Jana Harichová

Evaluation of different PCR-based approaches for the identification and typing of environmental enterococci

The aim of the work was the evaluation of different PCR-based methods to found an appropriate identification and typing strategy for environmental enterococci. Environmental enterococci were isolated mainly from surface- and waste-waters. Species identification was provided by combination of phenotypic (Micronaut System, Merlin) and molecular detection methods (fluorescent ITS-PCR, ddl-PCR, REP-PCR, AFLP). Very similar results were observed among molecular methods, however several discrepancies were recognized during comparison of molecular and biochemical identification. Seven enterococcal species (E. faecium, E. hirae, E. casseliflavus, E. mundtii, E. faecalis, E. durans and E. gallinarum) were identified within 166 environmental isolates. The results obtained in this work attest the importance of PCR-based methods for identification and typing of environmental enterococci. The fluorescent ITS-PCR (fITS-PCR) showed the best results in order to identify the enterococci strains, the method used the automated capillary electrophoresis to separate the PCR products in a very rapid and precise way. The AFLP method was suitable to identify and characterize the isolates, while the REP-PCR can be used for species identification.

The isolation of heavy-metal resistant culturable bacteria and resistance determinants from a heavy-metal-contaminated site

In this study we performed a phylogenetic analysis of a culturable bacterial community isolated from heavymetal-contaminated soil from southwest Slovakia using 16S rRNA (16S rDNA) and heavy-metal resistance genes. The soil sample contained high concentrations of nickel (2,109 mg/kg), cobalt (355 mg/kg) and zinc (177 mg/kg), smaller concentrations of iron (35.75 mg/kg) and copper (32.2 mg/kg), and a trace amount of cadmium (<0.25 mg/kg). A total of 100 isolates were grown on rich (Nutrient agar No. 2) or minimal (soil-extract agar medium) medium. The isolates were identified by phylogenetic analysis using partial sequences of their 16S rRNA (16S rDNA) genes. Representatives of two broad taxonomic groups, Firmicutes and Proteobacteria, were found on rich medium, whereas four taxonomic groups, Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria, were represented on minimal medium. Forty-two isolates grown on rich medium were assigned to 20 bacterial species, while 58 bacteria grown on minimal medium belonged to 49 species. Twenty-three isolates carried czcA- and/or nccA-like heavy-metal-resistance determinants. The heavy-metalresistance genes of nine isolates were identified by phylogenetic analysis of their protein sequences.

Diversity and PAH growth abilities of bacterial strains isolated from a contaminated soil in Slovakia

Different abandoned industrial areas contaminated by polycyclic aromatic hydrocarbons (PAHs) are present in Slovakia. These environmental burdens are very dangerous to the health of human and environment. The bioremediation, based on the use of hydrocarbons degrading microorganisms, is a promising strategy to sanitize these polluted sites. The aim of this investigation was to assess the bacterial diversity of a PAHs-contaminated soil and to select the potential hydrocarbonoclastic bacteria which can be used for different bioremediation approaches. The bacterial strains were isolated on minimal medium agar supplemented with a mixture of PAHs. Seventy-three isolated strains were grouped by ribosomal interspacer analysis in 15 different clusters and representatives of each cluster were identified by 16S rRNA sequencing. The PAHs degradation abilities of all bacterial isolates were estimated by the 2,6-dichlorophenol indophenol assay and by their growth on minimal broth amended with a mixture of PAHs. Different kinds of strains, members of the genus Pseudomonas, Enterobacter, Bacillus, Arthrobacter, Acinetobacter and Sphingomonas, were isolated from the contaminated soil. Four isolates (Pseudomonas putida, Arthrobacter oxydans, Sphingomonas sp. and S. paucimobilis) showed promising PAHs-degrading abilities and therefore their possible employing in bioremediation strategies.

Assessment of environmental enterococci: bacterial antagonism, pathogenic capacity and antibiotic resistance

The properties of 166 environmental strains belonging to the seven enterococcal species were studied. Enterococci originated mainly from surface- and waste-waters. They were screened for the presence of enterocins, virulence factors, and antibiotic resistance. The presence of different enterocin genes (entA, entB, entP, ent31, entL50AB) was frequently observed in our enterococcal isolates, 109 strains contained at least one enterocin gene. The distribution of enterocin genes varied according to the species, the genes were present mainly in E. hirae and E. faecium. By enterocin spot assay, 10 isolates inhibited the growth of Listeria strains. To evaluate the pathogenic ability of isolates, the distribution of selected virulence genes (cylA, gelE and esp) was investigated, eleven strains were positive in some of these genes, five of them belonged to E. faecalis. Regarding the antibiotic resistance of isolates, only two strains were multiresistant and two strains (E. hirae and E. casseliflavus) were resistant to vancomycin.

Structure analysis of bacterial community and their heavy-metal resistance determinants in the heavy-metal-contaminated soil sample

In this study we performed the phylogenetic analysis of non-cultivable bacteria from anthropogenically disturbed soil using partial sequences of the 16S rRNA (16S rDNA) and the heavy-metal resistance genes. This soil sample contained high concentrations of nickel (2,109 mg/kg), cobalt (355 mg/kg) and zinc (177 mg/kg), smaller concentrations of iron (35.75 mg/kg) and copper (32.2 mg/kg), and also a trace amount of cadmium (<0.25 mg/kg). The 16S rDNA sequences from a total of 74 bacterial clones were distributed into four broad taxonomic groups, Acidobacteria, Actinobacteria, Bacteroidetes and Gemmatimonadetes, and some of them were unidentified. Comparing our clone sequences with those from the GenBank database, only 9 clones displayed high similarity to known bacteria belongig to actinomycetes; others were identified as uncultured ones. Among clones evidently Actinobacteria predominated. Sixteen clones from soil sample carried only the nccA-like heavy-metal-resistance genes and all sequences showed too low similarity to known proteins encoded by these genes. However, our results suggested that the heavy-metal-contaminated soil is able to present very important reservoir of the new and until now unknown partly bacteria, partly heavy-metal-resistance determinants and their products. Bacteria and nccA-like genes identified in this study could represent the objects of interest as bioremediation agents because they can be potentially used in different transformation and immobilization processes.

Function of a novel cadmium-induced yodA protein in Escherichia coli.

Cells of Escherichia coli increase greatly the synthesis of a small primarily cytoplasmic protein as soon as the cell growth rate falls below the maximal growth rate supported by cadmium exposure, after which the mature product is exported to the periplasm. This protein was previously identified as the product of the E. coli yodA open reading frame. We now report the isolation of an E. coli mutant defective in YodA synthesis because of insertional inactivation of the corresponding gene. In experiments to test the ability of both the wild-type and yodA mutant E. coli cells to bind cadmium, we have used γ-labeled [109Cd]. Whereas the wild-type E. coli strain was able to bind metal, the yodA mutant strain failed to do so. In addition, analysis of such a mutant demonstrated that it grows at a rate distinguishable from that of the isogenic parent in the presence of cadmium ions. However, challenging cells with hydrogen peroxide and additional metals such as zinc, copper, cobalt, and nickel did not significantly affect the growth rate of the mutant. This growth phenotype was found to be the result of the loss of its ability to bind cadmium. These results suggest that the role of YodA protein might be to decrease the concentration level of cadmium ions in E. coli cells during cadmium stress by its ability to bind heavy metal.

Actinobacteria occurrence and their metabolic characteristics in the nickel-contaminated soil sample

Heavy metals are a significant source of pollution in soils that have been demonstrated to exert significant toxic effect on soil microbial assemblages. Here we investigate the occurrence and metabolic characteristics of actinobacteria, which form a predominated component of farmland bacterial community near the town of Sereď in southwest Slovakia, contaminated by close nickel ore facility. Actinobacteria occurred in this environment with high concentrations of nickel (2.109 mg/kg), slightly above the natural occurrence of cobalt (355 mg/kg) and zinc (177 mg/kg), even too low concentration of iron (35.75 mg/kg) for a normal soil and not a toxic amount of copper (32.2 mg/kg) and cadmium (<0.25 mg/kg). The phylogeny was reconstructed using partial sequences of 16S rDNA genes of both, actinobacterial isolates and clones. A total of 105 actinobacterial representatives were divided into 66 species belonging to one order, 7 genera and 5 uncategorized groups. The selected 14 morphologically distinct isolates were able to produce drop collapsing, haemolytic and lipase activities. Whereas only 4 isolates produced dark brown melanin pigment and only 5 of them produced decolourization of the azo dye, all isolates tested were capable of assimilating all 11 sugars tested. All these actinobacterial isolates were resistant to nickel, cobalt, zinc, cadmium, copper, but the level of resistance differed between the individual isolates, and the resistance profiles of antibiotics (gentamycin, ampicillin, ciprofloxacin, chloramphenicol, erythromycin, rifampicin, penicillin-G) varied among them to a certain extent. Our results suggested that actinobacteria in soil contaminated by nickel present a relatively divergent group inside of microbial assemblage.

Comparison of culturable Gram-negative bacterial community structures in the rhizosphere of three fruit plants

This research work was oriented to outlining the diversity of Gram-negative culturable portion of the bacterial community in three fruit plants rhizosphere. Rhizosphere samples were taken from European chestnut (Castanea sativa Mill), true service tree (Sorbus domestica L.) and cornelian cherry (Cornus mas L.) plants. Experiments were conducted for three years during the vegetation period, and the bacterial community structure was assessed with cultivation-dependent approach. Many Gram-negative isolates (n = 251) from the rhizosphere survived sub culturing and were identified by biochemical tests. A total of 57 species belonging to 29 genera were identified and assigned to four broad taxonomic groups (Bacteroidetes, Alpha-, Beta- and Gamma-proteobacteria). Several specific bacterial cluster communities were identified inside all the three rhizospheres. Most of the species belonged to the genera Moraxella, Pseudomonas, Pantoea, Enterobacter and Acinetobacter. In addition, while, using the plate count analysis, large discrepancies in numbers among physiological groups of bacteria cultured from three rhizosphere samples have not been revealed, more expressive distinctions among bacterial populations were obtained concerning the relative abundance of different genera, different taxonomic groups as well as different diversity indices. Furthermore, the number of cultured bacteria and their taxonomic distribution in the rhizosphere of all three plants changed not only explicitly during vegetation period but continually during the three years of investigation. It seems that rhizosphere bacterial populations of each plant are under the influence of the specific root-released materials.

Screening for alkyl sulfosuccinate degrading microorganisms

Publisher Summary This chapter describes screening for pure microbial cultures capable of rapid alkyl sulfosuccinate (ASS) primary biodegradation among the culture collection strains and naturally occurring microorganisms. ASS represents synthetic surfactants of a secondary alkane sulfonate type. The ASS solubilization capacities, wetting of solids, their capabilities of reduction of surface tension at the phase boundaries are the basis of commercial use of ASS in products for washing and cleaning, cosmetics, antifogging agents, inks and pesticide sprays. Searching for ASS biodegraders among culture collection strains is done by systematic screening of 85 strains for the ability of Di-n-hexyl sulphosuccinate (DHSS) primary biodegradation. Screening test shows that DHSS degraders are frequent among bacteria belonging to the genera Bacillus , Rhodococcus , Streptomyces and Nocardiae . High frequency of DHSS degrading actinomycetes confirms their reputation for potent biodegraders. Enrichment media used for cultivation of microbial cultures are designed to contain a limited number of specific substrates that preferentially promotes the growth of ASS degraders.

The Heavy-Metal Resistance Determinant of Newly Isolated Bacterium from a Nickel-Contaminated Soil in Southwest Slovakia

Abstract A bacterial isolate MR-CH-I2 [KC809939] isolated from soil contaminated mainly by high nickel concentrations in southwest Slovakia was previously found carrying nccA-like heavy-metal resistance determinant, marked as MR-CH-I2-HMR [KF218096]. According to phylogenetic analysis of short (696 bp) 16S rDNA (16S rRNA) sequences this bacterium was tentatively assigned to Uncultured beta proteobacterium clone GC0AA7ZA05PP1 [JQ913301]. nccA-like gene product was on the same base of its partial (581 bp) sequences tentatively assigned to CzcA family heavy metal efflux pump [YP_001899332] from Ralstonia picketii 12J with 99% similarity. In this study the bacterium MR-CH-I2 and its heavy-metal resistance determinant were more precisely identified. This bacterial isolate was on the base of phylogenetic analysis of almost the whole (1,500 bp) 16S rDNA (16S rRNA) sequence, MR-CH-I2 [MF102046], and sequence for gyrB gene and its product respectively, MR-CH-I2-gyrB [MF134666], assigned to R. picketii 12J [CP001068] with 99 and 100% similarities, respectively. In addition, the whole nccA-like heavy-metal resistance gene sequence (3,192 bp), marked as MR-CH-I2-nccA [KR476581], was obtained and on the base of phylogenetic analysis its assignment was confirmed to MULTISPECIES: cation efflux system protein CzcA [WP_004635342] from Burkholderiaceae with 98% similarity. Furthermore, although the bacterium carried one high molecular plasmid of about 50 kb in size, nccA-like gene was not located on this plasmid. Finally, the results from RT-PCR analysis showed that MR-CH-I2-nccA gene was significantly induced only by the addition of nickel.