IncFII Replicon Research Articles

Calcium-responsive plasmid copy number regulation is dependent on discrete YopD domains in Yersinia pseudotuberculosis

Yersinia pathogenicity depends mainly on a Type III Secretion System (T3SS) responsible for translocating effector proteins into the eukaryotic target cell cytosol. The T3SS is encoded on a 70 kb, low copy number virulence plasmid, pYV. A key T3SS regulator, YopD, is a multifunctional protein and consists of discrete modular domains that are essential for pore formation and translocation of Yop effectors. In Y. pseudotuberculosis, the temperature-dependent plasmid copy number increase that is essential for elevated T3SS gene dosage and virulence is also affected by YopD. Here, we found that the presence of intracellular YopD results in increased levels of the CopA-RNA and CopB, two inhibitors of plasmid replication. Secretion of YopD leads to decreased expression of copA and copB, resulting in increased plasmid copy number. Moreover, using a systematic mutagenesis of YopD mutants, we demonstrated that the same discrete modular domains important for YopD translocation are also necessary for both the regulation of plasmid copy number as well as copA and copB expression. Hence, Yersinia has evolved a mechanism coupling active secretion of a plasmid-encoded component of the T3SS, YopD, to the regulation of plasmid replication. Our work provides evidence for the cross-talk between plasmid-encoded functions with the IncFII replicon.

CpxR negatively regulates IncFII-replicon plasmid pEC011 conjugation by directly binding to multi-promoter regions

CpxAR is a global regulatory protein and has important roles in plasmid mating. However, except for traJ, the regulatory effect of CpxAR on other tra genes is unclear. The aim of this study was to explore the effects of CpxAR on conjugative transfer of the epidemic plasmid pEC011 (IncFII replicon) in Escherichia coli. The plasmid mating frequencies were significantly higher for the single deletion mutant strain FΔcpxR than for the parental strain F25922. Additionally, expression levels of traM, traJ and traY in FΔcpxR were significantly higher than those in F25922. Further investigations revealed that His6−CpxR protein could directly bind to the traM, traJ and traY promoter regions with the binding sites of 5′-TTTACATT-3′ (PM), 5′-ATAAGAAT-3′ (PJ), and 5′-AATTTTAT-3′ (PY), respectively. Taken together, our results demonstrate that CpxAR can downregulate the expression of traM, traJ and traY by directly binding to the CpxR box-like sites of promoters, thus significantly reducing the mating rates of IncFII replicon plasmid pEC011.

Whole-Genome Sequencing of Gram-Negative Bacteria Isolated From Bovine Mastitis and Raw Milk: The First Emergence of Colistin mcr-10 and Fosfomycin fosA5 Resistance Genes in Klebsiella pneumoniae in Middle East.

Antimicrobial resistance is a major concern in the dairy industry. This study investigated the prevalence, antimicrobial resistance phenotypes, and genome sequencing of Gram-negative bacteria isolated from clinical (n = 350) and subclinical (n = 95) bovine mastitis, and raw unpasteurized milk (n = 125). Klebsiella pneumoniae, Aeromonas hydrophila, Enterobacter cloacae (100% each), Escherichia coli (87.78%), and Proteus mirabilis (69.7%) were the most prevalent multidrug-resistant (MDR) species. Extensive drug-resistance (XDR) phenotype was found in P. mirabilis (30.30%) and E. coli (3.33%) isolates. Ten isolates (four E. coli, three Klebsiella species and three P. mirabilis) that displayed the highest multiple antibiotic resistance (MAR) indices (0.54–0.83), were exposed to whole-genome sequencing (WGS). Two multilocus sequence types (MLST): ST2165 and ST7624 were identified among the sequenced E. coli isolates. Three E. coli isolates (two from clinical mastitis and one from raw milk) belonging to ST2165 showed similar profile of plasmid replicon types: IncFIA, IncFIB, IncFII, and IncQ1 with an exception to an isolate that contained IncR, whereas E. coli ST7624 showed a different plasmid profile including IncHI2, IncHI2A, IncI1α, and IncFII replicon types. ResFinder findings revealed the presence of plasmid-mediated colistin mcr-10 and fosfomycin fosA5 resistance genes in a K. pneumoniae (K1) isolate from bovine milk. Sequence analysis of the reconstructed mcr-10 plasmid from WGS of K1 isolate, showed that mcr-10 gene was bracketed by xerC and insertion sequence IS26 on an IncFIB plasmid. Phylogenetic analysis revealed that K1 isolate existed in a clade including mcr-10-harboring isolates from human and environment with different STs and countries [United Kingdom (ST788), Australia (ST323), Malawi (ST2144), Myanmar (ST705), and Laos (ST2355)]. This study reports the first emergence of K. pneumoniae co-harboring mcr-10 and fosA5 genes from bovine milk in the Middle East, which constitutes a public health threat and heralds the penetration of the last-resort antibiotics. Hence, prudent use of antibiotics in both humans and animals and antimicrobial surveillance plans are urgently required.

1247. Molecular Epidemiology of Multi-drug Resistant Klebsiella pneumoniae and K. quasipneumoniae in Qatar

BackgroundThe molecular epidemiology of carbapenem-resistant Klebsiella species is not well investigated in Qatar. The objective of this work was to characterize the genetic context of carbapenemase-producing Klebsiella isolates recovered from clinical specimens.Methods Klebsiella isolates (n=100) were collected at 7 tertiary hospitals from 2015-2017. Identification and susceptibility testing were performed using MALDI-TOF MS and BD Phoenix system, respectively. Whole Genome Sequencing was performed on the Illumina NextSeq platform. Phylogenomic analysis, screening of resistance and virulence genes, and comparison of genetic environment of carbapenemase were carried out.Results Klebsiella pneumoniae was common (80), followed by K. quasipneumoniae (16), K. aerogenes (3) and K. oxytoca (1). The most prevalent were genes encoding NDM-1 (39), OXA-48 (20), OXA-232 (10) and OXA-181 (12). KPC-2 (3) and KPC-3 (2) were also identified; no carbapenemase-encoding genes could be identified in 15 isolates. Plasmid locations of 24 carbapenemase-encoding genes were determined; blaNDM-1 was localized on IncFII replicon, while blaOXA-181 and blaOXA-232 were commonly associated with ColKP3 plasmids. pOXA-48-like plasmid was detected in 17/20 isolates harboring blaOXA-48. blaKPC-3 was located on a contig with ‘traditional’ Tn4401a mobile genetic element. Sequence types (STs) were diverse and the ‘traditional’ clonal group (CG) 258 was rare. K. pneumoniae ST147 was predominant (13), followed by ST231 (7) and ST11 (5). Nine K. quasipneumoniae isolates belonged to ST196 and were highly clonal. The virulence loci such as yersiniabactin (ybt) and rmpA were not detected within the study’s K. quasipneumoniae isolates. Amongst K. pneumoniae, there were 50 ybt+ isolates; 8 isolates had rmpA, and of these, 3 belonged to ST383. K. pneumoniae serotype K2, the capsular serotype associated with invasive liver abscess syndrome, was detected in 5 isolates. Genetic relationship of carbapenem-resistant Klebsiella pneumoniae and K. quasipneumoniae isolates in Qatar inferred from core genome SNPs. The tree is overlaid with predicted antimicrobial resistance genes and virulence factors for each isolate.ConclusionThe predominant carbapenemases among clinical Klebsiella species isolates in Qatar are NDM and OXA-48 like enzymes, disseminated through various plasmids. The detection of carbapenemase-producing isolate bearing rmpA and serotype K2 reflect the presence of both multidrug resistance and hypervirulence in K. pneumoniae.Disclosures Yohei Doi, MD, PhD, AstraZeneca (Speaker’s Bureau)bioMerieux (Consultant)FujiFilm (Advisor or Review Panel member, Speaker’s Bureau)Gilead (Consultant)GSK (Consultant)Meiji (Consultant)MSD (Consultant)Shionogi (Consultant) Yohei Doi, MD, PhD, Astellas (Individual(s) Involved: Self): Grant/Research Support; AstraZeneca (Individual(s) Involved: Self): Speakers’ bureau; bioMerieux (Individual(s) Involved: Self): Consultant, Speakers’ bureau; Chugai (Individual(s) Involved: Self): Consultant; Entasis (Individual(s) Involved: Self): Consultant; FujiFilm (Individual(s) Involved: Self): Advisor or Review Panel member; Gilead (Individual(s) Involved: Self): Consultant; GSK (Individual(s) Involved: Self): Consultant; Kanto Chemical (Individual(s) Involved: Self): Grant/Research Support; MSD (Individual(s) Involved: Self): Speaking Fee; Pfizer (Individual(s) Involved: Self): Grant/Research Support; Shionogi (Individual(s) Involved: Self): Grant/Research Support, Speakers’ bureau; Teijin Healthcare (Individual(s) Involved: Self): Speakers’ bureau; VenatoRx (Individual(s) Involved: Self): Consultant

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

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. 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. 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. 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.

Coexistence of three blaKPC-2 genes on an IncF/IncR plasmid in ST11 Klebsiella pneumoniae.

Here we report the finding of three copies of the blaKPC-2 gene on a plasmid in ST11 Klebsiella pneumoniae. Carbapenem-resistant K. pneumoniae clinical strain WCHKP2 was subjected to whole-genome sequencing (WGS) using both a short-read Illumina HiSeq X10 platform and long-read MinION sequencer. Hybrid assembly was performed using Unicycler, and contigs were corrected using Pilon. Based on WGS, the sequence type (ST), capsular type, plasmid replicon type and plasmid multilocus sequence type were determined and virulence and antimicrobial resistance genes were identified. Mating was performed to identify a self-transmissible plasmid mediating carbapenem resistance. Strain WCHKP2 was resistant to imipenem [minimum inhibitory concentration (MIC)=64μg/mL] and meropenem (MIC=128μg/mL). Strain WCHKP2 had a 5477148-bp circular chromosome, two small ColRNAI-like plasmids (5596bp and 10060bp), and one large plasmid (177516bp, designated pKPC2_020002) containing an IncR and an IncFII replicon. Surprisingly, there were three copies of the blaKPC-2 carbapenemase gene on pKPC2_020002, which was not self-transmissible. Each of the blaKPC-2 genes was located in the same genetic context with insertion sequence ISKpn27 upstream and ISKpn6 downstream and bracketed by IS26. The three copies of the IS26-ISKpn27-blaKPC-2-ISKpn6-IS26 unit were present in tandem. Here we report the surprising co-existence of three copies of blaKPC-2 on an IncR/IncF plasmid due to the action of IS26. Multiple copies of IS26 are a key factor generating genetic plasticity and could mediate the multiplication of resistance genes.

Molecular characterization of clinical multidrug-resistant Klebsiella pneumoniae isolates.

BackgroundKlebsiella pneumoniae is a frequent nosocomial pathogen, with the multidrug-resistant (MDR) K. pneumoniae being a major public health concern, frequently causing difficult-to-treat infections worldwide. The aim of this study was to investigate the molecular characterization of clinical MDR Klebsiella pneumoniae isolates.MethodsA total of 27 non-duplicate MDR K. pneumoniae isolates with a CTX-CIP-AK resistance pattern were investigated for the prevalence of antimicrobial resistance genes including extended spectrum β-lactamase genes (ESBLs), plasmid-mediated quinolone resistance (PMQR) genes, 16S rRNA methylase (16S-RMTase) genes, and integrons by polymerase chain reaction (PCR) amplification and DNA sequencing. Plasmid replicons were typed by PCR-based replicon typing (PBRT). Multi-locus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) were carried out to characterize the strain relatedness.ResultsAll the isolates co-harbored 3 or more resistance determinants. OqxAB, CTX-M-type ESBLs and RmtB were the most frequent determinants, distributed among19 (70.4%),18 (66.7%) and 8 (29.6%) strains. Fourteen isolates harbored class 1 integrons, with orfD-aacA4 being the most frequent gene cassette array. Class 3 integrons were less frequently identified and contained the gene cassette array of blaGES-1-blaOXA-10-aac(6′)-Ib. IncFII replicon was most commonly found in this collection. One cluster was observed with ≥80% similarity among profiles obtained by PFGE, and one sequence type (ST) by MLST, namely ST11, was observed in the cluster.ConclusionK. pneumoniae carbapenemase (KPC)–producing ST11 was the main clone detected. Of particular concern was the high prevalence of multiple resistance determinants, classs I integrons and IncFII plasmid replicon among these MDR strains, which provide advantages for the rapid development of MDR strains.

Molecular characterization of multidrug-resistant extended-spectrum β-lactamase-producing Enterobacteriaceae isolated in Antananarivo, Madagascar

BackgroundWe investigated the molecular characteristics of multidrug-resistant, extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae isolated in community settings and in hospitals in Antananarivo, Madagascar.ResultsForty-nine E. coli, K. pneumoniae, K. oxytoca and E. cloacae ESBL-producing isolates were studied. In antimicrobial susceptibility analyses, many of the isolates exhibited resistance to aminoglycosides, fluoroquinolones and trimethoprim-sulfamethoxazole. Gene amplification analysis and sequencing revealed that 75.5% (n=37) of the isolates harbored blaCTX-M-15 and 38.7% (n=19) harbored blaSHV-12. The non-ESBLs resistance genes detected were blaTEM-1, blaOXA-1, aac(6′)-Ib,aac(6′)-Ib-cr, tetA, sul-1, sul-2, qnrA, qnrB and catB-3. We found dfrA and aadA gene cassettes in the class 1 integron variable regions of the isolates, and the combination of dfrA17-aadA5 to be the most prevalent. All blaCTX-M-15 positive isolates also contained the ISEcp1 insertion element. Conjugation and transformation experiments indicated that 70.3% of the antibiotic resistance genes resided on plasmids. Through a PCR based replicon typing method, plasmids carrying the blaSHV-12 or blaCTX-M-15 genes were assigned to either the IncFII replicon type or, rarely, to the HI2 replicon type. All isolates were subtyped by the rep-PCR and ERIC-PCR methods.Phylogenetic grouping and virulence genotyping of the E. coli isolates revealed that most of them belonged to group A1. One isolate assigned to group B2 harbored blaCTX-M-15 and five virulence genes (traT, fyuA, iutA, iha and sfa) and was related to the O25b-ST131 clone.ConclusionsOur results highlight the dissemination of multidrug resistant Enterobacteriaceae isolates in Antananarivo. These findings underline the need for a rational use of antibiotic and for appropriate methods of screening ESBL in routine laboratories in Antananarivo.

Recombination in IS 26 and Tn 2 in the Evolution of Multiresistance Regions Carrying bla CTX-M-15 on Conjugative IncF Plasmids from Escherichia coli

CTX-M-15 now appears to be the dominant extended-spectrum β-lactamase worldwide, and a number of different factors may contribute to this success. These include associations between bla(CTX-M-15) and particular plasmids (IncF) and/or strains, such as Escherichia coli ST131, as well as the genetic contexts in which this gene is found. We previously identified bla(CTX-M-15) as the dominant ESBL gene in the western Sydney area, Australia, and found that it was carried mainly on IncF or IncI1 plasmids. Here, we have mapped the multiresistance regions of the 11 conjugative plasmids with one or more IncF replicons obtained from that survey and conducted a limited comparison of plasmid backbones. Two plasmids with only an IncFII replicon appear to be very similar to the published plasmids pC15-1a and pEK516. The remaining nine plasmids, with multiple IncF replicons, have multiresistance regions related to those of pC15-1a and pEK516, but eight contain additional modules previously found in resistance plasmids from different geographic locations that carry a variety of different resistance genes. Differences between the multiresistance regions are largely due to IS26-mediated deletions, insertions, and/or rearrangements, which can explain the observed variable associations between bla(CTX-M-15) and certain other resistance genes. We found no evidence of independent movement of bla(CTX-M-15) or of a large multiresistance region between different plasmid backbones. Instead, homologous recombination between common components, such as IS26 and Tn2, appeared to be more important in creating new multiresistance regions, and this may be coupled with recombination in plasmid backbones to reassort multiple IncF replicons as well as components of multiresistance regions.

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.

Mosaic plasmids and mosaic replicons: evolutionary lessons from the analysis of genetic diversity in IncFII-related replicons.

The alpha replicons of the multi-replicon plasmids pGSH500 and pLV1402 have been characterized by DNA sequence analysis. Analysis of the DNA sequence of a 3672 bp HIN:dIII fragment from pFDT100, which contains the pGSH500 alpha replicon, revealed similarity to a number of replicons belonging to, or related to, those of the IncFII family. The replicon region contains copA, tapA, repA and oriR, and replication initiation and termination sites are related to those from the IncFII replicon of R1. A copB gene was found to lie upstream of the HIN:dIII site in the parental plasmid pGSH500. Downstream of oriR, a 707 bp region shows 72.6% identity to a region of the Escherichia coli chromosome at 43.3', suggesting this region of pGSH500 may have been incorporated into the plasmid during a past chromosomal recombination event. Oligonucleotide primers homologous to consensus regions in the copB and repA genes, and the oriR regions from a number of IncFII-related replicons were used to amplify replication regions from pLV1402. Analysis of the amplified regions has shown the presence of copB, copA, tapA and repA genes. Phylogenetic analysis of Rep protein sequences from the RepFIIA family of antisense-control-regulated replicons revealed the presence of three distinct subgroups of Rep proteins. Comparative analysis of DNA and protein sequences from members of the RepFIIA family provides evidence supporting the roles of both non-selective divergence in co-integrate (multi-replicon) plasmids and Chi-mediated-recombination in replicon evolution, and in particular, that such processes may have been widespread in the evolution of the RepFIIA family.

Isolation and properties of the RepA1 protein of the IncFII replicon, RepFIC

The initiator protein RepA1 of the IncFII replicon RepFIC derived from the enterotoxin plasmid EntP307 has been cloned under the control of the lambda PL promoter. This has enabled us to overproduce this protein and study its properties. Here we show that RepA1 is a soluble basic protein with an experimentally determined molecular weight of 40,000. Deletion analysis indicates that the overproduced protein originates from the open reading frame which we previously designated as coding for RepA1. We have also shown that the replication function of the replicon RepFIC depends on the intact RepA1 coding frame.

Second replicon in ColV2-K94 mediates the stable coexistence of two incompatible plasmids.

The colicin-producing plasmid pWS12, a Tn903 derivative of ColV2-K94, was found to be incompatible with the IncFI plasmids KLF1 and R386. It was compatible with the IncFII plasmids R538 and R100. Three overlapping mini-ColV derivatives, pWS15, pWS16 and pWS17, were obtained by restriction digestion of pWS12. Unlike pWS12, pWS16 exhibited incompatibility with both IncFI and IncFII plasmids, whereas the pWS15 and pWS17 plasmids expressed IncFII incompatibility but not the IncFI incompatibility of their parental ColV plasmid. We show that, although pWS12 has an IncFII replicon, Rep1, it does not normally express IncFII incompatibility because a second replicon, Rep2 (homologous to the secondary replicon of F), functions during the stable coexistence of the plasmid with IncFII plasmids. When Rep2 is deleted (as in the mini-ColV plasmids) or made nonfunctional (as in a PolA mutant strain), ColV then behaves as an IncFII plasmid.