Abstract
Colistin is one of the last-resort therapeutic agents to combat multidrug-resistant Gram-negative bacteria (GNB) including Klebsiella pneumoniae. Although it happens rarely, resistance to colistin has been reported for several GNB. A total of 20 colistin resistant (col-R) and three colistin susceptible (col-S) clinical isolates of K. pneumoniae were studied to explore the underlying mechanisms of colistin resistance. The presence of plasmid encoded resistance genes, mcr-1, mcr-2, mcr-3, and mcr-4 genes were examined by PCR. The nucleotide sequences of pmrA, pmrB, phoP, phoQ, and mgrB genes were determined. To evaluate the association between colistin resistance and upregulation of pmrHFIJKLM and pmrCAB operons, transcriptional level of the pmrK and pmrC genes encoding for lipopolysaccharide target modifying enzymes was quantified by RT-qPCR analysis. None of the plasmid encoded resistance genes were detected in the studied isolates. Inactivation of MgrB due to nonsense mutations and insertion of IS elements was observed in 15 col-R isolates (75%). IS elements (IS5-like and IS1-like families) most commonly targeted the coding region and in one case the promoter region of the mgrB. Complementation with wild-type MgrB restored colistin susceptibility in isolates with altered mgrB. All col-R isolates lacked any genetic alterations in the pmrA, phoP, and phoQ genes and substitutions identified in the pmrB were not found to be involved in resistance conferring determined by complementation assay. Colistin resistance linked with upregulation of pmrHFIJKLM and pmrCAB operons with the pmrK and pmrC being overexpressed in 20 and 11 col-R isolates, respectively. Our results demonstrated that MgrB alterations are the major mechanisms contributing to colistin resistance in the tested K. pneumoniae isolates from Iran.
Highlights
The emergence of MDR Enterobacteriaceae and the lack of new antibiotics coming to market to combat them bring us perilously to the end of the “Antibiotic era.” Among Enterobacteriaceae, Klebsiella pneumoniae, is found to be associated with the highest rates of carbapenem resistance (Marinelli and Genilloud, 2013)
Polymyxin antibiotics such as colistin are among the few Abbreviations: col-R KP, colistin resistant K. pneumoniae; col-S KP, colistin susceptible K. pneumoniae; CRE, carbapenemresistant Enterobacteriaceae; Gramnegative bacteria (GNB), Gram-negative bacteria; L-Ara4N, 4-amino-4-deoxy-L-arabinose; MDR, multidrugresistant; PEtN, phosphoethanolamine
To assess the role of plasmid encoded resistance genes, mcr-1, mcr-2, mcr-3, and mcr-4, polymerase chain reaction (PCR) was carried out using genomic DNA obtained from 20 col-R, three col-S KP clinical isolates and K. pneumoniae ATCC 700603. mcr genes were not detected in any of the studied isolates indicating that chromosomally mediated mechanisms might play a prominent role in resistance occurrence
Summary
The emergence of MDR Enterobacteriaceae and the lack of new antibiotics coming to market to combat them bring us perilously to the end of the “Antibiotic era.” Among Enterobacteriaceae, Klebsiella pneumoniae, is found to be associated with the highest rates of carbapenem resistance (Marinelli and Genilloud, 2013). A second TCRS, PhoP/PhoQ is known to contribute to polymyxin resistance by indirectly activating the PmrA/PmrB TCRS via PmrD linker protein Mutations in these TCRSs can cause constitutive expression of the pmrCAB and pmrHFIJKLM operons and transfer of PEtN and L-Ara4N, respectively, to lipid A (Tamayo et al, 2005; Olaitan et al, 2014b). Inactivation of the PhoQ/PhoP negative regulator encoded by mgrB gene has been identified to play a prominent role in polymyxin resistance in K. pneumoniae isolates (Cannatelli et al, 2014b; Olaitan et al, 2014b; Poirel et al, 2015) Genetic alterations such as Leu26Pro in PhoQ (Cheng et al, 2015), Leu82Arg (Cannatelli et al, 2014a), and Thr157Pro (Jayol et al, 2014) in PmrB, Q30stop, C28stop and insertion of IS elements in MgrB (Poirel et al, 2015) have been found to contribute to colistin resistance in K. pneumoniae isolates. Due to lack of information about the molecular mechanisms of colistin resistance in K. pneumoniae isolates in Iran, we aimed to explore these mechanisms in colistin resistant K. pneumoniae strains from this geographic region
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