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Transcriptional Landscape of a bla KPC-2 Plasmid and Response to Imipenem Exposure in Escherichia coli TOP10.

ABSTRACT: The diffusion of KPC-2 carbapenemase is closely related to the spread of Klebsiella pneumoniae of the clonal-group 258 and linked to IncFIIK plasmids. Little is known about the biology of multi-drug resistant plasmids and the reasons of their successful dissemination. Using E. coli TOP10 strain harboring a multi-replicon IncFIIK-IncFIB bla KPC-2-gene carrying plasmid pBIC1a from K. pneumoniae ST-258 clinical isolate BIC-1, we aimed to identify basal gene expression and the effects of imipenem exposure using whole transcriptome approach by RNA sequencing (RNA-Seq). Independently of the antibiotic pressure, most of the plasmid-backbone genes were expressed at low levels. The most expressed pBIC1a genes were involved in antibiotic resistance (bla KPC-2, bla TEM and aph(3')-I), in plasmid replication and conjugation, or associated to mobile elements. After antibiotic exposure, 34% of E. coli (pBIC1a) genome was differentially expressed. Induction of oxidative stress response was evidenced, with numerous upregulated genes of the SoxRS/OxyR oxydative stress regulons, the Fur regulon (for iron uptake machinery), and IscR regulon (for iron sulfur cluster synthesis). Nine genes carried by pBIC1a were up-regulated, including the murein DD-endopeptidase mepM and the copper resistance operon. Despite the presence of a carbapenemase, we observed a major impact on E. coli (pBIC1a) whole transcriptome after imipenem exposure, but no effect on the level of transcription of antimicrobial resistance genes. We describe adaptive responses of E. coli to imipenem-induced stress, and identified plasmid-encoded genes that could be involved in resistance to stressful environments.

SUBMITTER: Jousset AB

PROVIDER: S-EPMC6286996 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundAntibiotics exert an outstanding selective pressure on bacteria, forcing their chromosomal gene mutations and drug resistance genes to spread. The objective of this study is to evaluate the expression of the New Delhi Metallo-β-Lactamase-1 gene (blaNDM-1) in the clinical isolate (Klebsiella pneumoniae TH-P12158), transformant strains Escherichia coli BL21 (DE3)-blaNDM-1, and Escherichia coli DH5α- blaNDM-1 when exposed to imipenem.Methodsβ-Lactamase genes (blaSHV, blaTEM-1, blaCTX-M-9, blaIMP, blaNDM-1, blaKPC, blaOXA, blaGES, and blaDHA) from randomly selected carbapenems-sensitive K.pneumoniae (n = 20) and E.coli (n = 20) strains were amplified by PCR. The recombinant plasmid of pET-28a harboring blaNDM-1 was transformed into E.coli BL21 (DE3) and E.coli DH5α by electroporation. The resistance phenotype and higher blaNDM-1 expression in K.pneumoniae TH-P12158, transformant E.coli BL21 (DE3)-blaNDM-1, and E.coli DH5α-blaNDM-1 were observed when exposed to imipenem with grade increasing, decreasing, and canceling doses, respectively.ResultsAfter being exposed to different doses of imipenem, the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of antimicrobial drugs and blaNDM-1 expression of strains increased, which was positively correlated with doses of imipenem. On the contrary, with the decrease or cancellation of imipenem doses, the blaNDM-1 expression was deteriorated, while the MIC and MBC values remained relatively stable. These results demonstrated that low doses of imipenem (˂MIC) could press blaNDM-1 positive strains producing stable drug resistance memory and altered blaNDM-1 expression.ConclusionsLow doses of imipenem could press blaNDM-1 positive strains producing sustained resistance memory and altered blaNDM-1 expression. In particular, the positive correlation between the resistance genes expression and antibiotics exposure shows promising guiding significance for clinical medication.

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Transcriptional Landscape of a bla KPC-2 Plasmid and Response to Imipenem Exposure in Escherichia coli TOP10.

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