Expansion of plasmid mediated blaACT-2 among Pseudomonas aeruginosa associated with postoperative infection and its transcriptional response under cephalosporin stress

Objectives: Organisms harboring multiple plasmid mediated β-lactamases are major concerns in nosocomial infections. Among these plasmid mediated β-lactamases, ACT (EBC family) is a clinically important enzyme capable of hydrolyzing broad spectrum cephalosporins. Therefore, the present study was undertaken to determine the prevalence of ACT determinant along with other co-existing β-lactamase genes in P. aeruginosa strains. Methods: A total of 176 Pseudomonas isolates were phenotypically screened for the presence of AmpC β-lactamase by M3DET Method followed by Molecular detection using PCR assay. Transcriptional evaluation of blaACT-2 gene was analyzed by RT-PCR and its transferability was performed by transformation and conjugation. Results: Present study demonstrates the presence of ACT-2 allele among 12 strains of P. aeruginosa. Co-existence of other β-lactamase genes were encountered among ACT-2 harboring strains which includes CTX-M (n=2), SHV (n=3), TEM (n=2), VEB (n=2), OXA-10 (n=1), CIT (n=2) and DHA (n=3). Fingerprinting by REP PCR revealed the isolates harboring ACT-2 to be distinct and these isolates showed high resistance to expanded-spectrum cephalosporins and even to carbapenem group of drugs. This ACT-2 allele was encoded in the plasmid (L/M, FIA, FIB Inc. Group) and conjugatively transferable. Transcriptional analysis revealed a significant increase in ACT-2 expression (483 fold) when induced by ceftriaxone at 4 μg/ml followed by ceftazidime at 8 μg/ml (31 fold) and cefotaxime 4 μg/ml (8 fold). Conclusion: In this study detection of ACT-2 plasmid mediated AmpC β-lactamase along with other β-lactamase genes in clinical isolates of P. aeruginosa represents a serious therapeutic challenge. Therefore, revision in antimicrobial policy is required for effective treatment of patients infected with pathogen expressing this mechanism. J Microbiol Infect Dis 2017; 7(2): 75-82