Wastewater treatment plants discharges disseminated more Giardia than Cryptosporidium.

Parasitic protozoa Giardia intestinalis and Cryptosporidium parvum are causative agents for giardiasis and cryptosporidiosis, respectively. These infections are mostly associated with waterborne diseases. The discharges from wastewater treatment plants (WWTPs) that reach surface waters cause waterborne transmission because there are no regulations for monitoring these protozoa. This emphasizes how crucial the removal capacities of WWTPs to prevent the spread of infectious parasitic pathogens. For this reason, in this study, five different types of WWTPs including conventional activated sludge (CAS), biological nutrient removal (BNR), sequencing batch reactor (SBR), membrane bioreactor (MBR), and WWTP with coagulation-flocculation and UV disinfection (CoFlUV) units were investigated over a year, seasonally in terms of their G. intestinalis and C. parvum removal capacities. The seasonal abundances of these protozoa-specific genes in both the influents and effluents of each WWTP were determined by qPCR. The reduction of protozoan rDNA copies in the effluent wastewater samples compared with the influent wastewater samples was assessed as log10 reduction values (LRVs). LRVs >3 were reachable for C. parvum in all types of WWTPs tested. However, only LRVs 1-2 were reachable for G. intestinalis in CAS, SBR, CoFlUV, and MBR. Significant seasonal variations were just observed in SBR and CAS for G. intestinalis and C. parvum (p < 0.05), respectively. The findings depicted that WWTPs tested disseminated more giardiasis causative agents than cryptosporidiosis. Therefore, G. intestinalis needs to be monitored in WWTPs' discharges to reduce any potential damage of this parasite to public health. PRACTITIONER POINTS: Removal of G. intestinalis and C. parvum in WWTPs was affected by the process. LRV 2.92 was the highest LRV achieved for G. intestinalis. LRV >3 was reachable for C. parvum. WWTPs discharges disseminated more G. intestinalis than C. parvum. WWTPs effluents should be monitored in terms of G. intestinalis.

intI1 Type Mobile Genetic Elements Co-selected Antibiotic-Resistant Genes in Untreated Hospital Wastewaters.

Dissemination of antibiotic-resistant genes (ARGs) from hospital wastewaters (HWWs) is facilitated by the horizontal gene transfer (HGT) and involves association of ARGs with mobile genetic elements (MGEs). In our previous study, HWWs were found to have relatively high copy numbers of ARGs aadA, tetA, cmlA, sul1, and qnrS. In this study, therefore, the same HWWs were also monitored for 3 MGEs class 1 integron (intI1), insertion sequence common region 1 (ISCR1) and conjugative transposon Tn916/Tn1545 by using quantitative polymerase chain reaction. The gene intI1 with 7.4 × 102 average copy number/mL was found to be the most prevalent MGE and was up to two orders of magnitude higher than ISCR1 (5.5 × 100 average copy number/mL, p < 0.05) and Tn916/Tn1545 (2.3 × 100 average copy number/mL, p < 0.05) in all HWWs tested. Positive correlation between intI1 and the aadA, tetA, cmlA and sul1 genes indicated that the MGEs harbouring class1 integron most likely played major role in co-selecting all these ARGs together.

Untreated HWWs Emerged as Hotpots for ARGs.

Hospital wastewaters (HWWs) are reported to be hotspots for antibiotics and antibiotic-resistant bacteria. However, limited information involves the impact of these effluents on dissemination of antibiotic-resistance genes (ARGs). In this study, therefore, seasonally collected HWWs were monitored for overall bacterial load and seven ARGs aadA, tetA, cmlA, sul1, qnrS, ermB and bla CTX-M by using quantitative polymerase chain reaction method. Overall bacterial 16S rRNA copy number was found to be the lowest in winter with 103 copy number/mL, while the highest copy number, with 105 copy number/mL, was observed in both summer and spring. All hospitals tested displayed similar seasonal ARG copy number profile of aadA > tetA > cmlA ≈ sul1 > ermB ≈ qnrS > bla CTX-M. The results indicated that untreated HWWs were hotspots for ARGs and required attention before discharging into public sewer.

Removal of antibiotic resistance genes in various water resources recovery facilities.

Water resources recovery facilities (WWTPs) are hotspots for antibiotic resistance genes (ARGs) and pose a significant threat to environments. Therefore, ARG removal efficiencies of WWTPs are of great importance. In this study, conventional activated sludge (CAS), biological nutrient removal (BNR), sequencing batch reactor (SBR), membrane bioreactor (MBR), package MBR, and WWTP with coagulation-flocculation and UV disinfection units were investigated in terms of their removal efficiencies on overall bacterial genes with 16S rDNA and seven ARGs including the genes aadA, blaCTX-M , cmlA, ermB, sul1, tetA, and qnrS corresponding to commonly used antibiotics aminoglycosides, ß-lactams, amphenicols, macrolides-lincosamides-streptogramins, sulfonamides-trimethoprim, tetracyclines, and quinolones, respectively. Seasonal abundance of overall genes and ARGs in influents and effluents of each WWTPs was determined by quantitative polymerase chain reaction. Membrane bioreactor and package MBR systems showed the highest removal efficiency up to 5-log reductions. Seasonal changes affected ARG removal efficiencies of BNR and CAS significantly (p < 0.05). The lowest log reductions were determined in summer for BNR and in both autumn and winter for CAS. The abundance of the genes increased in sludge treatment of CAS. In all WWTPs tested, ARG removal rates were correlated with the 16S rDNA gene removal (p > 0.05). The results elucidated that the removal mechanism was not ARG-specific. PRACTITIONER POINTS: Removal of genes in WWTPs was found to be not ARG-specific. MBR and package MBR systems had the highest gene removal efficiencies. Effluents of the MBR systems still retained up to 104  gene copy number/ml. Removal of genes in CAS and BNR systems showed seasonal variations. The copy numbers of the genes increased in the sludge of CAS.

Diversity of the Aromatic-Ring-Hydroxylating Dioxygenases in the Monoaromatic Hydrocarbon Degraders Held by a Common Ancestor.

Aromatic ring hydroxylating dioxygenases (ARHDs), harboured by a variety of bacteria, catalyze the initial reaction in the degradation of a wide range of toxic environmental contaminants like aromatic and polycyclic aromatic hydrocarbons (PAHs). Regardless of the source, bacteria harbouring RHDs play major role in the removal of these toxic contaminants. The diversity of ARHDs in contaminated sites is supposed to be huge. However, most of the ARHD diversity studies are based on the PAH degraders and the ARHD diversity in the monoaromatic hydrocarbon degraders has not fully explored yet. In this study, therefore, the ARHD gene from nine different genara of the monoaromatic hydrocarbon degraders including Raoultella, Stenotrophomons, Staphylococcus, Acinetobacter, Pseudomonas, Serratia, Comamonas, Pantoea, and Micrococcus was analysed through polymerase chain reactions and sequencing. The sequence alignments of the ARHD amplicons with 81%-99% homologies were found to be highly related and held by divergent evolution from a common ancestor.

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.

VanA-Type MRSA (VRSA) Emerged in Surface Waters.

Due to the widespread occurrence of mecA-encoded methicillin resistance in Staphylococcus aureus (MRSA), treatment of staphylococcal infections is shifted to glycopeptide antibiotics like vancomycin and teicoplanin. The selective pressure of glycopeptides has eventually led to the emergence of staphylococci with increased resistance. Of great concern is vanA-encoded high level vancomycin and teicoplanin resistance in MRSA (VRSA). Therefore, this study aimed at investigating the occurrence of VRSA in surface waters. Out of 290, two staphylococcal isolates identified as MRSA Al11, Ba01, and one as MRS Co11 through 16S rRNA sequencing, also displayed high level resistance towards vancomycin and teicoplanin. These staphylococcal isolates were found to harbor vanA gene with sequence similarities of 99 %-100 % to the vanA gene extracted from vancomycin- and teicoplanin-resistant enterococcal (VRE) surface water isolates of Enterococcus faecalis Cr07, E07, Pb06 and E. faecium E330. High level glycopeptide resistance rendering protein encoded by the vanA gene, D-alanine-D-lactate ligase found in VRE, was also shown to be present in all vanA-type staphylococcal isolates through western blot. Current study elucidated that surface waters provide high potential for enterococcal vanA gene being transferred to MRSA, so called VRSA, and require special scientific consideration.

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.

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.

Multidrug and heavy metal-resistant Raoultella planticola isolated from surface water.

A surface water isolate of Raoultella sp. having both multidrug- and multimetal-resistant ability was isolated and identified as Raoultella planticola. R. planticola displayed resistance to 15 drugs like ampicillin, amoxicillin/clavulanic acid, aztreonam, erythromycin, imipenem, oxacillin, pefloxacin, penicillin, piperacillin, piperacillin/tazobactam, rifampin, sulbactam/cefoperazone, ticarsillin, ticarsillin/clavulanic acid, vancomycin, and to 11 heavy metals like aluminum, barium, copper, iron, lead, lithium, manganese, nickel, silver, strontium, and tin. The multidrug and multi-metal-resistant R. planticola may remain present in the environment for a long time. 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 lead-resistant river isolate Enterococcus faecalis and assessment of its multiple metal and antibiotic resistance.

Contamination of surface waters has a direct impact on the public health of entire communities. Microorganisms inhabiting contaminated surface waters have developed mechanisms of coping with a variety of toxic metals and drugs. Investigations were carried out to isolate and identify lead-resistant bacteria from the river Kizilirmak along the city of Kirikkale, Turkey. Of the 33 lead-resistant isolates, one isolate with a minimal inhibitory concentration of 1,200 mg L(-1) was isolated and identified as Enterococcus faecalis by using biochemical tests and 16S rRNA sequencing. Lead-resistant E. faecalis isolate was found out to be resistant to other heavy metals like aluminum, lithium, barium, chromium, iron, silver, tin, nickel, zinc, and strontium and to drugs like amikacin, aztreonam, and gentamicin. E. faecalis harbored four plasmids with the molecular sizes of 1.58, 3.06, 22.76, and 28.95 kb. Plasmid profile analyses of cured derivatives revealed that the lead resistance ability of E. faecalis was still existing despite the elimination of all the plasmids. Moreover, the antibiotic resistance pattern of the cured derivatives did not demonstrate any change from the parental strain. Our findings indicated that the lead resistance genes of E. faecalis were located on the chromosomal DNA rather than the plasmid.

Real-time PCR investigation of the dynamic expression of three "RNA processing and modification" genes of Phanerochaete chrysosporium exposed to lead.

The expression of three ribosome binding proteins namely; polyadenylate-binding protein, splicing factor RNPS1 and ATP-dependent RNA helicase of Phanerochaete chrysosporium exposed to lead were analyzed by real-time PCR. The mRNA level of splicing factor RNPS1 showed 2.7 (p < 0.05), 2.6 (p < 0.05) and 4.9-fold (p < 0.001) increase when the cells were exposed to 25, 50 and 100 µM lead, respectively. 50 and 100 µM lead exposure resulted in almost 2-fold (p < 0.01and p < 0.05, respectively) increase in the expression of ATP-dependent RNA helicase. Polyadenylate-binding protein mRNA levels did not reveal any significant increase when cells exposed to the concentrations of lead employed. However, the mRNA level of polyadenylate-binding protein and splicing factor RNPS1 within a period of 1 and 2 h temporal exposure to 100 µM lead showed 2.5 (p < 0.001) and 3.4-fold (p < 0.001) increase, respectively. Expression level of ATP-dependent RNA helicase was not affected from the period of exposure to this metal.