Antibiotic-Resistant Escherichia coli in Wastewaters, Surface Waters, and Oysters from an Urban Riverine System

The antibiotic resistance (AR) patterns of 462 Escherichia coli isolates from wastewater, surface waters, and oysters were determined. Rates of AR and multiple-AR among isolates from surface water sites adjacent to wastewater treatment plant (WWTP) discharge sites were significantly higher (P < 0.05) than those among other isolates, whereas the rate of AR among isolates from oysters exposed to WWTP discharges was low (<10%).     

Identification of sources of Escherichia coli in South Carolina estuaries using antibiotic resistance analysis

Fecal pollution from nonhuman (pets, livestock or wildlife) and human sources is often one of the major factors associated with urbanization that contribute to the degradation of water quality. Methods to differentiate animal from human sources of fecal coliform contamination could assist resource managers in developing strategies to protect shellfish harvesting areas and recreational waters. In this study, surface water samples were collected from both a developed and an undeveloped watershed in coastal South Carolina. Influent and effluent samples from several wastewater treatment plants (WWTPs) in the same area were also collected. Most Probable Numbers (MPNs) of fecal coliforms were determined for all samples. Escherichia coli isolates were analyzed for antibiotic resistance (AR) to 10 antibiotics. Then, AR indices (no. of resistant/total no. of antibiotics tested), were calculated for each isolate and site. Results indicated that MPNs from the WWTP samples were significantly higher than those from the developed watershed which were significantly higher than those from the undeveloped watershed (p<0.0001). The AR analyses suggested that there was a trend toward increased antibiotic resistance in samples for the urbanized Broad Creek (BC) watershed. In the Okatee River (OR), E. coli isolates from three sites (20%) showed resistance to a single antibiotic (penicillin) but in BC, isolates from seven sites (47%) were resistant to multiple antibiotics, and the predominant resistance pattern was chlortetracycline-oxytetracycline-tetracycline. Raw sewage isolates from most WWTPs contained E. coli that exhibited resistance to multiple antibiotics. Cluster analysis indicated that all resistant OR sites had antibiotic resistant isolates that matched AR patterns found in isolates from WWTPs. Similarly, six of the seven sites in BC had AR patterns that matched with resistance patterns in WWTPs. These results suggest that AR testing may be a useful tool for differentiating E. coli from human and wildlife sources. Further testing of bacterial isolates from known animal sources is necessary to better assess the utility of this approach.     

An evaluation of survival and detection of Campylobacter jejuni and C. coli in broiler caecal contents using culture‐based methods

Aims: (i) To cultivate methicillin‐resistant Staphylococcus aureus (MRSA) from a full‐scale wastewater treatment plant (WWTP), (ii) To characterize the indigenous MRSA‐flora, (iii) To investigate how the treatment process affects clonal distribution and (iv) To examine the genetic relation between MRSA from wastewater and clinical MRSA. Methods: Wastewater samples were collected during 2 months at four key sites in the WWTP. MRSA isolates were characterized using spa typing, antibiograms, SSCmec typing and detection of Panton–Valentine leukocidin (PVL). Conclusions: MRSA could be isolated on all sampling occasions, but only from inlet and activated sludge. The number of isolates and diversity of MRSA were reduced by the treatment process, but there are indications that the process was selected for strains with more extensive antibiotic resistance and PVL+ strains. The wastewater MRSA‐flora had a close genetic relationship to clinical isolates, most likely reflecting carriage in the community. Significance and Impact of the Study: This study shows that MRSA survives in wastewater and that the WWTP may be a potential reservoir for MRSA.     

Multidrug-Resistant and Extended Spectrum Beta-Lactamase-Producing Escherichia coli in Dutch Surface Water and Wastewater

Objective

The goal of the current study was to gain insight into the prevalence and concentrations of antimicrobial resistant (AMR) Escherichia coli in Dutch surface water, and to explore the role of wastewater as AMR contamination source.

Methods

The prevalence of AMR E. coli was determined in 113 surface water samples obtained from 30 different water bodies, and in 33 wastewater samples obtained at five health care institutions (HCIs), seven municipal wastewater treatment plants (mWWTPs), and an airport WWTP. Overall, 846 surface water and 313 wastewater E. coli isolates were analysed with respect to susceptibility to eight antimicrobials (representing seven different classes): ampicillin, cefotaxime, tetracycline, ciprofloxacin, streptomycin, sulfamethoxazole, trimethoprim, and chloramphenicol.

Results

Among surface water isolates, 26% were resistant to at least one class of antimicrobials, and 11% were multidrug-resistant (MDR). In wastewater, the proportions of AMR/MDR E. coli were 76%/62% at HCIs, 69%/19% at the airport WWTP, and 37%/27% and 31%/20% in mWWTP influents and effluents, respectively. Median concentrations of MDR E. coli were 2.2×10², 4.0×10⁴, 1.8×10⁷, and 4.1×10⁷ cfu/l in surface water, WWTP effluents, WWTP influents and HCI wastewater, respectively. The different resistance types occurred with similar frequencies among E. coli from surface water and E. coli from municipal wastewater. By contrast, among E. coli from HCI wastewater, resistance to cefotaxime and resistance to ciprofloxacin were significantly overrepresented compared to E. coli from municipal wastewater and surface water. Most cefotaxime-resistant E. coliisolates produced ESBL. In two of the mWWTP, ESBL-producing variants were detected that were identical with respect to phylogenetic group, sequence type, AMR-profile, and ESBL-genotype to variants from HCI wastewater discharged onto the same sewer and sampled on the same day (A₁/ST23/CTX-M-1, B2₃/ST131/CTX-M-15, D₂/ST405/CTX-M-15).

Conclusion

In conclusion, our data show that MDR E. coli are omnipresent in Dutch surface water, and indicate that municipal wastewater significantly contributes to this occurrence.     

Antibiotic-resistance profile in environmental bacteria isolated from penicillin production wastewater treatment plant and the receiving river

The antibiotic‐resistance characteristics of bacterial strains in antibiotic production wastewater treatment plants (WWTP) that contain high concentrations of antibiotics are unknown, as are the environmental effects of the discharge of wastewater from such facilities. In this study, 417 strains were individually isolated from the effluent of a WWTP that treated penicillin G production wastewater, as well as from downstream and upstream areas of the receiving river. The minimum inhibition concentrations (MICs) of 18 antibiotics representing seven classes were then determined for each of these strains. Relatively high similarity in the bacterial composition existed between the wastewater and downstream river samples when compared with the upstream sample. High resistance ratios and MIC values were observed for almost all antibiotics in wastewater isolates, followed by strains from downstream river, of which the resistance ratios and levels were still significantly higher than those of upstream strains. The resistance ratios and levels also significantly differed among strains belonged to different species in the penicillin production wastewater effluent and downstream river. In both samples, the resistances to β‐lactam antibiotics were more frequent, with much higher levels, than the other class antibiotics. Then five clinically important resistant genes mainly coding for extended‐spectrum β‐lactamases (ESBLs) were determined for all strains, only bla_TEM‐1 which did not belong to ESBL was detected in 17.3% and 11.0% of strains isolated from wastewater and downstream river respectively. Class I integrons were detected in 14% of wastewater isolates and 9.1% of downstream isolates, and primarily contained gene cassettes conferring resistance to aminoglycoside antibiotics. The unexpectedly high levels of multiple antibiotic resistance in strains from wastewater and downstream river were speculated to be mainly due to multidrug efflux systems.     

Tracking Down Antibiotic-Resistant Pseudomonas aeruginosa Isolates in a Wastewater Network

The Pseudomonas aeruginosa-containing wastewater released by hospitals is treated by wastewater treatment plants (WWTPs), generating sludge, which is used as a fertilizer, and effluent, which is discharged into rivers. We evaluated the risk of dissemination of antibiotic-resistant P. aeruginosa (AR-PA) from the hospital to the environment via the wastewater network. Over a 10-week period, we sampled weekly 11 points (hospital and urban wastewater, untreated and treated water, sludge) of the wastewater network and the river upstream and downstream of the WWTP of a city in eastern France. We quantified the P. aeruginosa load by colony counting. We determined the susceptibility to 16 antibiotics of 225 isolates, which we sorted into three categories (wild-type, antibiotic-resistant and multidrug-resistant). Extended-spectrum β-lactamases (ESBLs) and metallo-β-lactamases (MBLs) were identified by gene sequencing. All non-wild-type isolates (n = 56) and a similar number of wild-type isolates (n = 54) were genotyped by pulsed-field gel electrophoresis and multilocus sequence typing. Almost all the samples (105/110, 95.5%) contained P. aeruginosa, with high loads in hospital wastewater and sludge (≥3×10⁶ CFU/l or/kg). Most of the multidrug-resistant isolates belonged to ST235, CC111 and ST395. They were found in hospital wastewater and some produced ESBLs such as PER-1 and MBLs such as IMP-29. The WWTP greatly reduced P. aeruginosa counts in effluent, but the P. aeruginosa load in the river was nonetheless higher downstream than upstream from the WWTP. We conclude that the antibiotic-resistant P. aeruginosa released by hospitals is found in the water downstream from the WWTP and in sludge, constituting a potential risk of environmental contamination.     

Intraspecific Diversity of Vibrio vulnificus in Galveston Bay Water and Oysters as Determined by Randomly Amplified Polymorphic DNA PCR

Randomly amplified polymorphic DNA (RAPD) PCR was used to analyze the temporal and spatial intraspecific diversity of 208 Vibrio vulnificus strains isolated from Galveston Bay water and oysters at five different sites between June 2000 and June 2001. V. vulnificus was not detected during the winter months (December through February). The densities of V. vulnificus in water and oysters were positively correlated with water temperature. Cluster analysis of RAPD PCR profiles of the 208 V. vulnificus isolates revealed a high level of intraspecific diversity among the strains. No correlation was found between the intraspecific diversity among the isolates and sampling site or source of isolation. After not being detected during the winter months, the genetic diversity of V. vulnificus strains first isolated in March was 0.9167. Beginning in April, a higher level of intraspecific diversity (0.9933) and a major shift in population structure were observed among V. vulnificus isolates. These results suggest that a great genetic diversity of V. vulnificus strains exists in Galveston Bay water and oysters and that the population structure of this species is linked to changes in environmental conditions, especially temperature.     

Real-Time PCR Assays for Quantification and Differentiation of Vibrio vulnificus Strains in Oysters and Water

Vibrio vulnificus is an autochthonous estuarine bacterium and a pathogen that is frequently transmitted via raw shellfish. Septicemia can occur within 24 h; however, isolation and confirmation from water and oysters require days. Real-time PCR assays were developed to detect and differentiate two 16S rRNA variants, types A and B, which were previously associated with environmental sources and clinical fatalities, respectively. Both assays could detect 10² to 10³ V. vulnificus total cells in seeded estuarine water and in oyster homogenates. PCR assays on 11 reference V. vulnificus strains and 22 nontarget species gave expected results (type A or B for V. vulnificus and negative for nontarget species). The relationship between cell number and cycle threshold for the assays was linear (R² = >0.93). The type A/B ratio of Florida clinical isolates was compared to that of isolates from oysters harvested in Florida waters. This ratio was 19:17 in clinical isolates and 5:8 (n = 26) in oysters harvested from restricted sites with poor water quality but was 10:1 (n = 22) in oysters from permitted sites with good water quality. A substantial percentage of isolates from oysters (19.4%) were type AB (both primer sets amplified), but no isolates from overlying waters were type AB. The real-time PCR assays were sensitive, specific, and quantitative in water samples and could also differentiate the strains in oysters without requiring isolation of V. vulnificus and may therefore be useful for rapid detection of the pathogen in shellfish and water, as well as further investigation of its population dynamics.     

Wastewater Treatment Effluent Reduces the Abundance and Diversity of Benthic Bacterial Communities in Urban and Suburban Rivers

In highly urbanized areas, wastewater treatment plant (WWTP) effluent can represent a significant component of freshwater ecosystems. As it is impossible for the composition of WWTP effluent to match the composition of the receiving system, the potential exists for effluent to significantly impact the chemical and biological characteristics of the receiving ecosystem. We assessed the impacts of WWTP effluent on the size, activity, and composition of benthic microbial communities by comparing two distinct field sites in the Chicago metropolitan region: a highly urbanized river receiving effluent from a large WWTP and a suburban river receiving effluent from a much smaller WWTP. At sites upstream of effluent input, the urban and suburban rivers differed significantly in chemical characteristics and in the composition of their sediment bacterial communities. Although effluent resulted in significant increases in inorganic nutrients in both rivers, surprisingly, it also resulted in significant decreases in the population size and diversity of sediment bacterial communities. Tag pyrosequencing of bacterial 16S rRNA genes revealed significant effects of effluent on sediment bacterial community composition in both rivers, including decreases in abundances of Deltaproteobacteria, Desulfococcus, Dechloromonas, and Chloroflexi sequences and increases in abundances of Nitrospirae and Sphingobacteriales sequences. The overall effect of the WWTP inputs was that the two rivers, which were distinct in chemical and biological properties upstream of the WWTPs, were almost indistinguishable downstream. These results suggest that WWTP effluent has the potential to reduce the natural variability that exists among river ecosystems and indicate that WWTP effluent may contribute to biotic homogenization.     

Mortality and herpesvirus infections of the Pacific oyster Crassostrea gigas in Tomales Bay, California, USA

Seed losses of Pacific oysters Crassostrea gigas have been associated with an ostreid herpesvirus-1 (OsHV-1) in Europe, and in 2002, a similar OsHV was detected in Tomales Bay, California, USA. In May of 2003, 5 stocks of seed Pacific oysters were planted at 2 sites (Inner Bay and Outer Bay) in Tomales Bay and monitored for mortality, presence/prevalence of OsHV (using polymerase chain reaction [PCR] and histology), and growth. Temperature (degrees C) and salinity data were collected every half an hour at each site. OsHV was detected at both the Inner and Outer Bay sites on the same sample date and mean temperature predicted OsHV presence (p < 0.005). High levels of mortality occurred 2 wk (Inner Bay site) and 4 wk (Outer Bay site) after OsHV detection. OsHV presence predicted mortality (p = 0.01). Temperature maximums and overall temperature exposure were greater at the Inner Bay site and may explain why mortality affected these oysters sooner than oysters planted at the Outer Bay site. Differences in cumulative mortality were significant among stocks (p < 0.0001), but not between sites (p > 0.05). OsHV prevalence was similar among stocks (p > 0.05) and between sites (p > 0.05). No evidence of herpesvirus-induced Cowdry type A nuclear inclusions or other pathogens were observed. Changes in tissue and cellular architecture including dilation of the digestive tubules and nuclear chromatin margination and pycnosis were observed in OsHV-infected oysters, consistent with previously observed OsHV infections. Stocks with smaller oysters had higher mortality rates than those with larger oysters; growth rate did not correlate with mortalities (p > 0.05). Taken together, these data suggest that the OsHV may cause or act in synergy with temperature to kill Pacific oyster seed in Tomales Bay, but further investigation of OsHV etiology in seed oysters is needed.     

Isolation of (S)-2,3-dichloro-1-propanol assimilating bacterium,its characterization,and its use in preparation of (R)-2,3-dichloro-1-propanol and (S)-epichlorohydrin

Summary Linear alkylbenzene sulfonate (LAS) is a widely used anionic surfactant. Although approximately 1 million metric tons of LAS are produced annually, relatively little is known about the bacteria or the genetic factors that control LAS degradation in the environment. The objectives of this research were to: i) compare bacterial populations in wastewater and pristine pond systems; ii) determine the frequency of plasmids in bacteria from these sites; and iii) compare the frequency of DNA sequences coding for aromatic catabolism in isolates from these two sites. Plate counts indicated that exposure to wastewater resulted in higher levels of both heterotrophic bacteria and bacteria capable of growing on LAS containing medium (LAS/YEPG). In addition to higher numbers, a higher proportion of heterotrophs from the wastewater system were capable of growth on LAS/YEPG medium. Thus, the high levels of LAS in the wastewater system apparently selected fro organisms that were able to tolerate and/or degrade, it. Mineralization of¹⁴C-ring labelled LAS in any habitat related to the presence of organisms that grew on LAS/YEPG. Although may of these isolates could carry out primary degradation, no isolate, could mineralize¹⁴C-ring LAS in pure culture. A higher incidence of plasmids was found in bacteria from the wastewater pond and among bacteria that grew on LAS containing medium. However, the presence of plasmid, DNA did not necessarily confer the ability to degrade LAS nor was the ability to degrade LAS dependent on the presence of a plasmid. The incidence of selected genotypes for aromatic catabolism was similar among isolates on LAS/YEPG at both sites, suggesting that LAS ring degradation may be present in other populations or encoded by alternative sequences. In conclusion, LAS mineralization is mediated by a consortium and the evidence that initial attack of LAS is plasmid mediated is inconclusive.     

Longitudinal study of antimicrobial resistance among Escherichia coli isolates from integrated multisite cohorts of humans and swine

In a 3-year longitudinal study, we examined the relationship between the seasonal prevalence of antimicrobial-resistant (AR) Escherichia coli isolates from human wastewater and swine fecal samples and the following risk factors: the host species, the production type (swine), the vocation (human swine workers, non-swine workers, and slaughter plant workers), and the season, in a multisite, vertically integrated swine and human population representative of a closed agri-food system. Human and swine E. coli (n = 4,048 and 3,429, respectively) isolates from wastewater and fecal samples were tested for antimicrobial susceptibility, using the Sensititre broth microdilution system. There were significant (P < 0.05) differences among AR E. coli prevalence levels of (i) the host species, in which swine isolates were at higher risk for resistance to tetracycline, kanamycin, ceftiofur, gentamicin, streptomycin, chloramphenicol, sulfisoxazole, and ampicillin; (ii) the swine production group, in which purchased boars, nursery piglets, and breeding boars isolates had a higher risk of resistance to streptomycin and tetracycline; and iii) the vocation cohorts, in which swine worker cohort isolates exhibited lower sulfisoxazole and cefoxitin prevalence than the non-swine worker cohorts, while the slaughter plant worker cohort isolates exhibited elevated cefoxitin prevalence compared to that of non-swine workers. While a high variability was observed among seasonal samples over the 3-year period, no significant temporal trends were apparent. There were significant differences in the prevalence levels of multidrug-resistant isolates between host species, with swine at a higher risk of carrying multidrug-resistant strains than humans. Considering vocation, slaughter plant workers were at higher risk of exhibiting multidrug-resistant E. coli than non-swine workers.     

Environmental impact assessment of centralized municipal wastewater management in Thailand

Purpose

Urbanization and industrial development intensify water utilization and wastewater generation. The efficiency of wastewater treatment systems varies and depends on system design and wastewater condition. The research aims to examine seven existing centralized municipal wastewater treatment plants (WWTPs) in Bangkok to discover which system configuration yields the best environmental and economic performance. The degree of environmental impact and operational costs from different system designs were investigated to help select future wastewater treatment systems.

Methods

Life cycle assessment (LCA) has been conducted to evaluate environmental impacts from centralized municipal wastewater treatment systems. Life cycle impact assessment method based on endpoint modeling (LIME) was applied, with three major potential environmental impact categories including eutrophication, global warming, and acidification. All seven centralized municipal WWTPs in Bangkok were investigated as case studies. The system configurations are classified into five types of activated sludge (AS) systems. The contribution of impacts from individual processes in each type of AS system was analyzed. The methodology covered major on-site and off-site operational processes excluding construction and maintenance phases. Average annual data were calculated to develop an inventory dataset. JEMAI-Pro software was utilized in this study to analyze the life cycle impact of the systems.

Results and discussion

The level of environmental impact from a WWTP depends on the configuration of the AS system. The highest potential environmental impact from a municipal WWTP is eutrophication, which is obviously affected by ammonium and phosphorous discharges into water bodies. The vertical loop reactor activated sludge (VLRAS) system yielded the best treatment performance among the five AS sub-systems. The consumption of electricity used to operate the system contributed significantly to global warming potential and correlated considerably with operating costs. Comparing among three system sizes, the large-scale WWTP revealed inefficient electricity consumption, whereas the medium plant provided better performance in chemical use and operating costs.

Conclusions

Centralized municipal WWTPs with capacities ranging from 10 to 350?×?10³ m³/day were evaluated with respect to environmental performance and costs during the operating phase. Among all case studies, a medium-scale WWTP with a VLRAS system offered the best operating performance in terms of low environmental impact, resource consumption, and cost. To enhance WWTP management, it is vital to improve the efficiency of electricity consumption in primary and secondary treatment processes and increase wastewater collection efficiency to maximize the plant operating capacity and minimize overall environmental impacts.

     

Moderate acidification affects growth but not survival of 6-month-old oysters

Oyster populations periodically exposed to runoff from acid sulfate soils (ASS) are of depressed abundance and have fewer smaller individuals than unaffected populations, despite having similar recruitment levels to unaffected sites during dry periods. We examined how the timing and duration of exposure to ASS runoff influences the growth and survival of successfully settled oysters. We predicted that among 6-month-old oysters, growth and survival would be (1) lower among individuals continuously exposed to ASS-acidified waters than those that are episodically exposed, and (2) most negatively affected during rainfall events, which enhance transport of ASS runoff to estuaries. Six-month-old Sydney rock oysters, Saccostrea glomerata, were deployed at ASS-affected and unaffected sites within each of two south-east Australian estuaries. After 10 weeks, oysters were transplanted within and across sites in an estuary and maintained in situ for another 10 weeks. Oysters that remained for 20 weeks at ASS-affected sites grew at just over half the rate of oysters at reference sites. Oysters transplanted from acidified to reference sites grew more than oysters transplanted from reference to acidified sites or oysters that remained at reference sites. Unexpectedly, overall oyster mortality was low. Greater rainfall, and hence a lower pH, is likely to have accounted for the greater impact of acidification on growth during the second 10 weeks. Where oysters recruit to a 6-month age cohort, they may be able to tolerate subsequent, moderate, acidification events. Reduced growth during acidification periods may be offset by positive growth during intervening dry periods.     

Intraspecific diversity of Vibrio vulnificus in Galveston Bay water and oysters as determined by randomly amplified polymorphic DNA PCR

Randomly amplified polymorphic DNA (RAPD) PCR was used to analyze the temporal and spatial intraspecific diversity of 208 Vibrio vulnificus strains isolated from Galveston Bay water and oysters at five different sites between June 2000 and June 2001. V. vulnificus was not detected during the winter months (December through February). The densities of V. vulnificus in water and oysters were positively correlated with water temperature. Cluster analysis of RAPD PCR profiles of the 208 V. vulnificus isolates revealed a high level of intraspecific diversity among the strains. No correlation was found between the intraspecific diversity among the isolates and sampling site or source of isolation. After not being detected during the winter months, the genetic diversity of V. vulnificus strains first isolated in March was 0.9167. Beginning in April, a higher level of intraspecific diversity (0.9933) and a major shift in population structure were observed among V. vulnificus isolates. These results suggest that a great genetic diversity of V. vulnificus strains exists in Galveston Bay water and oysters and that the population structure of this species is linked to changes in environmental conditions, especially temperature.     

Antibiotic susceptibility profiles of some <Emphasis Type="Italic">Vibrio</Emphasis> strains isolated from wastewater final effluents in a rural community of the Eastern Cape Province of South Africa

Background  

To evaluate the antibiogram and antibiotic resistance genes of some Vibrio strains isolated from wastewater final effluents in a rural community of South Africa. V. vulnificus (18), V. metschnikovii (3), V. fluvialis (19) and V. parahaemolyticus (12) strains were isolated from final effluents of a wastewater treatment plant (WWTP) located in a rural community of South Africa. The disk diffusion method was used for the characterization of the antibiogram of the isolates. Polymerase chain reaction (PCR) was employed to evaluate the presence of established antibiotic resistance genes using specific primer sets.     

Response of Estuarine Biofilm Microbial Community Development to Changes in Dissolved Oxygen and Nutrient Concentrations

The information content and responsiveness of microbial biofilm community structure, as an integrative indicator of water quality, was assessed against short-term changes in oxygen and nutrient loading in an open-water estuarine setting. Biofilms were grown for 7-day periods on artificial substrates in the Pensacola Bay estuary, Florida, in the vicinity of a wastewater treatment plant (WWTP) outfall and a nearby reference site. Substrates were deployed floating at the surface and near the benthos in 5.4 m of water. Three sampling events covered a 1-month period coincident with declining seasonal WWTP flow and increasing dissolved oxygen (DO) levels in the bottom waters. Biomass accumulation in benthic biofilms appeared to be controlled by oxygen rather than nutrients. The overriding effect of DO was also seen in DNA fingerprints of community structure by terminal restriction fragment length polymorphism (T-RFLP) of amplified 16S rRNA genes. Ribotype diversity in benthic biofilms at both sites dramatically increased during the transition from hypoxic to normoxic. Terminal restriction fragment length polymorphism patterns showed pronounced differences between benthic and surface biofilm communities from the same site in terms of signal type, strength, and diversity, but minor differences between sites. Sequencing of 16S rRNA gene clone libraries from benthic biofilms at the WWTP site suggested that low DO levels favored sulfate-reducing prokaryotes (SRP), which decreased with rising oxygen levels and increasing overall diversity. A 91-bp ribotype in the CfoI-restricted 16S rRNA gene T-RFLP profiles, indicative of SRP, tracked the decrease in relative SRP abundance over time.     

Comparison of Methods to Identify Pathogens and Associated Virulence Functional Genes in Biosolids from Two Different Wastewater Treatment Facilities in Canada

The use of treated municipal wastewater residues (biosolids) as fertilizers is an attractive, inexpensive option for growers and farmers. Various regulatory bodies typically employ indicator organisms (fecal coliforms, E. coli and Salmonella) to assess the adequacy and efficiency of the wastewater treatment process in reducing pathogen loads in the final product. Molecular detection approaches can offer some advantages over culture-based methods as they can simultaneously detect a wider microbial species range, including non-cultivable microorganisms. However, they cannot directly assess the viability of the pathogens. Here, we used bacterial enumeration methods together with molecular methods including qPCR, 16S rRNA and cpn60 gene amplicon sequencing and shotgun metagenomic sequencing to compare pre- and post-treatment biosolids from two Canadian wastewater treatment plants (WWTPs). Our results show that an anaerobic digestion WWTP was unsuccessful at reducing the live indicator organism load (coliforms, generic E. coli and Salmonella) below acceptable regulatory criteria, while biosolids from a dewatering/pelletization WWTP met these criteria. DNA from other pathogens was detected by the molecular methods, but these species were considered less abundant. Clostridium DNA increased significantly following anaerobic digestion treatments. In addition to pathogen DNA, genes related to virulence and antibiotic resistance were identified in treated biosolids. Shotgun metagenomics revealed the widest range of pathogen DNA and, among the approaches used here, was the only approach that could access functional gene information in treated biosolids. Overall, our results highlight the potential usefulness of amplicon sequencing and shotgun metagenomics as complementary screening methods that could be used in parallel with culture-based methods, although more detailed comparisons across a wider range of sites would be needed.