Occurrence of Acinetobacter calcoaceticus- baumannii complex in municipal wastewater

Background: Acinetobacter calcoaceticus- baumannii complex (ABC) includes six genetically closely related but phenotypically undistinguishable species: A. calcoaceticus, A. baumannii, A. pittii, A. nosocomialis, genomic species “close to 13TU”, and genomic species “between 1 and 3”. A. baumannii is most often studied as an emerging hospital pathogen. Reports on the occurrence of ABC in wastewater treatment plants are scarce. The objective of this study was to screen the municipal wastewater of Zagreb, the capital of Croatia, for the presence of viable ABC and for its possible discharge into a natural recipient after the wastewater passage through the treatment process. Material/methods: The sampling was done at the secondary type of wastewater treatment plant of the City of Zagreb (capacity 1, 200.000 population equivalents) where the municipal wastewater treated consists of domestic, industrial, hospital and storm wastewaters. The composite 24h samples of the influent and effluent wastewater were collected during 6 months in 2014. The isolation of ABC from wastewater was performed at 42C/48h on CHROMagar Acinetobacter with or without the addition of commercial supplement CR102 (CHROMagar) allowing the growth of carbapenemresistant isolates. Presumptive ABC colonies were characterized phenotypically by routine bacteriological techniques and by using Vitek 2 system. Further identification was carried out by MALDI-TOF MS and RNA polymerase β-subunit (rpoB) gene analyses. Antibiotic resistance profiles were determined for carbapenems meropenem and imipenem by Vitek 2 system and interpreted according to EUCAST criteria. Results: On 6 sampling occasions 34 isolates belonging to ABC, as determined by Vitek 2 system, were recovered: 28 from influent and 6 from effluent wastewater. The comparison of ribosomal proteins by MALDI-TOF MS with strains of bacteria in MALDI Biotyper database gave the score values form 2.013 - 2.409 identifying the ABC isolates as A. baumannii or A. pittii. Phylogenetic analysis of the rpoB gene fragment confirmed the identity of isolates as A. baumannii or A. pittii and showed their close relatedness to the clinical isolates with 100% sequence ID. Molecular identification of ABC confirmed that 22 and 6 isolates of A. baumannii were recovered from influent and effluent wastewater, respectively, while 6 A. pittii were recovered only from influent wastewater. The 28/34 isolates (27 A. baumannii and 1 A. pittii) were resistant to carbapenems. Conclusions: Municipal wastewaters of Zagreb are continuously polluted with ABC probably due to the input of untreated hospital wastewaters. Among the ABC, only A. baumannii and A. pittii species are present with A. baumannii as a predominant species. More frequent isolation of A. baumannii from influent than from effluent suggests its moderate elimination, but also its persistence in the secondary type wastewater treatment system. The absence of A. pittii isolation from effluent suggests its complete elimination in the secondary type of wastewater treatment plant.