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Infections caused by extensively drug-resistant (XDR) Acinetobacter nosocomialis have become a challenging problem. The frequent use of colistin as the last resort drug for XDR bacteria has led to the emergence of colistin-resistant A. nosocomialis (ColRAN) in hospitals. The mechanism of colistin resistance in A. nosocomialis remains unclear. This study aimed to investigate the mechanisms underlying colistin resistance in clinical ColRAN isolates. We collected 36 A. nosocomialis isolates from clinical blood cultures, including 24 ColRAN and 12 colistin-susceptible A. nosocomialis (ColSAN). The 24 ColRAN isolates clustered with ST1272 (13), ST433 (eight), ST1275 (two), and ST410 (one) by multilocus sequence typing. There was a positive relationship between pmrCAB operon expression and colistin resistance. Further analysis showed that colistin resistance was related to an amino acid substitution, Ser253Leu in PmrB. By introducing a series of recombinant PmrB constructs into a PmrB knockout strain and protein structural model analyses, we demonstrated that the association between Ser253Leu and Leu244 in PmrB was coupled with colistin resistance in ColRAN. To the best of our knowledge, this is the first study demonstrating that the key amino acid Ser253Leu in PmrB is associated with overexpression of the pmrCAB operon and hence colistin resistance. This study provides insight into the mechanism of colistin resistance in A. nosocomialis .

This study aimed to investigate the mechanisms of colistin resistance in 64 Acinetobacter baumannii isolates obtained from patients with ventilator-associated pneumonia hospitalised in Greece, Italy and Spain. In total, 31 A. baumannii isolates were colistin-resistant. Several novel amino acid substitutions in PmrCAB were found in 27 colistin-resistant A. baumannii. Most substitutions were detected in PmrB, indicating the importance of the histidine kinase for colistin resistance. In two colistin-resistant isolates, 93 amino acid changes were observed in PmrCAB compared with A. baumannii ACICU, and homologous recombination across different clonal lineages was suggested. Analysis of gene expression revealed increased pmrC expression in isolates harbouring pmrCAB mutations. Complementation of A. baumannii ATCC 19606 and ATCC 17978 with a pmrAB variant revealed increased pmrC expression but unchanged colistin MICs, indicating additional unknown factors associated with colistin resistance. Moreover, a combination of PmrB and PmrC alterations was associated with very high colistin MICs, suggesting accumulation of mutations as the mechanism for high-level resistance. The pmrC homologue eptA was detected in 29 colistin-susceptible and 26 colistin-resistant isolates. ISAba1 was found upstream of eptA in eight colistin-susceptible and one colistin-resistant isolate and eptA was disrupted by ISAba125 in two colistin-resistant isolates. Whilst in most isolates an association of eptA with colistin resistance was excluded, in one isolate an amino acid substitution in EptA (R127L) combined with a point mutation in ISAba1 upstream of eptA contributed to elevated colistin MICs. This study helps to gain an insight into the diversity and complexity of colistin resistance in A. baumannii., (Copyright © 2019 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.)

Acinetobacter baumannii is a highly versatile nosocomial pathogen. Multidrug resistance among A. baumannii isolates led to the use of colistin, subsequently giving rise to colistin-resistant strains. In this study, the genetic and phenotypic profiles of two colistin-resistant A. baumannii isolates were investigated. Two A. baumannii isolates were obtained from Patient 1 (C071 and C440) and three isolates were obtained from Patient 2 (C080, C314 and C428). Susceptibility profiles were determined by VITEK ® 2 and Etest. Clonality was determined by RAPD analysis and trilocus multiplex PCR. The pmrCAB operon was sequenced and common carbapenemase genes were screened for by PCR. Doubling times, haemolysis, surface motility, biofilm formation, siderophore production and proteolytic activity were phenotypically determined. Finally, whole-genome sequencing was performed for all five isolates. Isolates C440 and C428 were resistant to colistin and were clonally identical to their sensitive counterparts. The cause of colistin resistance was traced to the previously described P233S mutation in pmrB of C440 and to a novel ΔI19 mutation in pmrB of C428. bla OXA-58-like and bla GES-5 from the strains of Patients 1 and 2, respectively, were also detected. C440 had attenuated proteolytic activity and was positive for siderophore production compared with C071. No difference in in vitro virulence was detected between isolates C080, C314 and C428. In conclusion, one common and one novel mutation were encountered in pmrB from two distinct colistin-resistant A. baumannii isolates. These mutations caused colistin resistance during therapy in two distinct clones, and only one of them had altered in vitro virulence., (Copyright © 2017 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.)