Biofilms within drinking water distribution systems serve as a habitat for drinking water microorganisms. However, biofilms can negatively impact drinking water quality by causing water discoloration and deterioration and can be a reservoir for unwanted microorganisms. In this study, we investigated whether indicator organisms for drinking water quality, such as coliforms, can settle in mature drinking water biofilms. Therefore, a biofilm monitor consisting of glass rings was used to grow and sample drinking water biofilms. Two mature drinking water biofilms were characterized by flow cytometry, ATP measurements, confocal laser scanning microscopy, and 16S rRNA sequencing. Biofilms developed under treated chlorinated surface water supply exhibited lower cell densities in comparison with biofilms resulting from treated groundwater. Overall, the phenotypic as well as the genotypic characteristics were significantly different between both biofilms. In addition, the response of the biofilm microbiome and possible biofilm detachment after minor water quality changes were investigated. Limited changes in pH and free chlorine addition, to simulate operational changes that are relevant for practice, were evaluated. It was shown that both biofilms remained resilient. Finally, mature biofilms were prone to invasion of the coliform, Serratia fonticola. After spiking low concentrations (i.e., ±100 cells/100 mL) of the coliform to the corresponding bulk water samples, the coliforms were able to attach and get established within the mature biofilms. These outcomes emphasize the need for continued research on biofilm detachment and its implications for water contamination in distribution networks.
OBJECTIVE: The revelation that even low concentrations of coliforms can infiltrate into mature drinking water biofilms highlights a potential public health concern. Nowadays, the measurement of coliform bacteria is used as an indicator for fecal contamination and to control the effectiveness of disinfection processes and the cleanliness and integrity of distribution systems. In Flanders (Belgium), 533 out of 18,840 measurements exceeded the established norm for the coliform indicator parameter in 2021; however, the source of microbial contamination is mostly unknown. Here, we showed that mature biofilms, are susceptible to invasion of Serratia fonticola. These findings emphasize the importance of understanding and managing biofilms in drinking water distribution systems, not only for their potential to influence water quality, but also for their role in harboring and potentially disseminating pathogens. Further research into biofilm detachment, long-term responses to operational changes, and pathogen persistence within biofilms is crucial to inform strategies for safeguarding drinking water quality.

The present study describes the development of a potentiometric sensor for microbial monitoring in water based on catalase activity. The sensor comprises a MnO2-modified electrode that responds linearly to hydrogen peroxide (H2O2) from 0.16 M to 3.26 M. The electrode potential drops when the H2O2 solution is spiked with catalase or catalase-producing microorganisms that decompose H2O2. The sensor is responsive to different bacteria and their catalase activities. The electrochemical sensor exhibits a lower limit of detection (LOD) for Escherichia coli at 11 CFU/ml, Citrobacter youngae at 12 CFU/ml, and Pseudomonas aeruginosa at 23 CFU/ml. The sensor shows high sensitivity at 3.49, 3.02, and 4.24 mV/cm2dec for E. coli, C. youngae, and P. aeruginosa, respectively. The abiotic sensing electrode can be used multiple times without changing the response potential (up to 100 readings) with a shelf-life of over six months. The response time is a few seconds, with a total test time of 5 min. Additionally, the sensor effectively tested actual samples (drinking and grey water), which makes it a quick and reliable sensing tool. Therefore, the study offers a promising water monitoring tool with high sensitivity, stability, good detection limit, and minimum interference from other water contaminants.

Domestic wastewater is a source of persistent organic pollutants and pathogens to the aquatic environment, including groundwater aquifers. Wastewater contaminants include a variety of personal care products, pharmaceuticals, endocrine disrupters, bacteria, and viruses. Groundwater from 22 wells completed in a semi-confined to confined, fractured Silurian dolostone aquifer in southern Wellington County, Ontario, Canada, was analyzed for 14 organic wastewater contaminants (4 artificial sweeteners, 10 pharmaceuticals) as well as E. coli, total coliforms, and 6 human enteric viruses. Enteric viruses were detected in 8.6% of 116 samples, and at least one organic wastewater contaminant was detected in 82% of the wells (in order of decreasing detection frequency: acesulfame, ibuprofen, sulfamethoxazole, triclosan, carbamazepine, and saccharin). Virus indicator metrics [positive and negative predictive values (PPV, NPV), sensitivity, specificity] were calculated at the sample and well level for the organic wastewater compounds, E. coli, and total coliforms. Fecal bacteria were not good predictors of virus presence (PPV = 0%-8%). Of the potential chemical indicators, triclosan performed the best at the sample level (PPV = 50%, NPV = 100%), and ibuprofen performed the best at the well level (PPV = 60%, NPV = 67%); however, no samples had triclosan or ibuprofen concentrations above their practical quantification limits. Therefore, none of the compounds performed sufficiently well to be considered reliable for assessing the potential threat of enteric viruses in wastewater-impacted groundwater in this bedrock aquifer. Future studies need to evaluate the indicator potential of persistent organic wastewater contaminants in different types of aquifers, especially in fractured rock where heterogeneity is strong.IMPORTANCEAssessing the potential risk that human enteric viruses pose in groundwater aquifers used for potable water supply is complicated by several factors, including: (i) labor-intensive methods for the isolation and quantification of viruses in groundwater, (ii) the temporal variability of these viruses in domestic wastewater, and (iii) their potentially rapid transport in the subsurface, especially in fractured rock aquifers. Therefore, aquifer risk assessment would benefit from the identification of suitable proxy indicators of enteric viruses that are easier to analyze and less variable in wastewater sources. Traditional fecal indicators (e.g., E. coli and coliforms) are generally poor indicators of enteric viruses in groundwater. While many studies have examined the use of pharmaceutical and personal care products as tracers of domestic wastewater and fecal pollution in the environment, there is a paucity of data on the potential use of these chemical tracers as enteric virus indicators, especially in groundwater.

Coliforms are relatively common in aquatic environments, but their concentrations can be increased by environmental changes and anthropogenic activities, thus impacting fisheries resources. To determine the microbiological quality in the octopus production chain (capture, post-capture, processing and commercialization), total (TC) and fecal (FC) coliforms were quantified in sea water, fresh octopus, fresh water, ice and octopus in two presentations: packed in ice and boiled. Samples came from fishing zones Enmedio, Chopa and La Gallega at the Veracruz Reef System (VRS) during dry, rainy and windy seasons. The coliforms were determined using the most probable number technique (MPN). The most relevant results indicated that octopus packed in ice coming from the commercialization stage had FC levels >540 MPN/100 g, which exceeded the permissible limits (230 MPN/100 g). Therefore, these products present a risk for human consumption. Differences in FC were observed in octopuses between the three fishing zones (H = 8.697; p = 0.0129) and among the three climatic seasons, increasing during the rainy season, highlighting La Gallega with 203.33 ± 63 MPN (H = 7.200; p = 0.0273). The results provide evidence of the environmental and anthropogenic influences on coliform concentrations and the urgent need to implement an efficient cold chain throughout octopus production stages with adequate handling practices to reverse this situation.

The aim of this study was to evaluate the effect of dry cow therapy (DCT) on the antimicrobial resistance (AMR) profile of mastitis pathogens post-calving. A repository of isolates based on a DCT trial was utilized for the current study. A stratified random survey sample of cows from the trial were identified within the strata of season, herd, and trial treatment resulting in 382 cows. All isolates from the 382 cows were selected for the current study, which identified 566 isolates from milk samples collected at dry off (S1), post-calving (S2), and at the first clinical mastitis event up to 150 days in milk (S3). The AMR profiles were determined using broth microdilution method. Less than 10% of the coagulase-negative Staphylococcus species (CNS) isolates (n = 421) were resistant to tetracycline, ceftiofur, penicillin/novobiocin or erythromycin, while higher proportions of resistance to sulfadimethoxine (72%) and penicillin (28%) were observed. All Staphylococcus aureus (S. aureus) isolates (n = 4) were susceptible to all tested AMD except sulfadimethoxine, to which all isolates were resistant. Similarly, all Streptococcus spp. (n = 37) were susceptible to penicillin, penicillin/novobiocin, and ampicillin while resistant to tetracycline (17%). All coliforms (n = 21) were susceptible to ceftiofur, but resistance was recorded for sulfadimethoxine (70%), cephalothin (56%), and tetracycline (43%). The increased resistance percent from S1 to S2 was observed in CNS isolates from AMD-treated cows, with the highest increase recorded for penicillin (12.2%). Parametric survival interval regression models were used to explore the association between antimicrobial drug (AMD) therapy at dry off and the AMR phenotype post-calving. The accelerated failure-time metric was adopted to minimum inhibitory concentration measurements to permit interpretation of model exponentiated coefficients. Models for cows with CNS isolated at both S1 and S2 showed increased resistance against cephalothin, oxacillin, and ceftiofur in cows that received DCT from the same drug class, or a class with a shared resistance mechanism. In contrast, resistance of CNS isolates to tetracycline were associated with any AMD therapy at dry off. Resistance of CNS isolates to Penicillin decreased in CNS isolates in cows that received any AMD therapy at dry off compared to those that didn\'t. The study provided evidence that dry-cow IMM AMD was associated with AMR post-calving.

Raw, ready-to-eat (RTE) seafood products, such as ceviche, poke, and sushi, have experienced growing demand globally; however, these products have the potential to be contaminated with foodborne pathogens. The objective of this study was to determine the prevalence of Escherichiacoli/coliforms, Salmonella, and Listeria in ceviche, poke, and sushi dishes sold at the retail level in Orange County, CA, USA. Additional organisms detected during testing were also considered in the results. A total of 105 raw, RTE samples of ceviche, poke, and sushi were collected from restaurants and grocery stores in Orange County, CA. Samples were tested for Salmonella and Listeria utilizing methods from the Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM). E. coli and total coliforms were enumerated utilizing 3 M Petrifilm plates. Overall, two samples (1.9%) were positive for generic E. coli, with a range of 5-35 CFU/g. Coliforms were detected in 85 samples (81%), with a range of 5-1710 CFU/g. The average coliform levels in ceviche samples (259 CFU/g) were significantly higher than the levels in sushi samples (95 CFU/g), according to a Kruskal-Wallis H test followed by the Dunn test (p < 0.05). The coliform levels in poke samples (196 CFU/g) were not significantly different from those in ceviche or sushi. All levels of E. coli and coliforms were considered acceptable or satisfactory/borderline according to standards for RTE seafood. None of the samples tested positive for Salmonella or Listeria monocytogenes; however, other microorganisms were detected in 17 samples, including Listeria spp., Proteus mirabilis, Providencia rettgeri, and Morganella morganii. The results of this study are novel in that they present data on the microbiological safety and quality of ceviche, poke, and sushi dishes sold at retail in the United States, as well as provide a comparison across the three categories of raw, RTE seafood.

With the onset of the coronavirus disease 2019 (COVID-19) pandemic, numerous countries imposed strict lockdown measures and travel bans, resulting in the closure of hotels. Over time, the opening of hotel units was gradually allowed, and new strict regulations and protocols were issued to ensure the hygiene and safety of swimming pools in the era of COVID-19. The present study aimed to evaluate the implementation of strict health COVID-19-related protocols in hotel units during the 2020 summer tourist season concerning microbiological hygiene and physicochemical parameters of water, and to compare the data with those from the 2019 tourist season. For this reason, 591 water samples from 62 swimming pools were analyzed, of which 381 samples were for the 2019 tourist season and 210 samples were for the 2020 tourist season. To examine the presence of Legionella spp, 132 additional samples were taken from 14 pools, of which 49 in 2019 and 83 in 2020. In 2019, 2.89% (11/381) of the samples were out of legislative limits (0/250 mg/l) regarding the presence of Escherichia coli (E. coli), 9.45% (36/381) were out of acceptable limits (0/250 mg/l) regarding the presence of Pseudomonas aeruginosa (P. aeruginosa) and 8.92% (34/381) had residual chlorine levels <0.4 mg/l. In 2020, 1.43% (3/210) of the samples were out of the legislative limits as regards the presence of E. coli, 7.14% (15/210) were out of acceptable limits regarding the presence of P. aeruginosa and 3.33% (7/210) of the samples measured residual chlorine levels <0.4 mg/l. The risk ratio (RR) in relation to the presence of E. coli due to incorrect compliance with the requirements for residual chlorine was calculated for 2019 at 8.50, while in 2020 it was calculated at 14.50 (P=0.008). The RR of the presence of P. aeruginosa due to inappropriate residual chlorine requirements was calculated in 2019 at 2.04 (P=0.0814), while in 2020 it was calculated at 2.07 (P=0.44). According to the microbiological hygiene and physicochemical parameters of the water samples studied, there was a significant improvement due to the strict protocols for the swimming pools in the summer season of 2020 compared to the tourist season of 2019, namely 72.72% (E. coli), 58.33% (P. aeruginosa), 79.41% (of residual chlorine <0.4 mg/l) in the three main parameters studied. Finally, an increased colonization by Legionella spp. detected in the internal networks of the hotels due to the non-operation of the hotels during the lockdown, the improper disinfection and stagnant water in the internal water supply networks. Specifically, in 2019, 95.92% (47/49) of the samples tested negative and 4.08% (2/49) tested positive (≥50 CFU/l) for Legionella spp., compared to 2020 where 91.57% (76/83) of the samples tested negative and 8.43% (7/83) tested positive.

In developing countries, an estimated 80% of the population use traditional herbal medicines as part of their primary health care. As the market for herbal medicine expands in many African countries, partly due to their use in the treatment of COVID-19, there is the need to address all the associated safety issues. The aim of the study was to evaluate the microbial contamination of locally prepared, as well as imported foreign herbal products sold in Accra. Standard microbiological methods were employed in the enumeration of coliforms and the identification of pathogenic microbes in 60 herbal preparations that were sampled. A larger proportion (76.7%) of local herbal preparations was contaminated with bacteria as compared with imported ones (63.3%). Bacillus species and Pseudomonas aeruginosa were the predominant bacteria obtained from foreign and locally manufactured herbal preparations, respectively. A proportion of 36.7% (11) of the local samples were positive for coliform and the coliform counts ranged from 3.0 × 101 cfu/ml to 2.0 × 104 cfu/ml. Two foreign herbal samples (6.7%) were positive for coliforms; one had a count of 1.7 × 105 cfu/g while the other had 2 × 104 cfu/g. Herbal preparations sold in markets of Accra harbour several microbial pathogens; the risk is relatively higher for locally produced herbal preparations compared to imported herbal preparations. As a result, it is recommended that quality assurance in the production of local herbal preparations should be thoroughly monitored from the beginning of production to the final selling of the preparations. There is also the need to strengthen microbiological safety monitoring of imported herbal preparations.

Greywater is an important alternative water resource which could be treated and reused in buildings, reducing the freshwater demand in drought affected areas. For the successful implementation of this solution, it is important to ensure the microbial safety of treated greywater. This study examined the microbiological quality of treated greywater produced by an emergent nature-based technology (green roofs) and a chlorination process. Specifically, the effect of substrate, substrate depth, and vegetation on the removal of total coliforms, Escherichia coli, and enterococci in experimental green roofs treating greywater was examined for a period of about 12 months. In addition, the ability of chlorination to inactivate the abovementioned pathogen indicators was evaluated and their potential regrowth was examined. Results shown that green roofs filled with 10 cm of perlite reduce total coliform concentration by about 0.4 log units while green roofs filled with 20 cm of vermiculite reduce total coliform concentration by about 1.2 log units. In addition, the use of vegetation in green roofs improves the removal of pathogenic bacteria by about 0.5 log units in comparison with unvegetated systems. In all cases, the effluents of green roofs failed to satisfy the criteria for indoor reuse of treated greywater for non-potable uses such as toilet flushing without a disinfection process. The addition of 3 mg/L of chlorine in the effluent provided safe greywater microbiological quality for storage periods of less than 24 h, while longer periods resulted in the significant regrowth of pathogens. In contrast, a chlorination dose of 7 mg/L completely secured inactivation of pathogen indicators for periods of up to 3 days.

Antimicrobial resistance is a global health challenge caused by the ability of microorganisms including bacteria, fungi, protozoans and viruses to survive the effects of drugs that hitherto were effective against them. This study sought to investigate the presence of antibiotic-resistant bacteria and their corresponding molecular determinants in fish farms of the Central and Western Regions of Ghana. Management practices and antibiotic use at the fish farms were obtained through the administration of a questionnaire. Coliform and Gram-positive bacterial loads of catfish (Clarias gariepinus), tilapia (Oreochromis niloticus) intestinal microbiota, and pond water samples recovered on MacConkey Agar and Mannitol Salt Agar were determined. Bacterial isolates were identified using various biochemical assays. Antibiotic resistance profiles and possible responsible genes of bacterial isolates were determined using the disc diffusion and Polymerase Chain Reaction (PCR) methods respectively. The study revealed that none of the fish farm managers admitted using antibiotics for prevention and treatment of diseases and no major disease outbreak had ever been recorded. Bacterial loads of pond water exceeded the acceptable level of ≤100 E. coli and <10 coliforms per mL for wastewater recommended for use in fish farming. In all, 145 bacterial isolates comprising 99 Gram negative and 46 Gram-positive bacteria were stored and identified. Most isolates were resistant to at least an antibiotic. Both Gram-negative and Gram-positive bacteria were highly resistant to beta-lactam antibiotics with a corresponding high percentage detection of the bla TEM gene compared to other classes of antibiotics. This study has revealed the presence of various molecular determinants of antibiotic resistance including bla TEM, cmIA, qnrS, tetB and bla CTX-M, in multidrug-resistant bacteria at some fish farms in Ghana. There is the need to increase awareness about risks associated with the misuse and overuse of antibiotics by humans and the potential risk of spread of multi-drug resistant-bacteria in the environment.