Higher alkyl sulfatase activity required by microbial inhabitants to remove anionic surfactants in the contaminated surface waters.

Biodegradation of anionic surfactants, like sodium dodecyl sulfate (SDS) are challenged by some bacteria through the function of the enzyme alkyl sulfatases. Therefore, identifying and characterizing bacteria capable of degrading SDS with high alkyl sulfatase enzyme activity are pivotal. In this study, bacteria isolated from surfactant contaminated river water were screened for their potential to degrade SDS. Primary screening carried out by the conventional enrichment culture technique and assessment of SDS-degrading ability through methylene blue active substance assay revealed 12, out of 290, SDS-degrading surface water bacteria with maximum SDS degrading abilities of 46-94% in 24-54 h. The isolates exhibited optimum growth at SDS concentration of 1 g/L, but tolerated up to 15-75 g/L. Eleven isolates were identified as the species of Pseudomonas and one isolate was identified as Aeromonas through 16S rRNA sequencing. Proteolytic activity of alkyl sulfatases in the identified isolates was shown by using native-PAGE analysis. The determined enzyme activities changed in between 1.32 and 2.90 U/mg in the crude extracts. Preliminary experiments showed that the isolates with the alkyl sulfatase enzyme activities ≥2.50 U/mg were strong gratuitous degraders. However, their relative importance in soil, sewage, and wastewater treatment plants remains to be assessed.

vanA Gene Harboring Enterococcal and Non-enterococcal Isolates Expressing High Level Vancomycin and Teicoplanin Resistance Reservoired in Surface Waters.

Untreated wastewaters and treated effluents even after final disinfection contain antibiotic resistant bacteria and resistance genes before they are released into surface waters. A correlation between resistant bacteria and antibiotics in surface waters has been found, as have antibiotic resistance genes. Of particular interest are vancomycin-resistant enterococci harboring vanA gene that confers high level of resistance to glycopeptide antibiotics including teicoplanin. Therefore, in this study, river water samples were analysed to investigate vancomycin- and teicoplanin-resistant bacterial isolates harboring vanA gene. Out of 290, 15 surface water isolates displayed resistance to both antibiotics. These glycopeptide resistant enterococcal and non-enterococcal isolates, identified by 16S rRNA sequencing, were found to harbor vanA gene with sequence similarities of 50 % to 100 %. The presence of D-alanine-D-lactate ligase encoded by vanA gene was also shown for all vancomycin- and teicoplanin-resistant isolates through western blot analysis. Due to reuse of treated wastewater and release of untreated wastewaters to water bodies, antibiotic resistant bacteria and resistance genes are being introduced into surface waters and present human health risks. Therefore, surface waters are not only hot spots for vanA harboring enterococcal isolates but also non-enterococcal isolates due to gene dissemination and require special scientific consideration.

vanA-targeted oligonucleotide DNA probe designed to monitor vancomycin- and teicoplanin-resistant bacteria in surface waters.

The glycopeptide antibiotics teicoplanin and vancomycin are common to treat severe Gram-positive bacterial infections. The gene vanA confers high-level resistance to these antibiotics, and these phenomena have been shown to be transferable. Release of vancomycin- and teicoplanin-resistant bacteria to surface waters is, therefore, of particular concern since they might proliferate and spread in different environments. Monitoring of the fate of vanA gene in these waters provides information on the exposure and potential threats of those bacteria for the environment and public health. Therefore, this study aimed at preparing a 25-mer-oligonucleotide DNA probe based on the 909 bp BamHI-ClaI fragment from Enterococcus faecium plasmids pVEF1 and pVEF2 through the use of Vector NTI Express Software. The newly designed vanA probe displayed highly specific hybridization with vanA-positive Enterococcus faecalis tested at 46 °C, 55 % formamide, and 0.020 M NaCl stringency conditions. In situ fluorescein hybridizations under the same stringency conditions were also used to monitor river water samples by using fluorescein microscopy. The results showed that the vanA-targeted oligonucleotide DNA probe prepared was not only highly specific but also quantitative tool for monitoring vancomycin- and teicoplanin-resistant bacteria in surface waters.

Methicillin-Resistant Bacteria Inhabiting Surface Waters Monitored by mecA-Targeted Oligonucleotide Probes.

Part of a 20-60 kb staphylococcal chromosome cassette called mecA encodes low-affinity penicillin-binding protein PBP2a and causes methicillin resistance. Among all methicillin-resistant bacteria, methicillin-resistant Staphylococcus aureus is a major pathogen and main concern worldwide. Although the origin of the mecA is not very well-defined, mecA homologues are also ubiquitous in methicillin-resistant non-staphylococcal bacteria. Due to the dissemination of methicillin resistance through the transmission of mecA gene among staphylococcal and non-staphylococcal bacteria inhabiting surface waters, there is a need to monitor mecA gene in these waters for public health safety. Therefore, this study aimed at monitoring mecA harboring bacteria inhabiting surface waters by using fluorescently labelled mecA-targeted oligonucleotide probes. Under the hybridization conditions of 55 % formamide and 0.020 M NaCl at 46°C, the oligonucleotide probe used in the study showed high hybridization stringency to the mecA gene targeted. The strong linear relationships observed between the signal intensity and the target gene were used to assess the population dynamics of mecA harboring isolates over a 2-year-period. The results indicated that mecA-targeted oligonucleotide probes can be effectively used for in situ monitoring of methicillin resistant isolates inhabiting surface waters.

Use of cadA-Specific Primers and DNA Probes as Tools to Select Cadmium Biosorbents with Potential in Remediation Strategies.

Biosorption, using cadmium-resistant bacterial isolates, is often regarded as a relatively inexpensive and efficient way of cleaning up wastes, sediments, or soils polluted with cadmium. Therefore, many efforts have been devoted to the isolation of cadmium-resistant isolates for the efficient management of cadmium remediation processes. However, isolation, identification and in situ screening of efficient cadmium-resistant isolates are primary challenges. To overcome these challanges, in this study, cadA, cadmium resistance coding gene, specific primers and DNA probes were used to identify and screen cadmium-resistant bacteria in the cadmium-polluted river waters through polymerase chain reaction (PCR) and fluorescein in situ hybridization (FISH). PCR amplification of the cadA amplicon coupled with 16S rRNA sequencing revealed various gram-positive and -negative bacterial isolates harboring cadA. Accordingly, a cadA-mediated DNA probe was prepared and used for in situ screening of cadmium-resistant isolates from water samples collected from cadmium-polluted river waters. The FISH analyses of cadA probe showed highly specific and efficient hybridization with cadA harboring isolates. The use of primers and DNA probes specific for cadA gene seems to be very helpful tools for the selection and screening of cadmium biosorbents with potential to be used in the remediation of cadmium-polluted sites.

Design a cadA-targeted DNA probe for screening of potential bacterial cadmium biosorbents.

Due to their metal removal ability, bacterial biosorbents can be effectively used for the treatment of wastewaters containing heavy metals. Searching for bacterial biosorbents for hazardous heavy metals like cadmium is a pivotal for remediation efforts. The gene cadA, that mediates resistance to cadmium over an ATP-dependent efflux mechanism, provides a good target for the selection of potential cadmium biosorbents. For this reason, in this study, a 36-mer-oligonucleotide DNA probe based on the entire 3.5-kb BglII-XbaI fragment of cadA operon from staphylococcal plasmid pI258 was prepared by using Vector NTI Express software. Under the hybridization conditions of 46 °C, 50 % formamide, and 0.028 M NaCl, the designed cadA probe appeared to be highly specific to the cadA-positive Staphylococcus warneri and Delftia acidovorans isolates tested. The results indicated that the newly designed cadA-targeted DNA probe has potential as a specific, sensitive, and quantitative tool in selecting and in situ screening of potential cadmium biosorbents.

mecA Gene Dissemination Among Staphylococcal and Non-staphylococcal Isolates Shed in Surface Waters.

Aquatic ecosystems represent important vehicles for the dissemination of antibiotic resistant bacteria and antibiotic resistance genes. Of particular interest are methicillin-resistant staphylococci (MRS) harboring mecA gene that confers their resistance to ß-lactams. Therefore, in this study, water samples collected from different locations of a river impacted by surrounding facilities and domestic effluents were analyzed to learn more about the occurrence of MRS and mecA gene. Out of 290, 12 surface water isolates displayed resistance to both cefoxitin and oxacillin antibiotics. Resistant staphylococcal and non-staphylococcal isolates, identified by 16S rRNA sequencing, were found to harbor mecA gene. The phylogenetic tree of partial mecA sequences obtained from staphylococcal and non-staphylococcal isolates showed sequence similarity values of 8 %-100 %. Surface water bodies receive contaminated waters via runoff, effluents from industrial, agricultural, and municipal discharges. Therefore, surface waters are not only hot spots for mecA harboring staphylococcal isolates but also non-staphylococcal isolates and require special scientific consideration.

Co-occurrence of antibiotic and heavy metal resistance in Kizilirmak River isolates.

Contamination of surface waters with antimicrobials has become an increasing public health concern because of the emergence of multi-resistant pathogens. For this reason, water samples collected from the Kizilirmak River-Kirikkale, Turkey were analysed to learn more about the co-occurrence of heavy metal and antibiotic resistance in bacteria. Resistant profiles were determined by using 26 antibiotics and 17 heavy metals. Out of 290, 24 isolates with both heavy metal and antibiotic resistance were determined. Resistance to heavy metals including lead, tin, nickel, barium, aliminum, strontium, silver and lithium varied between 50 and 92 %. More than 50 % of the isolates showed resistance to cephalosporin, quinolone, sulfonamide and aminoglycoside type of antibiotics. The discharge of antimicrobials to water bodies may cause a combined effect of selection and co-selection towards resistant bacteria. Therefore, surface waters may be potential hot spots of the evolution of heavy metal- and antibiotic-resistant bacteria and require special scientific consideration.

Detection and spread of oxa-48-producing Klebsiella oxytoca isolates in Istanbul, Turkey.

Five OXA-48 producing Klebsiella oxytoca strains isolated in April-July 2010 were analyzed. Antibiotic susceptibility tests were performed using disc diffusion method and VITEK 2 system. Carbapenemase activity was investigated using the Modified Hodge test. Beta-lactamase genes were detected by PCR and blaOXA-48 was sequenced. Genetic relatedness between K. oxytoca isolates was investigated by pulse-field gel electrophoresis (PFGE). Carbapenemase activity was detected in 5 isolates by Modified Hodge test. Although all strains were resistant to ertapenem and imipenem, only one strain was also resistant to meropenem. BlaOXA-48 in 4 isolates harbored 2 or 3 other ESBL types, namely, blaTEM, blaSHV, blaCTX-M, or blaVEB. PFGE revealed 3 different pulso-types among the K. oxytoca isolates. The presence of OXA-48 carbapenemase in other species of clinical isolates should also be considered.

Surface water isolates of hemolytic and non-hemolytic Acinetobacter with multiple drug and heavy metal resistance ability.

Acinetobacter in surface waters are a major concern because of their rapid development of resistance to a wide range of antimicrobials and their ability to persist in these waters for a very long time. Four surface water isolates of Acinetobacter having both multidrug- and multimetal-resistant ability were isolated and identified through biochemical tests and 16S rDNA sequencing. Based on these analyses, two hemolytic isolates were affiliated with Acinetobacter haemolyticus with an accession number of X81662. The other two non-hemolytic isolates were identified as Acinetobacter johnsonii and Acinetobacter calcoaceticus and affiliated with accession numbers of Z93440 and AJ888983, respectively. The antibiotic and heavy metal resistance profiles of the isolates were determined by using 26 antibiotics and 17 heavy metals. Acinetobacter isolates displayed resistance to ß-lactams, cephalosporins, aminoglycosides, and sulfonamides. The hemolytic isolates were found to show resistance to higher numbers of heavy metals than the non-hemolytic ones. Due to a possible health risk of these pathogenic bacteria, a need exists for an accurate assessment of their acquired resistance to multiple drugs and metals.

Characterization of SDS-degrading Delftia acidovorans and in situ monitoring of its temporal succession in SDS-contaminated surface waters.

Incomplete removal of sodium dodecyl sulfate (SDS) in wastewater treatment plants may result in SDS residues escaping and finding their way into receiving water bodies like rivers, lakes, and sea. Introduction of effective microorganisms into the aerobic treatment facilities can reduce unpleasant by-products and SDS residues. Selecting effective microorganisms for SDS treatment is a big challenge. Current study reports the isolation, identification, and in situ monitoring of an effective SDS-degrading isolate from detergent-polluted river waters. Screening was carried out by the conventional enrichment culture technique and the isolate was tentatively identified by using fatty acid methyl ester and 16S ribosomal RNA (rRNA) sequence analyses. Fatty acids produced by the isolate investigated were assumed as typical for the genus Comamonas. 16S rRNA sequence analysis also confirmed that the isolate had 95% homology with Delftia acidovorans known as Comamonas or Pseudomonas acidovorans previously. D. acidovorans exhibited optimum growth at SDS concentration of 1 g l(-1) but tolerated up to 10 g l(-1) SDS. 87% of 1.0 g l(-1) pure SDS was degraded after 11 days of incubation. The temporal succession of D. acidovorans in detergent-polluted river water was also monitored in situ by using Comamonas-specific fluorescein-labeled Cte probe. Being able to degrade SDS and populate in SDS-polluted surface waters, D. acidovorans isolates seem to be very helpful in elimination of SDS.

Surface water-borne multidrug and heavy metal-resistant Staphylococcus isolates characterized by 16S rDNA sequencing.

Four Staphylococcus isolates having both multidrug- and multimetal-resistant ability were isolated from surface water. Further identification of the isolates was obtained through biochemical tests and 16S rDNA gene sequencing. One methicillin-resistant and two methicillin-sensitive isolates were determined as Staphylococcus aureus. The other isolate was identified as Staphylococcus warneri. The antibiotic and heavy metal resistance profiles of the Staphylococcus isolates were determined by using 26 antibiotics and 17 heavy metals. S. aureus isolates displayed resistance to most of the ß-lactam antibiotics tested. All Staphylococcus isolates were resistant to heavy metals including silver, lithium, and barium. Due to a possible health risk of these pathogenic bacteria, a need exists for an accurate assessment of their acquired resistance to multiple drugs and metals.

Effects of cypermethrin on Allium cepa.

In this study, toxic effects of the cypermethrin in Allium cepa L. cells were investigated. For this aim, we investigated the changes in pigment contents, antioxidant enzymes, mitotic index and chromosomal abnormalities as indicators of toxicity. The seeds were treated with different doses (1.5, 3.0, 6.0 ppm) of cypermethrin for 72 h. The result showed that there was a significant alteration in the tested parameters depending on treatment dose in the seeds exposed to cypermethrin when compared to the control group. Cypermethrin exposure significantly reduced the carotenoid, chlorophyll a and b pigments in all treatment groups. The activity of superoxide dismutase showed a concentration-time dependent increase and the maximum increase was observed on day 15 of treatment at 6.0 ppm cypermethrin exposure. The activity of catalase increased gradually with increasing cypermethrin concentration, but a soft decrease in CAT activity was decreased after 15 days of 1.5 ppm and 3.0 ppm cypermethrin treatment. In the roots treated with 1.5, 3.0, and 6.0 ppm cypermethrin, the level of malondialdehyde was about 1.8, 2.4, and 3.4 times higher than the control group, respectively. It was also found that cypermethrin has a mitodepressive action on mitosis, and the MI was decreased depending on the dose of cyprmethrin. All of the concentrations of cypermethrin induced chromosomal abnormalities and the most common abnormality observed in the present study was chromosome bridges.