First evidence of KPC-producing ST258 Klebsiella pneumoniae in river water.

This paper reports the first case of Klebsiella pneumoniae carbapenemase (KPC)-2-producing K. pneumoniae in river water in Croatia. In total, four KPC-2-producing K. pneumoniae isolates were analysed. All isolates shared a similar genetic background, belonging to ST258. Isolates displayed uniform, multi-drug-resistant profiles susceptible to colistin. blaSHV-1, aac(3')-II, aac(6')-Ib and aph(3')-Ia genes were detected in all isolates. In all isolates, the blaKPC-2 gene was localized on a single non-conjugative IncFII plasmid that varied in size (∼140, ∼230, ∼225 and ∼220 kb). K. pneumoniae was viable in river water for up to 50 days, confirming its ability to survive and disseminate in the environment.

Emission of extensively-drug-resistant Acinetobacter baumannii from hospital settings to the natural environment.

BACKGROUND: Acinetobacter baumannii is a leading emerging pathogen that is frequently recovered from patients during hospital outbreaks. The role of environmental A. baumannii reservoirs is therefore of great concern worldwide. AIM: To investigate the connection between A. baumannii causing hospital outbreaks and environmental isolates from hospital wastewater, urban sewage and river water as the final natural recipient of wastewaters. METHODS: Clinical isolates from patients with hospital-acquired pneumonia and environmental isolates from water were collected during a two-month monitoring period. Recovery of A. baumannii was performed using CHROMagar Acinetobacter plates, incubated at 42°C for 48 h. Identification was performed by matrix-assisted laser desorption ionization-time of flight mass spectrometry and analyses of rpoB gene. The antibiotic resistance profiles were interpreted according to criteria given for clinical isolates of A. baumannii. The sequence types (ST) were retrieved by multi-locus sequence typing. RESULTS: Fourteen of 19 isolates recovered from patients, hospital wastewaters, urban sewage and river water belonged to ST-195. The remaining five isolates recovered from patients and river water were assigned to ST-1421. All isolates showed very strong relatedness and clustered into CC92, which corresponds to IC2. All isolates were non-susceptible to at least one agent in all but two or fewer antimicrobial categories, and thus were classified as 'extensively-drug-resistant' (XDR). Heteroresistance to colistin was found in two isolates from hospital wastewater. CONCLUSION: Close relatedness of clinical and environmental isolates suggests the emission of XDR A. baumannii via the untreated hospital wastewater in the natural environment.

Extensive polymorphism and evidence of selection pressure on major histocompatibility complex DLA-DRB1, DQA1 and DQB1 class II genes in Croatian grey wolves.

The genes of the major histocompatibility complex (MHC) are a key component of the mammalian immune system and have become important molecular markers for measuring fitness-related genetic variation in wildlife populations. Because of human persecution and habitat fragmentation, the grey wolf has become extinct from a large part of Western and Central Europe, and remaining populations have become isolated. In Croatia, the grey wolf population, part of the Dinaric-Balkan population, shrank nearly to extinction during the 20th century, and is now legally protected. Using the cloning-sequencing method, we investigated the genetic diversity and evolutionary history of exon 2 of MHC class II DLA-DRB1, DQA1 and DQB1 genes in 77 individuals. We identified 13 DRB1, 7 DQA1 and 11 DQB1 highly divergent alleles, and 13 DLA-DRB1/DQA1/DQB1 haplotypes. Selection analysis comparing the relative rates of non-synonymous to synonymous mutations (d(N)/d(S)) showed evidence of positive selection pressure acting on all three loci. Trans-species polymorphism was found, suggesting the existence of balancing selection. Evolutionary codon models detected considerable difference between alpha and beta chain gene selection patterns: DRB1 and DQB1 appeared to be under stronger selection pressure, while DQA1 showed signs of moderate selection. Our results suggest that, despite the recent contraction of the Croatian wolf population, genetic variability in selectively maintained immune genes has been preserved.

Selective immobilization of Acinetobacter junii on the natural zeolitized tuff in municipal wastewater.

The immobilization of desired bacteria onto material was usually performed in synthetic media. The aim of this study was to test the immobilization of phosphate (P)-accumulating bacteria Acinetobacter junii onto natural zeolitized tuff (NZ) in the raw or sterilized municipal wastewater containing the common bacteria Escherichia coli and Enterococcus faecalis and the performance of immobilized A. junii in the same type of wastewater. In the sterilized wastewater which contained the mixture of A. junii, E. coli and E. faecalis, the A. junii was selectively immobilized onto NZ in significantly higher numbers than E. coli and E. faecalis. The A. junii added in the form of bioparticles to the wastewater containing E. coli and E. faecalis, multiplied and removed P from wastewater. The P removal from wastewater was a function of biomass of P-accumulating bacteria and not the amount of NZ or bioparticles used. The performance of A. junii was significantly better in membrane filtered than in autoclaved wastewater. The experiments that were performed in raw non sterilized wastewater showed that A. junii can be successfully immobilized onto NZ in competition with natively present heterotrophic bacteria, retain its metabolic activity and successfully remove P from such water, which makes this technology feasible from biotechnological aspect.

Antimicrobial activity of commercial zeolite A on Acinetobacter junii and Saccharomyces cerevisiae.

The influence of three samples of commercially produced zeolite A (named A, M and R) in water medium on the bacterium Acinetobacter junii and yeast Saccharomyces cerevisiae was investigated. These microorganisms were used in the bioassay and are not specifically related to the use of zeolite A. All zeolite samples showed the negative influence on the survival and physiological status of A. junii and S. cerevisiae. The EC(50) values for the inhibition of CFU of A. junii were 0.328, 0.138 and 0.139 g l(-1) for zeolite sample A, M and R, respectively. The EC(50) values of tested zeolites for S. cerevisiae, estimated by fermentation and fluorescence microscopy assay, ranged from 2.88 to 5.47 g l(-1). The genotoxic effect of three samples of zeolite to S. cerevisiae was shown by the alkaline comet assay. When assuming all the aspects of zeolite toxicity to bacterium and yeast, the zeolite sample R appeared to be less toxic than the samples A and M. The hydrolysis of zeolite crystals, amorphous aluminosilicate and unreacted gel fraction in water medium and consecutive dissolution and leaching of aluminium and silicon in the form of aluminosilicate molecules (700-1300 Da) was detected.

Sorption phenomena of modification of clinoptilolite tuffs by surfactant cations.

The sorption of cationic surfactant hexadecyltrimethylammonium (HDTMA) onto the solid/liquid interfaces of different clinoptilolite rich tuffs (CT) is examined. Examined were CT from Serbia with 75% clinoptilolite, CT from Turkey with 70% clinoptilolite, and CT from Croatia with 60% clinoptilolite. The sorption of HDTMA cations increased in the following order: CT from Serbia>CT from Turkey>CT from Croatia. The maximum amounts of sorbed HDTMA cations, normalized with regard to external cation exchange capacities of tuffs, were 1.79, 1.70, and 1.14 for Serbian, Turkish, and Croatian CT. FTIR analysis of samples with the maximum amount of sorbed HDTMA cations showed that HDTMA chains on Serbian CT adopt mainly a stretched all-trans conformation, while at the surfaces of CT from Turkey and Croatia the amount of gauche conformations increased. The zeta potentials of CT samples with the maximum amount of sorbed HDTMA cations and the sorption of benzoate anions on these samples increased in the following order: CT from Turkey>CT from Serbia>CT from Croatia. It can be assumed that on the surface of CT from Turkey iron (hydr)oxide clusters or nanoparticles with positive surface sites are present, due to which the isoelectric point is sifted toward lower and the zeta potential toward higher values. Therefore, the sorption of benzoate anions on modified CT from Turkey is also higher.

The effect of mineral carrier composition on phosphate-accumulating bacteria immobilization.

The goal of this study was to determine the dynamics and yield of immobilization of the phosphate-accumulating bacterium Acinetobacter junii on mineral carriers. As mineral carriers natural clinoptilolite tuff from Turkey (T) and Serbia (S) and natural bentonite (TER), in original and magnesium (Mg)-exchanged form were used. The key feature which determined the extent of immobilization of A. junii was the type of carrier; the immobilization yield decreased in order T>TER>S. The number of immobilized cells was significantly higher for the Mg-exchanged carriers when compared to their original counterparts (95 and 75 x 10(8)CFU g(-1) for T, 74 and 58 x 10(8)CFU g(-1) for TER, 19 and 6 x 10(8)CFU g(-1) for S). The Mg-exchanged T and S displayed a prolonged biofilm growth up to 24h, while the original counterparts reached the mature biofilm after 12h of incubation. Both forms of TER reached the mature biofilm after 24h of incubation, due to swelling property of the material. The number of immobilized cells correlated significantly negatively with particle size of the carrier, indicating that particle size is another important feature which determined the extent of immobilization. The Mg-exchange of original carriers resulted in significant increase of the zeta potential. When all of the materials were compared, the increase of the zeta potential of carriers correlated negatively with the number of immobilized cells, suggesting that the zeta potential of material is not a crucial factor which determined the immobilization of cells.

Interaction of surfactant-modified zeolites and phosphate accumulating bacteria.

The aim of this study was to determine the interaction of surfactant-modified zeolites (SMZ) and orthophosphate (P)-accumulating bacteria in the process of P removal from wastewater. The SMZ were prepared from the natural zeolite (NZ) of size fractions <0.122 mm and 0.25-0.5 mm. The hexadecyltrimethylammonium (HDTMA) bromide was used to modify the NZ surface from partial monolayer to the bilayer coverage. The surface modification of NZ resulted in the change of zeta potential of particles from negative to positive and great enhancement of the P-adsorption capacity. Only in reactors containing <0.122 mm fraction of partial monolayer coverage of the SMZ, the P was efficiently removed from wastewater by combined adsorption onto the SMZ and bacterial uptake in the biomass. The SMZ with bilayer or patchy bilayer coverage showed the bactericidal effect. To enhance the P removal from wastewater in the aerated biological system, the SMZ can be used, but the special attention should be given to the configuration of sorbed HDTMA molecules and its potential desorption.

Percentage of gelatinolytic bacteria among heterotrophic bacteria as indicator of water quality.

The relationship between the physiological group of gelatinolytic bacteria and the abundance of heterotrophic bacteria in freshwater ecosystems was described, based on analysis of 1082 different freshwater samples collected in Croatia. Percentages of gelatinolytic bacteria among the population of heterotrophic bacteria showed a significant negative correlation with the abundance of heterotrophic bacteria. The relation between the physiological group (gelatinolytic bacteria) and heterotrophic bacteria can be considered to be an indicator of the pollution degree of freshwaters. A high relative content of gelatinolytic bacteria (> 76%) always indicates the colony-forming units of heterotrophic bacteria < 1000/mL, which corresponds to the high water quality; gelatinolytic bacteria < 11% indicate polluted waters. Isolated strains of aerobically grown gelatinolytic bacteria were Gram-negative rod-shaped or Gram-positive endospore-forming rod-shaped cells.

Rapid detection of sulfide-producing bacteria from sulfate and thiosulfate.

An original liquid medium and a field broth bottle method for the rapid detection of the most probable number of sulfide-producing bacteria (SPB) from sulfate and thiosulfate are described. The broth bottle method enables after inoculation with a sample (water, sediment) the growth of ubiquitous aerobic bacteria, causing oxygen depletion, required for the growth of the present various anaerobic and facultative anaerobic SPB. The medium regularly gives higher numbers of the SPB than the used control medium (Postgate's E) for detection of sulfate-reducing bacteria and the final results are obtained just 36 h after the medium inoculation. The method is simple and suitable for the estimation of the physiological group of SPB in fresh waters, saline waters, sediments and industrial waters.