Microplastics in aquatic environments raise concern about their role as potential carriers of pathogens and organic pollutants. This study investigates the survival of the extensively drug-resistant Acinetobacter baumannii in the presence of selected priority substances including benzene derivatives (trichlorobenzene, pentachlorobenzene, and hexachlorobenzene), trifluralin, and primary polyethylene microplastics. The results indicate that the amount of adsorbed priority substances on microplastics from a mixture solution is slightly lower than the adsorption from individual solutions. Furthermore, GC/MS analysis shows that other chemicals used in plastic manufacturing can be released from microplastics into the environment over time and should be taken into account when assessing the environmental impact of microplastics. The proliferation of the environmental Sava 4 A. baumannii strain was not affected by the presence of benzene derivatives and microplastic particles at concentrations of up to 10 g/L in the water medium, and microscopy confirmed that it can colonise and form a biofilm on microplastic particles with adsorbed benzene derivatives, which demonstrates that microplastics have the potential to spread pollutants and potentially harmful bacteria over long distances and introduce them into various aquatic environments.

Wastewater treatment plants (WWTPs) receive clinically relevant Acinetobacter species originating from hospitals and municipal sewage, yet their role in enhanced biological phosphorus removal (EBPR) remains unclear. This study evaluated the capacity of Acinetobacter spp. to accumulate orthophosphate (P) under conditions simulating the aerobic phase of EBPR and investigated the physiological and molecular determinants of P accumulation. Clinically relevant A. pittii and A. baumannii exhibited comparable growth to the model P-accumulating species A. junii and successfully proliferated in synthetic wastewater. However, A. junii maintained significantly higher P uptake rates, accumulated more intracellular polyP granules, and continued to remove P after entering the stationary phase, whereas P removal by A. pittii and A. baumannii reached a plateau. While A. junii and A. pittii contained a single ppk1 gene, two distinct ppk1 homologues and the corresponding PPK1 isoforms were identified in A. baumannii. Nevertheless, neither ppk1 copy number, ppk1 expression, nor the abundance of key enzymes involved in canonical polyP metabolism explained the enhanced P accumulation capacity of A. junii. Proteomic analyses suggested that continued P uptake in A. junii was associated with physiological adaptations that support the maintenance of cellular fitness during the stationary phase. Consequently, EBPR systems may favour the persistence and proliferation of clinically relevant Acinetobacter spp. without substantially benefiting from their contribution to P removal.

Carbapenem-resistant bacteria (CRB) pose a significant threat to public health due to their resistance to last-resort antibiotics. Even though they are widely studied in clinical settings, much less is known about their presence in environmental compartments with multiple pathways contributing to their dissemination, which raises a growing concern. The aim of this narrative review is to summarise the current knowledge about the occurrence, isolation, and characterisation of CRB in hospital and natural environments and to highlight their clinical relevance and environmental reservoirs. The CRB species pathogenic for humans - Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa - are often identified in hospital and urban sewage, wastewater treatment plants, water bodies, sediments, soil, animals, and plants. Their presence in these environments is largely attributed to anthropogenic factors such as the discharge of untreated or partially treated effluent from wastewater treatment plants. Suitable methods for CRB isolation include selective media, phenotypic assays, and molecular tools for species identification and resistance gene detection. This review also addresses the One Health approach, which stems from the interconnectedness of humans, animals, and environment in the spread of CRB. While the species-level transmission within the One Health framework is well-documented, further research is needed to establish strain-level dissemination patterns. Understanding the mechanisms of CRB persistence and transmission in diverse environments is crucial for developing effective mitigation strategies to curb their spread.

The Krapina River, located in the Hrvatsko Zagorje region, is exposed to anthropogenic pollution due to discharges of untreated urban and hospital wastewater. The aim of this research was to determine the bacteriological quality of water and surface sediments by analysing the presence of indicators of faecal pollution. Sampling was carried out from the river source and the sources of two tributaries, as well as downstream after the confluence of the Krapinica River. Intestinal enterococci and faecal coliforms were already recorded at the sources of the river and its tributaries, with their concentrations significantly increasing downstream due to the inflows of urban and hospital wastewater. The concentrations of indicator bacteria in the sediment were higher than in the corresponding free water. Based on the concentration of bacteria indicating faecal pollution, the Krapina River does not meet the criteria for bathing and recreation. Wastewater treatment prior to its discharges into the river is crucial for reducing anthropogenic impact on the ecosystem, while systematic water and sediment quality monitoring can contribute to the adoption of effective river protection measures.

Certain bacterial species are a persistent issue in hospital-acquired infections due to their fast and potent development of multi-drug resistance. This study aimed to evaluate the antibacterial activity of mono-substituted HAp, incorporating potentially antibacterial ions (Ag+, Ce3+, Cu2+, Ga3+, and Mn2+), against clinically-relevant bacteria Escherichia coli, Staphylococcus aureus, Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Samples were analyzed using X-ray diffraction, Rietveld refinement analysis, Fourier Transform Infrared Spectroscopy, scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy and zeta potential, alongside the cytotoxicity assays and antibacterial activity determined by disc diffusion and broth microdilution methods. All materials were non-cytotoxic to cells, except for Cu-substituted HAp. HAp substituted with Ag+ showed significant antibacterial activity against all tested bacteria. Ce-substituted HAp showed no antibacterial effect, while Ga- and Mn-substitution indicated potential antibacterial properties towards certain bacterial strains by inhibiting bacterial attachment to its surface. Judging by the SEM analysis, the gram-negative bacteria attached to the HAp surface in a lesser amount then the gram-positive ones. Given the variability in methodologies used for evaluating the antibacterial activity of HAp reported in the literature, we propose a standardized testing protocol that incorporates minimum bactericidal concentration (MBC) assessments and disc diffusion tests on finished materials or coatings, coupled with SEM analysis.

Natural zeolites are highly effective adsorbents that can remove various metal cations which would otherwise contaminate the environment. However, different metal cations (Cu, Zn, and Ag) within their lattice or quaternary long-chain surfactant cations on their surface modify their affinity towards hazardous anions and promote antibacterial activity in natural zeolites. Specifically, natural zeolites in their non-modified form lack intrinsic antibacterial characteristics. NZ is the most widespread natural zeolite. This review presents the antibacterial efficiency of NZ containing transition metals, nano oxides, and organics. This effect is nonspecific and primarily driven by the nutritional makeup of the medium rather than the species of pathogenic bacteria under study. Studies on using NZ-based disinfectants to clean up contaminated water and soil and using modified and purified NZ to protect health are also considered. By eliminating toxic ions and, when modified by these toxic cations, removing pathogens from the environment, natural NZ can serve a dual function, providing it with the distinctive characteristics of a sustainable material.

The aim of this retrospective study was to investigate the molecular characteristics and survival ability of clinical isolates of extended-spectrum beta-lactamase (ESBL)-producing strains of Klebsiella pneumoniae (K. pneumoniae) in a hospital setting. ESBL-producing K. pneumoniae clinical isolates were collected from hospitalized patients at the University Clinical Hospital of Mostar, Bosnia and Herzegovina. Identification and antibiotic susceptibility tests were performed according to established laboratory methods and Clinical and Laboratory Standards Institute standards. Phenotypic identification of the isolates was confirmed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Minimum inhibitory concentrations were determined using the Vitek (R) 2 Compact System. Clonal relationships between the different isolates were studied using pulsed-field gel electrophoresis. Molecular characterization of ESBL-coding genes was performed using multiplex polymerase chain reaction. The survival and pellicle formation abilities of ESBL-producing K. pneumoniae isolates were also investigated. Among 471 isolated K. pneumoniae strains, 149 isolates (31.6%) were ESBL-producing. The bla genes encoding CTX-M-1-group ESBLs were identified in almost all isolates, with the majority carrying blaCTX-M-15. Two isolates contained bla(CTX-M-3). All isolates carried bla(TEM-1). The blaSHV genes identified were bla(SHV-1), bla(SHV-5), bla(SHV-11), and bla(SHV-28). Six isolates harbored bla(OXA-1). ESBL-producing K. pneumoniae strains survived on dry cotton for up to 49 days.

The production of biogas is achieved during anaerobic digestion (AD) using organic matter as a substrate. In Mediterranean countries, a promising substrate is lignocellulose biomass of perennial grass Miscanthus x giganteus, due to its potentially high biogas yields, which could be comparable to maize silage. During AD, bacteria convert biomass into more minor compounds, which are further converted to methane by methanogenic archaea. The selection of appropriate microbes for the degradation of the substrate is crucial, and the enhancement of this step lies in the immobilization of microbes on biocarriers. Described here, a microbial consortium, de novo isolated and conditioned to degrade the Mischantus biomass, was immobilized onto several natural biocarriers: natural zeolitized tuff, ZeoSand® (Velebit Agro, Zagreb, Croatia), perlite, and corncob. There was no statistically significant difference in the number of immobilized bacteria across the different materials. Therefore, all proved to be suitable for the immobilization of the consortium. In the consortium, five bacterial species with different shares in the consortium were identified: Enterobacter cloacae, Klebsiella pneumoniae, Enterobacter asburiae, Leclercia adecarboxylata, and Exiguobacterium indicum. After immobilization on each carrier, the share of each species changed when compared to starting conditions, and the most dominant species was E. cloacae (71–90%), while the share for other species ranged from 2 to 23%. The share of E. indicum was 14% at the start. However, it diminished to less than 1% because it was overgrown during the competition with other bacterial species, not due to an inability to immobilize.

Adsorptive, catalytic, and antibacterial properties of clinoptilolite-rich tuffs (ZT) are presented here. ZT transformed into Fe-containing ZT (Fe-ZT) removes various organic and inorganic anions from water. Fe-ZT, which contains selenium, is beneficial for growing Pleurotus ostreatus mushrooms. The fungi convert inorganic Se from Fe-ZT into a more useful organically bonded form. ZT and Fe-ZT as supplements retain nitrogen and potassium in sandy, silty loam and silty clay soils. ZT shows an affinity toward toxic metal cations, which are essential for cleaning contaminated water. The adsorption of atenolol, acetylsalicylic, and salicylic acid onto M-ZT (M-Cu2+, Mn2+, Ni2+, or Zn2+) from water solutions suggests that both the natures of M and pharmaceuticals have a significant impact on the adsorption mechanism and determine the adsorption capability of the ZT. ZT is an excellent carrier for ultrafine (2-5 nm) nano oxide particles, which have been shown to have catalytic activity in different chemical processes and photodegradation reactions of organic pollutants. ZT can also be transformed into SO4-SnO2-ZT, which is catalytically active as a solid acid. M-ZT is an effective carrier of valuable bacteria. Ag-ZT possesses beneficial bactericidal activity in disinfecting water and soil remediation.

The aim of this study was to find the source of Acinetobacter baumannii in the intensive care unit (ICU) after an outbreak during the coronavirus disease 2019 (COVID-19) pandemic, as there was no A. baumannii detected on usually screened susceptible surfaces. The screening of the ICU environment was done in April 2021 when eleven different samples were taken. One A. baumannii isolate was recovered from the air conditioner and was compared with four clinical A. baumannii isolates obtained from patients hospitalized in January 2021. Isolates were confirmed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), minimum inhibitory concentrations (MICs) were determined, and the multilocus sequence typing (MLST) was performed. The molecular identification of A. baumannii isolates as ST208, the presence of the same blaOXA-23 carbapenemase gene, and the same antibiotic susceptibility profile suggest that the isolate recovered from the air conditioner is the same as the isolates recovered from hospitalized patients. The environmental isolate was recovered three months later than the clinical isolates, emphasizing the ability of A. baumannii to survive on dry abiotic surfaces. The air conditioner in the clinical environment is an important but undoubtedly neglected source of A. baumannii outbreaks, hence, frequent disinfection of hospital air conditioners with appropriate disinfectants is mandatory to mitigate the circulation of A. baumannii between patients and the hospital environment.

OBJECTIVES:To characterise 11 colistin- and carbapenem-resistant Acinetobacter baumannii isolates recently emerging in hospital settings.METHODS:A. baumannii isolates were collected from hospitalised patients under colistin treatment in three countries of Southeast Europe: Turkey, Croatia, and Bosnia and Herzegovina. Isolates were identified using molecular methods.RESULTS:Isolates from Turkey and Croatia belong to the sequence types ST195 or ST281 of the clone lineage 2, while the single isolate from Bosnia and Herzegovina belongs to the ST231 of clone lineage 1. All isolates turned out to be highly resistant to colistin (MIC ≥ 16 mg/L) and have point mutations in pmrCAB operon genes. The colistin-resistant isolate from Bosnia and Herzegovina had a unique P170L point mutation in the pmrB gene and the R125H point mutation in the pmrC gene. The L20S mutation in the pmrA gene was detected only in isolates from Croatia and has never been reported before in isolates from this country.CONCLUSION:Colistin resistance in A. baumannii in hospitalised patients receiving colistin treatment is a result of chromosomal mutations. The pattern of point mutations in pmrCAB genes suggests a spread of specific colistin-resistant isolates within the hospital.

Microplastic, an emerging pollutant in the aquatic environment, raises concerns about its role as a potential carrier of pathogens and organic pollutants. The study investigates the survival of the extensively drug-resistant bacterium Acinetobacter baumannii in the presence of priority substances, benzene derivates: trichlorobenzenes, pentachlorobenzene, hexachlorobenzene, trifluralin and primary polyethylene as microplastics. The results indicated that the amount of adsorbed priority substances applied to the microplastics as a mixture in solution is slightly lower than sorption from individual solutions. In addition, the results of GC/MS analysis of the non-target substances indicate that chemicals from plastic manufacturing or plastic-containing products can be released into the environment over time, underlining the importance of considering this factor when assessing the environmental impact of microplastics. The survival of A. baumannii was not affected by the presence of benzene derivates and microplastic particles up to a concentration of 10.000 mg/L in the water medium. Microscopic analysis confirmed that A. baumannii is able to colonise and form a biofilm on microplastic particles with sorbed benzene derivates. This indicates that microplastics can promote the spread of pollutants and potentially pathogenic bacteria in the environment.

A group of about 20 bacteria species called the Burkholderia cepacia complex (BCC) are widespread in the environment, but they are also the cause of severe infections in humans. In the Republic of Croatia, relatively little research has been conducted on the presence of these bacteria in different habitats and the impact these bacteria have on humans. The paper examines the long-term behaviour of five clinically significant BCC bacteria species in waters with different nutrient concentrations. The clinical isolate and four isolates extracted from the environment impacted by human liquid or solid waste survived in the long term and even multiplied in nutrient-poor or nutrient-rich water. After a 50-day monitoring, the concentration of four out of the five tested isolates in water was higher than the initial concentration. Considering the potential for a long-term retention of clinically significant BCC bacteria species in aquatic ecosystems, the presence of the BCC in water should be considered as a source of sporadic human infections. In order to avoid this public health risk, the BCC should be removed from human solid and liquid waste before its disposal in the environment. For monitoring of the BCC epidemiology, it is necessary to pay attention to the isolation and characterization of the BCC both in the clinics and the environment.

Little is known about the bonfire impact on microbial properties in soil. This work aimed to study moderate- to high-severity experimental burning (250 °C) compared to unburned Cambisol in a natural Mediterranean environment (Croatia) on selected soil properties. The soil was sampled immediately and 1, 2, 4, and 6 months after the fire. The fire increased the mean weight diameter, water stable aggregates, and water repellence in different soil fractions, and the observed effect was the strongest immediately after the fire. It also altered soil pH, electrical conductivity, total nitrogen carbon, and sulphur content, and completely destroyed carbapenem-resistant bacteria, but did not significantly affect the soil’s mineralogical properties. Six months after the fire, most microbial properties (save for pH) returned to near control values. Heterotrophic, sporogenic, and phosphate-solubilising bacteria started to recover after a month, whereas the population of carbapenem-resistant bacteria was destroyed initially, but recovered by the fourth month after the fire. Dehydrogenase activity was not significantly affected, but proper recovery started four months after the fire. Even though Cambisol showed some resilience to fire and its properties mostly returned to normal by the sixth month, and a full recovery is expected to occur later, as vegetation returns.

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.

Pipeline spills and pollution of the environment by crude oil pose a threat to natural resources, especially soil and water. One such incident occurred on 25 September 2018 in the area of Budrovac (Croatia; 46°00′14.6″ N 17°04′16.8″ E) on agricultural land as a pipeline spill. Bioremediation of the contaminated soil was carried out with organic pollutants using an environmentally safe absorbent Spill-Sorb (Canadian Sphagnum Peat Moss) and a mineral fertilizer—nitrogen. The experiment was conducted in the greenhouse of the Faculty of Agriculture, Croatia, during a six-month (October 2018–April 2019) study. Samples of agricultural soils contaminated with total petroleum hydrocarbons (TPHs) and polycyclic aromatic hydrocarbons (PAHs) were taken after the rupture of the local gas condensate pipeline. The experiment was conducted in five treatments in triplicate: I-control (clean soil); II-100% contaminated soil + organic absorbent + nitrogen; III-100% contaminated soil + organic absorbent; IV-50% clean soil + 50% contaminated soil + organic absorbent + nitrogen; and V-50% clean soil + 50% contaminated soil + organic absorbent. The soil properties studied were pH, organic matter content, carbon and nitrogen content and ratio, and changes in the concentration of potential organic contaminants—TPHs and individual PAHs. The results demonstrated that the mixture of organic absorbent and nitrogen efficiently removed organic pollutants from the contaminated soil within six months. However, the application of Spill-Sorb alone was more effective for the degradation of hydrocarbons. The effectiveness of the absorbent studied was dependent on the concentration of organic pollutants and nitrogen application.

For a long time, the production and processing of cowhide was based on the use of chrome tanning. However, the growing problem with chromium waste and its negative impact on human health and the environment prompted the search for more environmentally friendly processes such as vegetable tanning or aldehyde tanning. In the present study, we investigated the DNA-damaging effects induced in HepG2 cells after 24 h exposure to leather samples (cut into 1 × 1 cm2 rectangles) processed with different tanning agents. Our main objective was to determine which tanning procedure resulted in the highest DNA instability. The extent of treatment-induced DNA damage was determined using the alkaline comet assay. All tanning processes used in leather processing caused primary DNA damage in HepG2 cells compared to untreated cells. The effects measured in the exposed cells indicate that the leaching of potentially genotoxic chemicals from the same surface is variable and was highest after vegetable tanning, followed by synthetic tanning and chrome tanning. These results could be due to the complex composition of the vegetable and synthetic tanning agents. Despite all limitations, these preliminary results could be useful to gain a general insight into the genotoxic potential of the processes used in the processing of natural leather and to plan future experiments with more specific cell or tissue models.

Bacterium Acinetobacter baumannii is a leading cause of nosocomial infections. The occurrence of antibiotic-resistant A. baumannii isolates outside hospitals suggests that monitoring of this pathogen in environmental samples is needed. Survival of pandrug-resistant A. baumannii was followed on selective plates with and without carbapenems in water and soil. After a few days of starvation, A. baumannii lost the ability to be cultivated at 44°C on plates supplemented with carbapenems. Once cultivated on plates without carbapenems and/or at 36°C, A. baumannii could grow again at 44°C in the presence of carbapenems. Comparative proteomic analysis revealed that impaired membrane integrity and reduced function of efflux pumps due to elevated temperature combined with antibiotic exposure were the main reasons for this phenomenon. Loss of thermotolerance in the presence of antibiotics points to the need for temperature adjustment in long-term monitoring of A. baumannii in environmental samples, to avoid the underestimation of viable bacteria.

Novel composites based on natural zeolite—clinoptilolite and monoterpene phenols—thymol and carvacrol were prepared by supercritical solvent impregnation (SSI) in supercritical carbon dioxide (scCO2) at 30 MPa and 35 °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 μT 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.