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The carbapenem-resistant bacteria (CRB) are currently at the top of the WHO priority list of bacteria that pose the greatest threat to human health. Considering that soil is one of the important environments for the emergence of antibiotic-resistant bacteria, we isolated and quantified cultivable CRB in soils across Croatia, including ones affected by illegal dumps. We cultivated CRB at two temperatures, distinguishing between the intrinsically resistant CRB (37°C, mostly Stenotrophomonas spp.) and the ones that are presumably human-associated and clinically relevant (42°C, Acinetobacter sp., Enterobacteriaceae, Burkholderia spp.). Our study demonstrated that distinguishing between the two offers a better insight into the diversity of CRB in the environment. The ones cultivated at 37°C were found in almost all soil samples, while the presumably clinically relevant ones were absent from uncontaminated pasture and grassland, indicating that human-associated CRB are unlikely to be found in soils spared from anthropogenic influence.

Bacterium Acinetobacter baumannii is an emergent pathogen associated with nosocomial infections, which can be also found in natural waters. The impact of ecological factors on A. baumannii is insufficiently investigated. The aim was to examine the influence of temperatures (−20 to 80 °C) and pH values (2 to 12) on the survival of environmental and clinical isolates of A. baumannii in nutrient-deprived spring water (SW) and nutrient-rich diluted nutrient broth during 5 months. A. baumannii successfully survived at −20 to 44 °C and neutral pH for 5 months, which is consistent with the persistence of this pathogen in the hospital environment. At temperatures 50 to 80 °C the survival of A. baumannii ranged from 5 days to 5 min. The pH 2 was the most lethal with survival time up to 3 hours, suggesting that acidic conditions are promising for disinfection of water contaminated with A. baumannii. Although the type of media was not statistically significant for long-time survival, the extensively resistant or pandrug-resistant isolates survived better in SW than susceptible or multidrug-resistant isolates. Two distinct colony phenotypes were recorded at extreme temperatures and pH values. The results of this study provide insight into the behaviour of this emerging pathogen in the environment.

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Bacterium Acinetobacter baumannii is an emerging human pathogen whose presence in the aquatic environment raises the issue of public health risk. Fish colonization represents the potential route of pathogen transmission to humans. The aim was to examine the colonization of A. baumannii to freshwater fish Poecilia reticulata. An extensively drug-resistant A. baumannii was tested at three concentrations in natural spring water. Additionally, 70 fish from the Sava River (Croatia) were screened for the presence of A. baumannii, which was not found in gill swabs or analysed gut. The colonization potential of A. baumannii in freshwater fish is dependent upon its concentration in surrounding water. The low concentration of A. baumannii in natural waters represents low colonization potential of freshwater fish. The risk for public health exists in closed water bodies where there is constant inflow of water polluted by A. baumannii in concentrations above 3 log CFU mL.

Bacterium Acinetobacter baumannii is a leading cause of hospital infections. Over the last decade, its occurrence in natural environments outside hospital settings has been reported. The aim was to examine the survival of A. baumannii in water media exposed to different ranges of oxygen supply in order to predict its behaviour in the environment. The abundance of five A. baumannii isolates was monitored in nutrient-depleted and nutrient-rich water media in aerated, intermediate and anaerobic conditions (oxygen saturation 96, 56 and 0%, respectively). A. baumannii survived in both media in all tested oxygen concentrations for 50 days. In nutrient-rich water survival of A. baumannii was lowest in anaerobic conditions, while in nutrient-depleted water there was no difference in survival regardless of oxygen availability. A. baumannii formed translucent small colony variants as the fast response (after 1 day) and dormant cells as the prolonged response (after 14 days) to anaerobic conditions. Transmission electron microscopy (TEM) images showed the outer membrane of coccobacillus dormant cells was up to four times thicker than in regular cells. Once in the environment, A. baumannii is able to survive regardless of the availability of dissolved oxygen, which represents a serious public health concern.

Bacteriological pollution, especially that including clinically important bacteria, of the aquatic environment caused by anthropogenic pressure attracts much attention with regard to public health. Reports to date have not addressed the concentration of emerging carbapenem-resistant bacteria (CRB) in riverine ecosystems, and the source of this pollution is hard to track. We examined the impact of discharge of untreated wastewaters on the bacterial population in the riverine ecosystem, with emphasis on clinically important CRB using a small river in Croatia as a model. River sediments were analysed mineralogically and geochemically. Cultivation of CRB was performed at 37 and 42°C to distinguish the presumably environmental intrinsically resistant (CRB37) from the presumably clinically important acquired resistance (CRB42) species. The significantly positive correlation of CRB42 with CRB37 and total heterotrophs, but not with intestinal enterococci, suggests that entry of CRB42 in riverine ecosystem is not necessarily connected to faecal pollution. The numbers and prevalence of CRB42 are rather dependent on the type of pollution, and is connected to the discharge of wastewaters from different human and animal healthcare centres. Emerging hospital pathogens Acinetobacter baumannii and Klebsiella pneumoniae were exclusively isolated among CRB42 from river water after the discharge of wastewater of a general hospital. The CRB42, once discharged into the riverine ecosystem, behaves as part of the indigenous bacterial population, and could be spread through the natural water bodies or accumulate in river sediments. This implies the need for disinfection of hospital wastewater prior to its discharge into the natural environment in order to avoid the consequent public-health threat. The anthropogenic impact evidenced as bacteriological changes is accompanied by an increase in heavy metal concentrations in river sediments.