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If not properly treated, hospital wastewaters become a route for dissemination of antibiotic-resistant bacteria to the environment. Even with disinfection, many hospital bacteria survive classical methods such as chlorination or UV irradiation. A novel, effective and economically feasible methods for removal of such bacteria from wastewaters would be desirable. One of the prominent hospital pathogens is bacterium Acinetobacter baumannii. Due to its extraordinary resistance to many classes of antibiotics, especially to carbapenems that were for long time the “last resort” drug, it has become common to detect multiple -, extensive- and even pan-drug resistant isolates of A. baumannii worldwide, both in hospital settings but also in natural environments. Over the last few years, it has been shown without a doubt that in Croatia, A. baumannii along with other Gram-negative pathogens readily disseminates to surface waters via hospital wastewaters, and that it readily passes wastewater treatment plant. Therefore, we tested natural mineral zeolite, enriched with silver by ion-exchange (AgNZ) as a substrate for disinfection of wastewater. The antibacterial efficacy of AgNZ was tested in flow system (biofilter) with pure bacterial cultures, but also with real effluent wastewater from Zagreb wastewater treatment plant. In experiments with real wastewater, we measured the numbers of all carbapenem-resistant bacteria, not just A. baumannii. The biofilter filled with AgNZ and constantly fed with bacterial suspension at 30 ml h-1 flow rate eliminated all of the A. baumannii cells (100 % removal) during 4 days of the experiment and remained effective by removing up to 3 Log values for 14 days. In experiments with real wastewater, the biofilter was effective in removing 90-100 % of carbapenem-resistant bacteria during 4 days. By using X-Ray microtomography it was also shown that particle size of biofilter filling is important from technological point of view, as small sized particles caused biofilter breakthrough by causing preferential flow-paths to emerge. However, after regeneration the biofilter became active again suggesting possile long-time use of AgNZ as biofilter filling. Here presented study is a contribution to “One Health” approach as it proposes a sustainable step forward in reduction of antibiotic resistance dissemination from hospitals to the environment and vice-versa.

Acinetobacter baumannii is an emerging human pathogen. Biofilm formation is an important virulence factor which contributes to the pathogenesis of A. baumannii, but data regarding the isolates recovered from the natural environment are missing. The biofilm formation at the solid-liquid/air-liquid interfaces of environmental isolates of A. baumannii, as well as the influence of natural zeolitizied tuff (NZ) on its formation was determined. In total, 24 environmental isolates of A. baumannii (14 multi-drug resistant, 10 sensitive to antibiotics) were recovered from different stages of the secondary type of municipal wastewater treatment plant. Isolates sensitive to antibiotics were statistically significantly more hydrophobic and formed stronger biofilm than multi-drug resistant isolates. Biofilm formation was significantly positive correlated with hydrophobicity of cells. Biofilm formation at solid-liquid interface was significantly inhibited by the addition of 1% of NZ into the growth medium due to the immobilization of bacterial cells onto the NZ particles. Biofilm formation at air- liquid interface was inhibited only by the addition of 10% of NZ. NZ could find application in control of the biofilm formation of this pathogen on abiotic surfaces.

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.

Over the last decade hospital-acquired infections due to Acinetobacter baumannii are increasing worldwide. Clinical isolates of A. baumannii are usually multi-drug resistant (MDR), with resistance to carbapenems increasing drastically in Croatia. The most important mechanism of carbapenem resistance in A. baumannii is the enzymatic hydrolysis mediated by oxacillinases encoded by blaOXA genes. Although A. baumannii has been isolated from patients and hospital environment during outbreaks, its epidemiology remains incompletely understood. Here we report the finding of MDR carbapenem resistant A. baumannii in treated municipal wastewater related to clinical isolates. The isolate of A. baumannii named EF2 was recovered from the secondary treated municipal wastewater of the City of Zagreb. The isolation was performed at 42°C/48h on CHROMagar Acinetobacter supplemented with CR102 and cefsulodin sodium salt hydrate. By phenotypical analyses and Vitek 2 system the isolate was determined as A. calcoaceticus-baumannii complex. MALDI-TOF MS analysis gave the reliable score value of 2.352 identifying it as A. baumannii. Phylogenetic analysis of the RNA polymerase β-subunit (rpoB) gene fragment confirmed the identity as A. baumannii species and showed 100% sequence ID to the clinical isolates. According to MIC values determined by Vitek 2 system or E-test and interpreted according to EUCAST criteria, isolate was susceptible to amikacin, trimethoprim- sulfamethoxazole and colistin, but resistant to carbapenems (imipenem, meropenem), fluoroquinolones (ciprofloxacin, levofloxacin), aminoglycosides (gentamicin, tobramycin) and therefore could be classified as MDR. Multiplex PCR and sequencing confirmed the presence of both intrinsic blaOXA-51 and acquired blaOXA-23 genes. Phylogenetic analyses of blaOXA genes from environmental isolate showed association with those previously described from clinical isolates. Isolate multiplied in the batch system containing autoclaved water from which it was recovered up to 50 days of monitoring at 16.7°C, when its number was 9% higher than initial number. These results suggest that MDR A. baumannii recovered from treated municipal wastewater is most probably of clinical origin and is able to survive in environment outside hospital.