Disk diffusion

Ethical approval for this study was obtained from the Malaysian Ministry of Health’s National Medical Research Register (approval no. NMRR-14-1650-23625-IIR). A. baumannii AC1633 and A. nosocomialis AC1530 were isolated from Hospital Sultanah Nur Zahirah, Kuala Terengganu, Malaysia in 2016 and 2015, respectively. Species identification of both isolates was performed by sequencing of the rpoB gene as previously described (13 (link), 71 (link)). Antimicrobial susceptibility profiles of both isolates were determined using a panel of 22 antibiotics recommended for Acinetobacter spp. (26 (link)) and by disk diffusion (Oxoid Ltd., Basingstoke, UK) on Mueller-Hinton (MH) agar, except for colistin and polymyxin B, which were determined by obtaining the MIC values by the agar diffusion method (25 (link)). Carbapenem resistance was validated by determining the MIC values for imipenem, meropenem and doripenem using M.I.C. Evaluator strips (Oxoid Ltd., Basingstoke, UK). Results were interpreted according to the Clinical and Laboratory Standards Institute (CLSI) guidelines (72 ). Production of metalo-β-lactamases was determined using the Etest MBL kit (bioMérieux, La Balme-les-Grottes, France).

Antimicrobial susceptibility testing (AST) for A. baumannii isolates was carried out according to the standard disk diffusion method (Oxoid, Basingstoke, UK) on Mueller-Hinton agar plates (bioMérieux, Marcy-l’Étoile, France), during which, the following antibiotics were tested: aminoglycosides (gentamicin [10 µg disk], amikacin [30 µg disk]), carbapenems (imipenem [10 µg disk], meropenem [10 µg disk]), fluoroquinolones (ciprofloxacin [5 µg disk], levofloxacin [5 µg disk]), trimethoprim-sulfamethoxazole [23.75/1.25 µg disk] and colistin [10 µg disk]. With the exception of colistin, interpretation of the results was carried out according to the standards and breakpoints of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) v. 11.0 [30 ]. Results indicating “susceptible, increased exposure (I)” were grouped with and reported as susceptible (S) [31 ]. Susceptibility to colistin was assessed according to the provisional breakpoints, as advised by Galani et al. [32 (link)]. Classification of the isolates as MDR (i.e. resistant to at least one agent in ≥3 antibiotic groups) was based on the recommendations of Magiorakos et al. [33 (link)].

Susceptibilities to ampicillin, cefotaxime, ceftazidime, cefoxitin, ciprofloxacin, imipenem and meropenem were tested for all E. coli isolates by disk diffusion (Oxoid Ltd., Basingstoke, United Kingdom) according to the manufacturer’s instructions, and interpretation was done according to EUCAST criteria (http://www.eucast.org/clinical_breakpoints; v7.1). The phylogenetic group of the E. coli isolates was determined by a PCR-based assay [72 (link)]. The presence of β-lactamase genes (bla_TEM, bla_SHV, bla_{CTX-M-group-1-2-9}, and bla_CMY) was confirmed by PCR and sequencing as previously described [73 (link), 74 (link)]; only CMY-2 positive isolates were included in the present study.