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A sample of nickel (Ni)-rich clay from Gornje Orešje (POSILOVIĆ, 2009), Croatia was analysed mineralogically and bacteriologically. The light greyish-green sample of alteration product of peridotite mother rocks was recognized as a mixture of kaolinite, montmorillonite, saponite and negligible quantity of quartz and calcite. The abundance of total heterotrophic bacteria grown at 22°C was 7.0±0.8 log colony-forming unit (CFU)/mL of wet clay. The population of carbapenem-resistant bacteria grown at 35°C was present at abundance of 3.9±0.6 log CFU/mL. According to the Bruker MALDI-TOF MS analysis, the population of carbapenem-resistant bacteria was represented by Stenotrophomonas sp. with intrinsic resistance, and Pseudomonas sp. with acquired resistance to carbapenems. The results suggest that Ni-rich clay is a habitat of dense bacterial population, including those resistant to last-resort antibiotics. Scanning Electron Microscopy analysis (Fig. 1) confirmed that bacteria were mostly restricted to the edges of montmorillonite sheets, which probably represent the sites of easy access of bacteria to the cations present in mineral.

Lack of purification of urban wastewaters before its discharge to the natural recipient could represent the epidemiological route for spread of pathogenic bacteria. Currently bacteria resistant to carbapenems has become a worldwide problem (MELETIS, 2016). Three sediment samples were collected during autumn at the mouth of Rio de Minas in Punta Arenas, Chile, South America: two from river flow and one from the Pacific sea. Fine river sediments (sand to silt) were dark brown to black in color, without any smell. X-ray diffraction allowed the identification of mixture of montmorillonite and kaolinite with quartz and albite, while ilmenite was also identified in one sample. Heterotrophic bacteria were found in abundance of 6.2-6.6 log colony-forming unit (CFU)/mL in all three sediment samples. We also found as much as 1.9 and 2.7 log CFU/g of carbapenem-resistant bacteria in river sediments, but they were absent in seawater sediment. The prevalence of carbapenem-resistant bacteria among total heterotrophic bacteria was 29 % and 42 % log CFU. The results are comparable to the prevalence of carbapenem-resistant bacteria (49 % log CFU) reported in landfill leachate (DURN et al., 2014). As confirmed by Bruker MALDI-TOF MS analysis, the carbapenem-resistant bacteria were represented by species of the genus Pseudomonas, which are acquired resistant to carbapenems. Scanning Electron Microscopy confirmed the presence of randomly distributed bacteria (Fig. 1) in the sediment samples. The river sediments under anthropogenic influence of untreated urban and technological wastewaters reveal a significant source of potentially pathogenic carbapenem-resistant bacteria.

INTRODUCTION: Acinetobacter baumannii is a leading emerging pathogen of the 21st century, which is frequently recovered from patients during hospital outbreaks. Acute community-acquired human infections suggest a source of this pathogen outside of the hospital settings. The significance of environmental isolates in the epidemiology of A. baumannii is under a great concern worldwide. THE AIM: In this study the overview of the presence of A. baumannii in natural environment in Croatia is given. METHODS: The recovery of A. baumannii was performed on commercial CHROMagar Acinetobacter supplemented with cefsulodin sodium salt hydrate after incubation at 42C/48h. Identification was performed by routine bacteriological techniques and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) on cell extracts. The antibiotic resistance profile was determined by Vitek2 system and interpreted according to EUCAST and CLSI criteria for clinical isolates of A. baumannii. RESULTS: A single isolate of A. baumannii was incidentally recovered near the City of Pula from 0.1g of acid paleosol influenced by illegally disposed solid waste. The isolate showed multidrug-resistance (MDR) to fluoroquinolones, gentamicin and trimethoprim-sulfamethoxazole. Three isolates of A. baumannii were recovered from 0.01-1g of technosol at a dump site situated above City of Rijeka in a karst pit. All three isolates were MDR and shared the complete or intermediate resistance to carbapenems, fluoroquinolones, amikacin, ampicillin-sulbactam and ticarcillin- clavulanic acid. Four isolates of A. baumannii were recovered from 10mL of water from Sava River downstream of the City of Zagreb after discharge of the urban wastewaters into the natural recipient. All four isolates were MDR and shared the resistance to carbapenems, fluoroquinolones, aminoglycosides and ticarcillin-clavulanic acid. CONCLUSION: MDR A. baumannii were present in natural environment influenced by human solid and liquid waste. The proper management and disposal of human waste is mandatory to prevent the spread of MDR A. baumannii in nature.

Acinetobacter baumannii is well known emerging hospital pathogen of the 21st century. Multi- drug resistant (MDR) A. baumannii are found in untreated as well as in chemically treated hospital wastewaters. However, there is no clear evidence about their origin. The aim of this study was to screen the hospital wastewater for the presence of viable A. baumannii and to find the correlation between recovered wastewater and clinical isolates. The sampling of the hospital wastewater was done at the central manhole of the Special Hospital for Pulmonary Diseases in Zagreb, Croatia. The clinical isolate was recovered on the exact date from bronchial aspirate of patient hospital. The isolation of A. baumannii was performed on CHROMagar Acinetobacter supplemented after incubation at 42°C/48h. One clinical and six isolates from hospital wastewater gave reliable MALDI-TOF MS score values ranging from 2.021-2.271 confirming the identity of A. baumannii colonies. All isolates were MDR and shared the resistance to carbapenems and fluoroquinolones, but sensitivity to colistin. MLST analysis following Oxford scheme revealed that all isolates belong to the ST-195 clustering into the CC92 within the IC2. This study confirmed the occurrence of viable MDR A. baumannii in hospital wastewater in Zagreb. Close relatedness of isolates from hospital wastewater with the clinical isolates from the same hospital in the period of monitoring suggests the possible origin of recovered wastewater A. baumannii. This finding confirms the need for proper treatment and disposal of untreated hospital wastewaters in order to prevent the spread of MDR A. baumannii in nature.

Data on the presence of Acinetobacter baumannii in natural environment influenced by human solid waste are very scarce. There is only one literature report on the incidental finding of one multi-drug resistant (MDR) A. baumannii in paleosol influenced by illegally disposed solid waste. Here we report the finding of three isolates of A. baumannii recovered from technosol at dump site. The dump site is situated above City of Rijeka in Croatia in a karst pit. At this dump site the hazardous industrial waste was continuously disposed from 1956-1990 and further was periodically used as an illegal dump site. Three presumptive colonies were isolated from plates inoculated with 0.01-1g of the surface part of technosol collected at the edge of dump. MALDI-TOF MS score values ranged from 2.000-2.086 for A. baumannii. All three isolates were MDR. They shared the resistance to carbapenems, fluoroquinolones, ticarcillin/clavulanic acid and piperacillin/tazobactam, and resistance or intermediate resistance to amikacin and ampicillin/sulbactam. One isolate was resistant to trimethoprim/sulfamethoxazole and intermediate resistant to minocycline. All three isolates were sensitive to tobramycin, gentamicin and colistin. The proper management and disposal of human solid waste is mandatory to prevent the spread of MDR A. baumannii in nature.

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.

Acinetobacter baumannii is an emerging hospital pathogen causing outbreaks in Croatia since 2002 and is still present in Croatian hospitals. Clinical isolates of A. baumannii in Croatian hospitals are usually multi-drug resistant(MDR), with resistance to carbapenems dramatically increasing from 10% in 2008 to 82% in 2014. Although A. baumannii has been isolated from patients and hospital environment during outbreaks, crucial questions regarding its epidemiology remains incompletely understood. The aim of this study was to screen the hospital and municipal wastewater of the City of Zagreb for the presence of viable A. baumannii and carbapenems. Sampling of hospital wastewater was performed on 2 occasions in 2015 at the central manhole of one Zagreb’s hospital from which the clinical isolates of A. baumannii were recovered. Sampling of municipal wastewater was performed on 6 occasions in 2014/15 at the influent and effluent of the central Zagreb’s wastewater treatment plant. Concentration of carbapenems in wastewater was measured by UHPLC Q-TOF MS. The isolation of A. baumannii from wastewater was performed at 42C/48h on CHROMagar Acinetobacter without or with the addition of commercial supplement CR102 which allows the growth of carbapenem- resistant isolates. Presumptive A. baumannii colonies were characterized phenotypically, by using Vitek2 system, and MALDI-TOF MS. Antibiotic resistance profiles were determined by Vitek2 system and interpreted according to EUCAST criteria. Hospital and municipal wastewaters contained high concentrations of imipenem and meropenem which averaged: 894 and 129ng/L in hospital wastewater, 3060 and 380ng/L in raw and 497 and 311ng/L in treated municipal wastewater, respectively. From hospital wastewater, raw and treated municipal wastewater 8, 30 and 7 isolates of A. baumannii were recovered, respectively. All isolates from hospital wastewater and majority (33/37) of isolates from municipal wastewater were resistant to carbapenems and majority of tested antibiotics except colistin. The 7 MDR clinical isolates recovered in the same period showed comparable levels of antibiotic resistance to MDR isolates from hospital and municipal wastewater. These suggest that A. baumannii is able to survive in environment outside hospitals. However, 4 isolates from raw municipal wastewater were susceptible to carbapenems and other antibiotics. This finding opens the possibility that A. baumannii could have natural habitat in sewage system.

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.