First visualisation of bacterial biofilms in 3D porous media with neutron microtomography without contrast agent.

Characterising bacterial biofilm growth in porous media is important for developing reliable numerical models of biofouling in industrial biofilters. One of the promising imaging methods to do that has been a recent successful application of X-ray microtomography. However, this technique requires a contrast agent (1-chloronaphtalene, for example) to distinguish biofilm from the liquid phase, which raises concern about biofilm disruption and impaired image interpretation. To overcome these drawbacks, we tested a new approach based on neutron tomography (NT), which does not need a contrast agent, by imaging two types of porous media (polytetrafluoroethylene - PTFE - and clay beads of various diameters) in glass or PTFE tubes in which bacterial biofilms were grown for 7 days and by comparing these images with the ones obtained with X-ray microtomography. NT images showed that the biofilm formed preferentially around the beads and at bead/bead interface. Visual comparison of both imaging techniques showed consistent biofilm spatial distributions and that the contrasting agent did not significantly disrupt the biofilm. NT images, on the other hand, were still too noisy to allow quantitative measurements. Therefore, X-ray microtomography (provided it uses non-disruptive contrast agents) seems to provide more reliable microstructural descriptors.

Antibacterial Properties of Non-Modified Wool, Determined and Discussed in Relation to ISO 20645:2004 Standard.

Wool is considered to possibly exhibit antibacterial properties due to the ability of wool clothing to reduce the build-up of odor, which arises from the microbial activity of skin microbiota. Indeed, when tested with a widely used agar diffusion plate test method, even wool or other textiles not treated with any antimicrobial agent can be interpreted to show certain antibacterial effects due to the lack of growth under the specimen, as instructed in ISO 20645:2004 standard. Therefore, we analyzed in detail what happens to bacterial cells in contact with untreated wool and cotton fabric placed on inoculated agar plates by counting viable cells attached to the specimens after 1 and 24 h of contact. All wool and several cotton samples showed no growth under the specimen. Nevertheless, it was shown without a doubt that neither textile material kills bacteria or inhibits cell multiplication. A reasonable explanation is that bacterial cells firmly attach to wool fibers forming a biofilm during multiplication. When the specimen was lifted off the nutrient agar surface, the cells in the form of biofilm remained attached to the wool fibers, removing the biomass and resulting in a clear, no growth zone underneath it. By imaging the textile specimens with X-ray microtomography, we concluded that the degree of attachment could be dependent on surface topography. The results indicate that certain textiles, in this case, wool, could exhibit antibacterial properties by removing excess bacteria that grow on the textile/skin interface when taken off the body.

Presence of carbapenem-resistant bacteria in soils affected by illegal waste dumps.

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.

Impact of biotic interactions on the survival of emerging pathogen Acinetobacter baumannii in aquatic media.

Acinetobacter baumannii is an opportunistic pathogen causing infections in immunocompromised patients. Recent studies recorded its persistence in a variety of abiotic conditions, but data regarding the biotic interactions with other microorganisms are limited. The aim was to assess the interaction of clinically relevant A. baumannii with common faecal bacteria Escherichia coli and Enterococcus faecium. Additionally, the interaction with a bdelloid rotifer Adineta vaga as a potential agent for biological control of A. baumannii was examined. Experiments were conducted in nutrient-poor spring water (SW) and nutrient-rich diluted nutrient broth (DNB) at 22 °C. A. baumannii coexisted with E. coli and E. faecium in both media, suggesting the absence of inter-bacterial competition in long-term survival. No difference in the survival of pandrug-resistant, extensively drug-resistant or antibiotic sensitive isolates of A. baumannii was observed. Rotifers contributed to the removal of all tested bacteria, particularly in SW. Rotifers were able to remove 5.5 ± 1.3 log CFU/mL of A. baumannii in SW and 3.5 ± 1.7 log CFU/mL in DNB. Additionally, no intracellular growth of A. baumannii inside A. vaga was detected. In wastewater treatment plants and drinking water facilities, grazing by rotifers might be useful for the removal of emerging human pathogens such as A. baumannii from water.

Removal of emerging pathogenic bacteria using metal-exchanged natural zeolite bead filter.

Hospital wastewaters can become a route for dissemination of antibiotic-resistant bacteria to the environment if not properly treated. Some of these bacteria are able to survive conventional disinfection treatments (e.g. chlorination, UV irradiation), which evokes the need for novel disinfection methods. The metal-exchanged zeolites were tested as novel antibacterial agents for wastewater treatment. The natural zeolite clinoptilolite enriched with silver (AgNZ) showed far better antibacterial activity towards hospital pathogenic bacterium Acinetobacter baumannii when compared with copper-exchanged zeolite (CuNZ), with minimal bactericidal concentration of 0.25-2 (AgNZ) compared with 32-64 mg L-1 (CuNZ) in a batch system and respective log 5.6 reduction compared with log 0.5 reduction in a flow system with pure bacterial culture. In the flow system with real effluent wastewater from the treatment plant, the removal of carbapenem-resistant bacteria using AgNZ was 90-100% during the 4 days of the experimental run. These results indicate that the AgNZ efficiently removes pathogenic bacteria from the wastewater, including A. baumannii, and is promising as a disinfectant material in a bead filter system.

Emerging human pathogen Acinetobacter baumannii in the natural aquatic environment: a public health risk?

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-1.

Survival of ESKAPE pathogen Acinetobacter baumannii in water of different temperatures and pH.

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.

Development and evaluation of an experimental protocol for 3-D visualization and characterization of the structure of bacterial biofilms in porous media using laboratory X-ray tomography.

The development of a reliable model allowing accurate predictions of biofilm growth in porous media relies on a good knowledge of the temporal evolution of biofilm structure within the porous network. Since little is known about the real 3-D structure of biofilms in porous media, this work was aimed at developing a new experimental protocol to visualize the 3-D microstructure of the inside of a porous medium using laboratory X-ray microtomography. A reliable and reproducible methodology is proposed for (1) growing a biofilm inside a porous medium, and (2) X-ray tomography-based characterization of the temporal development of the biofilm at the inlet of the biofilter. The statistical analysis proposed here also validates the results presented in the literature based on a biofilm structure single measurement.

Resistance of bioparticles formed of phosphate-accumulating bacteria and zeolite to harsh environmental conditions.

Extreme environmental conditions, such as pH fluctuations, high concentrations of toxicants or grazing of protozoa, can potentially be found in wastewater treatment systems. This study was carried out to provide specific evidence on how 'bioparticles' can resist these conditions. The term 'bioparticle' is used to describe a particle comprising natural zeolitized tuff with a developed biofilm of the phosphate-accumulating bacterial species, Acinetobacter junii, on the surface. The bacteria in the biofilm were protected from the negative influence of extremely low pH, high concentrations of benzalkonium-chloride and grazing by Paramecium caudatum and Euplotes affinis, even under conditions that caused complete eradication of planktonic bacteria. During an incubation of 24 h, the biofilms were maintained and bacteria detached from the bioparticles, thus bioaugmenting the wastewater. The bioparticles provided a safe environment for the survival of bacteria in harsh environmental conditions and could be used for successful bioaugmentation in wastewater treatment plants.

Antibacterial activity of silver doped hydroxyapatite toward multidrug-resistant clinical isolates of Acinetobacter baumannii.

Bacteria Acinetobacter baumannii is a persistent issue in hospital-acquired infections due to its fast and potent development of multi-drug resistance. To address this urgent challenge, a novel biomaterial using silver (Ag+) ions within the hydroxyapatite (HAp) lattice has been developed to prevent infections in orthopedic surgery and bone regeneration applications without relying on antibiotics. The aim of the study was to examine the antibacterial activity of mono-substituted HAp with Ag+ ions and a mixture of mono-substituted HAps with Sr2+, Zn2+, Mg2+, SeO32- and Ag+ ions against the A. baumannii. The samples were prepared in the form of powder and disc and analyzed by disc diffusion, broth microdilution method, and scanning electron microscopy. The results from the disc-diffusion method have shown a strong antibacterial efficacy of the Ag-substituted and mixture of mono-substituted HAps (Sr, Zn, Se, Mg, Ag) toward several clinical isolates. The Minimal Inhibitory Concentrations for the powdered HAp samples ranged from 32 to 42 mg/L (Ag+ substituted) and 83-167 mg/L (mixture of mono-substituted), while the Minimal Bactericidal Concentrations after 24 h of contact ranged from 62.5 (Ag+) to 187.5-292 mg/L (ion mixture). The lower substitution level of Ag+ ions in a mixture of mono-substituted HAps was the cause of lower antibacterial effects measured in suspension. However, the inhibition zones and bacterial adhesion on the biomaterial surface were comparable. Overall, the clinical isolates of A. baumannii were effectively inhibited by substituted HAp samples, probably in the same amount as by other commercially available silver-doped materials, and such materials may provide a promising alternative or supplementation to antibiotic treatment in the prevention of infections associated with bone regeneration. The antibacterial activity of prepared samples toward A. baumannii was time-dependent and should be considered in potential applications.

Capillary bacterial migration on non-nutritive solid surfaces.

Here we describe an additional type of bacterial migration in which bacterial cells migrate vertically across a non-nutritive solid surface carried by capillary forces. Unlike standard motility experiments, these were run on a glass slide inserted into a Falcon tube, partly immersed in a nutrient medium and partly exposed to air. Observations revealed that capillary forces initiated upward cell migration when biofilm was formed at the border between liquid and air. The movement was facilitated by the production of extracellular polymeric substances (EPS). This motility differs from earlier described swarming, twitching, gliding, sliding, or surfing, although these types of movements are not excluded. We therefore propose to call it "capillary movement of biofilm". This phenomenon may be an ecologically important mode of bacterial motility on solid surfaces.

Toxicity prediction and effect characterization of 90 pharmaceuticals and illicit drugs measured in plasma of fish from a major European river (Sava, Croatia).

Chemical analysis of plasma samples of wild fish from the Sava River (Croatia) revealed the presence of 90 different pharmaceuticals/illicit drugs and their metabolites (PhACs/IDrgs). The concentrations of these PhACs/IDrgs in plasma were 10 to 1000 times higher than their concentrations in river water. Antibiotics, allergy/cold medications and analgesics were categories with the highest plasma concentrations. Fifty PhACs/IDrgs were identified as chemicals of concern based on the fish plasma model (FPM) effect ratios (ER) and their potential to activate evolutionary conserved biological targets. Chemicals of concern were also prioritized by calculating exposure-activity ratios (EARs) where plasma concentrations of chemicals were compared to their bioactivities in comprehensive ToxCast suite of in vitro assays. Overall, the applied prioritization methods indicated stimulants (nicotine, cotinine) and allergy/cold medications (prednisolone, dexamethasone) as having the highest potential biological impact on fish. The FPM model pointed to psychoactive substances (hallucinogens/stimulants and opioids) and psychotropic substances in the cannabinoids category (i.e. CBD and THC). EAR confirmed above and singled out additional chemicals of concern - anticholesteremic simvastatin and antiepileptic haloperidol. Present study demonstrates how the use of a combination of chemical analyses, and bio-effects based risk predictions with multiple criteria can help identify priority contaminants in freshwaters. The results reveal a widespread exposure of fish to complex mixtures of PhACs/IDrgs, which may target common molecular targets. While many of the prioritized chemicals occurred at low concentrations, their adverse effect on aquatic communities, due to continuous chronic exposure and additive effects, should not be neglected.

Reduced susceptibility to disinfectants of Acinetobacter baumannii biofilms on glass and ceramic.

The aim of this study was to determine the susceptibility of hospital and environmental Acinetobacter baumannii isolate biofilms on ceramics and glass to common disinfectants benzalkonium chloride and chlorhexidine. For this purpose we developed a new method for biofilm cultivation and quantification on ceramics. The biofilm bacteria were more resistant to disinfectants than the planktonic populations, as more than 50 % of the biofilm population and none of the planktonic population survived 5-minute exposure. Furthermore, biofilm populations on ceramic tiles were significantly more resistant than those on glass coverslips, even though the amount of biofilm was practically the same on ceramics and glass. The reason for reduced susceptibility of A. baumannii biofilms on ceramics may be related to surface/disinfection interactions. Our findings suggest that biofilms on ceramic surfaces can be an important source of A. baumannii infection in hospital environments.

The fate of carbapenem-resistant bacteria in a wastewater treatment plant.

Wastewater treatment plants have been considered potential sources of antibiotic resistance gene exchange and release into the environment. The aim of our study was to quantify environmental and human-associated carbapenem-resistant bacterial populations (CRBPs) across wastewater treatment stages and correlate bacterial counts to physicochemical and other bacteriological parameters in order to see their behaviour in wastewater and sludge and their potential dissemination in the environment. Samples were taken from five sites (treatment stages) of the largest Croatian wastewater treatment plant (20 per site) over 10 months of monitoring. CRBPs were found at all wastewater treatment stages save for the lime-treated, stabilised sludge, which underlines the importance of effluent and digested sludge disinfection. Secondary sludge settling removed 99% of CRBP from the effluent, but the relative proportion of CRBP in the total bacterial count significantly increased in the effluent (0.0020%) and digested sludge (0.0019%) compared to the influent (0.0006%), indicating selection for resistant bacteria in these settings. CRBP counts did not correlate with measured carbapenem concentrations in wastewater, which suggests that antibiotic concentrations were not the reason for CRBP selection. Negative correlation between activated sludge retention time and CRBP indicated that their number could be reduced by increasing the retention time during secondary treatment. Despite the indications that WWTPs select for antibiotic-resistant bacteria, wastewater treatment is very efficient in reducing their absolute numbers, and proper effluent and sludge disinfection can significantly reduce dissemination of antibiotic-resistant bacteria into the environment.

Alkaline disinfection of urban wastewater and landfill leachate by wood fly ash.

Wood fly ash is an industrial by-product of the combustion of different wood materials and is mostly disposed of as waste on landfills. In our preliminary experiments, wood ash exhibited antibacterial activity against urban wastewater bacteria and we focused on wood fly ash as a potential substrate for wastewater disinfection. The addition of ash at a concentration of 10 g L⁻¹ (1%) caused an instant increase of pH in urban wastewater and landfill leachate. High pH (10.1-12.7) inactivated bacterial populations in the wastewater and the removal of faecal coliforms and intestinal enterococci after 6 h of contact was 100% (below the detection limit; <1 CFU per mL) with the most efficient ash sample (ash from combustion of beech) both in urban wastewater and landfill leachate. Properly chosen wood fly ash, i.e. one that tends to increase the pH to the greatest extent, proved to be a very effective disinfection substrate. Considering that water treated with wood ash has a high pH and needs to be neutralised before discharge, ash would be suitable for disinfection of leachates when smaller volumes are treated.

Antibacterial activity of heavy metal-loaded natural zeolite.

The antibacterial activity of natural zeolitized tuffs containing 2.60wt.% Cu(2+), 1.47 Zn(2+) or 0.52 Ni(2+) were tested. Antibacterial activities of the zeolites against Escherichia coli and Staphylococcus aureus were tested after 1h and 24h of exposure to 1g of the zeolite in 100mL of three different media, namely Luria Bertani, synthetic wastewater and secondary effluent wastewater. The antibacterial activities of the zeolites in Luria Bertani medium were significantly lower than those in the other media and negatively correlated with the chemical oxygen demand of the media. The Ni-loaded zeolite showed high leaching of Ni(2+) (3.44-9.13wt.% of the Ni(2+) loaded) and weak antibacterial activity in the effluent water. Since Cu-loaded zeolite did not leach Cu(2+) and the leaching of Zn(2+) from Zn-loaded zeolite was low (1.07-1.61wt.% of the Zn(2+) loaded), the strong antibacterial activity classified the Cu- and Zn-loaded zeolite as promising antibacterial materials for disinfection of secondary effluent water.

Surfactants in the environment.

Surfactants are a diverse group of chemicals that are best known for their wide use in detergents and other cleaning products. After use, residual surfactants are discharged into sewage systems or directly into surface waters, and most of them end up dispersed in different environmental compartments such as soil, water or sediment. The toxic effects of surfactants on various aquatic organisms are well known. In general, surfactants are present in the environment at levels below toxicity and in Croatia below the national limit. Most surfactants are readily biodegradable and their amount is greatly reduced with secondary treatment in wastewater treatment plants. The highest concern is the release of untreated wastewater or wastewater that has undergone primary treatment alone. The discharge of wastewater polluted with massive quantities of surfactants could have serious effects on the ecosystem. Future studies of surfactant toxicities and biodegradation are necessary to withdraw highly toxic and non-biodegradable compounds from commercial use and replace them with more environmentally friendly ones.

Requirement of Acinetobacter junii for magnesium, calcium and potassium ions.

The goal of this study was to determine the concentrations of Mg, Ca and K ions required for the formation of metabolically active population of phosphate (P)-accumulating bacterium Acinetobacter junii. The availability of Mg, Ca and K originating from natural minerals in the conditions of severe shortage of these cations was tested. In the case of shortage of Mg, Ca and K ions in wastewater the P removal was absent due to the decay of A. junii. In the cases of Mg or K shortage in wastewater the P removal was negligible due to the decay of A. junii, while Ca was not essential for the examined bacterium. The minimal required concentrations of Mg and K in synthetic wastewater were 0.64 mg Mg/mg P and 0.50 mg K/mg P. The natural zeolitized tuffs and bentonite, either in Mg, Ca or K form, successfully replaced the lack of Mg, Ca, K and trace metals in wastewater. The requirement of A. junii for examined cations was in the order: Mg>K>Ca.