Accumulation of antibiotics in the environment: Have appropriate measures been taken to protect Canadian human and ecological health?

In Canada, every day, contaminants of emerging concern (CEC) are discharged from waste treatment facilities into freshwaters. CECs such as pharmaceutical active compounds (PhACs), personal care products (PCPs), per- and polyfluoroalkyl substances (PFAS), and microplastics are legally discharged from sewage treatment plants (STPs), water reclamation plants (WRPs), hospital wastewater treatment plants (HWWTPs), or other forms of wastewater treatment facilities (WWTFs). In 2006, the Government of Canada established the Chemicals Management Plan (CMP) to classify chemicals based on a risk-priority assessment, which ranked many CECs such as PhACs as being of low urgency, therefore permitting these substances to continue being released into the environment at unmonitored rates. The problem with ranking PhACs as a low priority is that CMP's risk management assessment overlooks the long-term environmental and synergistic effects of PhAC accumulation, such as the long-term risk of antibiotic CEC accumulation in the spread of antibiotic resistance genes. The goal of this review is to specifically investigate antibiotic CEC accumulation and associated environmental risks to human and environmental health, as well as to determine whether appropriate legislative strategies are in place within Canada's governance framework. In this research, secondary data on antibiotic CEC levels in Canadian and international wastewaters, their potential to promote antibiotic-resistant residues, associated environmental short- and long-term risks, and synergistic effects were all considered. Unlike similar past reviews, this review employed an interdisciplinary approach to propose new strategies from the perspectives of science, engineering, and law.

Widespread detection of antibiotic-resistant bacteria from natural aquatic environments in southern Ontario.

To elucidate how widespread antibiotic resistance is in the surface water environment, we studied the prevalence of antibiotic resistance bacteria at four locations in southern Ontario. We found that the percentage of bacteria resistant to the antibiotic tetracycline was higher at the river site, which flows through agricultural land, and lower at the lake sites. A total of 225 colonies were selected for further testing of antibiotic disc susceptibility to eight different antibiotics to calculate the multiple antibiotic resistance (MAR) score and the antibiotic resistance index for each site. Although the isolates from the lake site outside the city displayed resistance to fewer antibiotics, their MAR scores were not significantly different from that of the lake sites adjacent to urban beaches, showing that MAR was widespread in the natural water environments tested. Isolation of colonies under selection pressure to tetracycline was found to have a significant effect on the likelihood that the isolates would contain multiple resistance traits for other antibiotics. Identification of isolates selected on tetracycline was compared with that of isolates that were sensitive to tetracycline, and the community composition was found to be distinctly different, although isolates from the genera Chryseobacterium, Pseudomonas, and Stenotrophomonas were found in both communities.

Functional relationship between ammonia-oxidizing bacteria and ammonia-oxidizing archaea populations in the secondary treatment system of a full-scale municipal wastewater treatment plant.

The abundance of ammonia-oxidizing bacteria and archaea and their amoA genes from the aerobic activated sludge tanks, recycled sludge and anaerobic digesters of a full-scale wastewater treatment plant (WWTP) was determined. Polymerase chain reaction and denaturing gradient gel electrophoresis were used to generate diversity profiles, which showed that each population had a consistent profile although the abundance of individual members varied. In the aerobic tanks, the ammonia-oxidizing bacterial (AOB) population was more than 350 times more abundant than the ammonia-oxidizing archaeal (AOA) population, however in the digesters, the AOA population was more than 10 times more abundant. Measuring the activity of the amoA gene expression of the two populations using RT-PCR also showed that the AOA amoA gene was more active in the digesters than in the activated sludge tanks. Using batch reactors and ddPCR, amoA activity could be measured and it was found that when the AOB amoA activity was inhibited in the anoxic reactors, the expression of the AOA amoA gene increased fourfold. This suggests that these two populations may have a cooperative relationship for the oxidation of ammonia.

Wastewater surveillance for COVID-19 in shelters: A creative strategy for a complex setting.

People experiencing homelessness experience disproportionate rates of morbidity and mortality from coronavirus disease 2019 (COVID-19) compared to the general population and shelters for people experiencing homelessness are a major contributing factor to these negative outcomes. As a result of their unique structure, population and physical space, these settings pose several challenges to the prevention of COVID-19 infection that are not adequately addressed by conventional non-pharmaceutical public health interventions. Wastewater surveillance for COVID-19 is a viable strategy for health protection in shelters due to its ability to meet these unique challenges. Its passive nature does not depend on individual health-seeking behaviours, and it can provide useful epidemiological information early on in an outbreak setting. In this commentary, the authors examine a recent application of wastewater surveillance of COVID-19 in a men's shelter in Toronto. Further applications of wastewater surveillance for other infectious diseases of concern in shelters are proposed, and the need for the development of ethical frameworks governing the use of this technology is discussed.

SARS-CoV-2 viral titer measurements in Ontario, Canada wastewaters throughout the COVID-19 pandemic.

During the COVID-19 pandemic, the Province of Ontario, Canada, launched a wastewater surveillance program to monitor SARS-CoV-2, inspired by the early work and successful forecasts of COVID-19 waves in the city of Ottawa, Ontario. This manuscript presents a dataset from January 1, 2021, to March 31, 2023, with RT-qPCR results for SARS-CoV-2 genes and PMMoV from 107 sites across all 34 public health units in Ontario, covering 72% of the province's and 26.2% of Canada's population. Sampling occurred 2-7 times weekly, including geographical coordinates, serviced populations, physico-chemical water characteristics, and flowrates. In doing so, this manuscript ensures data availability and metadata preservation to support future research and epidemic preparedness through detailed analyses and modeling. The dataset has been crucial for public health in tracking disease locally, especially with the rise of the Omicron variant and the decline in clinical testing, highlighting wastewater-based surveillance's role in estimating disease incidence in Ontario.

Impact of perfluorooctanoic acid (PFOA) and perfluorooctane sulfonic acid (PFOS) on secondary sludge microorganisms: removal, potential toxicity, and their implications on existing wastewater treatment regulations in Canada.

Perfluorooctanoic acid (PFOA) and perfluorooctane sulfonic acid (PFOS) are two of the most commonly researched per- and polyfluoroalkyl substances (PFAS). Globally, many long-chain PFAS compounds including PFOS and PFOA are highly regulated and, in some countries, PFAS use in commercial products is strictly prohibited. Despite the legal regulation of these 'forever chemicals' under the Canadian Environmental Protection Act, PFOA and PFOS compounds are still found in high concentrations in discharges from wastewater treatment plants, both from liquid and sludge streams. Yet, their potential impact on wastewater treatment effectiveness remains poorly understood. The findings of this research show that: (1) PFOS and PFOA might be hindering the overall outcome treatment performance - calling into question the efficacy of Canada's existing wastewater treatment regulatory standard (Wastewater Systems Effluent Regulations, SOR/2012-139), and (2) specific microorganisms from the Thiobacillus and Pseudomonas genera seem capable of adsorbing PFOS and PFOA onto their cell wall and even degrading the chemicals, but it is unclear as to what extent degradation occurs. The results also raise questions whether existing wastewater regulations should be expanded to include the detection and monitoring of PFAS, as well as the establishment of a regulatory wastewater treatment plant discharge standard for PFAS that is protective of human and ecological health.

Early identification of a COVID-19 outbreak detected by wastewater surveillance at a large homeless shelter in Toronto, Ontario.

SETTING: Toronto (Ontario, Canada) is a large urban centre with a significant population of underhoused residents and several dozen shelters for this population with known medical and social vulnerabilities. A sizeable men's homeless shelter piloted a facility-level SARS-CoV-2 wastewater surveillance program. INTERVENTION: Wastewater surveillance was initiated at the shelter in January 2021. One-hour composite wastewater samples were collected twice weekly from a terminal sanitary clean-out pipe. The genetic material of the SARS-CoV-2 virus was extracted from the solid phase of each sample and analyzed using real-time qPCR to estimate the viral level. Wastewater results were reported to facility managers and Toronto Public Health within 4 days. OUTCOMES: There were 169 clients on-site at the time of the investigation. Wastewater surveillance alerted to the presence of COVID-19 activity at the site, prior to clinical detection. This notification acted as an early warning signal, which allowed for timely symptom screening and case finding for shelter managers and the local health unit, in preparation for the declaration of an outbreak. IMPLICATIONS: Wastewater surveillance acted as an advanced notification leading to the timely deployment of enhanced testing prior to clinical presentation in a population with known vulnerabilities. Wastewater surveillance at the facility level is beneficial, particularly in high-risk congregate living settings such as shelters that house transient populations where clinical testing and vaccination can be challenging. Open communication, established individual facility response plans, and a balanced threshold for action are essential to an effective wastewater surveillance program.

RéSUMé: LIEU: Toronto (Ontario, Canada) est un grand centre urbain qui compte une importante population de résidents mal logés et plusieurs douzaines de refuges pour cette population aux vulnérabilités médicales et sociales connues. Un assez gros refuge pour hommes sans-abri a mis à l'essai dans ses installations un programme de surveillance des eaux usées pour le SRAS-CoV-2. INTERVENTION: La surveillance des eaux usées du refuge a commencé en janvier 2021. Des échantillons composites d'une heure ont été prélevés deux fois par semaine à partir d'un regard de nettoyage à l'extrémité du drain sanitaire. Le matériel génétique du virus du SRAS-CoV-2 a été extrait du support solide de chaque échantillon et analysé par PCR quantitative en temps réel pour estimer le niveau du virus. Les résultats des eaux usées ont été déclarés aux gestionnaires du refuge et à Santé publique Toronto dans un délai de quatre jours. RéSULTATS: Il y avait 169 usagers sur place au moment de l'enquête. La surveillance des eaux usées a averti de la présence d'une activité de la COVID-19 sur les lieux avant sa détection clinique. Cet avertissement a servi de signal d'alerte précoce, ce qui a permis aux gestionnaires du refuge et au bureau de santé local de procéder au dépistage rapide des symptômes et à la recherche des cas en préparation pour la déclaration d'une éclosion. CONSéQUENCES: La surveillance des eaux usées a servi de notification préalable et entraîné le déploiement opportun d'un dépistage complémentaire avant la manifestation clinique dans une population qui présente des vulnérabilités connues. La surveillance des eaux usées d'une installation est avantageuse, surtout dans des milieux d'hébergement collectif à risque élevé comme les refuges qui hébergent des populations de passage, où le dépistage clinique et la vaccination peuvent être difficiles. Une communication ouverte, des plans d'intervention établis pour chaque installation et un seuil d'intervention équilibré sont essentiels à l'efficacité d'un programme de surveillance des eaux usées.

From individual response to population ecology: Environmental factors restricting survival of vegetative bacteria at solid-air interfaces.

Combating microbial survival on dry surfaces contributes to improving public health in indoor environments (clinical and industrial settings) and extends to the natural environment. For vegetative bacteria at solid-air interfaces, lack of water impacts cellular response, and acclimation depends on community support in response to ecological processes. Gaining insights about important ecological processes leading to inhibition of microbial survival under extreme conditions, such as vicinity of highly radioactive nuclear waste, is key for improving engineering designs. Canada plans to store used nuclear fuel and radioactive waste in a deep geological repository (DGR) with a multiple-barrier system constructed at an approximate depth of 500 m. Microorganisms in highly compacted bentonite surrounding used fuel containers will be challenged by high pressure, temperature, and radiation, as well as limited water and nutrients. Thus, it is difficult to estimate microbial activities, given that the prime concern for a microbial community is survival, and energy expenditure is regulated. To enable preventive measures and for risk evaluation, a deeper understanding of community-based survival strategies of bacterial cells exposed to air (gaseous phase) during prolonged periods of desiccation is required. An in-depth review of collective studies that assess microbial survival and persistence during desiccation is presented here to augment and direct our prior knowledge about tactics used by bacteria for survival at interfaces in hostile natural environments including and similar to a DGR.

The utility and application of real-time PCR and FISH in the detection of single-copy gene targets in Escherichia coli O157:H7 and Salmonella Typhimurium.

The ultimate specificity in molecular-based assays for pathogen detection relies on the design of the primers and probes. Their ability to hybridize to DNA sequences found only in pathogens can be realized by designing primers and probes that are complementary to pathogen-specific virulence genes. This study evaluates the detection and enumeration strengths of real-time PCR (qPCR) and fluorescent in situ hybridization (FISH) for selected waterborne pathogens and their ultimate applicability within a monitoring framework. Detection limits calculated in the qPCR assay were 150 tir (intimin protein receptor) gene copies for Escherichia coli O157:H7 and 2 x 103 invA (inner membrane invasive protein) gene copies for Salmonella enterica serovar Typhimurium. Detection limits were, however, at least 100-fold less sensitive in wastewater extracts, partly because of the inhibitory effect of the wastewater itself. Fluorescent signals from hybridized whole target cells were below the detection limit of the FISH assay. While this research demonstrates the potential detection strength of qPCR, it highlights the need for strong dependable primer and probe sets among PCR and FISH methodologies as well as the need for further signal amplification with DNA-targeted FISH for single-copy gene targets within environmental samples.

A closer look at the antibiotic-resistant bacterial community found in urban wastewater treatment systems.

The conventional biological treatment process can provide a favorable environment for the maintenance and dissemination of antibiotic-resistant bacteria and the antibiotic resistance genes (ARG) they carry. This study investigated the occurrence of antibiotic resistance in three wastewater treatment plants (WWTP) to determine the role they play in the dissemination of ARGs. Bacterial isolates resistant to tetracycline were collected, and tested against eight antibiotics to determine their resistance profiles and the prevalence of multiple antibiotic resistance. It was found that bacteria resistant to tetracycline were more likely to display resistance to multiple antibiotics compared to those isolates that were not tetracycline resistant. Polymerase chain reaction (PCR) was used to identify the tetracycline resistance determinants present within the bacterial communities of the WWTPs and receiving waters, and it was found that ARGs may not be released from the treatment process. Identification of isolates showed that there was a large diversity of species in both the tetracycline-resistant and tetracycline-sensitive populations and that the two groups were significantly different in composition. Antibiotic resistance profiles of each population showed that a large diversity of resistance patterns existed within genera suggesting that transmission of ARG may progress by both horizontal gene and vertical proliferation.

Utilization of multiple organisms in a proposed early-warning biomonitoring system for real-time detection of contaminants: preliminary results and modeling.

During past decades, biomonitors were deployed in lakes and rivers to rapidly detect hazardous chemicals by measuring the endpoints of a single aquatic species at defined short intervals. Most biomonitors, however, are only capable of indicating a departure from baseline water conditions without identifying the cause. In order to provide a more comprehensive assessment, a biomonitoring system which features a library of stereotyped responses of multiple aquatic species in various water conditions is proposed. A preliminary library was constructed by characterizing the behavioural and physiological responses of Daphnia magna, Hyalella azteca, Lumbriculus variegatus, and Pseudokirchneriella subcapitata to various concentrations of atrazine and tributyltin. By employing multivariate statistical tools such as principal component analysis (PCA) and discriminant analysis, this library (which contained responses after 6h of exposure to contaminants) was used as a template to classify and to model other sets of earlier measurements at 2 and 4h, resulting in an accuracy of 73 and 97%, respectively. These findings demonstrated the potential capability of the proposed early-warning biomonitoring system to provide real-time water quality assessment and early-warning contaminant detection.

Detection of melamine and cyanuric acid in vegetable protein products used in food production.

The multitude of food recalls in 2007 clearly demonstrated that total nitrogen-content (ΣN) determination by means of near-infrared spectroscopy (NIRS) and Kjeldahl-based measurements can be deceived, and should no longer be regarded as a complete quality assurance program for nutritive-protein evaluations. Furthermore, contemporary Canadian-employed analytical tools are precariously limited in their ability to effectively assure a product where there is no a priori knowledge of the environmental toxin(s) involved. In light of these challenges, this study explored a number of analytical techniques used to assess and furthermore assure the quality of vegetable protein products (VPPs). Using liquid chromatography with tandem mass spectrometry (LC/MS/MS) technologies, a combination of VPP-based samples was analyzed for the presence of nitrogen-bearing environmental toxicants. Of the 52 samples tested, involving an assortment of matrices, melamine and cyanuric acid were positively identified (>1 ng/mL) in 22 and 17 samples, respectively. Subsequent high pressure liquid chromatography with ultraviolet/visible (HPLC-UV) amino acid profiling further confirmed the adulteration of those materials contaminated with melamine and melamine-related compounds. Based on the evidence presented herein, LC/MS/MS in combination with HPLC-UV provides for a reliable food safety detection system as applied to VPPs. Moreover, HPLC-UV is indispensable as a stand-alone 1st level of screening to assess the integrity of a VPP or any nutritive protein-based sample.

Evaluation of low-copy genetic targets for waterborne bacterial pathogen detection via qPCR.

Recent developments in water quality research have highlighted difficulties in accurately predicting the incidence of pathogens within freshwater based on the viability, culturability and metabolic activity of indicator organisms. QPCR-driven assays are candidates to replace standard culture-based methods, however, protocols suitable for routine use have yet to be sufficiently validated. The objective of this study was to evaluate five oligonucleotide primers sets (ETIR, SINV, exoT, VS1 and ipaH2) for their potential applicability in qPCR assays to detect contamination from five waterborne bacterial pathogens (Escherichia coli O157:H7, Salmonella Typhimurium, Campylobacter jejuni, Pseudomonas aeruginosa, and Shigella flexneri). An enrichment-free qPCR protocol was also tested using S. Typhimurium-seeded source water, combining membrane filtration and mechanical, chemical and enzymatic lysis techniques to recover the bacterial cells. All five primer sets were found to have high specificity and sensitivity for the tested organisms. Four of the primers were able to detect pathogen loads as low as 10 cells/mL while 200 cells/mL of C. jejuni were detectable in pure culture. Although sensitivity decreased in an artificially contaminated environmental matrix, it was still possible to detect as few as 10 S. Typhimurium cells without enrichment. The primers and protocols evaluated in this study have demonstrated potential for further validation for possible application alongside traditional indicator techniques.

Impact of protozoan grazing on nitrification and the ammonia- and nitrite-oxidizing bacterial communities in activated sludge.

In activated sludge, protozoa feed on free-swimming bacteria and suspended particles, inducing flocculation and increasing the turnover rate of nutrients. In this study, the effect of protozoan grazing on nitrification rates under various conditions in municipal activated sludge batch reactors was examined, as was the spatial distribution of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) within the activated sludge. The reactors were monitored for ammonia, nitrite, nitrate, and total nitrogen concentrations, and bacterial numbers in the presence and absence of cycloheximide (a protozoan inhibitor), allylthiourea (an inhibitor of ammonia oxidation), and EDTA (a deflocculating agent). The accumulations of nitrate, nitrite, and ammonia were lower in batches without than with protozoa grazing. Inhibition of ammonia oxidation also decreased the amount of nitrite and nitrate accumulation. Inhibiting protozoan grazing along with ammonia oxidation further decreased the amounts of nitrite and nitrate accumulated. Induction of deflocculation led to high nitrate accumulation, indicating high levels of nitrification; this effect was lessened in the absence of protozoan grazing. Using fluorescent in situ hybridization and confocal laser scanning microscopy, AOB and NOB were found clustered within the floc, and inhibiting the protozoa, inhibiting ammonia oxidation, or inducing flocculation did not appear to lower the number of AOB and NOB present or affect their position within the floc. These results suggest that the AOB and NOB are present but less active in the absence of protozoa.

Nitrification in activated sludge batch reactors is linked to protozoan grazing of the bacterial population.

Protozoa feed upon free-swimming bacteria and suspended particles inducing flocculation and increasing the turnover rate of nutrients in complex mixed communities. In this study, the effect of protozoan grazing on nitrification was examined in activated sludge in batch cultures maintained over a 14-day period. A reduction in the protozoan grazing pressure was accomplished by using either a dilution series or the protozoan inhibitor cycloheximide. As the dilutions increased, the nitrification rate showed a decline, suggesting that a reduction in protozoan or bacterial concentration may cause a decrease in nitrification potential. In the presence of cycloheximide, where the bacterial concentration was not altered, the rates of production of ammonia, nitrite, and nitrate all were significantly lower in the absence of active protozoans. These results suggest that a reduction in the number or activity of the protozoans reduces nitrification, possibly by limiting the availability of nutrients for slow-growing ammonia and nitrite oxidizers through excretion products. Furthermore, the ability of protozoans to groom the heterotrophic bacterial population in such systems may also play a role in reducing interspecies competition for nitrification substrates and thereby augment nitrification rates.

A survey of the composition and diversity of bacterial populations in bleached kraft pulp-mill wastewater secondary treatment systems.

Bacterial community compositions from 10 pulp- and paper-mill treatment systems were compared using both traditional and molecular techniques. 16S-RFLP (Random Fragment Length Polymorphisms) analysis was used to examine the genotypic profiles of the whole bacterial community of each treatment system. Although all the communities shared approximately 60% of their DNA band pattern, as determined by computer-assisted cluster analysis, each community displayed a unique profile that was stable over time under normal operating parameters. Reverse Sample Genome Probing (RSGP) and 16S-RFLP were used to compare the culturable bacterial communities of several geographically separated pulp-mill biotreatment system communities. There was little overlap in the composition of the culturable community between mills at the genus level. Furthermore, RSGP variation was almost as high within a mill as between mills. Partial sequences of the 16S rRNA genes from culturable isolates identified Bacillus spp., Pseudomonas spp., and Xanthobacter as some of the dominant species. Finally, several 16S rRNA genes from two whole community 16S RNA gene libraries were partially sequenced and identified as similar to unknown alpha-, beta-, and gamma-Proteobacteria, Ralstonia, Alcaligenes, Nitrospira, Firmicutes, and clones representing the new Holophaga/Acidobacterium phylum. These findings suggest that although these pulp- and paper-mill biotreatment communities perform similar functions, they are populated by unique mixtures of species.