Multi-drug resistant gram negative Bacilli in the wastewater of a Lebanese hospital : profiles and mechanisims of resistance

Abstract

In the absence of efficient community sanitation and sewage disposal infrastructure, the risks for transmission of Multi-Drug-Resistant Organisms into the community, mainly hospital isolates, are high and pose a major public health concern. In addition, the inappropriate disposal of antimicrobial agents into the environment contributes to the emergence and spread of resistance. Our main objective was to study the occurrence of Multi-Drug-Resistant Gram Negative Bacilli in one Lebanese hospital sewage water and to explore the related mechanisms of resistance manifested by these isolates. Wastewater samples were collected twice between February and August 2011. Water filtration was performed using 0.45 µm Millipore membranes and screening for resistant Gram Negative Bacilli was done on VACC medium. Antimicrobial susceptibility tests were performed according to KirbyBauer technique. For the phenotypic characterization of the mechanisms of resistance, the following was used: Double Disc Synergy Test for the detection of ESBLs, Cefoxitin with Phenyl-Boronic Acid for the detection of AmpC, EDTA and PBA for the detection of MBL and KPC respectively. NaCl (0.2M ) was used as Oxacillinases inhibitor and Carbonyl cyanide m-chlorophenyl hydrazine (CCCP) as inhibitor of the efflux pump upregulation. For the genotypic characterization of the mechanisms of resistance, the genes blaOxa48, blaKPC, blaIMI-1, blaIMI-2, blaGES, blaVIM-1, blaVIM-2, and blaIMP were detected using PCR. A total of 30 MDR Gram Negative Bacilli were isolated during the period of the study. These included Enterobacter spp, as the most frequently isolated, followed by E.coli, Klebsiella, Citrobacter, Serratia, Pseudomonas and Acinetobacter spp. Eleven strains (36%) were ESBL producers with the highest occurrence in Klebsiella spp. followed by E.coli spp (4) and Enterobacter spp. (5) Chromosomal and plasmidic AmpC were detected respectively in 19 (63%) and 13 (44%) of the isolates. Among the 12 carbapenem resistant isolates (40%), Enterobacter spp. was the most common with 6 isolates producing MBL and 3 producing OXA carbapenemases. Co-existence of several mechanisms of resistance was detected in most isolates (19). Minimal inhibitory concentration of sodium hypochlorite necessary to clear collected wastewater was in some cases as high as 1377μg/ml. Understanding the local epidemiology of resistance in hospitals should include areas of potential resistance such as wastewater and hospital environment.