Role of multidrug efflux systems in antimicrobial resistance in Acinetobacter Baumannii clinical isolates from humans and animals

Abstract

In this study, we aimed to determine the role of multidrug efflux systems in antimicrobial resistance in Acinetobacter baumannii isolates from humans and animals. For the human isolates 100 Acinetobacter spp. were obtained from patients admitted at Siriraj hospital and King Chulalongkorn Memorial hospital. For the animal isolates total of 210 animal samples were collected from animal carcasses submitted for necropsy at Pathology unit, Faculty of Veterinary Science, Chulalongkorn University and 30 A. baumannii were obtained. All human (n=100) and animal (n=30) isolates were confirmed as the baumannii species by using Amplified Ribosomal DNA Restriction Analysis (ARDRA). i) All the isolates were tested for antimicrobial susceptibilities to 15 clinically important antimicrobials, the contribution of multidrug efflux pump in the presence and absence of reserpine and carbonyl cyanide m-chlorophenylhydrozone (CCCP) and expression of multidrug efflux pumps. Most human (98%) and animal (70%) isolates were MDR. Fifty three percent of the human isolates and 6.7% of the animal isolates were resistant to all drugs tested. Effect of CCCP was more potent than reserpine enhancing of antimicrobial activity and regaining antimicrobial susceptibility. AdeB-AdeG-AdeJ was the predominant expression pattern and no adeE expression was observed in neither human nor animal isolates. ii) Twenty-five clinical isolates (10 human and 15 animal isolates), which cover all expression pattern were selected for determine transcription level of resistance-nodulation-cell division (RND) efflux systems by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) and characterized for the regulatory mutations by DNA sequencing analysis. No overexpression of adeB was found in any human and animal isolates. Overexpression of adeG was observed in 2 and 10 of human and animal isolates, respectively. No mutation was identified in AdeL of all isolates overexpressing adeG. Expression level of adeJ was 0.4 to 5.0 fold and 0.1 to 16.7 fold in human and animal isolates, respectively. Therefore, the existence of additional regulatory mechanisms on AdeABC, AdeFGH and AdeIJK expression other that were not characterized in this study is suggested. iii) The isolates with low MIC values of most antimicrobial agents tested (10 human and 15 animal isolates) were selected for characterization of the contribution of multidrug efflux systems in biocide-antibiotic cross-resistance by step-wise passage to benzalkonium chloride, chlorhexidine and triclosan. Five and 12 triclosan-resistant mutants were obtained from the human and animal isolates, respectively. These triclosan-resistant mutant derivatives were additional resistant to several antibiotics. Expression level of efflux pumps was detected and was not corresponding to MIC level. The presence of mutation in AdeL and AdeN did not correlate to the expression of the corresponding efflux pumps. This suggested that transcription of RND efflux pumps could not be the sole mechanism for acquired resistance of triclosan in A. baumannii clinical isolates. In conclusion, the result showed that there is the wide spread of MDR A. baumannii and the wide distribution of RND efflux systems among the human and animal clinical isolates. Exposure to triclosan can promote cross-resistance to antibiotics, leading to multidrug resistance in A. baumannii. The data affirmed that the appropriate use of antimicrobials and biocides in human and veterinary medicine should be advocated.