IncF Plasmids Research Articles

Dissemination of the rmtB gene carried on IncF and IncN plasmids among Enterobacteriaceae in a pig farm and its environment

To investigate the prevalence and characterization of 16S rRNA methylase-producing bacteria in a pig farm and its environment in East China. Enterobacteriaceae isolates and metagenomic DNA from 102 pig faecal samples from a pig farm and 97 soil samples taken in or around the farm were screened for the presence of 16S rRNA methylase genes. The clonal relationships of 16S rRNA methylase-positive isolates, plasmid content and other associated resistance genes were also characterized. Fifty-six rmtB-positive Enterobacteriaceae isolates, including 54 Escherichia coli, 1 Morganella morganii and 1 Proteus mirabilis, were recovered from 55 pig faecal samples. Nineteen rmtB-positive bacteria, including 13 E. coli, 2 M. morganii, 2 Leclercia adecarboxylata, 1 Enterobacter aerogenes and 1 Enterobacter cloacae, were recovered from 16 soil samples. Among the 75 rmtB-positive isolates, 31 and 25 also carried the qepA and bla(CTX-M) genes, respectively. The qepA gene co-localized with rmtB on the F2:A-:B1 plasmids and the bla(CTX-M-65) gene co-localized with rmtB on the F33:A-:B- plasmids. The rmtB gene was also found to be associated with the IncN plasmids. Clonal transmission of rmtB-positive E. coli isolates was observed between different pig groups and soil samples. Both horizontal gene transfer and clonal spread could be responsible for the dissemination of the rmtB gene in the pig farm and its environment. To our knowledge, this study is the first report of rmtB-positive bacteria from farmland soils and indicates that these antibiotic-resistant bacteria and/or resistance genes could be acquired by humans through the food chain.

Multiple ST clonal complexes, with a predominance of ST131, of Escherichia coli harbouring blaCTX-M-15 in a tertiary hospital in Tanzania

The molecular epidemiology of 32 non-duplicate, CTX-M-15 extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli strains, isolated from clinical samples, was investigated. Multilocus sequence typing revealed multiple sequence type clonal complexes: ST131 (12), ST405 (4), ST638 (3), ST38 (2), ST827 (2), ST224 (1), ST648 (1), ST46 (1) and two new sequence type clonal complexes (1845 and 1848) in 22 pulsed field gel electrophoresis clusters. The blaCTX-M-15 gene was located on conjugative IncF plasmids. This is the first report of the worldwide emerging clonal complex ST131 linked to blaCTX-M-15 in Tanzania and demonstrates the need for constant surveillance in developing countries to prevent the spread of these multiresistant isolates.

F33:A−:B− and F2:A−:B− Plasmids Mediate Dissemination of rmtB-bla CTX-M-9 Group Genes and rmtB-qepA in Enterobacteriaceae Isolates from Pets in China

This study investigated the prevalence of 16S rRNA methylase genes in 267 Enterobacteriaceae isolates collected from pets. The rmtB gene was detected in 69 isolates, most of which were clonally unrelated. The coexistence of the rmtB gene with the bla(CTX-M-9) group genes and/or qepA within the same IncFII replicons was commonly detected. The two dominant types of IncF plasmids, F2:A-:B-, carrying rmtB-qepA, and F33:A-:B-, carrying the rmtB-bla(CTX-M-9) group genes (and especially bla(CTX-M-65)), shared restriction patterns within each incompatibility group.

INCF plasmids responsible by dissemination of blaKPC gene among enterobacteriaceae

In recent years, carbapenem resistance has emerged among Enterobacteriaceae in many geographical locations due to Klebsiella pneumoniae producing KPC carbapenemase. The aim of this study was to characterize the genetic elements involved in blaKPC gene mobilization and diffusion.

Analysis of bla CTX-M -Carrying Plasmids from Escherichia coli Isolates Collected in the BfT-GermVet Study

In this study, 417 Escherichia coli isolates from defined disease conditions of companion and farm animals collected in the BfT-GermVet study were investigated for the presence of extended-spectrum β-lactamase (ESBL) genes. Three ESBL-producing E. coli isolates were identified among the 100 ampicillin-resistant isolates. The E. coli isolates 168 and 246, of canine and porcine origins, respectively, harbored bla(CTX-M-1), and the canine isolate 913 harbored bla(CTX-M-15), as confirmed by PCR and sequence analysis. The isolates 168 and 246 belonged to the novel multilocus sequence typing (MLST) types ST1576 and ST1153, respectively, while isolate 913 had the MLST type ST410. The ESBL genes were located on structurally related IncN plasmids in isolates 168 and 246 and on an IncF plasmid in isolate 913. The bla(CTX-M-1) upstream regions of plasmids pCTX168 and pCTX246 were similar, whereas the downstream regions showed structural differences. The genetic environment of the bla(CTX-M-15) gene on plasmid pCTX913 differed distinctly from that of both bla(CTX-M-1) genes. Detailed sequence analysis showed that the integration of insertion sequences, as well as interplasmid recombination events, accounted for the structural variability in the bla(CTX-M) gene regions.

Characterization of IncF plasmids carrying the blaCTX-M-14 gene in clinical isolates of Escherichia coli from Korea

The purpose of this study was to investigate the molecular epidemiology of CTX-M-14-producing Escherichia coli clinical isolates from Korea. A total of 138 non-duplicate E. coli clinical isolates showing reduced susceptibility or resistance to ceftazidime and/or cefotaxime were included in the study. Resistance genes, genetic environment, R plasmid size and replicon type, sequence type (ST) and XbaI-macrorestriction patterns were determined. Among 138 isolates, 35 were found to carry the bla(CTX-M-14) gene. The ISEcp1 element was identified in the upstream region of the bla(CTX-M-14) gene in 32 isolates. The bla(CTX-M-14) gene was located on an IncF plasmid in 21 isolates, on an IncA/C plasmid in 1 isolate, on the chromosome in 8 isolates and on both the chromosome and an IncF plasmid in 5 isolates. The most prevalent ST was ST405 (n = 8), followed by ST354 (n = 4), ST38 (n = 3), ST69 (n = 3) and the intercontinental ST, ST131 (n = 3). PFGE and multilocus sequence typing experiments demonstrated no major clonal relationship among the CTX-M-14-producing isolates. The bla(CTX-M-14) gene was probably mobilized by IncF plasmids, which can readily spread in E. coli, causing horizontal dissemination of the resistance gene in Korea.

The transfer operon of plasmid R1 extends beyond finO into the downstream replication genes

finO is the final gene in the 35.4 kb transfer operon of IncFI plasmid F that is known to be involved in self-conjugative transfer. The genetic region distal to finO separates the conjugation and replication control modules of IncFII plasmid R100 and carries uncharacterized genes not found in plasmid F. However, comparison of the R100 gene organization with database entries of F-like plasmids suggests its broad conservation. We determined the DNA sequence of this region of IncFII plasmid R1 and studied its transcriptional organization. We find that transcription occurs through the entire gene region spanning from finO to the 5′ regulatory region of copB. The region lacks independent promoter activity and transcription is co-regulated with the adjacent transfer operon. We show that this extended transcriptional organization beyond finO is shared by plasmid R100. These findings support the hypothesis that gene products coexpressed from the finO-distal region in F-like plasmids are advantageous under some conditions when conjugative DNA is exchanged.

Functional analysis of the finO distal region of plasmid R1

The intergenic region linking conjugative transfer and replication copy control modules of IncF plasmids shows conservation of gene homology and organization. Genes distal to finO are coordinately expressed with the upstream transfer operon encoding the majority of conjugation genes in related plasmids. Here we investigate potential functions for these genes in copy number control and in processes related to conjugation: gene transfer, pilus specific phage infection and plasmid-promoted biofilm formation by an Escherichia coli host. We find that insertional inactivation of genes in the finO distal region reduced transcriptional read through into the downstream copB gene of plasmid R1. The mutant plasmid derivatives exhibited a reduced copy number compared to the wild type. Moreover all insertion mutant derivatives of plasmid R1–16 with aberrantly low copy numbers conferred poor biofilm forming ability to their hosts. The general mutagenesis thus identified plasmid stability genes as the only plasmid functions besides conjugation genes linked to plasmid-promoted biofilm production under these laboratory conditions. Our findings imply that a novel component of cis- or trans-regulation on the transcriptional level is important to normal R1 plasmid copy number regulation.

Molecular recognition determinants for type IV secretion of diverse families of conjugative relaxases

In preparation for transfer conjugative type IV secretion systems (T4SS) produce a nucleoprotein adduct containing a relaxase enzyme covalently linked to the 5' end of single-stranded plasmid DNA. The bound relaxase is expected to present features necessary for selective recognition by the type IV coupling protein (T4CP), which controls substrate entry to the envelope spanning secretion machinery. We prove that the IncF plasmid R1 relaxase TraI is translocated to the recipient cells. Using a Cre recombinase assay (CRAfT) we mapped two internally positioned translocation signals (TS) on F-like TraI proteins that independently mediate efficient recognition and secretion. Tertiary structure predictions for the TS matched best helicase RecD2 from Deinococcus radiodurans. The TS is widely conserved in MOB(F) and MOB(Q) families of relaxases. Structure/function relationships within the TS were identified by mutation. A key residue in specific recognition by T4CP TraD was revealed by a fidelity switch phenotype for an F to plasmid R1 exchange L626H mutation. Finally, we show that physical linkage of the relaxase catalytic domain to a TraI TS is necessary for efficient conjugative transfer.

Replicon sequence typing of IncF plasmids carrying virulence and resistance determinants

IncF plasmids are frequently encountered in clinical enterobacterial strains associated with the dissemination of relevant antimicrobial resistance and virulence genes. These plasmids are usually heterogeneous in size and carry multiple replicons, and technical difficulties can impair the comparison and detection of related plasmids by restriction fragment length polymorphism analysis. We devised a rapid sequence-based typing scheme to categorize the members of this plasmid family into homogeneous groups. We compared the available IncF replicon sequences, identifying the combination of the different IncF replicon alleles as the discriminating characteristic of these plasmid scaffolds. An IncF typing method based on PCR amplification and sequence typing of the IncF replicons was devised. A collection of IncF plasmids carrying resistance and/or virulence genes, identified in strains from different sources and geographical origins, was tested with this typing system. We devised a replicon sequence typing (RST) scheme discriminating IncF plasmid variants. This system was tested on the collection of IncF plasmids, demonstrating that it was useful for the discrimination of plasmids carrying the same resistance gene (i.e. the bla(CTX-M-15) gene), but also recognized strictly related virulence plasmids (i.e. IncFIme plasmids). The PCR-based replicon typing (PBRT) system was also updated by including new primer pairs to allow the identification of the Salmonella, Klebsiella and Yersinia IncF plasmids. The ability to recognize and sub-categorize IncF plasmids by RST in homogeneous groups on the basis of their phylogenetic relatedness can be helpful in analysing their distribution in nature and discovering their evolutionary origin.

Predicting Plasmid Promiscuity Based on Genomic Signature

Despite the important contribution of self-transmissible plasmids to bacterial evolution, little is understood about the range of hosts in which these plasmids have evolved. Our goal was to infer this so-called evolutionary host range. The nucleotide composition, or genomic signature, of plasmids is often similar to that of the chromosome of their current host, suggesting that plasmids acquire their hosts' signature over time. Therefore, we examined whether the evolutionary host range of plasmids could be inferred by comparing their trinucleotide composition to that of all completely sequenced bacterial chromosomes. The diversity of candidate hosts was determined using taxonomic classification and genetic distance. The method was first tested using plasmids from six incompatibility (Inc) groups whose host ranges are generally thought to be narrow (IncF, IncH, and IncI) or broad (IncN, IncP, and IncW) and then applied to other plasmid groups. The evolutionary host range was found to be broad for IncP plasmids, narrow for IncF and IncI plasmids, and intermediate for IncH and IncN plasmids, which corresponds with their known host range. The IncW plasmids as well as several plasmids from the IncA/C, IncP, IncQ, IncU, and PromA groups have signatures that were not similar to any of the chromosomal signatures, raising the hypothesis that these plasmids have not been ameliorated in any host due to their promiscuous nature. The inferred evolutionary host range of IncA/C, IncP-9, and IncL/M plasmids requires further investigation. In this era of high-throughput sequencing, this genomic signature method is a useful tool for predicting the host range of novel mobile elements.

Molecular characterization of addiction systems of plasmids encoding extended-spectrum β-lactamases in Escherichia coli

Escherichia coli producing CTX-M-type extended-spectrum beta-lactamases (ESBLs) are spreading worldwide. The aim of this work was to investigate the addiction systems carried by the replicons involved in the emergence and spread of ESBLs in relation to ESBL and replicon types. A collection of 125 TEM, SHV and CTX-M ESBL-producing E. coli isolates and their 125 transconjugants or transformants was analysed. Five plasmid protein antitoxin-regulated systems and three plasmid antisense RNA-regulated systems were sought by PCR. Two hundred and ninety-eight plasmid addiction systems were detected in the parental strains (mean 2.38, range 0-6 per strain) and 86 were detected in the recipient strains (mean 0.69, range 0-5 per strain). PemKI, CcdAB, Hok-Sok and VagCD were the most frequently represented systems in both recipient and parental strains. The parental SHV and CTX-M ESBL-producing strains had more addiction systems than the TEM ESBL producers. In the recipient strains, the frequency of addiction systems was significantly higher in IncF plasmids. Among the IncF replicons carrying CTX-M-type enzymes, the frequency of addiction systems was significantly higher in IncF plasmids carrying CTX-M-15 (mean 3.5) or CTX-M-9 (mean 4) than in those carrying CTX-M-14 (mean 0.6). In E. coli producing CTX-M-15 or CTX-M-9 ESBLs, plasmids bearing the bla(CTX-M) gene have multiple addiction systems that could contribute to their maintenance in host strains.

P39 Multi-locus sequencing analysis of conjugative IncF plasmids with bla CTX-M-15

P39 Multi-locus sequencing analysis of conjugative IncF plasmids with bla CTX-M-15

Conjugative IncFI plasmids carrying CTX-M-15 among Escherichia coliESBL producing isolates at a University hospital in Germany

BackgroundMulti-drug-resistant, extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae, constitute an emerging public-health concern. Little data on the molecular epidemiology of ESBL producing Escherichia coli is available in Germany. Here we describe the prevalence and molecular epidemiology of ESBL producing-Escherichia coli isolates at a German University hospital.MethodsWe analysed 63 non-duplicate clinical ESBL isolates obtained over an 8-month period using PCR and sequence-based ESBL allele typing, plasmid replicon typing, phylogenetic group typing. Pulsed-field gel electrophoresis (PFGE) based genotyping and plasmid profiling was performed, as well as confirmatory DNA-based hybridization assays.ResultsExamination of the 63 Escherichia coli isolates revealed an almost equal distribution among the E. coli phylogenetic groups A, B1, B2 and D. High prevalence (36/63) of the CTX-M-15 gene was observed and an analysis of PFGE-based patterns revealed the presence of this CTX-M allele in multiple clones. Resistance to cefotaxime was a transferable trait and a commonly occurring 145.5 kb conjugative IncFI plasmid was detected in 65% of E. coli carrying the CTX-M-15 allele. The rate of transferable antibiotic resistances for GM, SXT, TET, GM-SXT-TET, SXT-TET and GM-TET was 33%, 61%, 61%, 27%, 44% and 11%, respectively. The remaining strains did not have a common IncFI plasmid but harboured transferable IncFI plasmids with sizes that ranged from 97 to 242.5 kb.ConclusionOur data demonstrate the presence of IncFI plasmids within the prevailing E. coli population in a hospital setting and suggest that the dissemination of CTX-M-15 allele is associated to lateral transfer of these well-adapted, conjugative IncFI plasmids among various E. coli genotypes.

Mechanisms of resistance and mobility among multidrug-resistant CTX-M–producing Escherichia coli from Canadian intensive care units: the 1st report of QepA in North America

Abstract We studied the molecular mechanisms of resistance and mobility of 18 multidrug-resistant CTX-M–producing Escherichia coli isolates isolated from patients in Canadian intensive care units. Fluoroquinolone-resistant isolates (83.3%) had mutations in gyrA and parC. Plasmid-mediated quinolone resistance genes qnr ( A, B, and S), qepA, and aac(6′)-Ib-cr were detected in 0%, 5.6%, and 44.4%, respectively. Sulfamethoxazole/trimethoprim-resistant isolates (61.1%) carried a dfr gene, and 10 (90.9%) of the 11 carried 1 or more sul genes. Gentamicin-resistant isolates (27.8%) carried the aac(3′)-II gene, and doxycycline-resistant isolates (33.3%) carried 1 or more tet efflux genes. Both genetically related and unrelated groups of E. coli harboring extended-spectrum β-lactamases were observed. The bla CTX-M genes were primarily located on diverse IncF plasmids of multiple replicon types downstream of the IS Ecp1 element. The spread of the bla CTX-M genes among E. coli in Canada occurs through a diversity of different mechanisms and does not correspond to a single CTX-M determinant, or a single clone, or a single plasmid but rather through the combination of clonal spread of virulent strains and acquisition of diverse CTX-M–bearing plasmids. We report the 1st qepA-producing E. coli in North America.

Dissemination of Plasmid-mediated qnr, aac(6')-Ib-cr, and qepA Genes Among 16S rRNA Methylase Producing Enterobacteriaceae in Korea

Plasmid-mediated quinolone resistance (PMQR) genes: qnr, aac(6')-Ib-cr, and qepA were investigated among 153 armA and 51 rmtB-positive transconjugants and their 204 clinical isolates of Enterobacteriaceae. Overall, qnrB4 and aac(6')-Ib-cr genes were identified in 52.3% (63 K. pneumoniae, 10 E. coli, 4 E. cloacae, and 3 E. aerogenes) and 24.8% (16 K. pneumoniae, 8 E. coli, 6 S. marcescens, 4 E. cloacae, 3 C. freundii and 1 K. oxytoca) of 153 armA-positive isolates, respectively. Four isolates of K. pneumoniae and two isolates of E. coli positive for armA co-harbored both qnrB4 and aac(6')-Ib-cr. The qepA gene was detected in 11.8% (5 E. coli and 1 K. pneumoniae) of 51 rmtB-positive clinical isolates and their transconjugants. Southern hybridization confirmed the co-localization of qepA and rmtB on a large conjugative plasmid of size between 90 to 170 kb. Inc replicon typing showed that qnrB4/6, aac(6')-Ib-cr, and qepA genes were principally disseminated by IncFIIAs, IncL/M, and IncF plasmids, respectively. This study constitutes the first report of the three known PMQR genes among the 16S rRNA methylase producing Enterobacteriaceae isolates of human origin from Korea.

Replicon typing of plasmids in Escherichia coli producing extended-spectrum β-lactamases

Escherichia coli producing CTX-M-15 and CTX-M-14 extended-spectrum beta-lactamases (ESBLs) are spreading worldwide. The aim of this work was to investigate the replicons involved in the emergence and spread of ESBLs in relation to ESBL type. A collection of 125 TEM, SHV and CTX-M ESBL-producing E. coli strains was analysed. The replicons carrying the ESBLs and the total plasmid content of the strains have been characterized by PCR replicon typing in relation to the type of ESBL. The ESBL replicons were then compared with the replicon content of E. coli strains carrying TEM-1 or inhibitor-resistant TEM (IRT) beta-lactamases. IncF plasmids were the most frequently carried replicons in our collection, but none carried TEM ESBL. Of TEM ESBLs, 67% were carried on IncA/C replicons except for TEM-52 genes, which were carried preferentially on IncI1 replicons. Although CTX-M enzymes can be carried by various replicons, the great majority of genes encoding CTX-M-14 and CTX-M-15 ESBLs were carried by IncF replicons, as were TEM-1 and IRT beta-lactamases. Resistance genes borne by the narrow host-range IncF replicon spread readily as this replicon is well adapted to E. coli. This is observed for blaTEM-1 and blaCTX-M-15 and, to a lesser extent, for blaCTX-M-14. Transposition immunity seems to play an important role in the diffusion process.

Sequence of Conjugative Plasmid pIP1206 Mediating Resistance to Aminoglycosides by 16S rRNA Methylation and to Hydrophilic Fluoroquinolones by Efflux

Self-transferable IncFI plasmid pIP1206, isolated from an Escherichia coli clinical isolate, carries two new resistance determinants: qepA, which confers resistance to hydrophylic fluoroquinolones by efflux, and rmtB, which specifies a 16S rRNA methylase conferring high-level aminoglycoside resistance. Analysis of the 168,113-bp sequence (51% G+C) revealed that pIP1206 was composed of several subregions separated by copies of insertion sequences. Of 151 open reading frames, 56 (37%) were also present in pRSB107, isolated from a bacterium in a sewage treatment plant. pIP1206 contained four replication regions (RepFIA, RepFIB, and two partial RepFII regions) and a transfer region 91% identical with that of pAPEC-O1-ColBM, a plasmid isolated from an avian pathogenic E. coli. A putative oriT region was found upstream from the transfer region. The antibiotic resistance genes tet(A), catA1, bla(TEM-1), rmtB, and qepA were clustered in a 33.5-kb fragment delineated by two IS26 elements that also carried a class 1 integron, including the sulI, qacEDelta1, aad4, and dfrA17 genes and Tn10, Tn21, and Tn3-like transposons. The plasmid also possessed a raffinose operon, an arginine deiminase pathway, a putative iron acquisition gene cluster, an S-methylmethionine metabolism operon, two virulence-associated genes, and a type I DNA restriction-modification (R-M) system. Three toxin/antitoxin systems and the R-M system ensured stabilization of the plasmid in the host bacteria. These data suggest that the mosaic structure of pIP1206 could have resulted from recombination between pRSB107 and a pAPEC-O1-ColBM-like plasmid, combined with structural rearrangements associated with acquisition of additional DNA by recombination and of mobile genetic elements by transposition.

Biofilm Induced Tolerance towards Antimicrobial Peptides

Increased tolerance to antimicrobial agents is thought to be an important feature of microbes growing in biofilms. We address the question of how biofilm organization affects antibiotic susceptibility. We established Escherichia coli biofilms with differential structural organization due to the presence of IncF plasmids expressing altered forms of the transfer pili in two different biofilm model systems. The mature biofilms were subsequently treated with two antibiotics with different molecular targets, the peptide antibiotic colistin and the fluoroquinolone ciprofloxacin. The dynamics of microbial killing were monitored by viable count determination, and confocal laser microscopy. Strains forming structurally organized biofilms show an increased bacterial survival when challenged with colistin, compared to strains forming unstructured biofilms. The increased survival is due to genetically regulated tolerant subpopulation formation and not caused by a general biofilm property. No significant difference in survival was detected when the strains were challenged with ciprofloxacin. Our data show that biofilm formation confers increased colistin tolerance to cells within the biofilm structure, but the protection is conditional being dependent on the structural organization of the biofilm, and the induction of specific tolerance mechanisms.

F-Like Type IV Secretion Systems Encode Proteins with Thioredoxin Folds That Are Putative DsbC Homologues

F and R27 are conjugative plasmids of enteric bacteria belonging to the IncF and IncHI1 plasmid incompatibility groups, respectively. Based on sequence analysis, two genes of the F transfer region, traF and trbB, and three genes of the R27 transfer region, trhF, dsbC, and htdT, are predicted to encode periplasmic proteins containing a C-terminal thioredoxin fold. The C-X-X-C active-site motif of thioredoxins is present in all of these proteins except TraF(F). Escherichia coli carrying a dsbA mutation, which is deficient in disulfide bond formation, cannot synthesize pili and exhibits hypersensitivity to dithiothreitol (DTT) as monitored by mating ability. Overproduction of the E. coli disulfide bond isomerase DsbC, TrbB(F), DsbC(R27), or HtdT(R27), but not TraF(F) or TrhF(R27), reverses this hypersensitivity to DTT. Site-directed mutagenesis established that the C-X-X-C motif was necessary for this activity. Secretion into the periplasm of the C-terminal regions of TrbB(F) and DsbC(R27), containing putative thioredoxin folds, but not TrhF(R27), partially complemented the host dsbA mutation. A trbB(F) deletion mutant showed a 10-fold-lower mating efficiency in an E. coli dsbC null strain but had no phenotype in wild-type E. coli, suggesting redundancy in function between TrbB(F) and E. coli DsbC. Our results indicate that TrbB(F), DsbC(R27), and HtdT(R27) are putative disulfide bond isomerases for their respective transfer systems. TraF(F) is essential for conjugation but appears to have a function other than disulfide bond chemistry.