Enterobacterales are known for harboring resistance to various antibiotics, especially to carbapenems. Moreover, treated wastewater are hotspots for their dissemination into the environment. Therefore, this work aims to reveal the occurrence of carbapenemase-producing Enterobacterales from treated wastewater of a wastewater treatment plant (WWTP), in Zagreb, Croatia (Winter 2020).
200 presumptive Enterobacterales species were isolated from treated wastewater (secondary effluent) on CHROMagar mSuperCARBA plates and identified by MALDI-TOF MS. Antimicrobial susceptibility profiles and carbapenemase production was assessed. Carbapenemase and mcr genes were detected by PCR. Genetic relatedness was explored by puls-field gel electrophoresis (PFGE). Chosen isolates underwent whole genome sequencing (WGS) for the determination of resistance genes and multilocus-sequence typing (MLST).
148/200 Enterobacterales from treated wastewater, were confirmed to be carbapenemase-producing Enterobacterales. The predominant taxa included Klebsiella spp. (32 %), Citrobacter spp. (27 %), and Enterobacter spp. (24 %). All isolates were multi-drug resistant, with 29 % resistance to colistin. In a subset of 83 isolates, selected according to their resistance profiles, blaKPC-2, blaNDM-1, and blaOXA-48 were predominant genes, while blaVIM-1 and blaIMP-13 were detected sporadically. Colistin resistance gene mcr-4.3 was detected in three Klebsiella pneumoniae. PFGE revealed five pulsotypes in K. pneumoniae belonging to ST1803, ST3590, and ST1697, while two went undetermined. Four pulsotypes of Klebsiella oxytoca belonged to ST43, ST1697, ST17, and ST68. In Enterobacter spp, eight pulsotypes belonged to ST277, ST339, and ST32. All Escherichia coli belonged to ST8889. Eleven isolates (1 E. coli, 5 K. pneumoniae, 2 K. oxytoca, and 3 Enterobacter spp.) were chosen for WGS, according to their pulsotypes and detected genes. Ten isolates harbored at least one carbapenemase gene in co-occurrence with other β-lactamase genes. All isolates carried resistance genes to aminoglycosides, fluoroquinolones, trimethoprim, sulfonamides, macrolides, and/or biocides. The pan-drug K. pneumoniae harbored mcr-4.3 with co-occurrence of blaOXA-48, blaCTX-M-15, and blaSHV-81 genes.
This is the first report of K. pneumoniae harboring the plasmid-mediated mcr-4.3 gene with the co-occurrence of carbapenemase genes. The improvement of WWTP technologies is needed to minimize the release of highly resistant pathogens into the receiving water bodies which can contribute spread of the resistance.