Detection of microplastics in zebrafish housing systems: Can microplastic background contamination affect the final results of microplastic-related toxicological tests?

Concentrations of microplastics (MPs) were determined in three commonly used zebrafish housing systems to see if their levels could affect the final results of laboratory microplastic-related toxicology tests. MPs have received notable attention in the last few years, and their toxicology tests have also come to the fore. Zebrafish (Danio rerio), kept in fish housing systems, are widely used as models for MPs studies. Most of these systems contain a significant number of parts made of different polymers. As usage and amortization can erode these parts, MPs might appear in the keeping water or the fish body, which may represent a background load and possibly influence the results of microplastic-related toxicological tests. To take representative water samples from systems, two in-situ filtration techniques, a newly developed peristaltic pump-, and a jet pump-driven method were applied. The collected MP particles were analyzed with a Fourier-transform infrared microscope (detection limit 50 µm), and their possible origin was also investigated. The newly developed technique was more sufficient for sampling as it had a higher MPs recovery, especially in the smaller size range. Polyester, polyethylene and polypropylene were the most frequently detected polymers in the examined fish housing systems, the highest detected concentration was 0.31±0.12 particles/liter (0.22±0.16 µg/liter). These values are negligible compared to the literature data reporting enormously high applied MPs concentrations (104 - 2.21 × 108 particles/liter) during toxicology tests. The results also show that some detected MPs did not originate from the systems, their origin was presumed to be external.

The Cocktail Effects on the Acute Cytotoxicity of Pesticides and Pharmaceuticals Frequently Detected in the Environment.

Xenobiotics never appear as single, isolated substances in the environment but instead as multi-component mixtures. However, our understanding of the ecotoxicology of mixtures is far from sufficient. In this study, three active pharmaceutical ingredients (carbamazepine, diclofenac, and ibuprofen) and three pesticides (S-metolachlor, terbuthylazine, and tebuconazole) from the most frequently detected emerging micropollutants were examined for their acute cytotoxicity, both individually and in combination, by bioluminescence inhibition in Aliivibrio fischeri (NRRL B-11177). Synergy, additive effects, and antagonism on cytotoxicity were determined using the combination index (CI) method. Additionally, PERMANOVA was performed to reveal the roles of these chemicals in binary, ternary, quaternary, quinary, and senary mixtures influencing the joint effects. Statistical analysis revealed a synergistic effect of diclofenac and carbamazepine, both individually and in combination within the mixtures. Diclofenac also exhibited synergy with S-metolachlor and when mixed with ibuprofen and S-metolachlor. S-metolachlor, whether alone or paired with ibuprofen or diclofenac, increased the toxicity at lower effective concentrations in the mixtures. Non-toxic terbuthylazine showed great toxicity-enhancing ability, especially at low concentrations. Several combinations displayed synergistic effects at environmentally relevant concentrations. The application of PERMANOVA was proven to be unique and successful in determining the roles of compounds in synergistic, additive, and antagonistic effects in mixtures at different effective concentrations.

In vivo estrogenicity of glyphosate, its formulations, and AMPA on transgenic zebrafish (Danio rerio) embryos.

In this study, the disrupting effects of glyphosate (GLY), aminomethylphosphonic acid (AMPA), and three glyphosate-based herbicides (GBHs) on vitellogenesis in a non-concentration-dependent manner are reported for the first time in 120 h of acute exposure of zebrafish at environmentally relevant concentrations. GBHs are commonly used worldwide in weed control management. Due to their extensive application, they frequently occur in aquatic ecosystems and may affect various organisms. The active substance GLY and its major by-product, AMPA, are the most thoroughly studied chemicals; however, the adverse effects of the complex formulas of GBHs with diverse and unknown content of co-formulants are still not sufficiently researched. This study focused on the embryotoxicity, sublethal malformations, and estrogenic potency of GLY, AMPA, and four commonly used GBHs on zebrafish embryos using a wild type and an estrogen-sensitive, transgenic zebrafish line (Tg(vtg1:mCherry)). After 120 h of exposition, AMPA did not cause acute toxicity, while the LC50 of GLY was 160 mg/L. The GBHs were more toxic with LC50 values ranging from 31 to 111 GLY active equivalent (a.e.) mg/L. Exposure to 0.35-2.8 mg/L GBHs led to sublethal abnormalities: typical symptoms were structural deformation of the lower jaw and anomalies in the olfactory region. Deformity rates were 10-30% in the treated groups. In vivo, fluorescently expressed vtg1 mCherry protein in embryonic liver was detected by a non-invasive microscopic method indicating estrogenic action through vitellogenin production by GLY, AMPA, and GBHs. To confirm the in vivo findings, RT-qPCR method was performed to determine the levels of the estrogenicity-related vtg1 mRNA. After 120 h of exposure to GLY, AMPA, and three GBHs at a concentration of 0.35 mg/L, the expression of vtg1 gene was significantly up-regulated. Our results highlight the risk that short-term GLY and GBH exposure can cause developmental malformations and disrupt the hormonal balance in zebrafish embryos.

Evaluating the In Vivo Virulence of Environmental Pseudomonas aeruginosa Using Microinjection Model of Zebrafish (Danio rerio).

(1) Background: Microinjection of zebrafish (Danio rerio) embryos offers a promising model for studying the virulence and potential environmental risks associated with Pseudomonas aeruginosa. (2) Methods: This work aimed to develop a P. aeruginosa infection model using two parallel exposition pathways on zebrafish larvae with microinjection into the yolk and the perivitelline space to simultaneously detect the invasive and cytotoxic features of the examined strains. The microinjection infection model was validated with 15 environmental and clinical strains of P. aeruginosa of various origins, antibiotic resistance profiles, genotypes and phenotypes: both exposition pathways were optimized with a series of bacterial dilutions, different drop sizes (injection volumes) and incubation periods. Besides mortality, sublethal symptoms of the treated embryos were detected and analyzed. (3) Results: According to the statistical evaluation of our results, the optimal parameters (dilution, drop size and incubation period) were determined. (4) Conclusions: The tested zebrafish embryo microinjection infection model is now ready for use to determine the in vivo virulence and ecological risk of environmental P. aeruginosa.

Presence, variation, and potential ecological impact of microplastics in the largest shallow lake of Central Europe.

The presence of microplastics (MPs) in the global ecosystem has generated a rapidly growing concern worldwide. Although their presence in the marine environment has been well-studied, much less data are available on their abundance in freshwaters. MPs alone and in combination with different chemicals has been shown to cause acute and chronic effects on algae and aquatic invertebrate and vertebrate species at different biological levels. However, the combined ecotoxicological effects of MPs with different chemicals on aquatic organisms are still understudied in many species and the reported data are often controversial. In the present study, we investigated, for the first time, the presence of MPs in Lake Balaton, which is the largest shallow lake of Central Europe and an important summer holiday destination. Moreover, we exposed neonates of the well-established ecotoxicological model organism Daphnia magna to different MPs (polystyrene [3 µm] or polyethylene [≤ 100 µm]) alone and in combination with three progestogen compounds (progesterone, drospirenone, levonorgestrel) at an environmentally relevant concentration (10 ng L-1) for 21 days. The presence of 7 polymer types of MPs in the size range of 50-100 µm was detected in Lake Balaton. Similarly to the global trends, polypropylene and polyethylene MPs were the most common types of polymer. The calculated polymer-independent average particle number was 5.5 particles m-3 (size range: 50 µm - 100 µm) which represents the values detected in other European lakes. Our ecotoxicological experiments confirmed that MPs and progestogens can affect D. magna at the behavioral (body size and reproduction) and biochemical (detoxification-related enzyme activity) levels. The joint effects were negligible. The presence of MPs may lead to reduced fitness in the aquatic biota in freshwaters such as Lake Balaton, however, the potential threat of MPs as vectors for progestogens may be limited.

Glyphosate and glyphosate-based herbicides (GBHs) induce phenotypic imipenem resistance in Pseudomonas aeruginosa.

GBHs are the most widely used herbicides for weed control worldwide that potentially affect microorganisms, but the role of their sublethal exposure in the development of antibiotic resistance of Pseudomonas aeruginosa is still not fully investigated. Here, the effects of glyphosate acid (GLY), five glyphosate-based herbicides (GBHs), and POE(15), a formerly used co-formulant, on susceptibility to imipenem, a potent carbapenem-type antibiotic, in one clinical and four non-clinical environmental P. aeruginosa isolates were studied. Both pre-exposure in broth culture and co-exposure in solid media of the examined P. aeruginosa strains with 0.5% GBHs resulted in a decreased susceptibility to imipenem, while other carbapenems (doripenem and meropenem) retained their effectiveness. Additionally, the microdilution chequerboard method was used to examine additive/antagonistic/synergistic effects between GLY/POE(15)/GBHs and imipenem by determining the fractional inhibitory concentration (FIC) indexes. Based on the FIC index values, glyphosate acid and Total demonstrated a potent antagonistic effect in all P. aeruginosa strains. Dominator Extra 608 SL and Fozat 480 reduced the activity of imipenem in only one strain (ATCC10145), while POE(15) and three other GBHs did not have any effect on susceptibility to imipenem. Considering the simultaneous presence of GBHs and imipenem in various environmental niches, the detected interactions between these chemicals may affect microbial communities. The mechanisms of the glyphosate and GBH-induced imipenem resistance in P. aeruginosa are yet to be investigated.

Novel members of bacterial community during a short-term chilled storage of common carp (Cyprinus carpio).

This work aimed to identify the key members of the bacterial community growing on common carp (Cyprinus carpio) fillets during chilled storage with next-generation sequencing (NGS) and cultivation-dependent methods. Carp fillets were stored for 96 h at 2 °C and 6 °C with and without a vacuum package, and an additional frozen-thawed storage experiment was set for 120 days. Community profiles of the initial and stored fish samples were determined by amplicon sequencing. Conventional microbial methods were used parallelly for the enumeration and cultivation of the dominant members of the microbial community. Cultivated bacteria were identified with 16S rRNA sequencing and the MALDI-TOF MS method. Based on our results, the vacuum package greatly affected the diversity and composition of the forming microbial community, while temperature influenced the cell counts and consequently the microbiological criteria for shelf-life of the examined raw fish product. Next-generation sequencing revealed novel members of the chilled flesh microbiota such as Vagococcus vulneris or Rouxiella chamberiensis in the vacuum-packed samples. With traditional cultivation, 161 bacterial strains were isolated and identified at the species level, but the identified bacteria overlapped with only 45% of the dominant operational taxonomic units (OTUs) revealed by NGS. Next-generation sequencing is a promising and highly reliable tool recommended to reach a higher resolution of the forming microbial community of stored fish products. Knowledge of the initial microbial community of the flesh enables further optimization and development of processing and storage technology.

Dyadobacter subterraneus sp. nov., isolated from hydrocarbon-polluted groundwater from an oil refinery in Hungary.

A Gram-stain-negative, aerobic, non-spore-forming, rod-shaped bacterial strain (UP-52T) was isolated from hydrocarbon-polluted groundwater located near an oil refinery in Tiszaujvaros, Hungary. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the isolate belongs to the genus Dyadobacter in the family Cytophagaceae. Its closely related species are Dyadobacter frigoris (98.00 %), Dyadobacter koreensis (97.64 %), Dyadobacter psychrophilus (97.57 %), Dyadobacter ginsengisoli (97.56 %) and Dyadobacter psychrotolerans (97.20 %). The predominant fatty acids are summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω7c/C16 : 1 ω6c), C15 : 0 iso, C16 : 1 ω5c and C17 : 0 iso 3OH. The predominant respiratory quinone detected in strain UP-52T is quinone MK-7. The dominant polar lipids are glycolipid, phosphoaminolipid, phospholipid and aminolipid. The DNA G+C content is 40.0 mol%. Flexirubin-type pigment was present. Based on these phenotypic, chemotaxonomic and phylogenetic results, UP-52T represents a novel species of the genus Dyadobacter, for which the name Dyadobacter subterraneus sp. nov. is proposed. The type strain is UP-52T (=NCAIM B.02653T=CCM 9030T).

Groundwater, soil and compost, as possible sources of virulent and antibiotic-resistant Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a major public health concern all around the world. In the frame of this work, a set of diverse environmental P. aeruginosa isolates with various antibiotic resistance profiles were examined in a Galleria mellonella virulence model. Motility, serotypes, virulence factors and biofilm-forming ability were also examined. Molecular types were determined by pulsed-field gel electrophoresis (PFGE). Based on our results, the majority of environmental isolates were virulent in the G. mellonella test and twitching showed a positive correlation with mortality. Resistance against several antibiotic agents such as Imipenem correlated with a lower virulence in the applied G. mellonella model. PFGE revealed that five examined environmental isolates were closely related to clinically detected pulsed-field types. Our study demonstrated that industrial wastewater effluents, composts, and hydrocarbon-contaminated sites should be considered as hot spots of high-risk clones of P. aeruginosa.

Parvularcula mediterranea sp. nov., isolated from marine plastic debris from Zakynthos Island, Greece.

A Gram-negative, dark orange-pigmented, aerobic, non-spore-forming, coccoid-shaped bacterium designated as ZS-1/3T was isolated from a floating plastic litter (polypropylene straw) sample, collected from shallow seawater near the public beach of Laganas on Zakynthos island, Greece. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the isolate is affiliated with the genus Parvularcula in the family Parvularculaceae. Its closest relatives are Parvularcula lutaonensis (98.09  %) and Parvularcula oceanus (95.89  %). The pH and temperature ranges for growth are pH 5-10 and 20-38 °C (optima, pH 7.0 and 28 °C). The predominant fatty acids are C18 : 1 ω7c (56.84  %), C16 : 0 (27.51  %), C18 : 0 (2.25  %) and C12 : 0 (1.42  %). The predominant respiratory quinone detected in strain ZS-1/3T is quinone-10 (Q10); the majority of detected polar lipids are glycolipid. The DNA G+C content is 62.5  mol%. Physiological and chemotaxonomic data further confirmed the distinctiveness of strain ZS-1/3T from other members of the genus Parvularcula. Thus, strain ZS-1/3T is considered to represent a novel species of the genus, for which the name Parvularcula mediterranea. sp. nov. is proposed. The type strain is ZS-1/3T (=NCAIM B 02654T=CCM 9032T).

Planktonic and Benthic Bacterial Communities of the Largest Central European Shallow Lake, Lake Balaton and Its Main Inflow Zala River.

Lake Balaton is the largest European shallow lake, which underwent cultural eutrophication in the '70-80s. Therefore, strict pollution control measures were introduced and the water quality has become meso-eutrophic since the millennium. Due to the touristic significance and change in trophic levels of the lake, numerous ecological studies were carried out, but none of them was focused on both benthic and planktonic microbial communities at the same time. In our study, an attempt was made to reveal the spatial bacterial heterogeneity of the Lake Balaton and Zala River by 16S rDNA terminal restriction fragment length polymorphism fingerprinting and Illumina amplicon sequencing methods in the summer of 2017. According to the molecular biology results, mostly well-known freshwater microorganisms, adapted to nutrient-poor conditions were found in the pelagic water column. The LD12 subclade member Fonsibacter ubiquis, the cyanobacterial Synechococcus sp. and unknown Verrucomicrobia species were abundant in the less nutrient-dense basins, while the hgcI clade members showed various distribution. In the estuary and in the nutrient-dense western part of the lake, some eutrophic conditions preferring cyanobacteria (filamentous Anabaena and Aphanizomenon species) were also detectable. The benthic microbial community showed higher diversity, according to the observed appearance of microorganisms adapted to the deeper, less aerated layers (e.g. members of Desulfobacteraceae, Nitrosomonadaceae).

First isolation of carbapenem-resistant Acinetobacter beijerinckii from an environmental sample.

The emergence of opportunistic Acinetobacter spp. in healthcare settings poses a significant threat to public health. The major reasons for nosocomial spread of these species are their abilities to develop and transfer drug resistance against various classes of antibiotics. Considering that Acinetobacter spp. are ubiquitous in nature, can utilize several carbon sources, and reach humans via various pathways, our aim was to obtain information about the environmental strains of this genus. Our first step was to develop and test a multistep isolation procedure based on traditional scientific methods. Antibiotic resistance patterns of the isolated strains were determined, as susceptibility to 12 antibiotics of 7 classes was tested by MIC Test Strip method. Altogether 366 samples (groundwater, surface water, and soil) of 24 sites were investigated and a collection of 37 Acinetobacter isolates was obtained. Among others, clinically important human pathogen Acinetobacter spp., such as A. baumannii, A. johnsonii, and A. gyllenbergii were identified. Three environmental strains were determined as multidrug-resistant including a carbapenem-resistant, hemolytic Acinetobacter beijerinckii strain isolated from a hydrocarbon-contaminated groundwater sample. In summary, it has been found that the applied multistep isolation procedure is applicable to isolate various species of Acinetobacter genus. Based on the antibiotic resistance assay, we can conclude that environmental representatives of Acinetobacter spp. are able to develop multidrug resistance, but at a lower rate than their clinical counterparts.

Characterization of environmental Pseudomonas aeruginosa using multilocus sequence typing scheme.

PURPOSE: The objectives of this study were to examine environmental (hydrocarbon degrading) Pseudomonas aeruginosa isolates with Multilocus Sequence Typing (MLST) and to determine their relevant features, such as serotype, virulence genes, biofilm forming ability and hydrocarbon degrading capacity. METHODOLOGY: The diversity of environmental isolates was assessed with an MLST scheme. Investigation of virulence determinants included serotyping, hemolytic activity test and the detection of virulence genes exoS, exoY, exoT, exoU, exoA. Biofilm forming ability was examined in a modified microtiter assay, hydrocarbon degrading capacity was determined with gravimetric methods. RESULTS: The majority of environmental isolates shared the same MLST profiles with isolates of cystic fibrosis (CF). Virulence patterns and serotypes were slightly connected to the phylogenetic localization, but further clinically important features such as antibiotic resistance were not. At least one of the examined environmental isolates was multidrug-resistant, virulent and had biofilm forming ability such as nosocomial P. aeruginosa and retained its hydrocarbon degradation ability. CONCLUSION: The current theses that distinguish isolates originating from different sources are questionable; environmental P. aeruginosa can be a potential risk to public health and cannot be excluded as an external (non-nosocomial) source of infections, especially in patients with CF. Further studies such as pulsed-field gel electrophoresis (PFGE) and the determination of other clinically important virulence factors are needed to confirm these findings.

Pathogenic and phylogenetic features of 2 multiresistant Pseudomonas aeruginosa strains originated from remediated sites.

OBJECTIVES: To evaluate the possible occupational hazard of environmental strains of opportunistic Pseudomonas aeruginosa on hydrocarbon contaminated sites during remediation, 2 multidrug-resistant isolates originating from environmental (soil and groundwater) samples were examined. MATERIAL AND METHODS: Antibiotic resistance profiles of the examined 2 strains were determined by Etest® against 20 different agents. Virulence investigations included the hemolytic activity test, the detection of virulence-related gene sequences such as exoA, exoU, exoS, exoY, exoT and the determination of intraperitoneal LD50 (the lethal dose, 50%) values in a mouse model. The hydrocarbon-degrading ability was evaluated in a gravimetric experiment, in vitro. The phylogenetic relationship of the isolates was investigated with a multilocus sequence typing scheme. RESULTS: Multidrug resistant environmental strains of P. aeruginosa are strongly related to isolates that have proven effects on the infection of patients who suffer from cystic fibrosis, have a notable hemolytic activity, carry important virulence markers (exoS or exoU, respectively) and retain their hydrocarbon degradation ability (87.4% and 62.8% hydrocarbon degradation rate, respectively). CONCLUSIONS: Pseudomonas aeruginosa presumably raise considerable concerns for human health in the environment, already well known among nosocomial isolates, and the application of environmental strains of this species for environmental purposes is questionable.

Olivibacter oleidegradans sp. nov., a hydrocarbon-degrading bacterium isolated from a biofilter clean-up facility on a hydrocarbon-contaminated site.

A novel hydrocarbon-degrading, Gram-negative, obligately aerobic, non-motile, non-sporulating, rod-shaped bacterium, designated strain TBF2/20.2(T), was isolated from a biofilter clean-up facility set up on a hydrocarbon-contaminated site in Hungary. It was characterized by using a polyphasic approach to determine its taxonomic position. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the isolate is affiliated with the genus Olivibacter in the family Sphingobacteriaceae. It was found to be related most closely to Olivibacter ginsengisoli Gsoil 060(T) (93.3% 16S rRNA gene sequence similarity). Strain TBF2/20.2(T) grew at pH 6-9 (optimally at pH 6.5-7.0) and at 15-42 °C (optimally at 30-37 °C). The major fatty acids were iso-C(15:0) (39.4%), summed feature 3 (iso-C(15:0) 2-OH and/or C(16:1)ω7c; 26.0%), iso-C(17:0) 3-OH (14.5%) and C(16:0) (4.5%). The major menaquinone was MK-7 and the predominant polar lipid was phosphatidylethanolamine. The DNA G+C content of strain TBF2/20.2(T) was 41.2 mol%. Physiological and chemotaxonomic data further confirmed the distinctiveness of strain TBF2/20.2(T) from recognized members of the genus Olivibacter. Thus, strain TBF2/20.2(T) is considered to represent a novel species of the genus Olivibacter, for which the name Olivibacter oleidegradans sp. nov. is proposed. The type strain is TBF2/20.2(T) (=NCAIM B 02393(T) =CCM 7765(T)).

Antibiotic resistance profiles and virulence markers of Pseudomonas aeruginosa strains isolated from composts.

The aim of our work was to determine the presence of Pseudomonas aeruginosa in compost raw materials, immature and mature compost, and compost-treated soil. Twenty-five strains of P. aeruginosa were isolated from a raw material (plant straw), immature and mature compost and compost-treated soil samples. The strains were identified using the PCR method for the detection of species specific variable regions of 16S rDNA. Strains were examined for the presence of five different virulence-related gene sequences (exoA, exoU, exoT, exoS and exoY) and their antibiotic resistance profiles were determined. Based on our results, species P. aeruginosa can reach significant numbers (up to 10(6) MPN/g sample) during composting and 92.0% of the isolated strains carrying at least two gene sequences encoding toxic proteins. Various types of drug resistance were detected among compost originating strains, mainly against third generation Cephalosporins and Carbapenems. Six isolates were able to resist two different classes of antibiotics (third generation Cephalosporins and Carbapenems, wide spectrum Penicillins or Aminoglycosides, respectively). Based on our results, composts can be a source of P. aeruginosa and might be a concern to individuals susceptible to this opportunistic pathogen.

The occurrence of multidrug-resistant Pseudomonas aeruginosa on hydrocarbon-contaminated sites.

The main aim of this paper was the comprehensive estimation of the occurrence rate and the antibiotic-resistance conditions of opportunistic pathogen Pseudomonas aeruginosa in hydrocarbon-contaminated environments. From 2002 to 2007, 26 hydrocarbon-contaminated sites of Hungary were screened for the detection of environmental isolates. Altogether, 156 samples were collected and examined for the determination of appearance, representative cell counts, and antibiotic-resistance features of P. aeruginosa. The detected levels of minimal inhibitory concentrations of ten different drugs against 36 environmental strains were compared to the results of a widely used reference strain ATCC 27853 and four other clinical isolates of P. aeruginosa. Based on our long-term experiment, it can be established that species P. aeruginosa was detectable in case of 61.5% of the investigated hydrocarbon-contaminated sites and 35.2% of the examined samples that shows its widespread occurrence in polluted soil-groundwater systems. In the course of the antibiotic-resistance assay, our results determined that 11 of the examined 36 environmental strains had multiple drug-resistance against several clinically effective antimicrobial classes: cephalosporins, wide spectrum penicillins, carbapenems, fluoroquinolones, and aminoglycosides. The fact that these multiresistant strains were isolated from 8 different hydrocarbon-contaminated sites, mainly from outskirts, confirms that multiple drug-resistance of P. aeruginosa is widespread not only in clinical, but also in natural surroundings as well.