Insertional inactivation of oprD in carbapenem-resistant strains isolated from burn patients in Tehran, Iran.

In this study, we report the insertion sequence IS21 in the D porin gene of carbapenem-resistant isolates from burn patients in Tehran, Iran. Antibiotic susceptibility tests for isolates were determined. Production of metallo-β-lactamases (MBLs) and carbapenemase was evaluated and the β-lactamase-encoding and aminoglycoside-modifying enzyme genes were investigated by PCR and sequencing methods. The mRNA transcription level of and efflux pump genes were evaluated by real-time PCR. The outer membrane protein profile was determined by SDS-PAGE. The genetic relationship between the isolates was assessed by random amplified polymorphic DNA PCR. In all, 10.52% (10/95) of clinical isolates of harboured the IS21 insertion element in the D gene. The extended-spectrum β-lactamase-encoding gene in IS21-carrying isolates was . PCR assays targeting MBL and carbapenemase-encoding genes were also negative in all ten isolates. The A, A, B and A genes were positive in all IS21 harbouring isolates. The relative expression levels of the X, B, T and D genes in ten isolates ranged from 0.1- to 1.4-fold, 1.1- to 3.68-fold, 0.3- to 8.22-fold and 1.7- to 35.17-fold, respectively. The relative expression levels of the in ten isolates ranged from 0.57- to 35.01-fold, which was much higher than those in the control strain PAO1. Evaluation of the outer membrane protein by SDS-PAGE suggested that D was produced at very low levels by all isolates. Using random amplified polymorphic DNA PCR genotyping, eight of the ten isolates containing IS21 were shown to be clonally related. The present study describes a novel molecular mechanism, IS21 insertion of the D gene, associated with carbapenem resistance in clinical isolates.