Prevalence of antibiotic resistance and virulence genes in the biofilms from an aquifer recharged with stormwater.

An improved understanding of the diversity and composition of microbial communities carrying antibiotic resistance genes (ARGs) and virulence genes (VGs) in aquifers recharged with stormwater is essential to comprehend potential human health risks from water reuse. A high-throughput functional gene array was used to study the prevalence of ARGs and VGs in aquifer biofilms (n = 27) taken from three boreholes over three months. Bacterial genera annotated as opportunistic pathogens such as Aeromonas, Burkholderia, Pseudomonas, Shewanella, and Vibrio were ubiquitous and abundant in all biofilms. Bacteria from clinically relevant genera, Campylobacter, Enterobacter, Klebsiella, Mycobacterium, Mycoplasma, and Salmonella were detected in biofilms. The mean travel time of stormwater from the injection well to P1 and P3 boreholes was 260 and 360 days respectively. The presence of ARGs and VGs in the biofilms from these boreholes suggest a high spatial movement of ARGs and VGs in the aquifer. The ARGs with the highest abundance were small multidrug resistance efflux pumps (SMR) and multidrug efflux (Mex) followed by ß-lactamase C genes. ß- lactamase C encoding genes were primarily detected in Enterobacteriaceae, Pseudomonadaceae, Bacillaceae, and Rhodobacteraceae families. The VGs encoding siderophores, including aerobactin (iro and iuc genes), followed by pilin, hemolysin, and type III secretion were ubiquitous. Canonical correspondence analysis suggested that Total Organic Carbon (TOC), Dissolved Organic Carbon (DOC), turbidity, and Fe concentration has a significant impact on the microbial community structure of bacteria carrying ARGs and VGs. Post abstraction treatment of groundwater may be prudent to improve water security and reduce potential health risks.

Occurrence of antibiotic resistance genes and microbial source tracking markers in the water of a karst spring in Germany.

The emergence of antimicrobial resistances causes serious public health concerns worldwide. In recent years, the aquatic ecosystem has been recognized as a reservoir for antibiotic-resistant bacteria and antibiotic resistance genes (ARGs). The prevalence of 11 ARGs, active against six antibiotic classes (ß-lactams, aminoglycosides, tetracycline, macrolides, trimethoprim, and sulfonamides), was evaluated at a karst spring (Gallusquelle) in Germany, using molecular biological methods. In addition, fecal indicator bacteria (FIB), turbidity, electrical conductivity, spring discharge, and microbial source tracking markers specific for human, horse, chicken, and cow were determined. The ARGs most frequently detected were ermB (42.1%), tet(C) (40.8%), sul2 (39.5%), and sul1 (36.8%), which code for resistance to macrolides, tetracycline and sulfonamides, respectively. After a heavy rain event, the increase in FIB in the spring water was associated with the increase in ARGs and human-specific microbial source tracking (MST) markers. The determined correlations of the microbiological parameters, the observed overflow of a combined sewer overflow basin a few days before the increase of these parameters, and the findings of previous studies indicate that the overflow of this undersized basin located 9 km away from the spring could be a factor affecting the water quality of the karst spring. Our results provide a scientific basis for minimization of the input of fecal pollution and thus ARGs into the karst spring.

Association between antibiotic residues, antibiotic resistant bacteria and antibiotic resistance genes in anthropogenic wastewater - An evaluation of clinical influences.

The high use of antibiotics in human and veterinary medicine has led to a wide spread of antibiotics and antimicrobial resistance into the environment. In recent years, various studies have shown that antibiotic residues, resistant bacteria and resistance genes, occur in aquatic environments and that clinical wastewater seems to be a hot spot for the environmental spread of antibiotic resistance. Here a representative statistical analysis of various sampling points is presented, containing different proportions of clinically influenced wastewater. The statistical analysis contains the calculation of the odds ratios for any combination of antibiotics with resistant bacteria or resistance genes, respectively. The results were screened for an increased probability of detecting resistant bacteria, or resistance genes, with the simultaneous presence of antibiotic residues. Positive associated sets were then compared, with regards to the detected median concentration, at the investigated sampling points. All results show that the sampling points with the highest proportion of clinical wastewater always form a distinct cluster concerning resistance. The results shown in this study lead to the assumption that ciprofloxacin is a good indicator of the presence of multidrug resistant P. aeruginosa and extended spectrum ß-lactamase (ESBL)-producing Klebsiella spec., Enterobacter spec. and Citrobacter spec., as it positively relates with both parameters. Furthermore, a precise relationship between carbapenemase genes and meropenem, regarding the respective sampling sites, could be obtained. These results highlight the role of clinical wastewater for the dissemination and development of multidrug resistance.

Improving the identification of the source of faecal pollution in water using a modelling approach: From multi-source to aged and diluted samples.

The last decades have seen the development of several source tracking (ST) markers to determine the source of pollution in water, but none of them show 100% specificity and sensitivity. Thus, a combination of several markers might provide a more accurate classification. In this study Ichnaea® software was improved to generate predictive models, taking into account ST marker decay rates and dilution factors to reflect the complexity of ecosystems. A total of 106 samples from 4 sources were collected in 5 European regions and 30 faecal indicators and ST markers were evaluated, including E. coli, enterococci, clostridia, bifidobacteria, somatic coliphages, host-specific bacteria, human viruses, host mitochondrial DNA, host-specific bacteriophages and artificial sweeteners. Models based on linear discriminant analysis (LDA) able to distinguish between human and non-human faecal pollution and identify faecal pollution of several origins were developed and tested with 36 additional laboratory-made samples. Almost all the ST markers showed the potential to correctly target their host in the 5 areas, although some were equivalent and redundant. The LDA-based models developed with fresh faecal samples were able to differentiate between human and non-human pollution with 98.1% accuracy in leave-one-out cross-validation (LOOCV) when using 2 molecular human ST markers (HF183 and HMBif), whereas 3 variables resulted in 100% correct classification. With 5 variables the model correctly classified all the fresh faecal samples from 4 different sources. Ichnaea® is a machine-learning software developed to improve the classification of the faecal pollution source in water, including in complex samples. In this project the models were developed using samples from a broad geographical area, but they can be tailored to determine the source of faecal pollution for any user.

Comparative removal of antibiotic resistance genes during chlorination, ozonation, and UV treatment.

Efficient treatment methods for the removal of antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) from drinking water are needed to reduce health risks. However, there is a lack of empirical data on ARB and ARG removal during conventional water disinfection processes. In this study, the removal of ARB and ARGs by three disinfection processes (chlorination, ozonation, and UV treatment) was investigated on a laboratory scale using Escherichia coli and Enterococcus faecium carrying ARGs. Bacterial inactivation was determined by plate count methods, and ARG damage was quantified using real-time PCR. Only for ozone treatment, similar inactivation rates for bacterial cells and ARGs were observed when 1 mg*L-1 of ozone, with a contact time of 5 min, was used, which resulted in a 5.0 log reduction of bacterial cells and a 4.3-4.6 log reduction of ARGs. For chlorine and UV, inactivation of bacterial cells was observed at lower doses than those needed for the decrease of ARG copy numbers. The use of 0.5 mg*L-1 free chlorine (30 min contact time) led to a 3.8-5.6 log reduction of the bacterial numbers and to a 0.8-2.8 log reduction of ARGs. Ultraviolet light irradiation with 600 J*m-2 resulted in a 4.8-5.5 log reduction of bacterial cells, but in a negligible reduction (0-1.0 log) of ARGs. Although UV and chlorine treatments were effective in the inactivation of bacterial cells, incomplete degradation of ARGs was observed. Therefore, plasmid-borne ARGs can potentially be transferred to other bacteria even after the disinfection process. Our results provide important insights into the fate of ARGs during drinking water disinfection processes.

Distribution of clinically relevant antibiotic resistance genes in Lake Tai, China.

Lake Tai is China's third largest freshwater lake and an important water resource for agriculture, industrial sectors, and as drinking water for several large cities. In this study, the occurrence of five antibiotic resistance genes (sul1, blaTEM, blaNDM-1, blaCTX-M-32, mcr-1) was investigated in water and sediment samples collected from Lake Tai. Antibiotic resistances are currently increasing, posing a significant threat to public health. The sulfonamide resistance gene sul1 was highly abundant in all analyzed water and sediment samples. In addition, the two ß-lactamase genes blaTEMand blaNDM-1 - encoding clinically relevant antibiotic resistances - were detected in 67.1 and 7.3% of the water samples and in 70.7 and 15.4% of the sediment samples. The third ß-lactamase gene, blaCTX-M-32, was only detected in water samples (13.4%), while the colistin resistance gene mcr-1 was not detected in any of the samples. No significant variations between different sampling sites or time points could be observed. The investigation of drinking water treatment at Lake Tai, using lake water as influent, showed a significant reduction of the antibiotic resistance genes through the treatment process. Microbial source tracking showed only low fecal contamination by humans, ruminants, and pigs, indicating the relevance of other sources such as fish farms. Overall, our results provide important insights into the occurrence and abundance of antibiotic resistance genes in the Lake Tai water system and their elimination via drinking water treatment.

Antibiotic resistance and virulence genes in coliform water isolates.

Widespread fecal pollution of surface water may present a major health risk and a significant pathway for dissemination of antibiotic resistance bacteria. The River Rhine is one of the longest and most important rivers in Europe and an important raw water source for drinking water production. A total of 100 coliform isolates obtained from River Rhine (Germany) were examined for their susceptibility to seven antimicrobial agents. Resistances against amoxicillin, trimethoprim/sulfamethoxazole and tetracycline were detected in 48%, 11% and 9% of isolates respectively. The antibiotic resistance could be traced back to the resistance genes blaTEM, blaSHV, ampC, sul1, sul2, dfrA1, tet(A) and tet(B). Whereby, the ampC gene represents a special case, because its presence is not inevitably linked to a phenotypic antibiotic resistance. Multiple antibiotics resistance was often accompanied by the occurrence of class 1 or 2 integrons. E. coli isolates belonging to phylogenetic groups A and B1 (commensal) were more predominant (57%) compared to B2 and D groups (43%) which are known to carry virulent genes. Additionally, six E. coli virulence genes were also detected. However, the prevalence of virulence genes in the E. coli isolates was low (not exceeding 4.3% per gene) and no diarrheagenic E. coli pathotypes were detected. This study demonstrates that surface water is an important reservoir of ARGs for a number of antibiotic classes such as sulfonamide, trimethoprim, beta-lactam-antibiotics and tetracycline. The occurrence of antibiotic resistance in coliform bacteria isolated from River Rhine provides evidence for the need to develop management strategies to limit the spread of antibiotic resistant bacteria in aquatic environment.

Electrochemical degradation of perfluoroalkyl and polyfluoroalkyl substances (PFASs) in groundwater.

Perfluoroalkyl and polyfluoroalkyl substances (PFASs) represent hazardous pollutants and are frequently detected in the environment, e.g. in contaminated groundwater. PFASs are persistent to biodegradation and conventional oxidation processes such as ozonation. In this study electrochemical degradation of PFASs on boron-doped diamond (BDD) electrodes is demonstrated. Experiments were performed with model solutions and contaminated groundwater with a dissolved organic carbon (DOC) content of 13 mg/L. The perfluorinated carboxylic acids (PFCAs) perfluorobutanoate, perfluoropentanoate, perfluorohexanoate, perfluoroheptanoate and perfluorooctanoate, and the perfluorinated sulfonic acids (PFSAs) perfluorobutane sulfonate, perfluorohexane sulfonate, perfluorooctane sulfonate and 6:2 fluorotelomer sulfonate were detected in the groundwater samples. At PFAS concentrations ranging from 0.26 to 34 mg/L (0.7 to 79 µM), the degradation of PFASs was achieved despite of the high DOC background. Pseudo first-order kinetic constants of PFSA degradation increased with the increase of carbon chain length. Fluoride formation as well as the generation of PFCAs with shortened chain lengths was observed. Inorganic byproducts such as perchlorate were also formed and have to be considered in further process optimization.

Prevalence of clinically relevant antibiotic resistance genes in surface water samples collected from Germany and Australia.

The prevalence and proliferation of antibiotic resistant bacteria is profoundly important to human health, but the extent to which aquatic environments contribute toward the dissemination of antibiotic resistant genes (ARGs) is poorly understood. The prevalence of 24 ARGs active against eight antibiotic classes (ß-lactams, aminoglycosides, glycopeptides, chloramphenicols, tetracycline, macrolides, trimethoprim, and sulfonamides) was evaluated in surface water samples collected from Germany and Australia with culture independent methods. The ARGs most frequently detected both in Germany and Australia were sulI, sulII (77-100%), and dfrA1 (43-55%) which code for resistance to sulfonamide and trimethoprim. Macrolides resistance gene ermB was relatively more prevalent in the surface water from Germany (68%) than Australia (18%). In contrast, the chloramphenicol resistance gene catII was more frequently detected in Australia (64%) than Germany (9%). Similarly, ß-lactams resistance gene ampC was more prevalent in the samples from Australia (36%) than Germany (19%). This study highlights wide distribution of ARGs for sulfonamide, trimethoprim, macroline, ß-lactams and chloramphenicol in the aquatic ecosystems. Aquatic ecosystems can therefore be reservoirs of ARGs genes which could potentially be transferred from commensal microorganisms to human pathogens.

Consideration of emerging pollutants in groundwater-based reuse concepts.

Elimination of pathogens and emerging pollutants represents a key factor in integrated water resources management in arid regions. Within the SMART Jordan Valley project it is the objective of this study to assess the occurrence and examine the elimination of selected emerging pollutants and pathogens in waste water treatment and aquifer recharge. In batch and soil column studies non-chlorinated organophosphorous compounds (tri-n-butylphosphate, triphenylphosphate) and endocrine disruptors (e.g. 17-ß-estradiol, bisphenol A) proved to be biodegradable, while the X-ray contrast agents iomeprol and iopromide were eliminated in the soil columns only, and the chlorinated trialkylphosphates showed persistency. Treating waste water in a membrane bioreactor (MBR) in combination with powdered activated carbon (PAC) resulted in considerable removal rates also for the more persistent compounds such as the antiepileptic carbamazepine. Viruses were shown to be present in most of the Jordan Valley surface water samples. MBR treatment resulted in a decrease of MS2 bacteriophages used as model viruses.

The Yangtze-Hydro Project: a Chinese-German environmental program.

Water of good quality is one of the basic needs of human life. Worldwide, great efforts are being undertaken for an assured water supply. In this respect, one of the largest water technology projects worldwide is the Yangtze Three Gorges Dam in China. There is a need for extensive scientific and technical understanding of the challenges arising from this large hydrological engineering project. German and Chinese groups from various scientific fields are collaborating to provide knowledge for the sustainable management of the reservoir. In this project description, the Yangtze Three Gorges Dam Project, its goals and challenges, are described in brief, and the contributions of the German research projects are presented.

Development of a groundwater biobarrier for the removal of polycyclic aromatic hydrocarbons, BTEX, and heterocyclic hydrocarbons.

A full scale funnel-and-gate biobarrier has been developed for the removal of tar oil pollutants at an abandoned tar factory site near the city of Offenbach, Germany. Laboratory and on-site column studies were done to determine the operation parameters for microbiological clean-up of the groundwater polluted with 12,000 microg/L mono- aromatic hydrocarbons such as benzene and the xylenes, 4,800 microg/L polycyclic aromatic hydrocarbons such as naphthalene and acenaphthene, and 4,700 microg/L heterocyclic aromatic hydrocarbons such as benzofuran and benzothiophene. In the laboratory study, a residence time of approx. 70 h proved to be sufficient for aerobic pollutant biodegradation. Up to 180 mg/L H(2)O(2) were added and did not lead to any toxic effects to the degrading bacteria. The feasibility of the concept was confirmed in an on-site pilot study performed with a sedimentation tank (removal of ferric iron) and two bioreactors. In the bioreactors, >99.3% of the pollutants were degraded. Biodegradation activity corresponded to a significant increase in numbers of pollutant degrading bacteria. In the bioreactors, a fast dissociation of H(2)O(2) was observed resulting in losses of oxygen and temporary gas clogging. Therefore, a repeated addition of moderate concentrations of H(2)O(2) proved to be more favourable than the addition of high concentrations at a single dosing port. The full scale biobarrier consists of three separated bioreactors thus enabling extended control and access to the reactors. The operation of the funnel-and-gate biobarrier started in April 2007, and represents the first biological permeable reactive barrier with extended control (EC-PRB) in Germany.

Natural attenuation of chloroethenes: identification of sequential reductive/oxidative biodegradation by microcosm studies.

A different lines of evidence approach for investigation of biodegradation processes at a chloroethene contaminated site showed well corresponding results of pollutant profiles, redox zonation, characterisation of autochthonic microflora and microcosm studies. In particular microcosm studies allowed identification of the predominating degradation pathways. Perchloroethene and trichloroethene are reductively chlorinated to mainly cis-1,2-dichloroethene (cDCE) under anaerobic conditions. Further reductive degradation to vinyl chloride (VC) is restricted to a distinct strongly anaerobic zone in the plume. Addition of high amounts of sediment material (80 vol%) to groundwater microcosms enabled reductive dechlorination without amendment with further auxiliary substrates. Reductive dechlorination was not irreversibly hindered by initially high nitrate concentrations and initially high oxidation-reduction potential. The products of anaerobic degradation cDCE and VC are subsequently aerobically mineralised, even when only low oxygen concentrations are available. Anaerobic oxidative degradation could not be proven in this study.

Assessment of microbial natural attenuation in groundwater polluted with gasworks residues.

Intrinsic biodegradation, representing the key process in Natural Attenuation, was examined at a tar-oil polluted disposal site. Methods to assess microbial natural attenuation of BTEX and PAH included analysis of groundwater hydrochemistry, pollutant profiles, composition of the microflora, and microcosm studies. In the polluted groundwater downgradient the disposal site, oxygen and nitrate were only available adjacent to the groundwater table and at the plume fringes. In the anaerobic core of the plume, a sequence of predominating redox zones (methanogenic, sulphate-reducing, Fe(III)-reducing) was observed. Changing pollutant profiles in the plume indicated active biodegradation processes, e.g. biodegradation of toluene and naphthalene in the anaerobic zones. High numbers of microorganisms capable of growing under anaerobic conditions and of aerobic pollutant degrading organisms confirmed the impact of biodegradation at this site. In microcosm studies, the autochthonous microflora utilised toluene, ethylbenzene, and naphthalene under sulfate- and Fe(III)-reducing conditions. Additionally, benzene and phenanthrene were degraded in the presence of Fe(III). Under aerobic conditions, all BTEX and PAH were rapidly degraded. The microcosm studies in particular were suitable to examine the role of specific electron acceptors, and represented an important component of the multiple line of evidence concept to assess natural attenuation.

Ultrasonic waste activated sludge disintegration for improving anaerobic stabilization.

The pretreatment of waste activated sludge by ultrasonic disintegration was studied in order to improve the anaerobic sludge stabilization. The ultrasound frequency was varied within a range from 41 to 3217 kHz. The impact of different ultrasound intensities and treatment times was examined. Sludge disintegration was most significant at low frequencies. Low-frequency ultrasound creates large cavitation bubbles which upon collapse initiate powerful jet streams exerting strong shear forces in the liquid. The decreasing sludge disintegration efficiency observed at higher frequencies was attributed to smaller cavitation bubbles which do not allow the initiation of such strong shear forces. Short sonication times resulted in sludge floc deagglomeration without the destruction of bacteria cells. Longer sonication brought about the break-up of cell walls, the sludge solids were distintegrated and dissolved organic compounds were released. The anaerobic digestion of waste activated sludge following ultrasonic pretreatment causing microbial cell lysis was significantly improved. There was an increase in the volatile solids degradation as well as an increase in the biogas production. The increase in digestion efficiency was proportional to the degree of sludge disintegration. To a lesser degree the deagglomeration of sludge flocs also augmented the anaerobic volatile solids degradation.

Removal of chlorinated pollutants by a combination of ultrasound and biodegradation.

Chlorinated organic compounds are hazardous pollutants found in waste water, surface water, and ground water. Our study shows that a combination of ultrasonic pretreatment and biodegradation effectively removes the solvent chlorobenzene and the disinfectant 2,4-dichlorophenol, also reduces Adsorbable Organic Halogens (AOX) and Chemical Oxygen Demand (COD). In our experiments, the ultrasonic dechlorination was not influenced by the presence of other soluble organic compounds like acetate or glucose. Dechlorination of chlorobenzene by ultrasound did not lead to toxic or inhibiting reaction products. More than that, the ultrasonic pretreatment significantly reduced the toxicity of 2,4-dichlorophenol and biological activity was initiated after sonication. Residual organic pollutants after ultrasonic pretreatment were eliminated by biodegradation.

Degradation of polycyclic aromatic hydrocarbons in the presence of synthetic surfactants.

The biodegradation of polycyclic aromatic hydrocarbons (PAH) often is limited by low water solubility and dissolution rate. Nonionic surfactants and sodium dodecyl sulfate increased the concentration of PAH in the water phase because of solubilization. The degradation of PAH was inhibited by sodium dodecyl sulfate because this surfactant was preferred as a growth substrate. Growth of mixed cultures with phenanthrene and fluoranthene solubilized by a nonionic surfactant prior to inoculation was exponential, indicating a high bioavailability of the solubilized hydrocarbons. Nonionic surfactants of the alkylethoxylate type and the alkylphenolethoxylate type with an average ethoxylate chain length of 9 to 12 monomers were toxic to a PAH-degrading Mycobacterium sp. and to several PAH-degrading mixed cultures. Toxicity of the surfactants decreased with increasing hydrophilicity, i.e., with increasing ethoxylate chain length. Nontoxic surfactants enhanced the degradation of fluorene, phenanthrene, anthracene, fluoranthene, and pyrene.

Degradation of phenanthrene, fluorene, fluoranthene, and pyrene by a Mycobacterium sp.

Mycobacterium sp. strain BB1 was isolated from a former coal gasification site. It was able to utilize phenanthrene, pyrene, and fluoranthene as sole sources of carbon and energy and to degrade fluorene cometabolically. Exponential growth with solid phenanthrene, pyrene, and fluoranthene was obtained in fermentor cultures. The growth rates were 0.069, 0.056, and 0.040 h-1, respectively. Several metabolites of phenanthrene and fluorene metabolism were identified.