Bacterial Isolates
The study was conducted at a tertiary care hospital, South India, from January 2014 to July 2015. A total of 103 non-duplicate, consecutive, carbapenem-resistant isolates recovered from invasive infections of Acinetobacter species were included in this study. The isolates were obtained from invasive clinical specimens including blood and endotracheal aspirates (ETAs). The isolates were identified up to the species level as Acinetobacter baumannii calcoaceticus complex (ABCC) by standard biochemical tests and confirmed as Acinetobacter baumannii using blaOXA-51 PCR, which is intrinsic to this species [7].
Antimicrobial Susceptibility Testing
Susceptibility to different classes of antibiotics was determined by the Kirby Bauer disc diffusion method and interpreted according to the Clinical Laboratory Standard Institute guidelines [8, 9]. Antibiotics tested were ceftazidime (30 µg), cefepime (30 µg), piperacillin/tazobactam (100/10 µg), cefoperazone/Sulbactam (75/30 µg), amikacin (30 µg), netilmycin (30 µg), tobramycin (10 µg), aztreonam (30 µg), levofloxacin (5 µg), tetracycline (30 µg), co-trimoxazole (1.25/23.75 µg), imipenem (10 µg), meropenem (10 µg) and polymyxin B (10 µg).
Phenotypic Characterization
Phenotypic detection of carbapenemase was done by the CarbAcineto NP test. In brief, all the study isolates to be tested were grown on a Mueller-Hinton agar plate for 24 h, and the isolated colonies were re-suspended in two 1.5-ml centrifuge tubes (A and B) containing 100 µl NaCl (5 M). In tube A, 100 µl of solution A (phenol red solution with zinc sulfate) was added, and in tube B, 100 µl of solution A with imipenem (6 mg/ml) was added. The tubes were incubated at 37 °C for a maximum of 2 h. The carbapenemase activity was detected by color change in tube B resulting from hydrolysis of imipenem leading to a decrease in pH value [10]. Concurrently, BAA-1705 and BAA-1706 were included as positive and negative controls, respectively.
Molecular Characterization
PCR for the Detection of Carbapenemase Genes
Uniplex PCR was performed to detect the presence of the intrinsic blaOXA-51 gene. Multiplex PCR was performed to detect ESBL genes such as blaTEM, blaSHV, blaPER, blaVEB and blaGES. A second multiplex PCR was performed for MBL genes such as blaKPC, blaIMP, blaVIM, blaSIM, blaNDM and blaSPM. A third multiplex PCR was performed to detect the presence of acquired OXA genes, namely blaOXA-23, blaOXA-24 and blaOXA-58. Uniplex PCR was performed to detect the presence of the insertion sequence (IS) element, ISAba1 and PCR mapping was performed to map the position of the IS element relative to the blaOXA-23 gene.
Primers used in this study were described earlier [11–16]. Briefly, for PCR amplification, 2 µl of template DNA was added to 18 µl reaction containing 10 µl of Qiagen master mix, 2 µl of primer mix (1 µl each of the respective forward and reverse primers) and 6 µl of molecular-grade water. The cyclic conditions for the intrinsic OXA-51 gene were: initial denaturation at 94 °C for 3 min, 35 cycles of 94 °C for 45 s, 57 °C for 45 s and 72 °C for 1 min, followed by extension of 72 °C for 5 min. For ESBL and MBL genes, initial denaturation at 95 °C for 15 min, 30 cycles of 94 °C for 30 s, 59 °C for 1 min 30 s and 72 °C for 1 min 30 s were followed by extension of 72 °C for 10 min.
For acquired OXA genes, the initial denaturation was at 95 °C for 15 min, 30 cycles of 94 °C for 30 s, 52 °C for 1 min 30 s and 72 °C for 1 min 30 s, followed by extension of 72 °C for 1 min 30 s.
For the IS element PCR, briefly, 5 µl of template DNA was added to 20 µl reaction containing 12.5 µl of Hot Start Master Mix, 1 µl each of forward and reverse primer and 5.5 µl of molecular-grade water. For mapping PCR, 5 µl of template DNA was added to a 20-µl reaction containing 12.5 µl of Hot Start Master Mix, 1 µl each of IS forward and OXA-23 reverse primer, and 5.5 µl of molecular-grade water. The cyclic conditions for IS element PCR and mapping PCR were as follows: initial denaturation at 95 °C for 15 min, 35 cycles of 95 °C for 45 s, 56 °C for 45 s and 72 °C for 2 min, followed by extension of 72 °C for 10 min.
This article does not contain any new studies with human or animal subjects performed by any of the authors.