scholarly journals A novel plasmid entry exclusion system in pKPC_UVA01, a promiscuous conjugative plasmid carrying the bla KPC carbapenemase gene

2022 ◽  
Author(s):  
Muhammad Kamruzzaman ◽  
Amy J. Mathers ◽  
Jonathan R. Iredell
Keyword(s):  
Antibiotic Resistance Genes ◽  
Carbapenem Resistance ◽  
The United States ◽  
Recipient Strain ◽  
Conjugative Plasmid ◽  
Multi Drug Resistance ◽  
Conjugative Transfer ◽  
C Terminus ◽  
Carbapenemase Gene ◽  
Strict Specificity

Conjugative plasmids are the principal mediator in the emergence and spread of antibiotic resistance genes in Enterobacterales. Plasmid entry-exclusion (EEX) systems can restrict their transfer into the recipient bacteria carrying closely related plasmids. In this study, we have identified and characterized a novel plasmid entry exclusion system in a carbapenem resistance plasmid pKPC_UVA01, responsible for widespread dissemination of the bla KPC carbapenemase gene among Enterobacterales in the United States. The identified eex gene in the recipient strain of different Enterobacterales species inhibits the conjugation transfer of pKPC_UVA01 plasmids at a range of 200-400 fold, and this inhibition was found to be a dose-dependent function of the EEX protein in recipient cells. The C-terminus truncated version of eex or eex with an early termination codon at the C-terminus region alleviates inhibition of conjugative transfer. Unlike the strict specificity of plasmid exclusion by the known EEX protein, the newly identified EEX in the recipient strain can inhibit the transfer of IncP and IncN plasmids. The eex gene from the plasmid pKPC_UVA01 is not required for conjugative transfer but is essential in the donor bacteria for entry exclusion of this plasmid. This is a novel function of a single protein that is essential in both donor and recipient bacteria for entry exclusion of a plasmid. This eex gene is found to be distributed in multi-drug resistance plasmids similar to pKPC_UVA01 in different Enterobacterales species and may contribute to the stability of this plasmid type by controlling its transfer.

scholarly journals pSK41-Like Plasmid Is Necessary for Inc18-LikevanAPlasmid Transfer from Enterococcus faecalis to Staphylococcus aureusIn Vitro

2012 ◽  
Vol 57 (1) ◽  
pp. 212-219 ◽  
Author(s):  
Wenming Zhu ◽  
Nancye Clark ◽  
Jean B. Patel
Keyword(s):  
Enterococcus Faecalis ◽  
The United States ◽  
Conjugal Transfer ◽  
Conjugative Plasmid ◽  
Conjugative Transfer ◽  
Content Type ◽  
A Cell ◽  
Extracellular Factor ◽  
Vancomycin Resistant

ABSTRACTVancomycin-resistantStaphylococcus aureus(VRSA) is thought to result from thein vivoconjugative transfer of avanAplasmid from anEnterococcussp. toS. aureus. We studied bacterial isolates from VRSA cases that occurred in the United States to identify microbiological factors which may contribute to this plasmid transfer. First, vancomycin-susceptible, methicillin-resistantS. aureus(MRSA) isolates from five VRSA cases were tested for their ability to accept foreign DNA by conjugation in mating experiments withEnterococcus faecalisJH2-2 containing pAM378, a pheromone-response conjugative plasmid. All of the MRSA isolates accepted the plasmid DNA with similar transfer efficiencies (∼10−7/donor CFU) except for one isolate, MRSA8, for which conjugation was not successful. The MRSA isolates were also tested as recipients in mating experiments between anE. faecalisisolate with an Inc18-likevanAplasmid that was isolated from a VRSA case patient. Conjugative transfer was successful for 3/5 MRSA isolates. Successful MRSA recipients carried a pSK41-like plasmid, a staphylococcal conjugative plasmid, whereas the two unsuccessful MRSA recipients did not carry pSK41. The transfer of a pSK41-like plasmid from a successful MRSA recipient to the two unsuccessful recipients resulted in conjugal transfer of the Inc18-likevanAplasmid fromE. faecalisat a frequency of 10−7/recipient CFU. In addition, conjugal transfer could be achieved for pSK41-negative MRSA in the presence of a cell-free culture filtrate fromS. aureuscarrying a pSK41-like plasmid at a frequency of 10−8/recipient CFU. These results indicated that a pSK41-like plasmid can facilitate the transfer of an Inc18-likevanAplasmid fromE. faecalistoS. aureus, possibly via an extracellular factor produced by pSK41-carrying isolates.

scholarly journals Multicenter Clinical and Molecular Epidemiological Analysis of Bacteremia Due to Carbapenem-Resistant Enterobacteriaceae (CRE) in the CRE Epicenter of the United States

2017 ◽  
Vol 61 (4) ◽  
Author(s):  
Michael J. Satlin ◽  
Liang Chen ◽  
Gopi Patel ◽  
Angela Gomez-Simmonds ◽  
Gregory Weston ◽  
...  
Keyword(s):  
New York ◽  
Empirical Therapy ◽  
Carbapenem Resistance ◽  
The United States ◽  
Resistance Mechanisms ◽  
Rapid Diagnostics ◽  
Multicenter Studies ◽  
Content Type ◽  
Medical Centers ◽  
Carbapenem Resistant

ABSTRACT Although the New York/New Jersey (NY/NJ) area is an epicenter for carbapenem-resistant Enterobacteriaceae (CRE), there are few multicenter studies of CRE from this region. We characterized patients with CRE bacteremia in 2013 at eight NY/NJ medical centers and determined the prevalence of carbapenem resistance among Enterobacteriaceae bloodstream isolates and CRE resistance mechanisms, genetic backgrounds, capsular types (cps), and antimicrobial susceptibilities. Of 121 patients with CRE bacteremia, 50% had cancer or had undergone transplantation. The prevalences of carbapenem resistance among Klebsiella pneumoniae, Enterobacter spp., and Escherichia coli bacteremias were 9.7%, 2.2%, and 0.1%, respectively. Ninety percent of CRE were K. pneumoniae and 92% produced K. pneumoniae carbapenemase (KPC-3, 48%; KPC-2, 44%). Two CRE produced NDM-1 and OXA-48 carbapenemases. Sequence type 258 (ST258) predominated among KPC-producing K. pneumoniae (KPC-Kp). The wzi154 allele, corresponding to cps-2, was present in 93% of KPC-3-Kp, whereas KPC-2-Kp had greater cps diversity. Ninety-nine percent of CRE were ceftazidime-avibactam (CAZ-AVI)-susceptible, although 42% of KPC-3-Kp had an CAZ-AVI MIC of ≥4/4 μg/ml. There was a median of 47 h from bacteremia onset until active antimicrobial therapy, 38% of patients had septic shock, and 49% died within 30 days. KPC-3-Kp bacteremia (adjusted odds ratio [aOR], 2.58; P = 0.045), cancer (aOR, 3.61, P = 0.01), and bacteremia onset in the intensive care unit (aOR, 3.79; P = 0.03) were independently associated with mortality. Active empirical therapy and combination therapy were not associated with survival. Despite a decade of experience with CRE, patients with CRE bacteremia have protracted delays in appropriate therapies and high mortality rates, highlighting the need for rapid diagnostics and evaluation of new therapeutics.

scholarly journals First Description of KPC-2-ProducingKlebsiella oxytocaIsolated from a Pediatric Patient with Nosocomial Pneumonia in Venezuela

2014 ◽  
Vol 2014 ◽  
pp. 1-4 ◽  
Author(s):  
Indira Labrador ◽  
María Araque
Keyword(s):  
Pediatric Patient ◽  
Carbapenem Resistance ◽  
Control Measures ◽  
University Hospital ◽  
Conjugative Plasmid ◽  
Effective Control ◽  
Molecular Tests ◽  
The Andes ◽  
Clinically Significant ◽  
Significant Class

During the last decade, carbapenem resistance has emerged among clinical isolates of the Enterobacteriaceae family. This has been increasingly attributed to the production ofβ-lactamases capable of hydrolyzing carbapenems. Among these enzymes,Klebsiella pneumoniaecarbapenemases (KPCs) are the most frequently and clinically significant class-A carbapenemases. In this report, we describe the first nosocomial KPC-2-producingK. oxytocaisolated from a pediatric patient with pneumonia admitted to the intensive care unit at The Andes University Hospital, Mérida, Venezuela. This strain was resistant to several antibiotics including imipenem, ertapenem, and meropenem but remained susceptible to ciprofloxacin, colistin, and tigecycline. Conjugation assays demonstrated the transferability of all resistance determinants, except aminoglycosides. The isolate LMM-SA26 carried a ~21 kb conjugative plasmid that harbored theblaKPC-2,blaCTX-M-8, andblaTEM-15genes. Although carbapenem resistance in the Enterobacteriaceae is still unusual in Venezuela, KPCs have a great potential to spread due to their localization on mobile genetic elements. Therefore, rapid detection of KPC-carrying bacteria with phenotypic and confirmatory molecular tests is essential to establish therapeutic options and effective control measures.

scholarly journals Functional Characterization of IS1999, an IS4 Family Element Involved in Mobilization and Expression of β-Lactam Resistance Genes

2006 ◽  
Vol 188 (18) ◽  
pp. 6506-6514 ◽  
Author(s):  
Daniel Aubert ◽  
Thierry Naas ◽  
Claire Héritier ◽  
Laurent Poirel ◽  
Patrice Nordmann
Keyword(s):  
Resistance Genes ◽  
Consensus Sequence ◽  
Functional Characterization ◽  
Antibiotic Resistance Genes ◽  
Conjugative Plasmid ◽  
Transposition Frequency ◽  
Transposase Gene ◽  
Reverse Transcription Pcr ◽  
Mutant Derivative ◽  
Rapid Amplification

ABSTRACT IS1999 and a point mutant derivative, IS1999.2, have been described inserted upstream of emerging antibiotic resistance genes bla VEB-1 and bla OXA-48. 5′ Rapid amplification of cDNA ends experiments revealed that expression of these β-lactamase genes was driven by the outward-directed promoter, Pout, located in the IS1999 elements. These findings led us to study IS1999-mediated gene mobilization. Thus, the transposition properties of IS1999 and of IS1999-based composite transposons, made of two copies of IS1999 in different orientations, were investigated. IS1999 or IS1999-based composite transposons were capable of transposing onto the conjugative plasmid pOX38-Gen. Sequence analysis of the insertion sites revealed that IS1999 inserted preferentially into DNA targets containing the consensus sequence NGCNNNGCN. Transposition was more efficient when at least one left inverted repeat end was located at an outside end of the transposon. The transposition frequency of IS1999.2 was 10-fold lower than that of IS1999, and transposition frequencies of the putative natural transposon, Tn1999, were below detection limits of our transposition assay. This reduced transposition frequency of IS1999.2-based elements may result from a lower transcription of the transposase gene, as revealed by reverse transcription-PCR analyses.

scholarly journals Transposase-Mediated Excision, Conjugative Transfer, and Diversity of ICE6013 Elements in Staphylococcus aureus

2017 ◽  
Vol 199 (8) ◽  
Author(s):  
Emily A. Sansevere ◽  
Xiao Luo ◽  
Joo Youn Park ◽  
Sunghyun Yoon ◽  
Keun Seok Seo ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Sequence Analysis ◽  
Integration Site ◽  
Consensus Sequence ◽  
Open Reading Frames ◽  
Conjugative Plasmid ◽  
Site Preference ◽  
Conjugative Transfer ◽  
Content Type ◽  
Integrative Conjugative Elements

ABSTRACT ICE6013 represents one of two families of integrative conjugative elements (ICEs) identified in the pan-genome of the human and animal pathogen Staphylococcus aureus. Here we investigated the excision and conjugation functions of ICE6013 and further characterized the diversity of this element. ICE6013 excision was not significantly affected by growth, temperature, pH, or UV exposure and did not depend on recA. The IS30-like DDE transposase (Tpase; encoded by orf1 and orf2) of ICE6013 must be uninterrupted for excision to occur, whereas disrupting three of the other open reading frames (ORFs) on the element significantly affects the level of excision. We demonstrate that ICE6013 conjugatively transfers to different S. aureus backgrounds at frequencies approaching that of the conjugative plasmid pGO1. We found that excision is required for conjugation, that not all S. aureus backgrounds are successful recipients, and that transconjugants acquire the ability to transfer ICE6013. Sequencing of chromosomal integration sites in serially passaged transconjugants revealed a significant integration site preference for a 15-bp AT-rich palindromic consensus sequence, which surrounds the 3-bp target site that is duplicated upon integration. A sequence analysis of ICE6013 from different host strains of S. aureus and from eight other species of staphylococci identified seven divergent subfamilies of ICE6013 that include sequences previously classified as a transposon, a plasmid, and various ICEs. In summary, these results indicate that the IS30-like Tpase functions as the ICE6013 recombinase and that ICE6013 represents a diverse family of mobile genetic elements that mediate conjugation in staphylococci. IMPORTANCE Integrative conjugative elements (ICEs) encode the abilities to integrate into and excise from bacterial chromosomes and plasmids and mediate conjugation between bacteria. As agents of horizontal gene transfer, ICEs may affect bacterial evolution. ICE6013 represents one of two known families of ICEs in the pathogen Staphylococcus aureus, but its core functions of excision and conjugation are not well studied. Here, we show that ICE6013 depends on its IS30-like DDE transposase for excision, which is unique among ICEs, and we demonstrate the conjugative transfer and integration site preference of ICE6013. A sequence analysis revealed that ICE6013 has diverged into seven subfamilies that are dispersed among staphylococci.

scholarly journals 1348. In vitro Activity of Plazomicin, a Next-Generation Aminoglycoside, Against Carbapenemase-Producing Klebsiella pneumoniae

2018 ◽  
Vol 5 (suppl_1) ◽  
pp. S412-S413
Author(s):  
Michael R Jacobs ◽  
Caryn E Good ◽  
Ayman M Abdelhamed ◽  
Daniel D Rhoads ◽  
Kristine M Hujer ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Carbapenem Resistance ◽  
Vitro Activity ◽  
In Vitro Activity ◽  
Next Generation ◽  
Aminoglycoside Resistance ◽  
Carbapenem Resistant ◽  
Carbapenemase Gene ◽  
Research Grant

Abstract Background Plazomicin is a next-generation aminoglycoside with in vitro activity against multidrug-resistant Gram-negative species, including carbapenem-resistant isolates. The Consortium on Resistance Against Carbapenems in Klebsiella and other Enterobacteriaceae (CRACKLE) is a federally funded, prospective multicenter consortium of 20 hospitals from nine US healthcare systems to track carbapenem-resistant Enterobacteriaceae. Methods Minimum inhibitory concentrations (MICs) of plazomicin were determined by broth microdilution according to current CLSI guidelines against a collection of 697 carbapenem-resistant Klebsiella pneumoniae with defined carbapenem resistance mechanisms, including KPC and OXA carbapenemases. Isolates were submitted by participating CRACKLE centers. Results Carbapenemases present in study isolates included KPC-2 (n = 323), KPC-3 (n = 364), KPC-4 (n = 2), OXA-48 like (n = 7), and NDM (n = 1). Plazomicin MICs ranged from ≤0.12 to >32 mg/L, with MIC50 and MIC90 values of 0.25 and 1 mg/L, respectively (figure). MICs of 689 (98.8%) isolates were ≤4 mg/L, while MICs of the remaining eight isolates were >32 mg/L. Plazomicin MICs were related to specific carbapenemases present in isolates: of eight isolates with MICs >32 mg/L, seven contained OXA-48 like and one contained KPC-3, suggesting that these isolates possess an aminoglycoside-resistance mechanism on the same plasmid as their carbapenemase gene, such as a 16S ribosomal RNA methyltransferase, against which plazomicin is not active. Conclusion Plazomicin has good in vitro potency against a collection of carbapenemase-producing K. pneumoniae, with MIC90 value of 1 mg/L and MICs of ≤4 mg/L for 98.9% of isolates. Disclosures M. R. Jacobs, Achaogen: Investigator, Research grant. Shionogi: Investigator, Research grant. L. Connolly, Achaogen, Inc.: Consultant, Consulting fee. K. M. Krause, Achaogen: Employee, Salary. S. S. Richter, bioMerieux: Grant Investigator, Research grant. BD Diagnostics: Grant Investigator, Research grant. Roche: Grant Investigator, Research grant. Hologic: Grant Investigator, Research grant. Diasorin: Grant Investigator, Research grant. Accelerate: Grant Investigator, Research grant. Biofire: Grant Investigator, Research grant. D. Van Duin, achaogen: Scientific Advisor, Consulting fee. shionogi: Scientific Advisor, Consulting fee. Allergan: Scientific Advisor, Consulting fee. Astellas: Scientific Advisor, Consulting fee. Neumedicine: Scientific Advisor, Consulting fee. Roche: Scientific Advisor, Consulting fee. T2 Biosystems: Scientific Advisor, Consulting fee.

scholarly journals Complete Nucleotide Sequence of the pCTX-M3 Plasmid and Its Involvement in Spread of the Extended-Spectrum β-Lactamase Gene blaCTX-M-3

2007 ◽  
Vol 51 (11) ◽  
pp. 3789-3795 ◽  
Author(s):  
M. Gołębiewski ◽  
I. Kern-Zdanowicz ◽  
M. Zienkiewicz ◽  
M. Adamczyk ◽  
J. Żyliǹska ◽  
...  
Keyword(s):  
Nucleotide Sequence ◽  
Resistance Genes ◽  
Antibiotic Resistance Genes ◽  
Conjugative Plasmid ◽  
Bacterial Strains ◽  
Extended Spectrum ◽  
Gene Coding ◽  
Kluyvera Ascorbata ◽  
Almost All ◽  
Lactamase Gene

ABSTRACT Here we report the nucleotide sequence of pCTX-M3, a highly conjugative plasmid that is responsible for the extensive spread of the gene coding for the CTX-M-3 extended-spectrum β-lactamase in clinical populations of the family Enterobacteriaceae in Poland. The plasmid belongs to the IncL/M incompatibility group, is 89,468 bp in size, and carries 103 putative genes. Besides bla CTX-M-3, it also bears the bla TEM-1, aacC2, and armA genes, as well as integronic aadA2, dfrA12, and sul1, which altogether confer resistance to the majority of β-lactams and aminoglycosides and to trimethoprim-sulfamethoxazole. The conjugal transfer genes are organized in two blocks, tra and trb, separated by a spacer sequence where almost all antibiotic resistance genes and multiple mobile genetic elements are located. Only bla CTX-M-3, accompanied by an ISEcp1 element, is placed separately, in a DNA fragment previously identified as a fragment of the Kluyvera ascorbata chromosome. On the basis of sequence analysis, we speculate that pCTX-M3 might have arisen from plasmid pEL60 from plant pathogen Erwinia amylovora by acquiring mobile elements with resistance genes. This suggests that plasmids of environmental bacterial strains could be the source of those plasmids now observed in bacteria pathogenic for humans.

Sign in / Sign up

Export Citation Format

Share Document