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Comparative genomics of five different resistance plasmids coexisting in a clinical multi-drug resistant Citrobacter freundii isolate.

ABSTRACT: Background:Plasmid-mediated multi-drug resistance (MDR) has been widely found in Citro-bacter freundii. C. freundii P10159 was isolated from a human case of postoperative urinary tract infection in a Chinese teaching hospital. Methods:The complete nucleotide sequences of five resistance plasmids pP10159-1, pP10159-2, pP10159-3, pP10159-4 and pP10159-5 from C. freundii P10159 were determined through high-throughput genome sequencing, and then compared with related plasmids sequences. Plasmid transfer, CarbaNP test of carbapenemase activity, and bacterial antimicrobial susceptibility test were performed to characterize resistance phenotypes mediated by these plasmids. Results:pP10159-1 carrying blaNDM-1and pP10159-2 harboring blaIMP-4 plus qnrS1 were almost identical to IncX3 plasmid pNDM-HN380 and IncN1 plasmid pP378-IMP, respectively. The blaKPC-2-carrying plasmids pP10159-3, pHS062105-3 and pECN49-KPC were highly similar to each other, and constituted a novel group of plasmids belonging to an unknown incomparability group. The MDR plasmids pP10159-4 and pP10159-5 had the backbones highly similar to IncHI4 plasmid pNDM-CIT and type 2 IncC plasmid pR55, respectively, but their accessory resistance regions differed from pNDM-CIT and pR55, respectively. The five plasmids from the P10159 isolate contained a total of 24 different genes or gene loci, which contributed to resistance to 13 distinct antibiotic molecules or toxic compounds. Conclusion:This is the first report of co-occurrence of five different resistance plasmids, with determination of their complete sequences. Data presented here provide a deeper insight into co-selection and maintenance of multiple plasmids and an extremely large number of resistance genes in a single bacterial isolate.

SUBMITTER: Ouyang J

PROVIDER: S-EPMC6140695 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Comparative genomics of five different resistance plasmids coexisting in a clinical multi-drug resistant <i>Citrobacter freundii</i> isolate.

Ouyang Jing J Sun Fengjun F Zhou Dongsheng D Feng Jiao J Zhan Zhe Z Xiong Zhijian Z Yang Bo B Liu Zhirui Z Li Tingbo T Tong Yigang Y Xia Peiyuan P

Infection and drug resistance 20180912

<h4>Background</h4>Plasmid-mediated multi-drug resistance (MDR) has been widely found in <i>Citro-bacter freundii</i>. <i>C. freundii</i> P10159 was isolated from a human case of postoperative urinary tract infection in a Chinese teaching hospital.<h4>Methods</h4>The complete nucleotide sequences of five resistance plasmids pP10159-1, pP10159-2, pP10159-3, pP10159-4 and pP10159-5 from <i>C. freundii</i> P10159 were determined through high-throughput genome sequencing, and then compared with rela ...[more]

PMID: 30254476

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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:BackgroundCitrobacter freundii is the most common class of pathogens in the genus Citrobacter and is an important pathogen associated with certain underlying diseases or immune dysfunction. The aim of this study was to elucidate the resistance mechanism of clinically derived carbapenem-resistant C. freundii isolate and to characterize the genetic environment and delivery pattern of the IncN1 plasmid carrying the bla IMP-4 gene from C. freundii isolate.Materials and methodsWe identified a clinical isolate of C. freundii L91 carrying bla IMP-4 and performed phylogenetic analysis by whole-genome sequencing. The complete genomic sequence of L91 was obtained using the Illumina HiSeq 4000-PE150 and PacBio RS II platforms. Antimicrobial susceptibility testing was determined by the VITEK 2 system. Plasmid characteristics were presented by S1-pulsed-field gel electrophoresis (PFGE), Southern blotting and conjugation experiments.ResultsS1-PFGE, Southern blot and conjugation assay confirmed the presence of bla IMP-4 genes on a conjugative plasmid in this isolate. C. freundii L91 and transconjugant L91-E. coli 600 strains both showed resistance to carbapenems. In silico analysis further showed that pIMP-4-L91 is an IncN1 plasmid with a length of 51,042 bp. Furthermore, bla IMP-4 gene was found encoded in the bla IMP-4-qacG2-aacA4-catB3 cassette array within a class 1 integron. A conserved structure sequence (ΔISKpn27-bla IMP-4-ΔISSen2-hp-hp-IS6100) was found in the upstream and downstream of the bla IMP-4.ConclusionWe performed a comprehensive phylogenetic analysis of carbapenemase-resistant C. freundii and elucidated the resistance mechanism of clinically derived C. freundii L91. Not only that, we also found that the bla IMP-4 gene is located on the IncN1 plasmid and has a horizontal transfer function and a certain ability to spread. To lower the risk of the dissemination of such C. freundii isolates in clinical settings, more surveillance is needed in the future.

Dataset Information

Comparative genomics of five different resistance plasmids coexisting in a clinical multi-drug resistant Citrobacter freundii isolate.

Publications

Comparative genomics of five different resistance plasmids coexisting in a clinical multi-drug resistant <i>Citrobacter freundii</i> isolate.

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