Filling the Knowledge Gap Regarding Microbial Occupational Exposure Assessment in Waste Water Treatment Plants: A Scoping Review

Background: Wastewater treatment plants (WWTPs) are crucial in the scope of European Commission circular economy implementation. However, bioaerosol production may be a hazard for occupational and public health. A scoping review regarding microbial contamination exposure assessment in WWTPs was performed. Methods: This study was performed through PRISMA methodology in PubMed, Scopus and Web of Science. Results: 28 papers were selected for data extraction. The WWTPs’ most common sampled sites are the aeration tank (42.86%), sludge dewatering basin (21.43%) and grit chamber. Air sampling is the preferred sampling technique and culture-based methods were the most frequently employed assays. Staphylococcus sp. (21.43%), Bacillus sp. (7.14%), Clostridium sp. (3.57%), Escherichia sp. (7.14%) and Legionella sp. (3.57%) were the most isolated bacteria and Aspergillus sp. (17.86%), Cladosporium sp. (10.71%) and Alternaria sp. (10.71%) dominated the fungal presence. Conclusions: This study allowed the identification of the following needs: (a) common protocol from the field (sampling campaign) to the lab (assays to employ); (b) standardized contextual information to be retrieved allowing a proper risk control and management; (c) the selection of the most suitable microbial targets to serve as indicators of harmful microbial exposure. Filling these gaps with further studies will help to provide robust science to policy makers and stakeholders.


Introduction
The European Commission (EC) strongly recommends circular economy implementation aiming at a zero-waste strategy, by instigating water innovations technologies for water reuse and recycling [1].In this scope, wastewater treatment plants (WWTPs) are designed to maximize energy and water recovery, becoming of pivotal importance for the achievement of the EC's goals [2].
On WWTPs, the wastewater of domestic, hospital and industrial uses undergoes preliminary, primary, secondary, and in some cases, tertiary biological treatments [3,4].During these treatments, bioaerosol formation is higher throughout discharging, mixing and aerating processes, as well as during the spraying of sewage [3][4][5][6].The bioaerosols contain microorganisms, such as fungi, viruses, bacteria, and their metabolites, including endotoxins and mycotoxins, which may be potentially pathogenic to humans.Infection can occur through ingestion, dermal contact, or inhalation, and it is highly possible that due to prolonged exposure, a decline in the health status of WWTPs workers may be observed [5][6][7].In fact, several negative health outcomes associated with bioaerosol occupational exposure have been reported, including respiratory and gastrointestinal effects or allergies [4,6].In addition, WWTPs are recognized as key emission sources for the discharge of antimicrobial-resistant (AMR) bacteria and antibacterial resistance genes (ARGs) [8].
Although it is crucial to assess occupational exposure to bioaerosols in WWTPs, there is a lack of consensus regarding sampling approaches and analyses that should be performed, as well as the targets that can be used as surrogates to identify harmful microbial contamination, which is a common problem in settings where (micro)biologic agents need to be assessed.However, suggestions regarding the procedures to be employed from the field to lab have already been described in different occupational environments [9][10][11].Thus, this study aims to perform a scoping review to provide a broad overview of the stateof-the-art methods (sampling and analyses) applied to perform microbial contamination assessments in WWTPs, as well as to identify the most suitable targets to be used as indicators of hazardous microbial contamination.

Quality Assessment
The assessment of the risk of bias was performed by 4 investigators (BR, MR, LM, and CV).Within each research article, an evaluation of the risk of bias was performed across two parameters divided as key criteria ("Sampling methods" and "Assays").Each parameter's risk of bias was rated as "low" "medium" "high", or "not applicable".The studies for which all the key criteria and most of the other criteria were characterized as "high" were removed.

Results
The workflow illustrated in Figure 1 was used for selecting studies.Initially, 191 studies were found in the database search, from which 105 abstracts were analyzed, and 40 complete texts were deemed eligible for further examination.A total of 12 papers were rejected for not satisfying the inclusion and exclusion criteria, mostly because they did not have any information regarding microbial occupational exposure in WWTPs.Overall, the selection process yielded 28 studies on microbiologic contamination occupational exposure assessment.
Microorganisms 2024, 12, x FOR PEER REVIEW 3 of 33 while another (MR) examined the results.The data that follows were manually extracted: Database, Title, Country, Type of WWTP, Sampling Strategies and Methods, Assays applied, Main Findings, and References.

Quality Assessment
The assessment of the risk of bias was performed by 4 investigators (BR, MR, LM, and CV).Within each research article, an evaluation of the risk of bias was performed across two parameters divided as key criteria ("Sampling methods" and "Assays").Each parameter's risk of bias was rated as "low" "medium" "high", or "not applicable".The studies for which all the key criteria and most of the other criteria were characterized as "high" were removed.

Results
The workflow illustrated in Figure 1 was used for selecting studies.Initially, 191 studies were found in the database search, from which 105 abstracts were analyzed, and 40 complete texts were deemed eligible for further examination.A total of 12 papers were rejected for not satisfying the inclusion and exclusion criteria, mostly because they did not have any information regarding microbial occupational exposure in WWTPs.Overall, the selection process yielded 28 studies on microbiologic contamination occupational exposure assessment.

Extracted Data
After the selection of the 28 studies on microbiologic contamination occupational exposure assessment, the relevant data were extracted; the key findings are summarized in Table 2.

Culture-based methods
A high fecal coliform concentration was observed in the WWTPs.Enteric viruses were also detected, peaking in summer/autumn.There was a high risk for farmers (EV infection and disease burden) and risk for lettuce consumers, exceeding WHO guidelines.

Discussion
WWTPs are crucial for the implementation of the zero-waste strategy which is in the scope of the EC's circular economy management.Interestingly, the geographical distribution of the analyzed studies corroborated the urge for tackling WWTPs' pollution threat and to answer to the determined environmental goals worldwide.In agreement with previous reviews held in different settings, such as poultries [9] and sawmills [10], no standardization was observed in the sampling campaigns performed, as well as in the assays employed.Furthermore, the lack of standardized contextual information retrieved through the developed studies hinders the possibility to identify the environmental variables that contribute effectively to the occupational exposure assessment, as well as to propose suitable recommendations to avoid microbial exposure and dissemination [38].In fact, the contextual information (e.g., implemented occupational health measures, training on safety issues related to the working tasks, cleaning practices, ventilation conditions, number of workers in each workstation, protection devices used by workers), when retrieved, should allow the identification of the most critical scenario and, thus, the selection of proper sampling sites following the "worst case scenario" approach as a first step for exposure assessment.In those sampling sites considered as the most critical, besides the environmental sampling campaign, nasopharyngeal swabs should be collected from the workers' nose to obtain additional information regarding workers' exposure.In previous studies, nasopharyngeal swabs were also taken to assess MRSA prevalence in workers from different occupational settings [39] or to corroborate the predominant fungi present in the Portuguese cork industry and, more specifically, exposure to Penicillium section Aspergilloides [40].In addition, this approach can help occupational health services to prioritize multiple interventions in workers' education or even in personal protection device (e.g., gloves, respiratory protection devices) selection and replacement frequency.
The assessment of microbial dynamics in WWTPs is critical for ensuring public health and environmental safety.Seasonal evaluation plays a crucial role in this assessment, particularly given the influence of global warming and human activities, such as intensive agriculture, on microbial ecology [41,42].In fact, recent studies [43,44] suggest that these factors contribute to the emergence of new fungal species, underscoring the need for comprehensive monitoring strategies.Recognizing the prevalence of research in specific regions and climatic periods is vital for contextualizing findings and understanding their implications for human health.Moreover, linking environmental exposure to health outcomes emphasizes the importance of establishing regulatory limits based on health considerations.This underscores the interconnectedness of the environment, exposure, and health outcomes, necessitating comprehensive regulatory frameworks.
Most of the selected papers (78.57%) exclusively applied active sampling methods, with impaction being the most frequently used method (67.86%).This sampling strategy is based on culture-based methods, which only allows the evaluation of culturable microorganisms, and thus microorganisms' cells that are potentially damaged due to the high velocity of the airflow are not isolated [10,37,45].Furthermore, it is critical to emphasize that air is not uniform in place or time and that it is always subject to change based on the kind and intensity of the activities occurring there or other environmental variables (e.g., climate conditions) [36,46].Thus, the sampling period must match the setting of the research and the work being developed in that specific environment.Passive sampling methods were applied in only a few of the analyzed studies as a stand-alone method (14.29%).However, passive sampling methods are expected to be more reliable than active sampling methods since they can collect contamination over longer periods, allowing to cover all the changes that may happen in the environment [47] such as the ventilation, environmental features [48], water infiltrations and damage [49], as well as the type of task being developed in that workplace [10,50,51].Additionally, passive sampling methods allow the combination of different assays such as culture-based methods and molecular tools increasing the accuracy of obtained results [52].Although only two papers (7.14%) used active and passive sampling methods together, this should be the trend to follow, since this allows each sampling methods' drawbacks to be overcome [10].
The fact that culture-based methods are primarily used for microbial characterization as standard methods for microbial assessment [53,54] might justify its frequent use among the selected papers (71.43%).This methodology is crucial to estimate health risks, since microorganisms' viability can limit microorganisms' inflammatory and/or cytotoxic potential [10,54,55].Despite the advantages, conventional approaches may underestimate results since incubation temperatures and culture conditions may favor specific species.Plus, typical procedures may not always be effective in cultivating certain common microorganisms [53].Furthermore, a recent study [53] highlights the importance of culture media selection and its significant impact on fungal counts and species diversity.Although some studies (17.86%) did not mention what culture media were employed, accurate culture media selection is critical for exposure assessment in different environments, particularly when targeting Aspergillus sp.[53].Overall, three cultural media were employed for fungal assessment (MEA, DG18, and SDA).MEA and SDA are the most used non-selective media for fungi and yeasts, whereas DG18 is a fast-growing fungi inhibitor, allowing more diversity in the growth of fungal strains [56].MEA and DG18 have both been used alongside and have proven to be useful in the growth of Aspergillus species according to the matrix, sampling method employed, and indoor environment assessed [57].For bacterial assessment, TSA was the most non-selective media related to the growth of fastidious bacteria, while MAC was the most used selective and differential media related to the growth of Gram-negative bacteria, useful for the identification of enteric bacteria [58].MYP allows the identification of Gram-positive bacteria as Bacillus cereus [59].The use of multiple culture media is fundamental for the isolation and identification of a wider spectrum of microorganisms.Also, the integration of multiple culture media and different incubation temperatures in culturomics methods (such as MALDI-TOF) permits a more precise identification of unknown isolates [60,61].This approach allows accurate microbial characterization, particularly the rapid identification of potential pathogens.In fact, culturomics methods bridge the gap between culture-based methods and molecular techniques, providing a comprehensive assessment of bioaerosols [38].
Recently, culture-independent techniques such as PCR and genome sequencing have been demonstrated to be useful for various bioaerosol measurements [52].Indeed, PCR and sequencing were frequently performed by the authors in the selected papers.These techniques enable the detection of non-viable microorganisms as well as their potentially allergenic components [52,62], providing more information regarding microbial diversity in the evaluated environment [9].Molecular techniques along with culture-based methods were applied by some papers (39.29%).This strategy is highly supported, since both viable and non-viable microorganisms are considered, providing a wider microbial characterization [9,10,52], and a more accurate characterization of the exposure scenario [14].
Furthermore, molecular techniques development has also enabled the assessment of Antibiotic Multidrug Resistance (AMD), including resistance genes associated with bacteria contamination.Recently, the World Health Organization (WHO) released an updated Bacterial Priority Pathogens List (BPPL) 2024, in which 15 families of antibiotic-resistant bacteria were grouped into critical, high and medium categories in order to allow an effective prioritization [63].Additionally, the European Food Safety Authority (EFSA) panel on Biological Hazards recently emitted a Scientific Opinion in which the highest priority antimicrobial-resistant bacteria (ARB) and antibiotic resistance genes (ARG) were identified in different sources, including water.Among the most relevant ARB, the panel indicated carbapenem or extended-spectrum cephalosporin and/or fluoroquinolone-resistant Enterobacterales, fluoroquinolone-resistant Campylobacter sp., Methicillin-resistant Staphylococcus aureus and glycopeptide-resistant Enterococcus faecium and E. faecalis.Regarding the highest priority ARGs, the panel reported blaCTX-M, blaVIM, blaNDM, blaOXA-48-like, blaOXA-23, mcr, armA, vanA, cfr and optrA genes.The EFSA report also evidenced the existence of several data gaps regarding sources and the relevance of transmission routes and diversity of ARB and ARGs [64].The data analyzed in this review demonstrate that antibiotic resistance profiling, including MRSA, mecA gene [31], sulfonamide, sul1, sul2, sul3, tetracycline, tetA, tetC, tetO, tet W, integrons, intl1, intl2 and intl3 [4], and Carbapenem-Resistant blaNDM, blaKPC, blaOXA-48, blaIMP, and blaVIM genes [26] is already a reality.Moreover, despite the fact that the quantitative microbial risk assessment (QMRA) of WWTPs has been classically focused on risk-based monitoring targets, it is accepted that the expansion of QMRA methodologies, to include ARG, may be key for the assessment of the relative risk of these contaminants [65].The assessment of ARG units is crucial for the identification of relevant/high-priority sources and natural reservoirs of AMR, allowing the establishment of effective mitigation strategies in a One Health approach.Despite the fact that microbial assessment in water samples and sewage treatment plants has been carried out, the development of official monitoring strategies and effective risk assessment in sewage treatment plants is crucial.In agreement with the newly updated WHO-BPPL, which demonstrates the highly dynamic nature of AMR, increasing evidence and expert reports clearly highlight the urge to promote a comprehensive public health approach and international coordination to engage innovation and mitigation strategies [63].
On the other hand, it is important to note that ARGs identification may be influenced by the different methods employed and divergences in the measuring process from sampling to wet-lab differences, among others [66].In addition to the multi-criteria decision analysis (MCDA) method developed by the WHO in the 2017 WHO BPPL, which is still currently applied in the 2024 WHO BPPL [63] and EFSA Panel on Biological Hazards (BIOHAZ) risk assessment monitoring (https://www.efsa.europa.eu/en/topics/topic/biological-hazards), other international multi-disciplinary networks, such as NEREUS COST Action ES1403 [67], created to access the current challenges related to wastewater reuse and high-priority concerns regarding public health and environmental protection, concluded that scientific research and environmental management should follow system-atic, quantitative, and comparable ARG datasets, and reported that the research community should adopt "ARG copy per cell" [66].Thus, the development of effective mitigation measures including new monitoring technologies, such as on-line sensors that are able to detect and quantify bacterial pathogens, ARB and ARG, is crucial, as is the implementation and improvement of links between research and policy [65].
The identification of the most suitable fungal indicators in WWTPs is also critical for assessing treatment efficacy, environmental impacts, and public and occupational health risks [68].Commonly used fungal species such as Aspergillus sp. and Penicillium sp.serve as markers for organic matter removal and microbial contamination [69].Monitoring fungal indicators enables the identification of seasonal variations, climate influences, and anthropogenic impacts on wastewater quality, essential for tailoring treatment strategies.Additionally, their presence aids in the early detection of potential health hazards, such as opportunistic pathogens or allergenic molds, ensuring the safety of both workers and the public [70].Aspergillus sp. was recurrent and also the most prevalent in the selected papers; the prevalence of this genera in waste management industries has already been recognized, highlighting the need for further research regarding occupational exposure [14].In fact, Aspergillus section Fumigati was already suggested as an indicator of harmful fungal exposure in the waste management industry [71][72][73][74] and listed by the WHO as a critical priority, considering specific criteria such as antifungal resistance, mortality, evidence-based treatment, access to diagnostics, annual incidence and complications and sequelae [75].However, the WHO list did not consider the toxicologic potential from fungal species, neglecting the possible occupational exposure to mycotoxins, as was already reported in different occupational environments [76].
In agreement with bacteria contamination analysis, fungal assessment should also cover the resistance profile.Indeed, antifungal drug resistance is a growing global concern in both space and time.This includes newly emerging species that are resistant to multiple antifungal drugs (like the yeast Candida auris), as well as novel resistant variants of previously susceptible pathogens (such as the ubiquitous mold Aspergillus fumigatus) [77].Because of the selection of resistant strains triggered by the growing use of triazole drugs, azole resistance in Aspergillus fumigatus is currently seen as an emerging hazard to global public health [78,79].In Aspergillus fumigatus, azole resistance can evolve through two different pathways.First, in the setting of chronic pulmonary aspergillosis, as in individuals with cystic fibrosis, resistant strains may be chosen during or following a long-term azole therapy [79,80].Second, the prolonged use of azole antifungals in agriculture may be connected to azole resistance [79,[81][82][83][84]. Relevantly, it is reported that several antifungals cause inherent resistance in Fumigati cryptic species.However, selected pressure brought on by the prolonged azole therapy of patients with chronic aspergillosis or environmental selection pressures are the reasons behind the emergence of resistance acquisition in Aspergillus fumigatus sensu stricto.Mutations in genes engaged in the Aspergillus fumigatus ergosterol pathway are frequently linked to the mechanisms of azole resistance, especially in the cyp51A gene that encodes cytochrome P450 14-lanosterol demethylase, the primary target of azole antifungals [79,85,86], highlighting the relevance of using these mutations as an indicator for fungal resistance.
Considering the above, further research should be performed to select the most suitable indicators of harmful microbial contamination for this occupational setting.The lists provided by the WHO regarding fungi [86] and bacteria [87] should be considered for this endeavor, but the resistance and toxicological potential from fungi and bacteria should not be neglected.

Conclusions
Overall, this scope review concluded what is needed to provide robust science for the guidance of occupational exposure assessments: (a) common protocol from the field (sampling campaign) to the lab (assays to employ) when aiming to perform exposure assessment in WWTPs; (b) standardized contextual information to be retrieved, allowing a proper risk control and management; (c) the selection of the most suitable microbial targets to serve as indicators of harmful microbial exposure.Filling these gaps with further studies will allow robust science to be provided to policy makers and stakeholders.

Table 2 .
Data extracted from the chosen papers.
Micrococcus spp.and Staphylococcus spp.had the highest emission of bacteria in the winter and summer, respectively.Cladosporium spp., Penicillium spp., Aspergillus spp.and Alternaria spp.were the dominant fungi.