Investigation of efflux-mediated tetracycline resistance in Shigella isolates using the inhibitor and real time polymerase chain reaction method

Abstract

Background: Shigella spp. are gram negative bacteria, which are of global public health importance. The growing of multidrug-resistant Shigella isolates are a major problem around the world. Methods: Overall, 50 isolates of Shigella spp. from children diarrheic stools were studied. The isolates were identified and confirmed using biochemical, serological and molecular methods (ipaH, wbgZ and rfc genes). Antimicrobial susceptibility test was done according to the Clinical and Laboratory Standards Institute (CLSI) guidelines against minocycline, tetracycline, doxycycline, ampicillin, streptomycin, trimethoprim-sulfamethoxazole, nalidixic acid, norfloxacin, ciprofloxacin and levofloxacin. Also, the role of efflux pump in defense of Shigella against tetracycline was investigated by Minimum Inhibitory Concentration (MIC) with and without an efflux pump inhibitor. Detection of tetA, tetB, tetC and tetD genes in Shigella was evaluated by conventional Polymerase Chain Reaction (PCR) and real time PCR. Results: Molecular identification revealed a prevalence of 14 for Shigella flexneri and 86 for Shigella sonnei. Minimum Inhibitory Concentration (MIC) of 90 of resistant isolates was changed in the presence CCCP. Results of conventional PCR exhibited that 66 of isolates were positive for tetA, while according to real time PCR method, 90 of isolates carried tetA. Positive results for tetB were 12 and 18 by conventional and real time PCR methods, respectively. No positive results were detected for tetC and tetD. Also, tetB was detected only in S. flexneri while tetA was detected in both S. flexneri and S. sonnei. Conclusion: It seems that efflux-mediated tetracycline resistance to tetracycline in S. flexneri can be related to tetB, however resistance in S. sonnei can be related to the expression of tetA. © 2016, IRANIAN JOURNAL OF PATHOLOGY