Irene Xagoraraki

Release of antibiotic resistant bacteria and genes in the effluent and biosolids of five wastewater utilities in Michigan

The purpose of this study was to quantify the occurrence and release of antibiotic resistant genes (ARGs) and antibiotic resistant bacteria (ARB) into the environment through the effluent and biosolids of different wastewater treatment utilities including an MBR (Membrane Biological Reactor) utility, conventional utilities (Activated Sludge, Oxidative Ditch and Rotatory Biological Contactors-RBCs) and multiple sludge treatment processes (Dewatering, Gravity Thickening, Anaerobic Digestion and Lime Stabilization). Samples of raw wastewater, pre- and post-disinfected effluents, and biosolids were monitored for tetracycline resistant genes (tetW and tetO) and sulfonamide resistant gene (Sul-I) and tetracycline and sulfonamide resistant bacteria. ARGs and ARB concentrations in the final effluent were found to be in the range of ND(non-detectable)-2.33 × 10(6) copies/100 mL and 5.00 × 10(2)-6.10 × 10(5) CFU/100 mL respectively. Concentrations of ARGs (tetW and tetO) and 16s rRNA gene in the MBR effluent were observed to be 1-3 log less, compared to conventional treatment utilities. Significantly higher removals of ARGs and ARB were observed in the MBR facility (range of removal: 2.57-7.06 logs) compared to that in conventional treatment plants (range of removal: 2.37-4.56 logs) (p < 0.05). Disinfection (Chlorination and UV) processes did not contribute in significant reduction of ARGs and ARB (p > 0.05). In biosolids, ARGs and ARB concentrations were found to be in the range of 5.61 × 10(6)-4.32 × 10(9) copies/g and 3.17 × 10(4)-1.85 × 10(9) CFU/g, respectively. Significant differences (p < 0.05) were observed in concentrations of ARGs (except tetW) and ARB between the advanced biosolid treatment methods (i.e., anaerobic digestion and lime stabilization) and the conventional dewatering and gravity thickening methods.

Correlation of tetracycline and sulfonamide antibiotics with corresponding resistance genes and resistant bacteria in a conventional municipal wastewater treatment plant

Antibiotics and corresponding resistance genes and resistant bacteria have been considered as emerging pollutants worldwide. Wastewater treatment plants (WWTPs) are potential reservoirs contributing to the evolution and spread of antibiotic resistance. In this study, total concentrations of tetracycline and sulfonamide antibiotics in final effluent were detected at 652.6 and 261.1ng/L, respectively, and in treated sludge, concentrations were at 1150.0 and 76.0μg/kg dry weight (dw), respectively. The quantities of antibiotic resistance genes and antibiotic resistant bacteria in final effluent were quantified in the range of 9.12×10(5)-1.05×10(6) gene abundances /100mL (genomic copies/100mL) and 1.05×10(1)-3.09×10(3)CFU/mL, respectively. In treated sludge, they were quantified at concentrations of 1.00×10(8)-1.78×10(9) gene abandances/100mL and 7.08×10(6)-1.91×10(8)CFU/100mL, respectively. Significant reductions (2-3 logs, p<0.05) of antibiotic resistance genes and antibiotic resistant bacteria were observed between raw influent and final effluent. The gene abundances of tetO and tetW normalized to that of 16S rRNA genes indicated an apparent decrease as compared to sulI genes, which remained stable along each treatment stage. Significant correlations (R(2)=0.75-0.83, p<0.05) between numbers of resistant bacteria and antibiotic concentrations were observed in raw influent and final effluent. No significance (R(2)=0.15, p>0.05) was found between tet genes (tetO and tetW) with concentration of tetracyclines identified in wastewater, while a significant correlation (R(2)=0.97, p<0.05) was observed for sulI gene and total concentration of sulfonamides. Correlations of the quantities of antibiotic resistance genes and antibiotic resistant bacteria with corresponding concentrations of antibiotics in sludge samples were found to be considerably weak (R(2)=0.003-0.07).

Quantitative Detection of Human Adenoviruses in Wastewater and Combined Sewer Overflows Influencing a Michigan River

ABSTRACT Enteric viruses are important pathogens found in contaminated surface waters and have previously been detected in waters of the Great Lakes. Human adenoviruses were monitored because of their high prevalence and persistence in aquatic environments. In this study, we quantified adenoviruses in wastewater, surface water, and combined sewer overflows (CSOs) by real-time PCR. Between August 2005 and August 2006, adenovirus concentrations in raw sewage, primary-treated effluent, secondary-treated effluent, and chlorinated effluent from a wastewater treatment plant in Michigan were examined. CSO samples (n = 6) were collected from a CSO retention basin in Grand Rapids, MI. Adenoviruses were detected in 100% of wastewater and CSO discharge samples. Average adenovirus DNA concentrations in sewage and CSOs were 1.15 × 106 viruses/liter and 5.35 × 105 viruses/liter, respectively. Adenovirus removal was <2 log10 (99%) at the wastewater treatment plant. Adenovirus type 41 (60% of clones), type 12 (29%), type 40 (3%), type 2 (3%), and type 3 (3%) were isolated from raw sewage and primary effluents (n = 28). Six of 20 surface water samples from recreational parks at the lower Grand River showed virus concentrations above the real-time PCR detection limit (average, 7.8 × 103 viruses/liter). This research demonstrates that wastewater effluents and wastewater-impacted surface waters in the lower Grand River in Michigan contain high levels of viruses and may not be suitable for full-body recreational activities. High concentrations of adenovirus in these waters may be due to inefficient removal during wastewater treatment and to the high persistence of these viruses in the environment.

Occurrence of pharmaceuticals in a municipal wastewater treatment plant: mass balance and removal processes.

Occurrence and removal efficiencies of fifteen pharmaceuticals were investigated in a conventional municipal wastewater treatment plant in Michigan. Concentrations of these pharmaceuticals were determined in both wastewater and sludge phases by a high-performance liquid chromatograph coupled to a tandem mass spectrometer. Detailed mass balance analysis was conducted during the whole treatment process to evaluate the contributing processes for pharmaceutical removal. Among the pharmaceuticals studied, demeclocycline, sulfamerazine, erythromycin and tylosin were not detected in the wastewater treatment plant influent. Other target pharmaceuticals detected in wastewater were also found in the corresponding sludge phase. The removal efficiencies of chlortetracycline, tetracycline, sulfamerazine, acetaminophen and caffeine were >99%, while doxycycline, oxytetracycline, sulfadiazine and lincomycin exhibited relatively lower removal efficiencies (e.g., <50%). For sulfamethoxazole, the removal efficiency was approximately 90%. Carbamazepine manifested a net increase of mass, i.e. 41% more than the input from the influent. Based on the mass balance analysis, biotransformation is believed to be the predominant process responsible for the removal of pharmaceuticals (22% to 99%), whereas contribution of sorption to sludge was relatively insignificant (7%) for the investigated pharmaceuticals.

Evaluation of public health risks at recreational beaches in Lake Michigan via detection of enteric viruses and a human-specific bacteriological marker

UNLABELLED Each year the National Resource Defense Council addresses the quality of US beaches by routine bacterial indicators. In the Great Lakes region the indicator used is Escherichia coli and for 2007 more beaches were closed and impacted than ever before. In this study, water quality was addressed at two Lake Michigan Beaches over the 2004 swimming season by monitoring infectious enteric viruses by cell culture and integrated PCR and for a human sewage marker based on the Enterococcal Surface Protein (esp). Our goals for this study were to 1) examine the occurrence and variety of human enteric viruses present during peak usage of the beaches 2) determine key variables for development of predictive models for viruses; and 3) use quantitative risk assessment to estimate the potential health impact. Our results demonstrate that for both beaches predictive models of virus pollution were best described utilizing physical parameters like wind speed, wind direction and water temperature. The esp marker was not predictive of human viruses. The daily risk of acquiring a viral infection at either of the beaches ranged from 0.2 to 2.4/1000 swimmers using a quantitative microbial risk assessment model, with three swims during a day at the beach for children and over the season, the risk was 9-15/1000 swimmers using adenovirus as the model. CONCLUSIONS Lake Michigan recreational beaches are being adversely impacted by human fecal pollution. Monitoring for the traditional indicators of water quality does not address viral risks and models can be developed and potentially used as real-time water quality forecasting tools.

Occurrence of human adenoviruses at two recreational beaches of the great lakes

ABSTRACT Human adenoviruses (HAdVs) have been related to several waterborne diseases such as acute gastroenteritis, conjunctivitis, and respiratory illness, and it has been shown that an important human exposure pathway is through recreational waters. However, HAdV occurrence at recreational freshwater beaches has not been previously investigated. In this study, a total of 58 water samples were collected from two recreational beaches on Lake Michigan (i.e., Silver Beach and Washington Park Beach) during the summer of 2004. Occurrences of HAdVs in these lake samples were determined using two hexon-based real-time PCR assays (one for monitoring all 51 serotypes of HAdVs and another for specifically detecting F species HAdVs, i.e., serotypes 40 and 41) and compared to an integrated cell culture (ICC) PCR method. The real-time PCR results showed that 8 of 30 Silver Beach samples and 6 of 28 Washington Park Beach samples contained HAdVs, and F species HAdVs were detected in three of these positive samples. The concentrations of HAdVs ranged from (1.7 ± 0.7) × 101 to (3.4 ± 0.8) × 102 and from (7 ± 2) × 100 to (3.8 ± 0.3) × 103 virus particles/liter for Silver Beach and Washington Park Beach, respectively. F species HAdVs were detected at levels ranging from (4.8 ± 0.8) × 101 to (4.6 ± 1.5) × 102 virus particles/liter. Approximately 60% of the ICC-PCR analyses agreed with the real-time PCR results. This study revealed the occurrence of HAdVs at Lake Michigan recreational beaches. Given the potential health risks, further assessment regarding sources, virus transport, and survival is needed to improve the safety of the region.

Assessment of human adenovirus removal in a full-scale membrane bioreactor treating municipal wastewater

Human adenoviruses (HAdVs) in wastewater samples taken from four different treatment stages of a full-scale municipal wastewater treatment plant (i.e., incoming raw sewage, primary sedimentation effluent, membrane bioreactor (MBR) influent, and MBR effluent) were quantified by real-time PCR assays to further estimate removal efficiency of the HAdVs. Based on hexon gene sequence comparisons, HAdV species A, C, and F were consistently found in the wastewater samples. In general, all three identified HAdV species were detected in most of the wastewater samples using the real-time PCR assays. Overall HAdV concentrations were rather stable over the entire 8-month study period (January-August, 2008) (approximately 10(6)-10(7)viral particles/L of wastewater for the raw sewage and primary effluent; 10(8)-10(9)viral particles/L for the MBR influent; and, 10(3)-10(4)viral particles/L for the MBR effluent). No significant seasonal differences were noticed for the HAdV abundances. Removal efficiencies of the viral particles in the full-scale MBR process were assessed and showed an average HAdV removal of 5.0+/-0.6logs over the study period. The removal efficiencies for F species (average log removal of 6.5+/-1.3logs) were typically higher (p-value <0.05) than those of the other two species (average of 4.1+/-0.9 and 4.6+/-0.5logs for species A and C, respectively). These results demonstrate that the full-scale MBR system efficiently removed most HAdV from the wastewater leaving about 10(3)viral particles/L in the MBR effluent.

Factors affecting bulk to total bacteria ratio in drinking water distribution systems

Bacteria in drinking water systems can grow in bulk water and as biofilms attached to pipe walls, both causing regrowth problems in the distribution system. While studies have focused on evaluating the factors influencing the bacteria in bulk water and in biofilms separately, there is a need for understanding biofilm characteristics relative to the bulk water phase. The current study evaluated the effects of chlorine and residence time on the presence of culturable bacteria in biofilms relative to that in bulk water. The results showed that when no chlorine residual was present in the system, the median ratio of bulk to total bacteria was 0.81, indicating that 81% of the bacteria were present in bulk water, whereas only 19% were present in the biofilm. As chlorine concentration increased to 0.2, 0.5, and 0.7 mg/L, the median percentage of bacteria present in bulk water decreased to 37, 28, and 31, respectively. On the other hand, as the residence times increased to 8.2, 12, 24, and 48h, the median percentage of bacteria present in bulk water increased to 7, 37, 58, and 88, respectively, in the presence of a 0.2mg/L chlorine residual. The common notion that biofilms dominate the distribution system is not true under all conditions. These findings suggest that bulk water bacteria may dominate in portions of a distribution system that have a low chlorine residual.

Determination of pharmaceuticals in biosolids using accelerated solvent extraction and liquid chromatography/tandem mass spectrometry.

An analytical method was developed to quantitatively determine pharmaceuticals in biosolid (treated sewage sludge) from wastewater treatment plants (WWTPs). The collected biosolid samples were initially freeze dried, and grounded to obtain relatively homogenized powders. Pharmaceuticals were extracted using accelerated solvent extraction (ASE) under the optimized conditions. The optimal operation parameters, including extraction solvent, temperature, pressure, extraction time and cycles, were identified to be acetonitrile/water mixture (v/v 7:3) as extraction solvent with 3 extraction cycles (15 min for each cycle) at 100 °C and 100 bars. The extracts were cleaned up using solid-phase extraction followed by determination by liquid chromatography coupled with tandem mass spectrometry. For the 15 target pharmaceuticals commonly found in the environment, the overall method recoveries ranged from 49% to 68% for tetracyclines, 64% to 95% for sulfonamides, and 77% to 88% for other pharmaceuticals (i.e. acetaminophen, caffeine, carbamazepine, erythromycin, lincomycin and tylosin). The developed method was successfully validated and applied to the biosolid samples collected from WWTPs located in six cities in Michigan. Among the 15 target pharmaceuticals, 14 pharmaceuticals were detected in the collected biosolid samples. The average concentrations ranged from 2.6 μg/kg for lincomycin to 743.6 μg/kg for oxytetracycline. These results indicated that pharmaceuticals could survive wastewater treatment processes, and accumulate in sewage sludge and biosolids. Subsequent land application of the contaminated biosolids could lead to the dissemination of pharmaceuticals in soil and water environment, which poses potential threats to at-risk populations in the receiving ecosystems.

Removal of human enteric viruses by a full-scale membrane bioreactor during municipal wastewater processing.

In the US, human enteric viruses are the main etiologic agents of childhood gastroenteritis, resulting in several hospitalizations and deaths each year. These viruses have been linked to several waterborne diseases, such as acute gastroenteritis, conjunctivitis and respiratory illness. The removal of human enterovirus (EV) and norovirus genogroup II (NoV GGII) was studied in a full-scale membrane bioreactor (MBR) wastewater treatment plant (WWTP) and compared with the removal of human adenovirus (HAdV). In total, 32 samples were quantified using real-time reverse transcription-PCR (RT-PCR) from four separate locations throughout the treatment process; influent, primary settling effluent, membrane influent (which includes the MLSS) and membrane effluent. EV was detected in all 32 samples (100%) with an average concentration of 1.1 × 10(7) and 7.8 × 10(1) viruses/L for the membrane influent and membrane effluent, respectively. NoV GGII was detected in 20 of 32 samples (63%) with an average membrane influent and membrane effluent concentration of 2.8 × 10(5) and 1.2 × 10(1) viruses/L, respectively. HAdV was detected in all 32 samples with an average membrane influent concentration of 5.2 × 10(8) and 2.7 × 10(3) viruses/L in the membrane effluent. Our findings indicate that this particular full-scale MBR treatment was able to reduce the viral loads by approximately 5.1 and 3.9 log units for EV and NoV GGII as compared to 5.5 log units for HAdV. This full-scale MBR system outperformed the removal observed in previous pilot and bench scale studies by 1 to 2 log units. To the best of our knowledge, this is the first study focusing on the removal of EV in a full-scale MBR WWTP using real-time RT-PCR, and on the solid-liquid distribution of EV and NoV GII in secondary biological treatment.