Molecular Analysis of bla KPC-2 -Harboring Plasmids: Tn 4401a Interplasmid Transposition and Tn 4401a -Carrying ColRNAI Plasmid Mobilization from Klebsiella pneumoniae to Citrobacter europaeus and Morganella morganii in a Single Patient

Plasmid transfer plays an important role in the interspecies spread of carbapenemase genes, including the Klebsiella pneumoniae carbapenemase (KPC)-coding gene, bla KPC . We conducted whole-genome sequencing (WGS) analysis and transmission experiments to analyze bla KPC-2 -carrying mobile genetic elements (MGEs) between the bla KPC-2 -harboring K. pneumoniae , Citrobacter europaeus , and Morganella morganii strains isolated from a single patient. bla KPC-2 was contained within an MGE, Tn 4401a .

Mobilization of a Kluyvera-like arnBCADTEF operon, besides mcr genes, confers colistin resistance to Escherichia coli isolated from healthy animals

The use of colistin as a last resort antimicrobial is compromised by the emergence of resistant enterobacteria with acquired determinants like mcr genes, mutations that activate the PmrAB two-component system and also by some other(s) still unknown mechanism(s). This work analyzed 74 E. coli isolates from healthy swine, turkey or bovine animals, characterizing their colistin resistance determinants. The mcr-1 gene, detected in 69 isolates, was the main determinant found among which 45% were carried by highly mobile plasmids, followed by four strains lacking previously known resistance determinants or two with mcr-4 (one in addition to mcr-1), whose phenotypes were not transferred by conjugation. Although a fraction of isolates carrying mcr-1 or mcr-4 genes also presented missense polymorphisms in pmrA or pmrB, constitutive activation of PmrAB was not detected, in contrast to control strains carrying mutations that confer colistin resistance. The expression of mcr genes negatively controls arnBCADTEF expression, a down-regulation that was also observed in the four isolates lacking known resistance determinants, three of them sharing the same macrorestriction and plasmid profiles. Genomic sequencing of one of these strains, isolated from a bovine in 2015, revealed a IncFII plasmid of 60 Kb encoding an arnBCADTEF operon closely related to Kluyvera ascorbata homologs. This element, named pArnT1, was cured by ethidum bromide and lost in parallel to colistin resistance. This work reveals that, besides mcr genes and chromosomal mutations, mobilization of arnBCADTEF operon represents a colistin resistance mechanism whose spread and relevance for public health should be carefully surveyed.Abstract ImportanceColistin is an old antibiotic that has returned to first-line fighting against (Gram negative) microorganisms after pandemic rising of antimicrobial resistance. However, low susceptibility to colistin is also becoming spread, mainly by plasmid mobilization of one of the enzymes (encoded by mcr genes) that modify covalently the external layer (the lipid A component of the lipopolysaccharide) of bacterial envelope, interfering antibiotic effectiveness. The second enzymatic system that performs envelope modification and confers colistin resistance when overexpressed is encoded by arnBCADTEF operon, a set of seven genes with location restricted (up to now) to the chromosome of Gram negative bacteria. This work describes plasmid mobilization of this operon between enterobacteria, from Kluyvera to Escherichia coli, where a Kluyvera-like arnBCADTEF operon carried by pArnT1 might represent, besides mcr genes, a potential risk for antimicrobial therapy and might require careful surveillance.

Comparative analysis of MOBQ4 plasmids demonstrates that MOBQ is a cis-acting-enriched relaxase protein family

ABSTRACTA group of small mobilizable plasmids is increasingly being reported in epidemiology surveys of enterobacteria. Some of them encode colicins, while others are cryptic. All of them encode a relaxase belonging to a previously non-described group of the MOBQ class, MOBQ4. While highly similar in their mobilization module, two families with unrelated replicons can be distinguished, MOBQ41 and MOBQ42. Members of both groups were compatible between them and stably maintained in E. coli. MOBQ4 plasmids were mobilized by conjugation. They contain two transfer genes, mobA coding for the MOBQ4 relaxase and mobC, which was non-essential but enhanced the plasmid mobilization frequency. The origin of transfer was located between these two divergently transcribed mob genes. MPFI conjugative plasmids were the most efficient helpers for MOBQ4 conjugative transmission. No interference in mobilization was observed when both MOBQ41 and MOBQ42 were present in the same donor cell. Remarkably, MOBQ4 relaxases exhibited a cis-acting preference for their oriTs, a feature already observed in other MOBQ plasmids. These findings indicate that MOBQ4 plasmids can efficiently spread among enterobacteria aided by coresident IncI1, IncK and IncL/M plasmids, while ensuring their self-dissemination over highly-related elements.IMPORTANCEPlasmids are key vehicles of horizontal gene transfer and contribute greatly to bacterial genome plasticity. A group of plasmids, called mobilizable, is able to disseminate aided by helper conjugative plasmids. Here, we studied a group of phylogenetically-related mobilizable plasmids, MOBQ4, commonly found in clinically-relevant enterobacteria, uncovering the helper plasmids responsible for their dissemination. We found that the two plasmid species encompassed in the MOBQ4 group can coexist and transfer orthogonally, despite origin-of-transfer cross-recognition by their relaxases. Specific discrimination among their highly similar oriT sequences is guaranteed by the preferential cis activity of the MOBQ4 relaxases. Such strategy would be biologically relevant in a scenario of co-residence of non-divergent elements to favor self-dissemination.

Characteristics of the Conjugative Transfer System of the IncM Plasmid pCTX-M3 and Identification of Its Putative Regulators

ABSTRACTPlasmid conjugative transfer systems comprise type IV secretion systems (T4SS) coupled to DNA processing and replication. The T4SSs are divided into two phylogenetic subfamilies, namely, IVA and IVB, or on the basis of the phylogeny of the VirB4 ATPase, into eight groups. The conjugation system of the IncM group plasmid pCTX-M3, fromCitrobacter freundii, is classified in the IVB subfamily and in the MPFIgroup, as are the conjugation systems of IncI1 group plasmids. Although the majority of the conjugative genes of the IncM and IncI1 plasmids display conserved synteny, there are several differences. Here, we present a deletion analysis of 27 genes in the conjugative transfer regions of pCTX-M3. Notably, the deletion of either of two genes dispensable for conjugative transfer, namely,orf35andorf36, resulted in an increased plasmid mobilization efficiency. Transcriptional analysis of theorf35andorf36deletion mutants suggested an involvement of these genes in regulating the expression of conjugative transfer genes. We also revised the host range of the pCTX-M3 replicon by finding that this replicon is unable to support replication inAgrobacterium tumefaciens,Ralstonia eutropha, andPseudomonas putida, though its conjugation system is capable of introducing plasmids bearingoriTpCTX-M3into these bacteria, which are representatives ofAlpha-,Beta-, andGammaproteobacteria, respectively. Thus, the conjugative transfer system of pCTX-M3 has a much broader host range than its replicon.IMPORTANCEHorizontal gene transfer is responsible for rapid changes in bacterial genomes, and the conjugative transfer of plasmids has a great impact on the plasticity of bacteria. Here, we present a deletion analysis of the conjugative transfer system genes of the pCTX-M3 plasmid of the IncM group, which is responsible for the dissemination of antibiotic resistance genes inEnterobacteriaceae. We found that the deletion of either of theorf35andorf36genes, which are dispensable for conjugative transfer, increased the plasmid mobilization efficiency. Real-time quantitative PCR (RT-qPCR) analysis suggested the involvement oforf35andorf36in regulating the expression of transfer genes. We also revised the host range of pCTX-M3 by showing that its conjugative transfer system has a much broader host range than its replicon.

Green Fluorescent Protein-Labeled Monitoring Tool To Quantify Conjugative Plasmid Transfer between Gram-Positive and Gram-Negative Bacteria

ABSTRACTOn the basis of pIP501, a green fluorescent protein (GFP)-tagged monitoring tool was constructed for quantifying plasmid mobilization among Gram-positive bacteria and between Gram-positiveEnterococcus faecalisand Gram-negativeEscherichia coli. Furthermore, retromobilization of the GFP-tagged monitoring tool was shown fromE. faecalisOG1X into the clinical isolateE. faecalisT9.

Distribution of Intrinsic Plasmid Replicase Genes and Their Association with Carbapenem-Hydrolyzing Class D β-Lactamase Genes in European Clinical Isolates of Acinetobacter baumannii

ABSTRACTNinety-six genetically diverse multidrug-resistant clinical isolates ofAcinetobacter baumanniifrom 25 hospitals in 17 European countries were screened by PCR for specific carbapenemase-hydrolyzing class D β-lactamase (CHDL) genes and by PCR-based replicon typing for the presence of 19 different plasmid replicase (rep) gene homology groups (GRs). Results were confirmed by DNA sequencing where necessary. All 96 isolates contained at least 1 (with a maximum of 4) of the 19 groups ofrepgenes. Groups detected were GR6 (repAci6; 93 isolates), GR2 (includingrepAci1 [67 isolates] andrepAci2 [3 isolates]), GR16 (repApAB49; 12 isolates), GR12 (p2ABSDF0001; 10 isolates), GR3 (repAci3; 4 isolates), GR4 (repAci4; 3 isolates), GR10 (repAciX; 1 isolate), and GR14 (repp4AYE; 1 isolate). Variations inrepgene content were observed even among epidemiologically related isolates. Genes encoding OXA-58-like CHDLs (22 isolates) were associated with carriage of therepAci1,repAci3,repAci4, andrepAciX genes, genes encoding OXA-40-like CHDLs (6 isolates) were associated withrepAci2 and p2ABSDF0001, and genes encoding OXA-23-like CHDLs (8 isolates) were associated withrepAci1. Most intrinsicAcinetobacterplasmids are non-self-transferable, but the almost ubiquitousrepAci6 gene was strongly associated with a potentialtralocus that could serve as a general system for plasmid mobilization and consequent horizontal transmission of plasmids and their associated antibiotic resistance genes among strains ofA. baumannii.

Two type IV secretion systems with different functions in Burkholderia cenocepacia K56-2

Bacterial type IV secretion systems (T4SS) perform two fundamental functions related to pathogenesis: the delivery of effector molecules to eukaryotic target cells, and genetic exchange. Two T4SSs have been identified in Burkholderia cenocepacia K56-2, a representative of the ET12 lineage of the B. cepacia complex (Bcc). The plant tissue watersoaking (Ptw) T4SS encoded on a resident 92 kb plasmid is a chimera composed of VirB/D4 and F-specific subunits, and is responsible for the translocation of effector(s) that have been linked to the Ptw phenotype. The bc-VirB/D4 system located on chromosome II displays homology to the VirB/D4 T4SS of Agrobacterium tumefaciens. In contrast to the Ptw T4SS, the bc-VirB/D4 T4SS was found to be dispensable for Ptw effector(s) secretion, but was found to be involved in plasmid mobilization. The fertility inhibitor Osa did not affect the secretion of Ptw effector(s) via the Ptw system, but did disrupt the mobilization of a RSF1010 derivative plasmid.