Environmental Carbapenem-Resistant Acinetobacter baumannii Isolated from Municipal Wastewater Treatment Plant in South Korea

Abstract

The emergence and dissemination of antibiotic resistant bacteria and genes (ARB and ARGs, respectively) due to worldwide overuse of antibiotics poses a major threat to human health. Especially, carbapenem-resistant Acinetobacter baumannii (CRAB) grows attention as one of the leading hazardous pathogens. The aim of this study is to compare the sequence types, antibiotic resistance genes and resistant patterns of clinical and environmental A. baumannii (CAB and EAB, respectively) and environmental non-baumannii (ENBA) isolates. The CAB isolates were provided from the National Culture Collection for Pathogens (NCCP) isolated from patients from Korean hospitals and EAB and ENBA isolates were recovered from municipal wastewater treatment plants (WWTPs) in South Korea. A total of three Acinetobacter species isolates were isolated from three major municipal WWTPs (1st and 2nd Gwangju WWTP and Yeongcheon WWTP) in Gwangju and Yeongcheon region in South Korea, respectively from 2016 to 2018. EAB and ENBA isolates were recovered from effluent samples from 1st Gwangju WWTP in 2016, influent samples from 2nd Gwangju WWTP in 2017 and effluent sample of Yeongcheon WWTP in 2018, respectively. Identification and molecular characterization of CAB and EAB isolates by 16S rRNA sequencing, gyrB and rpoB gene alignment, multilocus sequence typing (MLST) and biochemical test confirmed two environmental and four clinical isolates as A. baumannii. Every isolate was determined as multidrug resistant including carbapenems and possesses at least one virulence gene. Although minimum inhibition concentrations (MICs) of other antibiotics excluding carbapenems were lower in environmental isolates, MIC of carbapenems was the highest in WWTP influent isolate, strain 17GI. Among seven isolates tested for detection of blaOXA genes, only A. baumannii isolates contained chromosomal blaOXA-51-like gene. Also, detection of blaOXA genes resulted that strain 17GI possess blaOXA-72 gene which were first found from both CAB and EAB isolated in South Korea. Phenotypically, production of carbapenemases were detected by modified Hodge test and Triton Hodge test, resulting production of OXA-type carbapenemases were only detected by Triton Hodge test. Moreover, whole genome sequencing of strain 17GI showed three same blaOXA-72 genes located in the plasmid and RT-qPCR result indicated that these genes were expressed even in carbapenem-free conditions. In summary, our finding suggested that WWTPs can be a possible dissemination source of carbapenem-resistant nosocomial Acinetobacter and the aquatic environment can play a role as a reservoir for carbapenemases from Acinetobacter species. For instance, carbapenemase can be transfer to other pathogenic bacteria by horizontal gene transfer causing severe public health problem. Therefore, these results indicate that multi-drug resistant A. baumannii can be spread not only from hospital environments but also from WWTPs and environment. Thus, continuous surveillance of dissemination of species in the aquatic environment is urgent.