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Novel composites based on natural zeolite-clinoptilolite and monoterpene phenols-thymol and carvacrol were prepared by supercritical solvent impregnation (SSI) in supercritical carbon dioxide (scCO(2)) at 30 MPa and 35 degrees C during 18 h. The content of phenols in composites (thymol-23.0 and carvacrol-19.1 wt%) was determined using both C,H,N and thermal analysis (TGA); interactions of the phenols with the zeolite lattice studied by Fourier transform infrared spectroscopy (FTIR) and powder X-ray diffraction (PXRD) showed that the zeolite lattice was preserved in the composites. The composites exhibited antibacterial activity toward Gram-negative bacteria Escherichia coli DSM 498 and Gram-positive bacteria Staphylococcus aureus ATCC 25923 in different water media (phosphate buffer solution, spring water and lake water). After 1 h carvacrol-containing clinoptilolite showed better antibacterial activity than composite with thymol. Both composites expressed bactericidal effect after 24 h of contact. Leaching of the phenols from composite in water was more pronounced from thymol (22.7-32.1%) than for carvacrol (6.2-8.9%) suggesting influence of steric effect on the desorption process.

The order of magnitude of increased growth, multiplication rate, and decreased sporulation of Bacillus subtilis after exposure to nanotesla magnetic fields (MFs) relative to control samples were observed experimentally. Earth’s total magnetic field intensity was reduced from 47.9 +– 0.4 mT to cover the range from 97.5 +– 1.7 nT to 1115 +– 158 nT in eight subsequent experiments by using three pairs of Helmholtz coils combined with Mu-metal shielding. The growth, multiplication rate, sporulation, and potassium content were measured in the probe and control containing B. subtilis cultures after 24 h of exposure to nanotesla and Earth’s magnetic fields, respectively. The observed effect is discussed with regard to its possible repercussions on Earth’s living species during geomagnetic reversals that occurred when the magnetic field was much weaker than the field that exists today. In addition, effects on future manned voyages into deep space, an environment with reduced magnetic field intensity, are considered.

The natural environment is a primary source of infections caused by members of Burkholderia cepacia complex (BCC), but the release of human waste may in return enrich the natural environment with clinically relevant BCC. Seven BCC isolates from environment influenced by human liquid or solid waste across Croatia, and one clinical isolate were characterised. B. multivorans recovered from the soil at illegal dumpsite belonged to sequence type (ST)19 ; B. ambifaria from the agricultural soil fertilized with swine or poultry manure to ST927 or new ST ; B. cenocepacia from creek sediment, river water and wound swab to new STs. Antimicrobial susceptibility of isolates ranged from sensitive to multidrug-resistant. A variety of blaTEM genes was confirmed in isolates. Isolates expressed the virulence factors and survived in river water during 50 days. The BCC present in natural environments influenced by the human waste are of clinical relevance and a potential source of sporadic infections.

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.

Bacterium

International audience; Novel hyper-resistant bacteria were isolated from the Crven Dol mine (Allchar, North Macedonia), arsenic-rich extreme environment. Bacteria were recovered from a secondary mineral mixture, an alteration of hydrothermal realgar rich in arsenates (pharmacolite, hornesite, and talmessite). The sample was recovered from the dark part of the mine at 28 m depth. Three bacterial strains and a bacterial consortium were isolated for their capacity to survive exposure to 32 g/L (209 mM) of arsenite, and 176 g/L (564 mM) of arsenate. The 16S rRNA gene analysis identified bacterial isolates as Stenotrophomonas sp. and two Microbacterium spp. This analysis also revealed that bacterial consortium comprise two Bacteriodetes exhibiting similarity to Olivibacter ginsengisoli and to uncultured bacterium, and one γ-proteobacteria with similarity to Luteimonas sp. Among all isolates Stenotrophomonas sp. exhibited the highest tolerance to As compound as well as the capacity to accumulate As inside the cells. Analysis of genes involved in As-resistance showed that recovered isolates possess the genes encoding the ArsB, Acr3(1) and Acr3(2) proteins, indicating that at least a part of their resistance could be ascribed to As-efflux systems described in isolates obtained from human-polluted environments.

Although the molecular mechanisms of carbapenem resistance of environmental isolates of Acinetobacter baumannii are well described, data on the mechanisms of colistin resistance are scarce. In this study, we report the molecular mechanisms of colistin resistance in environmental isolates of A. baumannii. Seven clinically relevant isolates of A. baumannii belonging to ST-2Pasteur were recovered from hospital wastewater and wastewater treatment plant. The phenotypic resistance to colistin was confirmed by broth microdilution with minimum inhibitory concentration values ranging from 20 to 160 mg/L. Colistin sulfate and colistimethate sodium showed bactericidal activity against two colistin-heteroresistant isolates in vitro, but substantially recovery of population was observed after prolonged incubation. In silico genome analysis revealed nucleotide variations resulting in amino acid changes in LpxC (N286D), LpxD (E117K), PmrB (A138T, R263S, L267W, Q309P, and A444V), and EptA (F166L, I228V, R348K, A370S, and K531T). According to reverse transcription quantitative PCR, all isolates had increased levels of eptA mRNA and decreased levels of lpxA and lpxD mRNA. Isolates expressed low hydrophobicity, biofilm, and pellicle formation, but showed excellent survival in river water during 50 days of monitoring. Colistin- and pandrug-resistant A. baumannii disseminated in the environment could represent the source for the occurrence of serious community-acquired infections.

According to the World Health Organization, bacterium Acinetobacter baumannii is the first on the critical priority list of pathogens in urgent need for new antibiotics. The increasing resistance of A. baumannii to the last-line treatment options, including carbapenems, is a global problem. We report the molecular epidemiology of 12 carbapenem-resistant clinical isolates of A. baumannii collected from hospitalised patients in three neighbouring countries in Southeast Europe: Croatia, Serbia, and Bosnia and Herzegovina, giving an insight into the molecular characterisation and evolutionary history of the acquisition of resistance genes. Besides the blaOXA-23 gene, the endemic presence of OXA-72 oxacillinase of the same origin for more than a decade as the leading mechanism of carbapenem resistance in Southeast Europe was confirmed. To the best of our knowledge, this is the first paper that investigates and analyses the phylogenetic association of the most common mechanisms of resistance to carbapenems in clinical isolates of A. baumannii originating from three neighbouring countries in Southeast Europe.

Increasingly difficult treatment of multidrug-resistant (MDR) bacteria has become a global problem of the 21st century. Within a group of multiresistant bacteria, the Acinetobacter baumannii convincingly occupies the position at the top of the group designated as ESKAPE pathogens. In this study, 61 isolates of A. baumannii were recovered from different samples originating from various departments of the University Clinical Hospital Mostar during 2018. All of the isolates were identified using conventional phenotypic methods and the VITEK® 2 Compact System, and were confirmed by MALDI-TOF mass spectrometry. The minimum inhibitory concentrations (MICs) were determined by the microbroth dilution method using MICRONAUT-S MDR MRGN-Screening and VITEK 2 Compact System. All strains were resistant to carbapenems and classified in eight different resistotypes according to their antibiotic resistance and macrorestriction pulsed-field gel electrophoresis profiles, with all belonging to IC II. One isolate displayed resistance to colistin (MIC ≥16 mg/L). The presence of blaOXA genes encoding OXA-type carbapenemases was investigated by multiplex PCR and the Eazyplex® SuperBugAcineto system and showed 100% compatibility with the detection of acquired oxacillinases. Molecular characterization of the isolates tested in this study revealed the OXA-23- and OXA-40-like groups of acquired oxacillinases. Sequencing of two PCR products of the OXA-40-like group confirmed the presence of OXA-72. Survival assays with two selected isolates of A. baumannii encoding different mechanisms of carbapenem resistance revealed that one isolate was able to survive on a fragment of white laboratory coat during 90 days of monitoring. To the best of our knowledge, this is the first article to present the results of a comprehensive phenotypic, genotypic, and molecular analysis of A. baumannii isolates from the leading clinical hospital center in the southwestern part of Bosnia and Herzegovina, including data for the survival of this pathogen on the white laboratory coats used as compulsory medical clothing.

The order of magnitude of increased growth, multiplication rate, and decreased sporulation of

Increasingly difficult treatment of multidrug-resistant (MDR) bacteria has become a global problem of the 21st century. Within a group of multiresistant bacteria, the

Antibacterial activity of a natural zeolite, clinoptilolite (Z), enriched with about 0.4-0.5 mmol of Ag (Ag-Z), Cu (Cu-Z) or Zn (Zn-Z) was tested toward two strains of Gram-negative bacteria - Escherichia coli and Acinetobacter baumannii - in natural seawater from Adriatic Sea (Croatia). The Ag-Z and Cu-Z exhibited bactericidal activity toward E. coli and A. baumannii after 3 and 6 h of exposure, respectively. The Zn-Z showed antibacterial activity toward both strains, but lacking the bactericidal effect even after 24 h of exposure. All studied samples released metal cations in seawater. Concentration of Ag and Zn leached from Ag-Z and Zn-Z was higher than maximum allowable concentrations (MAC) for drinking water, whereas concentration of Cu was below MAC. Due to high concentration of Na+ in seawater (about 1.2 g Na+ dm(-3)) an ion-exchange process can be responsible for the leaching of the studied cations. The lowest extent of leaching from Cu-Z was ascribed to different position and geometry of sites occupied by Cu(II) in the clinoptilolite lattice. The antibacterial activity of M-Z was attributed to the metal cations leached from the zeolite lattice into seawater. The obtained results indicate that Cu-Z can be considered as a promising disinfectant for treatment of seawater.

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.

The extensively- and pandrug-resistant isolates of Acinetobacter baumannii are a leading cause of hospital acquired infections. Aside from reports of A. baumannii in wastewaters influenced by hospital effluents, only two studies reported its detection in soils. The lack of data hinders the assessment of soil as the possible environmental reservoir of A. baumannii, prediction of the behaviour of this emergent pathogen in soils and their potential consequences. This study examined the survival of clinically relevant isolates of A. baumannii in the sterilized and fresh soils of different moisture and pH values. In the alkaline, weakly acid, and strongly acid soil A. baumannii survived for five months, up to 15 days, and up to one hour, respectively. Decrease of moisture below 16 wt% can help in the elimination, but the long-term maintenance of viable A. baumannii was detected in completely dry alkaline soil. Linear regression model confirmed that the pH value of soil is the most important environmental factor, which determines the survival of A. baumannii in soils. Acidification of soil seems a promising method in the remediation of soils contaminated with emerging human pathogen A. baumannii.

Due to the development of resistance to antimicrobial agents, bacterium Acinetobacter baumannii is nowadays a leading cause of nosocomial outbreaks. Clinically relevant A. baumannii outside hospital settings including natural soils affected by human waste represents a public-health risk for humans and animals. The aim of this study was to investigate the potential of metal-loaded zeolites to eliminate viable A. baumannii from artificially contaminated natural soils. A. baumannii isolate was subjected to the activity of natural zeolitised tuff (NZ) and Cu-modified (CuNZ) or Ag-modified zeolite (AgNZ) in wet, slightly acidic terra rossa and slightly alkaline red palaeosol. A. baumannii survived in terra rossa and red palaeosol supplemented with 1 wt% of NZ for seven days and four months, respectively. The addition of 1 wt% of CuNZ to terra rossa and red palaeosol shortened the survival of A. baumannii to three and 14 days, respectively. The addition of 0.1 wt% of AgNZ to both soils resulted in complete removal of viable A. baumannii within 1 h of contact, while the total native heterotrophic bacterial counts remained high. Since AgNZ is prepared with a simple modification of cost-effective and environmentally friendly natural zeolite, it is a promising material for the remediation of soils contaminated with pandrug-resistant A. baumannii., Bakterija Acinetobacter baumannii u današnje je vrijeme vodeći uzročnik bolničkih infekcija, ponajprije zbog brzoga razvoja otpornosti te bakterije na antimikrobne lijekove. Klinički značajni A. baumannii izvan bolničkoga okružja, kao što su prirodna tla pod utjecajem ljudskoga otpada, javnozdravstveni su rizik za ljude i životinje. Cilj ovoga rada bio je istražiti potencijal zeolita modificiranih metalima za uklanjanje vijabilnih A. baumannii iz umjetno zagađenih prirodnih tala. Izolat A. baumannii u vlažnoj blago kiseloj terra rossa (pH 5,40) i blago lužnatom crvenom paleotlu (pH 8,43) podvrgnut je djelovanju prirodnoga zeolitnoga tufa (NZ) te NZ-a modificiranoga bakrom (CuNZ) ili srebrom (AgNZ). U slučaju dodatka 1 wt% NZ-a, A. baumannii je preživio u terra rossa sedam dana, a u crvenom paleotlu čak četiri mjeseca. Dodatak 1 wt% CuNZ-a u tla skratio je preživljavanje A. baumannii na tri dana u terra rossa i 14 dana u crvenom paleotlu. Dodatak samo 0,1 wt% AgNZ-a u tla rezultirao je kompletnim uklanjanjem vijabilnih A. baumannii tijekom jednoga sata kontakta, pri čemu je brojnost ukupnih heterotrofnih bakterija, prirodno prisutnih u tlu, ostala visoka. Čestice AgNZ-a pripremaju se jednostavnom modifikacijom jeftinoga i okolišno prihvatljivoga prirodnoga zeolita. Mala koncentracija (1 g AgNZ-a po 1 kg tla) mogla bi se jednostavno raspršiti po tlu koje je zagađeno bakterijom A. baumannii. Stoga je AgNZ obećavajući materijal za remedijaciju tala zagađenih tom bakterijom koja je otporna na sve dostupne antibiotike.

Višestruko rezistentne bakterije, uzročnici hospitalnih infekcija, kao što su Acinetobacter baumannii i Klebsiella pneumoniae, predstavljaju globalni problem 21. stoljeća. Osobito zabrinjava sve učestalija pojava otpornosti ovih mikroorganizama na komercijalno dostupne dezinficijense. Cilj našeg istraživanja bio je utvrditi učinkovitost dezinfekcije proizvoda „Sanosil super25“, u odnosu na vodikov peroksid i to na višestruko rezistentne izolate A. baumannii i K. pneumoniae. Ispitane su vrijednosti MTC (maksimalno tolerirajuća koncentracija); MIC (minimalna inhibirajuća koncentracija) i MBC (minimalna baktericidna koncentracija). MTC Sanosila (, Multi-resistant bacteria, the causes of nosocomial infections, such as Acinetobacter baumannii and Klebsiella pneumoniae, represent a global problem of the 21st century. The increasing resistance of these microorganisms to commercially available disinfectants is of a particular concern. The aim of our study was to determine disinfection effectiveness of the product "Sanosil super25", in relation to hydrogen peroxide, to multiple resistant isolates of A. baumannii and K. pneumoniae. The tested parameters were: MTC values (maximum tolerated concentration); MIC (minimum inhibitory concentration) and MBC (minimum bactericidal concentration). The MTC of Sanosil (

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., Primijećen je i prikazan dosad neopisan način pokretljivosti bakterija u obliku biofilma. Pokazano je kako se bakterijske stanice iz biofilma formiranoga na razmeđi tekućine i zraka gibaju okomito po staklenom predmetnom stakalcu, inertnoj, nehranjivoj ali djelomično vlažnoj površini. Takva površina, izložena zraku, uvelike se razlikuje od površine hranjivog agara, ili od površine prekrivene hranjivim agarom, kakve se koriste u standardnim testovima za ispitivanje bakterijske pokretljivosti u biofilmu. Sudeći prema opažanjima, stanice se prvotno gibaju posredstvom kapilarnih sila, a kada se formira biofilm, stvaranje izvanstanične polimerne tvari (EPS) potiče daljnju migraciju bakterija. Takva pokretljivost opažena je kod svih testiranih a fiziološki bitno različitih bakterijskih vrsta. Kako se eksperimentalni postav značajno razlikuje od standardnih testova praćenja bakterijske pokretljivosti, kao što su rojenje (swarming), trzanje (twitching) ili klizanje po površini (gliding, sliding ili surfing), predložen je generički naziv “kapilarna pokretljivost biofilma”. Ovdje opisani fenomen mogao bi biti značajan ekološki čimbenik pokretljivosti bakterija u okolišu.

La aparicion y propagacion de la resistencia a los antibioticos se considera un proceso muy complejo, con muchos factores que afectan a los mecanismos de resistencia, incluidos los metales. Con el fin de comprender plenamente la propagacion de la resistencia a los antibioticos en diferentes entornos, es imprescindible realizar estudios combinados que incluyan geoquimica y microbiologia. Hasta ahora, se han realizado muy pocos estudios de este tipo y ninguno de ellos afecta a las regiones mas meridionales. El objetivo de este estudio fue, por lo tanto, examinar las caracteristicas biogeoquimicas de los sedimentos del extremo sur de Chile. Se investigo la geoquimica de los sedimentos, asi como la presencia de bacterias heterotrofas y resistentes a los carbapenemas (BRC) en el Rio de las Minas y el Estrecho de Magallanes en Punta Arenas (Chile), para evaluar el impacto antropogenico de las aguas residuales urbanas no tratadas sobre la acumulacion de metales y contaminantes microbianos en este sistema costero. La geoquimica de los sedimentos indicaba la influencia tanto de los procesos naturales como de las actividades antropogenicas. Aunque los niveles de metales en los sedimentos actualmente no representan un efecto toxico sobre los organismos, no se puede negar la influencia antropogenica sobre la composicion de los sedimentos en el area de estudio. Se encontraron CCR clinicamente relevantes en los sedimentos de los rios (1,9 y 2,7 UFC logaritmicos/ ml). Nuestros hallazgos sugieren que el Rio de las Minas esta bajo el impacto antropogenico de las aguas residuales urbanas no tratadas y representa una fuente significativa de CRB potencialmente patogena para el medio ambiente local.

International audience; A mercury-resistant bacterial strain has been isolated from a rock of the Idrija mercury mine in Slovenia. The rock had 19 g carbon and 2952 mg mercury (Hg) per kg. Mass spectrometry and DNA sequencing showed that the bacterium belongs to the Pseudomonas genus. It is called Pseudomonas idrijaensis. This bacterial strain is sensitive to methylmercury (MeHg) like the reference P. aeruginosa strain PAO1, and is resistant to divalent mercury (Hg(II)) in contrast to PAO1. This difference could be attributed to the presence of the mer operon yet deprived of the merB gene encoding the organomercurial lyase, on the basis of whole genome sequencing. The P. idrijaensis mer operon displays the RTPCADE organization and is contained in the Tn5041 transposon. This transposon identified here occurs in other Gram-negative Hg-resistant strains isolated from mercury ores, aquatic systems and soils, including Pseudomonas strains from 15,000 to 40,000 years old Siberian permafrost. When P. idrijaensis was exposed to mercury chloride, two intracellular Hg species were identified by high energy-resolution XANES spectroscopy, a dithiolate Hg(SR)2 and a tetrathiolate Hg(SR)4 complex. P. idrijaensis had a much higher [Hg(SR)2]/[Hg(SR)4] molar ratio than bacteria lacking the mer operon when exposed to 4 µg Hg2+/L - resulting in an intracellular accumulation of 4.3 µg Hg/g dw. A higher amount of the Hg(SR)2 complex provides a chemical signature for the expression of the dicysteinate Mer proteins in response to mercury toxicity.

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.Keywords: bacteria, carbapenems, water, sediment, environment

Six carbapenem-resistant isolates of Acinetobacter baumannii were recovered from untreated and treated municipal wastewater of the capital city of Zagreb, Croatia. Molecular identification of environmental isolates of A. baumannii was performed by amplification, sequencing, and phylogenetic analyses of rpoB gene. The presence of blaOXA genes encoding OXA-type carbapenemases (OXA-51-like, OXA-23, and OXA-40-like) was confirmed by multiplex PCR and sequencing. Phylogenetic analyses corroborated the affiliation of detected blaOXA genes to three different clusters and showed association of environmental OXAs with those described from clinical isolates. This result suggests that isolates recovered from municipal wastewater are most probably of clinical origin. Furthermore, the presence of OXA-40-like (OXA-72) in an environmental A. baumannii isolate is reported for the first time. Persistence of A. baumannii harboring the clinically important OXAs in the wastewater treatment process poses a potentially signifi...

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.

Recently, concerns have been raised about the possibility of Acinetobacter baumannii transmission between animals and humans. So far, A. baumannii has been reported in animals with which people can come into contact. The presence of this pathogen in animal manure presents an equally important public health risk. In this study, we report the finding of two A. baumannii isolates in swine manure from a Croatian pig farm. Both isolates shared features with the widespread human clinical isolates: affiliation to the international clonal lineage 2 (ST-195), carbapenem, and extensive drug resistance and the plasmid-located acquired blaOXA-23 gene. These two A. baumannii isolates survived anaerobic conditions, competition with other microorganisms, and elevated concentrations of heavy metals in the stagnant swine manure for at least 2 weeks. These findings call for bacteriological analysis and disinfection of liquid swine manure before its application as a fertilizer in traditional extensive agriculture.

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.

In the previous studies OXA-23-like and OXA-24- like β-lactamase were reported among Acinetobacter baumannii in both hospitals and long-term care facilities (LTCF) in Croatia. The aim of this study was to analyze clinical and sewage A. baumannii isolates from two nursing homes in Zagreb, with regard to antibiotic susceptibility and resistance mechanisms, to determine the route of spread of carbapenem- resistant isolates. Nine clinical isolates were collected from February to May 2017 whereas in April 2017, ten A. baumannii isolates were collected from sewage of two nursing homes in Zagreb. Antibiotics susceptibility was determined by broth microdilution method. The presence of carbapenemase and extended spectrum β- lactamases (ESBL) encoding genes was explored by PCR. Conjugation and transformation experiments were performed as previously described. Genotyping was performed by SG determination, PFGE and MLST. Seven clinical isolates were positive for blaOXA24-like whereas two clinical and environmental carbapenem- resistant isolates, respectively, were found to possess blaOXA-23-like genes. Attempts to transfer imipenem resistance were unsuccessful indicating chromosomal location of blaOXA-23 gene. All carbapenem-resistant isolates belonged to SG- 1 (IC-2) whereas the rest of the isolates susceptible to carbapenems were allocated to SG- 2 (IC-1). PFGE analysis revealed low degree of genetic variability within both IC- I and IC- II. MLST corroborated that two environmental OXA-23 isolates belong to the ST- 195. This study showed dissemination of OXA-23 producing A. baumannii from the nursing home into the urban sewage. Disinfection of nursing home sewage should be recommended in order to prevent the spread of resistance genes into the community sewage.

Allchar ore deposit, a well-known polymetallic mineralisation, is drained by the Majdanska River characterized by elevated concentrations of heavy metals and metalloids, particularly As, Tl and Sb. The present study was conducted at the spot of the confluence of spring water, heavily loaded with toxic metals, which joins the Majdanska River by a few meters short stream. The spring water is outflow of underground water drained by passage through polymetallic mineralisation. The chemical characteristics of the Majdanska River water and the associated periphyton were used to evaluate and explain the abiotic response to the intrusion of toxicants and its impact on biota. At the site of the stream inflow, thorough mixing of Majdanska River water and incoming spring water resulted in an increase of Eh and pH close to the average river value. This geochemical barrier causes significant changes in the mobility of metal species. Moreover, the output composition of water is further modified by biological processes. The examined biota community was found to consist of algae Audouinella sp. and Spirogyra sp., with coexisting freshwater snails Radix labiata, diatoms and bacteria. While the carbonate bedrock controls the pH of the river water by effective buffering, it seems that bioconcentration and biomineralisation of algae exerted control on toxicants along the entire river flow. The highest bioconcentration factors were obtained for Fe, Mn and Ti which implies the biomineralisation of iron as a product of extracellular deposition on the cell walls on the organic matrices. Bioconcentration of As, Cr, Cs, Cu and Se was more important in Audouinella sp., while Cd, Co, Sr, and Zn preferentially accumulated in Spirogyra sp. High accumulation of Ba (3 mg/g d.w.) in Spirogyra sp. was associated with the intracellular biomineralisation. These findings suggest the potential use of Spirogyra sp. in the remediation of waters polluted by barium or as the biological pathfinder indicator for metal deposits associated with barite.

Every surface that is immersed in seawater becomes rapidly covered with an unavoidable biofilm. Such biofilm formation, also known as fouling, is a complex multistage process and not yet thoroughly investigated. In this study, the succession of diatoms and bacteria was investigated during a one month exposure on an artificial substrate of plexiglass (polymer of methyl methacrylate) mounted above the seafloor at a depth of 5 m. For biofilm analyses, the fouling was investigated using selective agar plates, epifluorescence, light and electronic microscopy, as well as high performance liquid chromatography (HPLC) pigment analysis. During biofilm development, the abundance of all biofilm components increased and reached maximum values after a one month exposure. In the bacterial community, heterotrophic marine bacteria were dominant and reached 1.96 ± 0.79 × 104colony forming units (CFU) cm-2. Despite the fact that faecal coliforms and intestinal enterococci were detected in the water column, faecal coliforms were not detected in the biofilm and intestinal enterococci appeared after one month of exposure but in the negligible number of 60 ± 10 CFU cm-2. The phototrophic component of the biofilm was dominated by diatoms and reached a concentration of 6.10 × 105cells cm-2, which was supported by pigment analysis with fucoxanthin as dominant pigment in a concentration up to 110 ng cm-2. The diatom community was dominated by Cylindrotheca closterium and other pennate benthic diatoms. A detailed taxonomic analysis by electronic microscopy revealed 30 different taxa of diatoms. The study confirmed that a plexiglass surface in a marine environment is susceptible to biofouling within 30 days of contact. Furthermore, the co lonization process sequence firstly involved bacteria and cyanobacteria, and secondly diatoms, which together formed a primary biofilm in the sea.

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.

In this study, biodegradability aspects of UV thermochromic leuco dye print on three different paper materials (synthetic, recycled, and bulky) were studied using the soil burial test under anaerobic conditions. Biodegradation of UV curable thermochromic prints were evaluated for changes by visual examination, microbial growth assay, weight loss measurements, Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy and colorimetric measurements. Results showed a better absorption of ink into the bulky paper structure, followed by recycled paper. Synthetic paper is not absorbent. The ink binder altogether with classic pigment and microcapsules penetrates into the structure of bulky paper. Recycled paper absorbs ink binder mostly with classic pigment. Better adsorption of binder into the bulky paper structure results in thinner layer of ink binder on the paper surface. On non-absorbent synthetic paper, microcapsules are covered with thicker layer of ink binder. In the case of bulky paper, the highest rate of biodegradation was observed, resulting in a higher number of bacteria, higher weight loss, higher changes in colour (destruction of almost all microcapsules) and almost complete reduction in thermochromic effect of the prints. Results show that microcapsules, which penetrate into the structure of bulky paper, are promoting the rate of bulky paper biodegradation. The opposite behaviour was noticed for the penetration of classic pigment into the structure of recycled paper, which resulted in remarkable reduction of recycled paper biodegradation rate. The thicker layer of ink binder (containing classic pigment) on the surface of microcapsules on recycled paper resulted in slower rate of microcapsules degradation and smaller colour change of the print. The thickest layer of ink binder and classic pigment on synthetic paper surface causes the slowest rate of biodegradation of print.

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.

The elimination of potentially pathogenic bacteria in wastewater treatment plants (WWTPs) attracts much attention in public health. Reports on the occurrence of the emerging hospital pathogen Acinetobacter baumannii in wastewaters do not include a continuous monitoring at all WWTP stages. The objective of this study was to characterize A. baumannii recovered from the water and sludge line of the secondary WWTP in Zagreb, Croatia over the period of one year. Recovery of A. baumannii was performed using CHROMagar Acinetobacter plates. Antimicrobial susceptibility testing was performed with broth microdilution and results interpreted using EUCAST breakpoints for clinical isolates of A. baumannii. Molecular characterization was performed by WGS and cgMLST. The secondary WWTP treating the urban wastewater is constantly receiving viable A. baumannii along with genes encoding carbapenem resistance, and emitting them via effluent into the environment. Furthermore, A. baumannii from influent are incorporated into activated sludge flocs in aeration basin. A. baumannii can survive the technological process of anaerobic mesophilic sludge digestion, and is finally destroyed in alkaline lime-treated stabilized sludge. The majority (102/119) of A. baumannii isolates were carbapenem-resistant, while antibiotic-susceptible isolates (17/119) were rarely recovered from all WWTP stages. Carbapenem-resistant isolates belonged to international clonal lineage IC2 carrying OXA-23 and IC1 carrying OXA-72, while the susceptible isolates belonged to IC5 or were unclustered. Increased resistance to antibiotics, together with the appearance of carbapenem- and even pandrug-resistant isolates in effluent as compared to influent wastewater, suggests the need of additional disinfection of effluent prior to its discharge into the natural recipient.

The multidrug resistant bacteria Acinetobacter baumannii cause serious hospital infections. Commercial poly(vinyl chloride) (PVC) used for endotracheal tubes was modified in order to obtain the composite with antibacterial effect towards clinical isolate of A. baumannii ST145. The composites were prepared by addition of different amounts of copper-containing zeolite tuff (CuZ) and by successive impregnation with D-Tyrosine (D-Tyr) solution. The composites which were obtained by addition of CuZ (CuZ-PVC) only did not exhibit antibacterial effect. The impregnation of the CuZ-PVC by D-Tyr resulted in an antibacterial effect which is explained by a synergistic effect of CuZ and D-Tyr. Rheological tests confirmed that the modification of PVC by CuZ does not affect its processability and reformability. [Projekat Ministarstva nauke Republike Srbije, br. 172018]

Bacterial resistance to carbapenems is an emerging problem of this century. A carbapenem-resistant bacterial population (CRBP) grown at 42°C was monitored in the influent and effluent of a secondary municipal wastewater treatment plant over 10 months. The municipal wastewater consisted of domestic, industrial, hospital and storm wastewaters. Median numbers of CRBP in influent and effluent water were 3.5 and 1.3 log CFU/mL, with its prevalence among total heterotrophic bacteria at 47% and 26%, respectively. Correlation of CRBP with physico-chemical and other bacteriological parameters of wastewater was estimated. Higher numbers of CRBP in influent and effluent were found in cases of nutrient-rich wastewater with higher concentrations of total heterotrophic bacteria and intestinal enterococci. Reduction of CRBP in the wastewater treatment process of 54% was comparable to the reduction of intestinal enterococci. Despite the significant elimination of CRBP in the secondary type of wastewater treatment plant, substantial numbers of CRBP are released through the effluent into the natural receiving waters. Since the CRBP grown at 42°C was not found in natural water samples beyond the vicinity of hospitals, these bacteria may be used as an indicator of hospital wastewaters. Keywords: environmental bacteria, carbapenem-resistant bacteria, public health, wastewater

Summary 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.

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.

Due to their susceptibility to bacterial biofilm formation, commercial tubes for medical use are one of the main sources of hospital infections with Acinetobacter baumannii. The anti-biofouling activity of novel composites against the clinical isolate of the multi-drug resistant A. baumannii is reported here. The composites were prepared by addition of micronised silver-exchanged natural zeolite (Ag-NZ) into poly(vinyl chloride) (PVC), followed by coating of the composites with D-Tyrosine (D-Tyr). The Ag-NZ composites (containing 1-15 wt% of Ag-NZ) coated with D-Tyr (Ag-NZ-Tyr) showed a bactericidal effect (100% or a 6.9 log CFU reduction) towards immobilised bacterial cells. The uncoated Ag- NZ composites showed a reduction of up to 70% (4.4 log CFU) of immobilised bacteria in comparison with the original PVC. Rheological testing of the composites revealed that the addition of Ag-NZ slightly affected processability and formability of the PVC and increased the elasticity of the polymer matrix.

Three samples of terra rossa were shown to be efficient adsorbents of phosphate [P(V)] from wastewater and removed 29.9–32.6% of P(V). The total iron content in terra rossa was the key factor which determined the P(V) removal from wastewater. The original samples of terra rossa were effective support materials for the immobilization of metabolically active P(V)-accumulating bacteriaAcinetobacter junii(0.56–2.47×1010CFU g–1). The removal of oxalate-extractable iron from original sample of terra rossa increased the number of immobilized bacteria to 1.34×10–11CFU g–1, which is the largest number of immobilized bacteria reported in the literature so far. In reactors containing theA. juniiand terra rossa P(V) was removed from wastewater by simultaneous adsorption onto terra rossa and accumulation inside bacterial cells, resulting in 40.5–62.5% of P(V) removal. Terra rossa is a promising substrate for biological P(V) removal from wastewater, acting both as adsorbent of P(V) and carrier of P(V)-accumulating bacteria.

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.

The antibacterial activity of natural zeolite (NZ) containing 2.03 wt.% of Cu 2+ (CuNZ), 5.07 of Ag + (AgNZ) or 8.50 of benzalkonium ion (BCNZ) were tested against clinical isolates of Acinetobacter baumannii belonging to European clone I and II. Isolate from clone II was more sensitive to CuNZ and AgNZ with minimum bactericidal concentration (MBC) 125 and 31.2 mg L −1 , respectively than isolate from clone I with MBC of 250 mg L −1 for both CuNZ and AgNZ. Isolate from clone I was more sensitive to BCNZ with MBC 250 mg L −1 than isolate from clone II with MBC 500 mg L −1 . The minimum inhibitory concentration (MIC) values were the same or slightly lower than the MBC values. The antibacterial activity of CuNZ, AgNZ and BCNZ against the A. baumannii European clone I and II was due to the activity of cations loaded onto NZ while not to the NZ used as support material. The antibacterial activity of CuNZ, AgNZ and BCNZ opens a possibility of future clinical investigations and its application.

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.