Monitoring of enteropathogenic Gram-negative bacteria in wastewater treatment plants: a multimethod approach.

The wastewater treatment processes are associated with the emission of microbial aerosols, including enteropathogenic bacteria. Their presence in this work environment poses a real threat to the health of employees, both through the possibility of direct inhalation of the contaminated air and indirectly through the pollution of all types of surfaces with such bioaerosol particles. This study aimed to investigate the prevalence of enteropathogenic bacteria in the air, on surfaces, and in wastewater samples collected in four wastewater treatment plants (WWTPs). The effectiveness of conventional culture-biochemical, as well as spectrometric and molecular methods for the rapid detection of enteropathogenic bacteria at workstations related to particular stages of wastewater processing, was also evaluated. Bioaerosol, surface swab, and influent and effluent samples were collected from wastewater plants employing mechanical-biological treatment technologies. The air samples were collected using MAS-100 NT impactor placed at a height of 1.5 m above the floor or ground, simulating aspiration from the human breathing zone. Surface samples were collected with sterile swabs from different surfaces (valves, handles, handrails, and coveyor belts) at workplaces. The raw influent and treated effluent wastewater samples were aseptically collected using sterile bottles. The identification of bacterial entheropathogens was simultaneously conducted using a culture-based method supplemented with biochemical (API) tests, mass-spectrometry (MALDI TOF MS), and molecular (multiplex real-time PCR) methods. This study confirmed the common presence of bacterial pathogens (including enteropathogenic and enterotoxigenic Escherichia coli, Salmonella spp., Campylobacter spp., and Yersinia enterocolitica) in all air, surface, and wastewater samples at studied workplaces. Higher concentrations of enteropathogenic bacteria were observed in the air and on surfaces at workplaces where treatment processes were not hermetized. The results of this study underline that identification of enteropathogenic bacteria in WWTPs is of great importance for the correct risk assessment at workplaces. From the analytical point of view, the control of enteropathogenic bacterial air and surface pollution using rapid multiplex-PCR method should be routinely performed as a part of hygienic quality assessment in WWTPs.

Engineering surgical face masks with photothermal and photodynamic plasmonic nanostructures for enhancing filtration and on-demand pathogen eradication.

The shortage of face masks and the lack of antipathogenic functions has been significant since the recent pandemic's inception. Moreover, the disposal of an enormous number of contaminated face masks not only carries a significant environmental impact but also escalates the risk of cross-contamination. This study proposes a strategy to upgrade available surgical masks into antibacterial masks with enhanced particle and bacterial filtration. Plasmonic nanoparticles can provide photodynamic and photothermal functionalities for surgical masks. For this purpose, gold nanorods act as on-demand agents to eliminate pathogens on the surface of the masks upon near-infrared light irradiation. Additionally, the modified masks are furnished with polymer electrospun nanofibrous layers. These electrospun layers can enhance the particle and bacterial filtration efficiency, not at the cost of the pressure drop of the mask. Consequently, fabricating these prototype masks could be a practical approach to upgrading the available masks to alleviate the environmental toll of disposable face masks.

Production of pro-inflammatory mediators stimulated by exposure of poultry house workers to airborne dust particulates.

INTRODUCTION AND OBJECTIVE: Poultry house employees spend a significant part of their work shift being exposed to airborne particulate pollutants. The aim of this study was to assess their exposure at different stages of chicken production cycle, based on quantification of pro-inflammatory mediators (IL-1ß, IL-6, IL-8, and TNFα) in nasal lavage (NAL) samples. MATERIAL AND METHODS: The concentrations of airborne dust at 3 different stages of the production cycle (i.e. empty poultry house, with 7- and 42-day-old chickens) were stationary measured using Grimm spectrometer, as well as CIS and Button samplers. The dust collected by the latter 2 samplers was analyzed for endotoxin and (1→3)-ß-D-glucan content. NAL samples were collected from employees after their work shift to determine the pro-inflammatory mediator levels. RESULTS: The maximum particulate aerosol, endotoxin, and (1→3)-ß-D-glucan concentrations at workplaces reached the levels of 4.12 mg/m3, 45.21 ng/m3, and 56.54 ng/m3, respectively. The IL-1ß, IL-6, and IL-8 concentrations in NAL samples ranged between 0.62-18.12 pg/mL, <0.70-25.37 pg/mL, and <3.50-259.5 pg/mL, respectively. All TNFα levels were below 4 pg/mL. There were no significant differences between these cytokine concentrations in NAL samples collected at different stages of chicken breeding in either 'winter' or 'summer' seasons. CONCLUSIONS: Inhalation stimulation with poultry dust containing endotoxins and (1→3)-ß-D-glucans resulted in the production of pro-inflammatory mediators, which proves the course of immunological processes in the exposed employees that may lead to adverse effects. The use of nasal lavage fluid in the control of such exposure confirms that NAL analysis is a reliable laboratory tool for assessing the impact of poultry dust on exposed farm workers.

Filters of automobile air conditioning systems as in-car source of exposure to infections and toxic moulds.

The main component of an air conditioning system is air filters. Over time, the filters of an air conditioning system in cars can turn into sources of emission of microbiological hazards. The aim of this study was to quantitatively and qualitatively assess the presence of infectious and toxic fungi in the AC filters in passenger cars. The studied non-woven filters were removed from passenger cars during the "winter"/"summer" seasons. The taxonomic identification of the fungi isolated from the filters was performed using both the culture-based and molecular methods. RT-PCR was applied to assess the presence of gene fragments regulating aflatoxin biosynthesis in the isolates obtained from fungal cultures. The average fungal concentrations in the filter samples collected during the summer/winter season were 5.4 × 104 cfu/m2 and 2.4 × 104 cfu/m2, respectively. Most of the filter samples, collected in both the studied seasons, revealed the presence of Aspergillus species including A. fumigatus, A. niger, A. terreus and/or A. flavus. The recorded levels of fungal contamination of AC filters in passenger cars indicate the necessity for more frequent filter replacement in this type of vehicle. Occupational exposure to moulds and the resulting health problems that may be experienced by professional drivers should be properly recognised in order to undertake effective preventive measures.

Seasonal prevalence of potentially infectious enteric viruses in surface waters below treated wastewater discharge.

INTRODUCTION AND OBJECTIVE: Enteric viruses are widely distributed in the natural water environment. The aim of the study was to assess the prevalence of potentially infectious adenoviruses (AdV) and rotaviruses (RoV) in surface water near treated wastewater discharge. MATERIAL AND METHODS: Water samples were collected from surface water below the treated wastewater effluent discharge located near a wastewater treatment plant receiving sewage from an urban area. Water samples were concentrated by ultrafiltration and treated with propidium monoazide dye, followed with v-qPCR/v-RT-qPCR analysis. Simultaneously, the temperature and pH of the collected samples were measured to check the influence of these parameters on the concentrations of potentially infectious viruses. RESULTS: The average concentrations of potentially infectious AdV and RoV particles in collected samples ranged between log10 1.86 ÷ 3.94 gc/L and log10 2.39 ÷ 3.82 gc/L in the winter season, and between log10 2.18 ÷ 3.59 gc/L and log10 1.85 ÷ 2.10 gc/L in the summer season, respectively. In general, AdVs were detected more often than RoVs, while RoV-positive samples were more frequent in the winter than in the summer season (Chi2: p = 0.028; Fisher's Exact test p = 0.033). Negative correlations between log10 concentration of viral particles and temperature and pH for both viruses were observed. CONCLUSIONS: The presence of potentially infectious AdVs and RoVs in the surface waters may constitute a health risk for the local population. Application of v-PCR-based methods and considering AdV as a viral contamination indicator should be introduced into virological water quality monitoring for estimations of public health risks.

Across-Shift Changes in Viable Nasal Bacteria among Waste-Incineration Plant Workers-A Pilot Study.

The aim of this pilot study was to assess the time-related changes in viable nasal bacteria concentrations among waste-incineration plant (WIP) workers compared to a group of office building (OB) workers outside the plant. In total, 20 volunteers participated in the study, including 14 WIP and 6 OB workers. WIP workers were divided into two sub-groups: supervisory staff (SVS) and maintenance and repair workers (MRW). Nasal swabs were collected before and after the morning work shift. Airborne bacteria were sampled with a six-stage impactor to assess the bioaerosol size distribution. The analysis showed that a significant, almost three-fold increase in nasal bacterial concentration was found only among WIP workers, and this referred mainly to anaerobic species. The load of anaerobic bacteria at the beginning of work was 12,988 CFU/mL, and after work shift 36,979 CFU/mL (p < 0.01). Significant increases in microbial concentrations was found only in the MRW subgroup, among non-smoking workers only. The results showed increased bacterial concentration in WIP nasal samples for as many as 12 bacterial species, including, e.g., Streptococcus constellatus, Peptostreptococcus spp., E. coli, and P. mirabilis. These preliminary data confirmed that the nasal swab method was helpful for assessment of the workers' real-time exposure to airborne bacteria.

Detection and identification of potentially infectious gastrointestinal and respiratory viruses at workplaces of wastewater treatment plants with viability qPCR/RT-qPCR.

This study aimed to qualitatively and quantitatively assess the prevalence of the most common respiratory and gastrointestinal viruses in the air, surface swab, and influent/effluent samples collected in wastewater treatment plants (WWTPs). Application of qPCR/RT-qPCR (quantitative polymerase chain reaction/reverse-transcription quantitative polymerase chain reaction) assays combined with PMA (propidium monoazide) dye pretreatment allowed detecting the potentially infectious and disintegrated viral particles in collected samples. In the air at workplaces in WWTPs, the most frequent isolation with the highest concentrations (reaching up to 103 gc/m3 of potentially infectious intact viral particles) were observed in case of adenoviruses (AdVs) and rotaviruses (RoVs), followed by noroviruses (NoVs). Viruses were significantly more often detected in the air samples collected with Coriolis µ impinger, than with MAS-100NT impactor. The temperature negatively (Spearman correlation: -1 < R < 0; p < 0.05), while RH (relative humidity) positively (0 < R < 1; p < 0.05) affected airborne concentrations of potentially infectious viral particles. In turn, the predominant viruses on studied surfaces were RoVs and noroviruses GII (NoV GII) with concentrations of potentially infectious virions up to 104 gc/100 cm2. In the cases of SARS-CoV-2 and presumptive SARS-CoV-2 or other coronaviruses, their concentrations reached up to 103 gc/100 cm2. The contamination level of steel surfaces in WWTPs was similar to this on plastic ones. This study revealed that the qualitative and quantitative characteristics of respiratory and gastrointestinal viruses at workplaces in WWTPs is important for proper exposure assessment and needs to be included in risk management in occupational environment with high abundance of microbial pollutants derived from wastewater.

Occupational exposure to anaerobic bacteria in a waste sorting plant.

The study focused on exposure assessment to bacterial aerosols and organic dust in waste sorting plant. Samples were collected at different workplaces of waste sorting cycle i.e.: waste press, reloading area, loading of conveyor belt, sorting cabin, sorting hall, and control room. A quantitative analysis of aerobic and anaerobic bacteria was supplemented by qualitative analysis of anaerobic biota with the use of culture-based methods and biochemical tests. In addition, inhalable dust concentrations were also evaluated. To confirm the presence of Clostridium genus, the PCR reaction with specific primers (Chis150f and ClostIr) was performed. The average concentration of total bacteria in waste sorting plant was 4347 CFU m-3 (SD = 2439), of which 66% were anaerobic strains (2852 CFU m-3; SD = 2127). It was found that about 24% of anaerobic bacteria belonged to Clostridium genus (682 CFU m-3; SD = 633). The highest contamination with anaerobic bacteria was observed near the waste reloading plant (3740 CFU m-3), and the lowest in the control room (850 CFU m-3). The average concentration of inhalable dust in the waste sorting plant was 0.81 mg m-3 (SD = 0.59). The correlation analysis showed that the presence of anaerobic bacteria, including clostridia was significantly determined by the microclimate parameters. Qualitative analysis showed the presence of 16 anaerobic species belonging to 9 genera, of which Actinomyces, Clostridium, and Gemella were present at all workplaces. The molecular analysis confirmed the presence of Clostridium genus in both bioaerosol and settled dust samples.Implications: The study showed that anaerobic bacteria should be taken into account as an important component of this microbiota when assessing the exposure of waste sorting workers to biological agents. However, future studies should investigate more precisely how the composition of sorted waste as well as the season can affect the diversity of anaerobic bacteria in this working environment. More attention should be paid to regular cleaning of equipment surfaces in the plant, as deposited organic dust is an important reservoir of anaerobic bacteria, including those of a potentially pathogenic nature.

Microbial contamination of money sorting facilities.

INTRODUCTION: Money is the most common item with which we have daily contact. Circulated banknotes and coins can become microbiologically contaminated and act as both a source and a means of spreading such pollutants. MATERIAL AND METHODS: The study was carried out in three money sorting facilities in Poland. Bioaerosol samples were collected using a 6-stage Andersen impactor, and microorganisms deposited on tabletop surfaces were sampled using the swab method. Bacterial and fungal concentrations were calculated and all isolated species were taxonomically identified. RESULTS: The study confirmed that means of payment are active sources of microbial emission in money sorting facilities. The bioaerosol concentrations did not exceed the threshold limit values proposed for this type of office premises. It confirms that ventilation systems in these facilities worked efficiently, protecting them from the migration of microbial contaminants present in both indoor and outdoor (atmospheric) background air. On the other hand, the average concentrations of bacteria and fungi on tabletop surfaces in banknote and coin sorting rooms were above the proposed purity levels for indoor surfaces and should be treated as microbiologically contaminated. Microbiota isolated from the air and surfaces were very diverse and among those strains were bacterial and fungal pathogens that can pose a health threat to exposed individuals. CONCLUSIONS: The results showed that employees in money sorting facilities were exposed to microorganisms that may contribute to the development of adverse health outcomes. To protect them, highly efficient hygienic measures should be introduced in this working environment, to prevent both unwanted pollution and subsequent secondary emission of microbial contaminants from sorted means of payment and tabletop surfaces.

Prevalence of Bovine Leukemia Virus (BLV) and Bovine Adenovirus (BAdV) genomes among air and surface samples in dairy production.

We aimed to assess the occurrence of bovine viruses (bovine leukemia virus-BLV and bovine adenovirus-BAdV) at workplaces in traditional dairies and to evaluate the potential role of airborne and surface contamination in spreading of these viruses derived from raw milk. The total amount of 122 samples-including 37 air (bioaerosol), 40 surface, and 45 milk samples-were checked for the presence of BLV and BAdV genomes using RT-qPCR/qPCR method. The study showed that the viruses were present in 7 air (among them 71.4% were BLV-positive and 28.6% were BAdV-positive), 14 surface (among them 85.7% were BLV-positive and 14.3% were BAdV-positive), and 34 milk (all were BLV-positive only) samples. Statistical analysis revealed that both the air and surfaces in studied occupational environment were more frequently contaminated with BLV than with BAdV (Chi-square test: p = 0.002, Fisher's Exact test: p = 0.002). Kruskal-Wallis tests showed significant differences in BLV genome concentrations in the air (p = 0.045) as well as in BLV and BAdV genome concentrations on surfaces (p = 0.005 and p = 0.040, respectively) between studied processing areas. In units of genome copies (gc) per area, the highest concentrations of BLV and BAdV genomes in the air (9.8 × 101 ± 1.14 × 102 gc/m3 and 5.4 × 101 ± 9.1 × 101 gc/m3, respectively) and on surfaces (9.83 × 102 ± 7.41 × 102 gc/100cm2 and 2.30 × 102 ± 3.8 × 102 gc/100cm2, respectively) were observed in milk reception area. The air and surfaces of pre-production zones were also significantly more contaminated with BAdV genomes compared to production areas (Mann-Whitney test: p = 0.039 and p = 0.029, respectively). This study showed that dairy workers may be exposed to bovine viruses through the inhalation of bioaerosols and contact with contaminated surfaces. To reduce the probability of virus transmission from the raw milk to humans, efficient surface cleaning procedures degrading viral particles should be introduced and the use of personal protection equipment, especially within pre-production zones, should be required. As the raw milk may be a source of bovine viruses, the development of strategies for both the control and eradication of BLV and BAdV among cattle seems to be also urgently needed.

Microbial contamination level and microbial diversity of occupational environment in commercial and traditional dairy plants.

OBJECTIVE: The aim of this study was to assess microbial contamination of the air and surfaces at workplaces in commercial (CD) and traditional (TD) dairies. MATERIAL AND METHODS: Bioaerosol (impactor) and surface (swab) samples were collected in CD and in TD. Bacterial and fungal concentrations in the air and on surfaces were calculated and all isolated microorganisms taxonomically identified, based on their morphological, biochemical and molecular features. RESULTS: Average concentrations of bacterial aerosol ranged between 70-860 CFU/m3 and 265-14639 CFU/m3, while for fungal aerosol were between 50-290 CFU/m3 and 55-480 CFU/m3 in CD and TD, respectively. Average bacterial concentrations on surfaces ranged between 1.0-49.7 CFU/cm2 and 0.2-60.4 CFU/cm2, whereas average fungal surface contamination ranged between 0-2.7 CFU/cm2 and 0-4.6 CFU/cm2 in CD and TD, respectively. Qualitative analysis revealed mainly the presence of saprophytic microorganisms; however, several pathogenic strains (Staphylococcus aureus, Streptococcus intermedius, Clostridium perfringens, Actinomyces spp., Streptomyces spp., Candida albicans) were also isolated from both the air and surface samples in the studied dairies. CONCLUSIONS: The air and surfaces in TD were more polluted than those in CD; however, in both types of dairies, the levels of microbial contaminants did not exceed respective threshold limit values. Nevertheless, the presence of pathogenic microorganisms may increase health risk for dairy workers and influence the quality of products. Hence, proper hygienic measures should be introduced and performed to guarantee high microbial quality of both production processes and milk products.

Nasal lavage as analytical tool in assessment of exposure to particulate and microbial aerosols in wood pellet production facilities.

Occupational exposure to wood dust and bioaerosols may lead to numerous respiratory tract diseases. We aimed to assess a degree of workplace contamination with dust, bacteria, fungi, endotoxins, and (1 → 3)-ß-D-glucans released into the air during wood processing in pellet production facilities and to check against this background the usefulness of nasal lavage (NAL) as analytical tool for assessment of combined workers' exposure to airborne dust and microbiological contaminants. In 10 pellet plants, the particulate (wood dust) aerosol concentrations were determined by using Grimm aerosol spectrometer and CIS filter sampler. The collected CIS samples were subsequently used to evaluate endotoxin and (1 → 3)-ß-D-glucan concentrations. Simultaneously with particulate aerosol, bioaerosol samples were collected by using 6-stage Andersen and single-stage MAS impactors. Bacterial and fungal aerosol concentrations were calculated and all isolated microorganisms were taxonomically identified. NAL fluid samples were collected from workers exposed to studied aerosols and the concentrations of proinflammatory mediators (IL-1ß, IL-6, IL-8, and TNFα) and cytological image of nasal mucosa (expressed as cell counts) were established. The dynamics of production activities resulted in wide range of observed wood dust, microorganism, endotoxin and (1 → 3)-ß-D-glucan concentrations reaching periodically extremely high values up to 65 mg m-3, 19,320 CFU m-3, 215 ng m-3 and 1525 ng m-3, respectively. Environmental stress caused by exposure to particulate and microbial aerosols stimulated immune response among workers of pellet production facilities. Correlation analysis revealed that interleukin levels and the number of cells in NAL were significantly affected by both wood dust and bioaerosol concentrations. As nasal mucosa serves as the primary barrier against inhaled pollutants, NAL seems a reliable analytical material to assess work-related adverse respiratory health outcomes derived from such exposure.

Across-shift changes in upper airways after exposure to bacterial cell wall components.

INTRODUCTION: To assess the across-shift changes of cytokine concentrations in nasal lavage (NAL) samples were collected from workers exposed to bacterial cell wall components present in organic dust in three different occupational environments. MATERIAL AND METHODS: The study was conducted in 38 employees including 10 workers from a municipal waste sorting plant (WSP), 20 from a sewage treatment plant (STP) and 8 from an office building (OB), who were established as a reference group, not exposed to organic dust. Interleukins 1ß (IL-1ß), 6 (IL-6), 8 (IL-8) and tumour necrosis factor alpha (TNF-α) were examined in NAL before and after work shift on Wednesdays. Bioaerosol exposure was determined by personal measurements and analysed for organic dust, endotoxins (END) and peptidoglycans (PGN). RESULTS: The analysis included the results for IL-8 only, because for the other cytokines their concentrations in 80% of cases were below the detection level. The most polluted were the workplaces in WSP with average concentrations of organic dust - 3.47 mg/m3, END - 96.31 ng/m3 and PGN - 571.88 ng/m3. The results of IL-8 showed a significant difference between the studied groups after the work shift (p=0.007). Among WSP workers concentrations of IL-8 increased also significantly (p=0.015) during the work shift. Multivariate analysis showed that organic dust and END were the factors that in the most distinct way (p<0.001) influenced changes of IL-8 levels in NAL. CONCLUSIONS: Each alteration in the composition of bioaerosols will probably determine the changes in the mechanisms responsible for both formation and modulation of inflammatory reactions in exposed workers.

Anaerobic bacteria in wastewater treatment plant.

PURPOSE: The objective of this study was to assess exposure to anaerobic bacteria released into air from sewage and sludge at workplaces from a wastewater treatment plant (WWTP). METHODS: Samples of both sewage and sludge were collected at six sampling points and bioaerosol samples were additionally collected (with the use of a 6-stage Andersen impactor) at ten workplaces covering different stages of the technological process. Qualitative identification of all isolated strains was performed using the biochemical API 20A test. Additionally, the determination of Clostridium pathogens was carried out using 16S rRNA gene sequence analysis. RESULTS: The average concentration of anaerobic bacteria in the sewage samples was 5.49 × 104 CFU/mL (GSD = 85.4) and in sludge-1.42 × 106 CFU/g (GSD = 5.1). In turn, the average airborne bacterial concentration was at the level of 50 CFU/m3 (GSD = 5.83) and the highest bacterial contamination (4.06 × 103 CFU/m3) was found in winter at the bar screens. In total, 16 bacterial species were determined, from which the predominant strains belonged to Actinomyces, Bifidobacterium, Clostridium, Propionibacterium and Peptostreptococcus genera. The analysis revealed that mechanical treatment processes were responsible for a substantial emission of anaerobic bacteria into the air. In both the sewage and air samples, Clostridium perfringens pathogen was identified. CONCLUSIONS: Anaerobic bacteria were widely present both in the sewage and in the air at workplaces from the WWTP, especially when the technological process was performed in closed spaces. Anaerobic bacteria formed small aggregates with both wastewater droplets and dust particles of sewage sludge origin and as such may be responsible for adverse health outcomes in exposed workers.