Susceptibility profile of bla OXA-23 and metallo-β-lactamases co-harbouring isolates of carbapenem resistant Acinetobacter baumannii (CRAB) against standard drugs and combinations

Abstract

Background: The rapid emergence of carbapenem resistant Acinetobacter baumannii (CRAB) has resulted in an alarming situation worldwide. Realizing the dearth of literature on susceptibility of CRAB in genetic context in the developing region, this study was performed to determine the susceptibility profile against standard drugs/combinations and the association of in-vitro drug synergy with the prevalent molecular determinants.

Methods and findings: A total of 356 clinical isolates of A. baumannii were studied. Confirmation of the isolates was done by amplifying recA and ITS region genes. Susceptibility against standard drugs was tested by Kirby Bauer disc diffusion. Minimum inhibitory concentration (MIC), MIC₅₀ and MIC₉₀ values against imipenem, meropenem, doripenem, ampicillin/sulbactam, minocycline, amikacin, polymyxin B, colistin and tigecycline was tested as per guidelines. Genes encoding enzymes classes A (bla _GES, bla _IMI/NMC-A, bla _SME, bla _KPC), B (bla _IMP, bla _VIM, bla _NDM) and D (bla _OXA-51, bla _OXA-23 and bla _OXA-58) were detected by multiplex polymerase chain reaction. Synergy against meropenem-sulbactam and meropenem-colistin combinations was done by checkerboard MIC method. Correlation of drug synergy and carbapenemase encoding genes was statistically analyzed.

Results: Of the total, resistance above 90% was noted against gentamicin, ciprofloxacin, levofloxacin, ceftazidime, cefepime, ceftriaxone, cotrimoxazole and piperacillin/tazobactam. By MIC, resistance rates from highest to lowest was seen against imipenem 89.04% (n=317), amikacin 80.33% (n=286), meropenem 79.49% (n=283), doripenem 77.80% (n=277), ampicillin/sulbactam 71.62% (n=255), tigecycline 55.61% (n=198), minocycline 14.04% (n=50), polymyxin B 10.11% (n=36), and colistin 2.52% (n=9). CRAB was 317 (89.04%), 81.46% (n=290) were multidrug resistant and 13.48% (n=48) were extensively drug resistant. All the CRAB isolates harboured bla _OXA-51 gene (100%) and 94% (n=298) bla _OXA-23 gene. The bla _IMP gene was most prevalent 70.03% (n=222) followed by bla _NDM, 59.62% (n=189). Majority (87.69%, 278) were co-producers of classes D and B carbapenemases, bla _OXA-23 with bla _IMP and bla _NDM being the commonest. Synergy with meropenem-sulbactam and meropenem-colistin was 47% and 57% respectively. Reduced synergy (p= <0.0001) was noted for those harbouring bla _OXA-51+bla_OXA-23with bla _NDM gene alone or co-producers.

Conclusion: Presence of bla _NDM gene was a significant cause of synergy loss in meropenem-sulbactam and meropenem-colistin. In bla _NDM endemic regions, tigecycline, minocycline and polymyxins could be viable options against CRAB isolates with more than one carbapenemase encoding genes.

Keywords: blaNDM; endemic; meropenem; minocycline; synergy.

Figure 1

Sunburst chart showing distribution of A. baumannii isolates among different departments and clinical specimen.

Figure 2

Representative gel image showing (A) recA and ITS genes in A baumannii; (B) bla _OXA-51 and bla _OXA-23 genes; (C) multiple class B carbapenemases. (A) Lane M: Marker 100 bp; Lane 1-5,6-10: recA (425bp) & ITS gene (208bp); NC: negative control PCR-grade water; PC: positive control A. baumannii ATCC 19606. (B) class D carbapenemase genes; Lane M: Marker 100 bp; Lane 1-12: bla _OXA-51 (353 bp) & Lane 1-12: bla _OXA-23 (501 bp); NC: negative control PCR-grade water; PC: previously confirmed & published isolate positive for bla _OXA-51 & bla _OXA-23 genes (C) class B carbapenamse genes; Lane M: Marker 100 bp; Lane 1-3,6,8, 13, 14: bla _IMP (232 bp), Lane 7-8,10-11, 14: bla _IMP (390 bp) & Lane 2-13, 15: bla _NDM (621 bp); PC: previously confirmed & published isolate positive for bla _IMP, bla _VIM and bla _NDM genes.