Source of bacterial isolates and identification as A.baumannii
Bacterial isolates identified as A.baumannii on the basis of biochemical tests were subjected to PCR assay for the amplification of gyrB and rpoB genes to confirm the clinical isolates genotypically. All isolates revealed the presence of 294 and 490 bp and 350 bp bands compatible to the presence of gyrB and rpoB genes respectively and confirming them as A.baumannii. The source of these isolates was: wound (33.9%), endotracheal aspirate (28.6%), blood (19.6%), urine (10.7%), broncho-alveolar lavage (4.5%) and IV catheter (2.7%) obtained from patients admitted in various wards: urology (n=5), burn (n=5), infectious diseases (n=5) and different ICU wards (n=97) (Supplementary Table 2).
Disk agar diffusion assay and phenotypic detection of antibiotic resistant profile
When results of disk agar diffusion were analyzed, all A.baumannii isolates could be grouped in four antimicrobial resistant profiles (Figure 1). Noteworthy, all A.baumannii isolates were found resistant to ceftazidime, cefotaxime, ceftriaxone, imipenem, meropenem, doripenem, ciprofloxacin, levofloxacin, trimethoprim-sulfamethoxazole, and piperacillin/tazobactam. Thus, all isolates were considered MDR and CRAB strains. In addition, resistance to gentamicin, and amikacin, tobramycin and ampicillin/sulbactam was observed in 72.3% (n=81), 64.3% (n=72), 48.2% (n=54) isolates, respectively. Among them, 51.8% (n=58) isolates showed resistant to all classes of antibiotics except colistin and ampicillin/sulbactam, and thus, were designated as XDR strains.
Antimicrobial susceptibility testing by MIC assay
To determine the lowest concentration of carbapenems and ampicillin-sulbactam that prevents visible growth of a microorganism, MIC assay was performed using E-test strips. All A.baumannii isolates were confirmed resistant to imipenem, meropenem, and doripenem (MIC>32µg/mL) thus defining them as CRAB while all isolates retained their susceptibility towards ampicillin- sulbactam (MIC<2µg/mL). When A.baumannii isolates were checked for their susceptibility towards colistin by broth dilution method, all isolates were observed susceptible (MIC<2µg/mL).
Screening of CRAB strains for carbapenemase genes and insertion sequence ISAba1
All CRAB strains were screened for the presence of OXA genes (blaOXA-51-like, blaOXA-23-like, blaOXA-24/40-like, blaOXA-58-like, blaOXA-143-like and blaOXA-235-like), MBLs genes (blaIMP, blaVIM, blaSIM, blaGIM, blaSPM and blaNDM) and the insertion sequence ISAba1 so as to assess the origin of carbapenem resistance in the above mentioned strains by performing either uniplex or multiplex PCRs depending upon the PCR amplification conditions.
All CRAB strains were positive for the blaOXA-51-like gene. Presence of blaOXA-23-like and blaOXA-24/40-like genes was observed in 82.1% (n=92) and 36.6% (n=41) CRAB strains respectively. No CRAB strain was positive for blaOXA-58-like, blaOXA-143-like or blaOXA-235-like. Co-existence of blaOXA-23-like, blaOXA-51-like and blaOXA-24/40-like was detected in 29 (25.8%) CRAB strains. Additional screening for MBLs genes detected presence of blaNDM (6.2%; n=7) and blaIMP (4.5%; n=5) while, no CRAB strain was observed positive for blaVIM, blaSIM, blaGIM and blaSPM. The ISAba1 element was present in the majority (95.5%; n=107) of CRAB strains and found to be upstream of blaOXA-23-like in 64 (69.5%) blaOXA-23-like -producing CRAB strains. However, the ISAba1 element was not found upstream of blaOXA-51-like positive CRAB strains.
Quantitative real time experiment for adeB and adeJ efflux pump genes expression
CRAB strains were also looked up for the expression of efflux pump adeB and adeJ genes by quantitative real time PCR. The assay showed higher expression of adeB and adeJ genes (1.1-5.6 and 1.0-2.6 fold respectively) in 66% (n=74) and 42.8% (n=48) CRAB strains respectively, compared to A.baumannii ATCC 19606 strain (Figure 2).
Quantitative real time experiment for the expression of carO, omp33-36, oprD porin genes
When CRAB strains were analyzed for the expression of porin genes, 84 (75%), 74 (66%) and 81 (72.3%) CRAB strains showed decreased carO, omp33-36 and oprD expression level respectively [expression levels being 0.06-0.90 fold, 0.01-0.93 fold and 0.01-0.97 fold, respectively] than that observed in A.baumannii ATCC 19606 strain (Figure 2).
Correlation between altered expression of efflux pumps and porin genes with oxacillinase and metallo-β-lactamases
All isolates with increased expression of adeB (n=72) and adeJ (n=48) were resistant to carbapenems. Among the isolates with increased of adeB expression, 87.8% (n=65) encoded blaOXA-23-like, 44.6% (n=33) blaOXA-24/40-like, 1.3% (n=1) blaIMP and 8.1% (n=6) blaNDM. Similar result was observed among isolates with adeJ over-expression, with 85.4% (n=41) encoding blaOXA-23-like, 43.7% (n=21) blaOXA-24/40-like, 0% (n=0) blaIMP and 10.4% (n=5) blaNDM, respectively (Figure 3). However, no significant correlation was observed between over expression (>1 fold) of adeB and adeJ genes and resistance to carbapenems, when using either χ2 test or studentʼs t test.
All isolates with decreased expression of carO (n=84), omp33-36 (n=74) and oprD (n=81) were resistant to carbapenems. Among isolates with decreased of carO expression, 82.1% (n=69) carried blaOXA-23-like, 31% (n=26) blaOXA-24/40-like, 5.9% (n=5) blaIMP and 5.9% (n=5) blaNDM genes. Alike outcome was observed among isolates with reduced omp33-36 expression, with 79.8% (n=59) encoding blaOXA-23-like, 31% (n=23) blaOXA-24/40-like, 6.7% (n=5) each blaIMP and blaNDM genes. Among isolates with decreased expression of oprD 81.4% (n=66) carried blaOXA-23-like, 28.3% (n=23) blaOXA-24/40-like, 6.1% (n=5) each blaIMP and blaNDM genes (Figure 4). Compatible to outcomes of efflux pump genes, no significant correlation was observed between decreased expression (<1 fold) of carO, omp33-36, oprD genes and resistance to carbapenems when using either χ2 test or Studentʼs t test.
Sequence typing of CRAB strains
CRAB strains were typed for knowing the epidemiological type of origin. Multiplex PCR performed for the identification of sequence groups (SGs) revealed 57 (50.9%) CRAB strains belonged to (sequence group 1) SG1 [European clone (EU) clone II], majority (85.9%; n=49) being recovered from ICU patients. Seven (6.3%) isolates belonged to SG2 (EU clone I) while, six (5.4%) isolates belonged to the SG3 (EU clone III). Furthermore, 42 (37.5%) isolates belonged to new variants of SGs (SG4-SG-9). No significant correlation was observed between carbapenemase genes distribution and the three main sequence groups (Table 1).