Dataset Information

Interplay among IncA and bla KPC-Carrying Plasmids in Citrobacter freundii.

ABSTRACT: We report two KPC-producing Citrobacter freundii isolates from unrelated patients. In one case, bla KPC-2 was harbored on a novel variant of a Tn4401 transposon of an IncN plasmid conjugated together with a coresident IncA plasmid, whereas in the other one, bla KPC-3 was on a Tn4401a transposon located on an IncX3-IncA self-conjugative plasmid fusion. The interplay among plasmids carrying bla KPC and the coresident IncA plasmids offers new information on plasmids coresident within clinically relevant enterobacteria.

SUBMITTER: Bitar I

PROVIDER: S-EPMC6496092 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Interplay among IncA and blaKPC-Carrying Plasmids in Citrobacter freundii.

Bitar Ibrahim I Caltagirone Mariasofia M Villa Laura L Mattioni Marchetti Vittoria V Nucleo Elisabetta E Sarti Mario M Migliavacca Roberta R Carattoli Alessandra A

Antimicrobial agents and chemotherapy 20190425 5

We report two KPC-producing Citrobacter freundii isolates from unrelated patients. In one case, blaKPC-2 was harbored on a novel variant of a Tn4401 transposon of an IncN plasmid conjugated together with a coresident IncA plasmid, whereas in the other one, blaKPC-3 was on a Tn4401a transposon located on an IncX3-IncA self-conjugative plasmid fusion. The interplay among plasmids carrying blaKPC and the coresident IncA plas ...[more]

PMID: 30858205

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Project description:A bla KPC-2-carrying Citrobacter freundii isolate developed ceftazidime-avibactam resistance during treatment with this agent. The initial and follow-up isolates exhibited ceftazidime-avibactam MICs of 4 and 64?µg/ml, respectively. Overexpression of AcrAB-TolC and porin alterations were detected in both isolates, but no other resistance mechanism was observed. After passaging the initial clinical isolate in ceftazidime-avibactam at a fixed concentration of 4?µg/ml and a 4:1 ratio, resistance to all ?-lactams was noted, and a percentage of the bla KPC-2 sequencing reads had mutations leading to the alterations D176Y (bla KPC-2-D176Y [78%]) or R164S plus P147L (bla KPC-2-R164S + P147L [82%]). Further investigation of the follow-up isolate showed that 11% of the bla KPC-2 reads had mutations leading to D179Y substitution (bla KPC-2-D179Y). In the absence of selective pressure, ceftazidime-avibactam MICs of the passaged and follow-up isolates revealed that 7 or 8 out of 20 screened colonies reverted to susceptible and possessed bla KPC-2 wild-type sequences. Recombinant plasmids carrying the bla KPC-2 alterations observed were transformed in Escherichia coli, and MIC values for ceftazidime ± avibactam were elevated. Lower MICs for ceftriaxone, cefepime, aztreonam, meropenem, and imipenem for the mutated KPC-2-producing isolates were observed compared to those of the isolates producing a wild-type KPC-2. Avibactam at a fixed concentration of 4?µg/ml restored the activity of all ?-lactams tested for the recombinant strains. The heterogenous population of wild-type and mutated bla KPC-2 and the reversibility of the genotypes observed suggest a significant challenge for managing KPC-producing isolates that develop ceftazidime-avibactam resistance during therapy.IMPORTANCE The development of ceftazidime-avibactam resistance among KPC-producing isolates during treatment with this agent has been reported. Usually isolates that become resistant have a mutated bla KPC gene that confers resistance to ceftazidime-avibactam and susceptibility to meropenem. We report a Citrobacter freundii isolate that developed ceftazidime-avibactam resistance due to mutations within the coding region of the bla KPC-2 ?-loop previously reported; however, in this case, only 11% of the whole-genome sequencing reads had mutations, making this alteration difficult to detect and the treatment of these isolates more challenging. In addition to bla KPC, the initial and the follow-up patient isolates displayed hyperexpression of the AcrAB-TolC efflux system and disruption of the outer membrane protein (OMP) OmpF, which contribute to carbapenem resistance. Experiments performed to confirm our findings included generating mutant isolates from the initial patient isolate, passaging the isolates for purity in drug-free medium, resulting in a reversible phenotype, and cloning the mutations to demonstrate the resistance conferred.

Project description:Klebsiella pneumoniae carbapenemase (KPC) producers are an emerging threat to global health, and the hospital water environment is considered an important reservoir of these life-threatening bacteria. We characterized plasmids of KPC-2-producing Citrobacter freundii and Klebsiella variicola isolates recovered from hospital sewage in Japan. Antimicrobial susceptibility testing, whole-genome sequencing analysis, bacterial conjugation, and transformation experiments were performed for both KPC-2 producers. The blaKPC-2 gene was located on the Tn3 transposon-related region from an IncP-6 replicon plasmid that could not be transferred via conjugation. Compared to the blaKPC-2-encoding plasmid of the C. freundii isolate, alignment analysis of plasmids with blaKPC-2 showed that the blaKPC-2-encoding plasmid of the K. variicola isolate was a novel IncP-6/IncF-like hybrid plasmid containing a 75,218-bp insertion sequence composed of IncF-like plasmid conjugative transfer proteins. Carbapenem-resistant transformants harboring blaKPC-2 were obtained for both isolates. However, no IncF-like insertion region was found in the K. variicola donor plasmid of the transformant, suggesting that this IncF-like region is not readily functional for plasmid conjugative transfer and is maintained depending on the host cells. The findings on the KPC-2 producers and novel genetic content emphasize the key role of hospital sewage as a potential reservoir of pathogens and its linked dissemination of blaKPC-2 through the hospital water environment. Our results indicate that continuous monitoring for environmental emergence of antimicrobial-resistant bacteria might be needed to control the spread of these infectious bacteria. Moreover, it will help elucidate both the evolution and transmission pathways of these bacteria harboring antimicrobial resistance. IMPORTANCE Antimicrobial resistance is a significant problem for global health, and the hospital environment has been recognized as a reservoir of antimicrobial resistance. Here, we provide insight into the genomic features of blaKPC-2-harboring isolates of Citrobacter freundii and Klebsiella variicola obtained from hospital sewage in Japan. The findings of carbapenem-resistant bacteria containing this novel genetic context emphasize that hospital sewage could act as a potential reservoir of pathogens and cause the subsequent spread of blaKPC-2 via horizontal gene transfer in the hospital water environment. This indicates that serial monitoring for environmental bacteria possessing antimicrobial resistance may help us control the spread of infection and also lead to elucidating the evolution and transmission pathways of these bacteria.

Dataset Information

Interplay among IncA and bla KPC-Carrying Plasmids in Citrobacter freundii.

Publications

Interplay among IncA and <i>bla</i><sub>KPC</sub>-Carrying Plasmids in <i>Citrobacter freundii</i>.

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