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).

Cytotoxicity and hormonal activity of glyphosate-based herbicides.

Glyphosate-based herbicides (GBHs) are the most widely used pesticides for weed control. In parallel with the renewal of the active ingredient, polyethoxylated POE(15) containing GBHs were banned in the EU in 2016. Since then, co-formulants were changed and numerous GBHs are marketed with different excipients declared as inert substances. In our study, we focused to determine acute and chronic cytotoxicity (by Aliivibrio fischeri assay) and direct hormonal activity (estrogenic and androgenic effects measured by Saccharomyces cerevisiae BLYES/BLYAS strains, respectively) of glyphosate, AMPA, polyethoxylated POE(15) and 13 GBHs from which 11 formulations do not contain polyethoxylated POE(15). Among the pure substances, neither glyphosate nor AMPA had any effects, while polyethoxylated POE(15) exhibited pronounced toxicity and was also estrogenic but not androgenic. Regarding the acute and chronic cytotoxicity and hormonal activity of GBHs, dilution percentages calculated from EC50 values were in the most cases by one or two order of magnitude lower than the minimum recommended dilution for agricultural and household use. Relation could not be observed between the biological effects and type of glyphosate-salts; hence toxicity could be linked to the co-formulants, which are not even declared in 3 GBHs. Toxicological evaluation must focus on these substances and free accessibility of GBHs should be reconsidered.

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.

Aerobic and oxygen-limited enrichment of BTEX-degrading biofilm bacteria: dominance of Malikia versus Acidovorax species.

Due to their high resistance against environmental challenges, bacterial biofilms are ubiquitous and are frequently associated with undesired phenomena in environmental industry (e. g. biofouling). However, because of the high phylogenetic and functional diversity, bacterial biofilms are important sources of biotechnologically relevant microorganisms, e.g. those showing bioremediation potential. In our previous work, the high phylogenetic and metabolic diversity of a clogging biofilm, developed in a simple aromatic hydrocarbon (BTEX)-contaminated groundwater well was uncovered. The determination of relationships between different groups of biofilm bacteria and certain metabolic traits has been omitted so far. Therefore, by setting up new biofilm-based enrichment microcosms, the research goal of the present study was to identify the aerobic/hypoxic BTEX-degrading and/or prolific biofilm-forming bacteria. The initial bacterial community composition as well as temporal dynamics due to the selective enrichment has been determined. The obtained results indicated that the concentration of dissolved oxygen may be a strong selective force on the evolution and final structure of microbial communities, developed in hydrocarbon-contaminated environments. Accordingly, members of the genus Malikia proved to be the most dominant community members of the aerobic BTEX-degrading enrichments. Acidovorax spp. dominated the oxygen-limited/hypoxic setup. During the study, a strain collection of 23 different bacterial species was obtained. Non-pathogenic members of this strain collection, with outstanding biodegradation (e.g. Pseudomonas, Variovorax isolates) and biofilm-forming potential (e.g. Rhizobium), may potentially be applied in the development of biofilm-based semipermeable reactive biobarriers.

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.

Taibaiella coffeisoli sp. nov., isolated from the soil of a coffee plantation.

A Gram-stain-negative, obligately aerobic, non-motile, non-sporulating, rod-shaped bacterium, designated TZCO2T, was isolated from the soil of an irrigated coffee plantation in Arusha, Tanzania, East Africa. Phylogenetic analysis, based on 16S rRNA gene sequences, indicated that the isolate is affiliated with the genus Taibaiella in the family Chitinophagaceae. Its closest relative is Taibaiella koreensis THG-DT86T (96.7%). The pH and temperature ranges for growth were pH 6.0-8.5 (optimum 7.0-7.5) and 10-35 °C (optimum 30 °C, respectively. The predominant fatty acids were iso-C15:0 (32.4%), iso-C15:1 G (22.6%), iso-C17:0 (15.1%) and iso-C17:0 3-OH (10.0%) The only isoprenoid quinone detected in strain TZCO2T was menaquinone-7 (MK-7); the major polar lipids were phosphoaminolipid, phosphatidylethanolamine, unidentified aminolipids and lipids. The DNA G+C content was 51.9 mol%. Physiological and chemotaxonomic data further confirmed that strain TZCO2T is distinct from other members of the genus Taibaiella. Thus, strain TZCO2T is considered to represent a novel species of the genus, for which the name Taibaiella coffeisoli sp. nov. is proposed. The type strain is TZCO2T (=NCAIM B 02601T=CCM 8601T).