escherichia coli, five Klebsiella oxytoca, five Enterobacter cloacae, three Klebsiella pneumoniae and one Enterobacter aerogenes were obtained. All five isolates of K. oxytoca had similar PFGE profiles which suggested clonal transmission. However, PFGE profiles of E. coli, E. cloacae and K. pneumoniae isolates were diverse. Plasmid analysis revealed that all 22 isolates shared ca. 50 kb IncN plasmid with blaIMP-6 which implies interspecies transmission of it The case–control study which adjusted by days of hospitalization with 11 cases and 24 controls revealed that pancreato-duodenectomy (adjusted odds ratio (aOR) = 6.4, 95% confidence interval (CI) 1.3–32.4) and enteric fistula (aOR = 8.0, 95% CI 1.5–41.9) were associated with IMP-CPE acquisition. Use of endoscopy within the past six months was not associated with IMP-CPE (aOR = 0.8 95% CI 0.2–4.2). With a bundled infection control with Osaka City Public Health Office, the outbreak was contained in July 2016. Conclusion Dissemination of carbapenemase gene by transmissible plasmid can play a critical role to complicate epidemiology of CPE outbreak and made it difficult to control. Plasmid analysis using WGS technology is a promising tool to untangle it. Disclosures All authors: No reported disclosures.

Sequence Type 273 Carbapenem-Resistant Klebsiella pneumoniae Carrying bla NDM-1 and bla IMP-4

ABSTRACT A carbapenem-resistant Klebsiella pneumoniae isolate was recovered from human blood. Its whole-genome sequence was obtained using Illumina and long-read MinION sequencing. The strain belongs to sequence type 273 (ST273), which was found recently and caused an outbreak in Southeast Asia. It has two carbapenemase genes, bla NDM-1 (carried by an ST7 IncN self-transmissible plasmid) and bla IMP-4 (located on a self-transmissible IncHI5 plasmid). Non-KPC-producing ST237 may represent a lineage of carbapenem-resistant K. pneumoniae , which warrants further monitoring.

Coexistence of Two bla NDM-5 Genes on an IncF Plasmid as Revealed by Nanopore Sequencing

ABSTRACT In a carbapenem-resistant Escherichia coli clinical isolate of sequence type 167, two copies of bla NDM-5 were found on a 144,225-bp IncF self-transmissible plasmid of the F36:A4:B − type. Both bla NDM-5 genes were located in 11,065-bp regions flanked by two copies of IS 26 . The two regions were identical in sequence but were present at different locations on the plasmid, suggesting a duplication of the same region. This study highlights the complex genetic contexts of bla NDM-5 .