Antibiotic resistance monitoring in wastewater in the Nordic countries: A systematic review.

The Nordic countries (Denmark, Finland, Iceland, Norway, and Sweden) have effectively kept lower antibiotic-resistant bacterial (ARB) pathogen rates than many other countries. However, in recent years, these five countries have encountered a rise in ARB cases and challenges in treating infections due to the growing prevalence of ARB pathogens. Wastewater-based surveillance (WBS) is a valuable supplement to clinical methods for ARB surveillance, but there is a lack of comprehensive understanding of WBS application for ARB in the Nordic countries. This review aims to compile the latest state-of-the-art developments in WBS for ARB monitoring in the Nordic countries and compare them with clinical surveillance practices. After reviewing 1480 papers from the primary search, 54 were found relevant, and 15 additional WBS-related papers were included. Among 69 studies analyzed, 42 dedicated clinical epidemiology, while 27 focused on wastewater monitoring. The PRISMA review of the literature revealed that Nordic countries focus on four major WBS objectives of ARB: assessing ARB in the human population, identifying ARB evading wastewater treatment, quantifying removal rates, and evaluating potential ARB evolution during the treatment process. In both clinical and wastewater contexts, the most studied targets were pathogens producing carbapenemase and extended-spectrum beta-lactamase (ESBL), primarily Escherichia coli and Klebsiella spp. However, vancomycin-resistant Enterococcus (VRE) and methicillin-resistant Staphylococcus aureus (MRSA) have received more attention in clinical epidemiology than in wastewater studies, probably due to their lower detection rates in wastewater. Clinical surveillance has mostly used culturing, antibiotic susceptibility testing, and genotyping, but WBS employed PCR-based and metagenomics alongside culture-based techniques. Imported cases resulting from international travel and hospitalization abroad appear to have frequently contributed to the rise in ARB pathogen cases in these countries. The many similarities between the Nordic countries (e.g., knowledge exchange practices, antibiotic usage patterns, and the current ARB landscape) could facilitate collaborative efforts in developing and implementing WBS for ARB in population-level screening.

Epidemiological and microbiological investigation of a large increase in vibriosis, northern Europe, 2018.

BackgroundVibriosis cases in Northern European countries and countries bordering the Baltic Sea increased during heatwaves in 2014 and 2018.AimWe describe the epidemiology of vibriosis and the genetic diversity of Vibrio spp. isolates from Norway, Sweden, Denmark, Finland, Poland and Estonia in 2018, a year with an exceptionally warm summer.MethodsIn a retrospective study, we analysed demographics, geographical distribution, seasonality, causative species and severity of non-travel-related vibriosis cases in 2018. Data sources included surveillance systems, national laboratory notification databases and/or nationwide surveys to public health microbiology laboratories. Moreover, we performed whole genome sequencing and multilocus sequence typing of available isolates from 2014 to 2018 to map their genetic diversity.ResultsIn 2018, we identified 445 non-travel-related vibriosis cases in the study countries, considerably more than the median of 126 cases between 2014 and 2017 (range: 87-272). The main reported mode of transmission was exposure to seawater. We observed a species-specific geographical disparity of vibriosis cases across the Nordic-Baltic region. Severe vibriosis was associated with infections caused by Vibrio vulnificus (adjOR: 17.2; 95% CI: 3.3-90.5) or Vibrio parahaemolyticus (adjOR: 2.1; 95% CI: 1.0-4.5), age ≥ 65 years (65-79 years: adjOR: 3.9; 95% CI: 1.7-8.7; ≥ 80 years: adjOR: 15.5; 95% CI: 4.4-54.3) or acquiring infections during summer (adjOR: 5.1; 95% CI: 2.4-10.9). Although phylogenetic analysis revealed diversity between Vibrio spp. isolates, two V. vulnificus clusters were identified.ConclusionShared sentinel surveillance for vibriosis during summer may be valuable to monitor this emerging public health issue.

Categorical performance characteristics of method ISO 7899-2 and indicator value of intestinal enterococci for bathing water quality monitoring.

Intestinal enterococci indicate the fecal contamination of bathing waters. This study defines the performance characteristics of the reference method ISO 7899-2:2000 with water samples collected from inland and coastal bathing areas in Finland. From a total of 341 bacterial isolates grown on Slanetz and Bartley medium, 63.6% were confirmed as intestinal enterococci on bile aesculin agar. The partial 16S rRNA gene sequences showed that Enterococcus faecium and Enterococcus faecalis clades accounted for 93.1% of the confirmed isolates. The range of the false positive and false negative rate of the ISO 7899-2 was 0.0-18.5% and 5.6-57.1%, respectively, being affected by the presumptive colony count on the membrane. The analysis of multiple sample volumes is proposed to reach 10-100 colonies per membrane when 47 mm diameter membranes are used to prevent overestimation of low counts and underestimation of the high counts.

On-line detection of Escherichia coli intrusion in a pilot-scale drinking water distribution system.

Improvements in microbial drinking water quality monitoring are needed for the better control of drinking water distribution systems and for public health protection. Conventional water quality monitoring programmes are not always able to detect a microbial contamination of drinking water. In the drinking water production chain, in addition to the vulnerability of source waters, the distribution networks are prone to contamination. In this study, a pilot-scale drinking-water distribution network with an on-line monitoring system was utilized for detecting bacterial intrusion. During the experimental Escherichia coli intrusions, the contaminant was measured by applying a set of on-line sensors for electric conductivity (EC), pH, temperature (T), turbidity, UV-absorbance at 254 nm (UVAS SC) and with a device for particle counting. Monitored parameters were compared with the measured E. coli counts using the integral calculations of the detected peaks. EC measurement gave the strongest signal compared with the measured baseline during the E. coli intrusion. Integral calculations showed that the peaks in the EC, pH, T, turbidity and UVAS SC data were detected corresponding to the time predicted. However, the pH and temperature peaks detected were barely above the measured baseline and could easily be mixed with the background noise. The results indicate that on-line monitoring can be utilized for the rapid detection of microbial contaminants in the drinking water distribution system although the peak interpretation has to be performed carefully to avoid being mixed up with normal variations in the measurement data.

Comparison of Colilert-18 with miniaturised most probable number method for monitoring of Escherichia coli in bathing water.

The purpose of this equivalence study was to compare an alternative method, Colilert-18 Quanti-Tray (ISO 9308-2) with the European bathing water directive (2006/7/EC) reference method, the miniaturised most probable number (MMPN) method (ISO 9308-3), for the analysis of Escherichia coli. Six laboratories analysed a total of 263 bathing water samples in Finland. The comparison was carried out according to ISO 17994:2004. The recovery of E. coli using the Colilert-18 method was 7.0% and 8.6% lower than that of the MMPN method after 48 hours and 72 hours of incubation, respectively. The confirmation rate of presumptive E. coli-positive wells in the Colilert-18 and MMPN methods was high (97.8% and 98.0%, respectively). However, the testing of presumptive E. coli-negative but coliform bacteria-positive (yellow but not fluorescent) Colilert-18 wells revealed 7.3% false negative results. There were more false negatives in the naturally contaminated waters than in the samples spiked with waste water. The difference between the recovery of Colilert-18 and the MMPN method was considered not significant, and subsequently the methods are considered as equivalent for bathing water quality monitoring in Finland. Future bathing water method equivalence verification studies may use the data reported herein. The laboratories should make sure that any wells showing even minor fluorescence will be determined as positive for E. coli.

Distribution of human-specific bacteroidales and fecal indicator bacteria in an urban watershed impacted by sewage pollution, determined using RNA- and DNA-based quantitative PCR assays.

The identification of fecal pollution sources is commonly carried out using DNA-based methods. However, there is evidence that DNA can be associated with dead cells or present as "naked DNA" in the environment. Furthermore, it has been shown that rRNA-targeted reverse transcription-quantitative PCR (RT-qPCR) assays can be more sensitive than rRNA gene-based qPCR assays since metabolically active cells usually contain higher numbers of ribosomes than quiescent cells. To this end, we compared the detection frequency of host-specific markers and fecal bacteria using RNA-based RT-qPCR and DNA-based qPCR methods for water samples collected in sites impacted by combined sewer overflows. As a group, fecal bacteria were more frequently detected in most sites using RNA-based methods. Specifically, 8, 87, and 85% of the samples positive for general enterococci, Enterococcus faecalis, and Enterococcus faecium markers, respectively, were detected using RT-qPCR, but not with the qPCR assay counterpart. On average, two human-specific Bacteroidales markers were not detected when using DNA in 12% of the samples, while they were positive for all samples when using RNA (cDNA) as the template. Moreover, signal intensity was up to three orders of magnitude higher in RT-qPCR assays than in qPCR assays. The human-specific Bacteroidales markers exhibited moderate correlation with conventional fecal indicators using RT-qPCR results, suggesting the persistence of nonhuman sources of fecal pollution or the presence of false-positive signals. In general, the results from this study suggest that RNA-based assays can increase the detection sensitivity of fecal bacteria in urban watersheds impacted with human fecal sources.

Sand filters for removal of microbes and nutrients from wastewater during a one-year pilot study in a cold temperate climate.

Onsite wastewater treatment systems (OWTS) are recognised as potential threats to groundwater or other water environments subject to discharged effluents. In this study, the microbiological and nutrient removal properties of three different pilot-scale sand filters (SFs) were followed over a one-year period. Moreover, a separate phosphorus removal unit was tested for six months. For the best treatment system, the average log removals were 2.2-3.5 for pathogenic human noro- and adenoviruses and 4.3-5.2 and 4.6-5.4 for indicator viruses and bacteria, respectively. The system that effectively removed microbes was also efficient at removing nutrients. However, the poorest treatment system yielded substantially lower removals. The remarkable differences noted between the studied SFs highlights the importance of construction materials and the careful planning of the filters. Moreover, seasonal conditions appear to have a clear effect on purification efficiencies, emphasising the vulnerability of these systems especially in cold climates.

Key considerations for pathogen surveillance in wastewater.

Wastewater surveillance (WWS) has received significant attention as a rapid, sensitive, and cost-effective tool for monitoring various pathogens in a community. WWS is employed to assess the spatial and temporal trends of diseases and identify their early appearances and reappearances, as well as to detect novel and mutated variants. However, the shedding rates of pathogens vary significantly depending on factors such as disease severity, the physiology of affected individuals, and the characteristics of pathogen. Furthermore, pathogens may exhibit differential fate and decay kinetics in the sewerage system. Variable shedding rates and decay kinetics may affect the detection of pathogens in wastewater. This may influence the interpretation of results and the conclusions of WWS studies. When selecting a pathogen for WWS, it is essential to consider it's specific characteristics. If data are not readily available, factors such as fate, decay, and shedding rates should be assessed before conducting surveillance. Alternatively, these factors can be compared to those of similar pathogens for which such data are available.

Avian Influenza outbreaks: Human infection risks for beach users - One health concern and environmental surveillance implications.

Despite its popularity for water activities, such as swimming, surfing, fishing, and rafting, inland and coastal bathing areas occasionally experience outbreaks of highly pathogenic avian influenza virus (HPAI), including A(H5N1) clade 2.3.4.4b. Asymptomatic infections and symptomatic outbreaks often impact many aquatic birds, which increase chances of spill-over events to mammals and pose concerns for public health. This review examined the existing literature to assess avian influenza virus (AIV) transmission risks to beachgoers and the general population. A comprehensive understanding of factors governing such crossing of the AIV host range is currently lacking. There is limited knowledge on key factors affecting risk, such as species-specific interactions with host cells (including binding, entry, and replication via viral proteins hemagglutinin, neuraminidase, nucleoprotein, and polymerase basic protein 2), overcoming host restrictions, and innate immune response. AIV efficiently transmits between birds and to some extent between marine scavenger mammals in aquatic environments via consumption of infected birds. However, the current literature lacks evidence of zoonotic AIV transmission via contact with the aquatic environment or consumption of contaminated water. The zoonotic transmission risk of the circulating A(H5N1) clade 2.3.4.4b virus to the general population and beachgoers is currently low. Nevertheless, it is recommended to avoid direct contact with sick or dead birds and to refrain from bathing in locations where mass bird mortalities are reported. Increasing reports of AIVs spilling over to non-human mammals have raised valid concerns about possible virus mutations that lead to crossing the species barrier and subsequent risk of human infections and outbreaks.

Scenario-based assessment of fecal pathogen sources affecting bathing water quality: novel treatment options to reduce norovirus and Campylobacter infection risks.

Wastewater discharge and runoff waters are significant sources of human and animal fecal microbes in surface waters. Human-derived fecal contamination of water is generally estimated to pose a greater risk to human health than animal fecal contamination, but animals may serve as reservoirs of zoonotic pathogens. In this study, quantitative microbial risk assessment (QMRA) tools were used to evaluate the hygienic impact of sewage effluents and runoff water from municipalities and animal farms on surface and bathing waters. The human-specific microbial source tracking (MST) marker HF183 was used to evaluate the dilution of fecal pathogens originating from the sewage effluent discharge to the downstream watershed. As novel risk management options, the efficiency of UV-LED disinfection and wetland treatment as well as biochar filtration was tested on-site for the contamination sources. According to the dilution pattern of the MST marker HF183, microbes from wastewater were diluted (2.3-3.7 log10) in the receiving waters. The scenario-based QMRA revealed, that the health risks posed by exposure to human-specific norovirus GII and zoonotic Campylobacter jejuni during the bathing events were evaluated. The risk for gastroenteritis was found to be elevated during wastewater contamination events, where especially norovirus GII infection risk increased (1-15 cases per day among 50 bathers) compared with the business as usual (BAU) situation (1 case per day). The noted C. jejuni infection risk was associated with animal farm contamination (1 case per day, versus 0.2-0.6 cases during BAU). Tertiary treatment of wastewater with wetland treatment and UV-LED disinfection effectively reduced the waterborne gastroenteritis risks associated with bathing. Based on the experiences from this study, a QMRA-based approach for health risk evaluations at bathing sites can be useful and is recommended for bathing site risk assessments in the future. In case of low pathogen numbers at the exposure sites, the MST marker HF183 could be used as a pathogen dilution coefficient for the watershed under evaluation. The full-scale implementation of novel tertiary treatment options at wastewater treatment plants (WWTPs) as well as on-site runoff water treatment options should be considered for infection risk management at locations where scenario-based QMRA implies elevated infection risks.

Wastewater-based surveillance is an efficient monitoring tool for tracking influenza A in the community.

Around the world, influenza A virus has caused severe pandemics, and the risk of future pandemics remains high. Currently, influenza A virus surveillance is based on the clinical diagnosis and reporting of disease cases. In this study, we apply wastewater-based surveillance to monitor the amount of the influenza A virus RNA at the population level. We report the influenza A virus RNA levels in 10 wastewater treatment plant catchment areas covering 40 % of the Finnish population. Altogether, 251 monthly composite influent wastewater samples (collected between February 2021 and February 2023) were analysed from supernatant fraction using influenza A virus specific RT-qPCR method. During the study period, an influenza A virus epidemic occurred in three waves in Finland. This study shows that the influenza A virus RNA can be detected from the supernatant fraction of 24 h composite influent wastewater samples. The influenza A virus RNA gene copy number in wastewater correlated with the number of confirmed disease cases in the Finnish National Infectious Diseases Register. The median Kendall's τ correlation strength was 0.636 (min= 0.486 and max=0.804) and it was statistically significant in all 10 WTTPs. Wastewater-based surveillance of the influenza A virus RNA is an independent from individual testing method and cost-efficiently reflects the circulation of the virus in the entire population. Thus, wastewater monitoring complements the available, but often too sparse, information from individual testing and improves health care and public health preparedness for influenza A virus pandemics.

Clinically relevant sequence types of carbapenemase-producing Escherichia coli and Klebsiella pneumoniae detected in Finnish wastewater in 2021-2022.

BACKGROUND: Antimicrobial resistance (AMR) is a critical threat to human health. Escherichia coli and Klebsiella pneumoniae are clinically the most important species associated with AMR and are the most common carbapenemase-producing (CP) Enterobacterales detected in human specimens in Finland. Wastewater surveillance has emerged as a potential approach for population-level surveillance of AMR, as wastewater could offer a reflection from a larger population with one sample and minimal recognized ethical issues. In this study, we investigated the potential of wastewater surveillance to detect CP E. coli and K. pneumoniae strains similar to those detected in human specimens. METHODS: Altogether, 89 composite samples of untreated community wastewater were collected from 10 wastewater treatment plants across Finland in 2021-2022. CP E. coli and K. pneumoniae were isolated using selective culture media and identified using MALDI-TOF MS. Antimicrobial susceptibility testing was performed using disk diffusion test and broth microdilution method, and a subset of isolates was characterized using whole-genome sequencing. RESULTS: CP E. coli was detected in 26 (29.2%) and K. pneumoniae in 25 (28.1%) samples. Among E. coli, the most common sequence type (ST) was ST410 (n = 7/26, 26.9%), while ST359 (n = 4/25, 16.0%) predominated among K. pneumoniae. Globally successful STs were detected in both E. coli (ST410, ST1284, ST167, and ST405) and K. pneumoniae (ST512, ST101, and ST307). K. pneumoniae carbapenemases (KPC) were the most common carbapenemases in both E. coli (n = 11/26, 42.3%) and K. pneumoniae (n = 13/25, 52.0%), yet also other carbapenemases, such as blaNDM-5, blaOXA-48, and blaOXA-181, were detected. We detected isolates harboring similar ST and enzyme type combinations previously linked to clusters in Finland, such as E. coli ST410 with blaKPC-2 and K. pneumoniae ST512 with blaKPC-3. CONCLUSIONS: Our study highlights the presence of clinically relevant strains of CP E. coli and K. pneumoniae in community wastewater. The results indicate that wastewater surveillance could serve as a monitoring tool for CP Enterobacterales. However, the specificity and sensitivity of the methods should be improved, and technologies, like advanced sequencing methods, should be utilized to distinguish data with public health relevance, harness the full potential of wastewater surveillance, and implement the data in public health surveillance.

Detection of SARS-COV-2 variants and their proportions in wastewater samples using next-generation sequencing in Finland.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variants may have different characteristics, e.g., in transmission, mortality, and the effectiveness of vaccines, indicating the importance of variant detection at the population level. Wastewater-based surveillance of SARS-CoV-2 RNA fragments has been shown to be an effective way to monitor the COVID-19 pandemic at the population level. Wastewater is a complex sample matrix affected by environmental factors and PCR inhibitors, causing insufficient coverage in sequencing, for example. Subsequently, results where part of the genome does not have sufficient coverage are not uncommon. To identify variants and their proportions in wastewater over time, we utilized next-generation sequencing with the ARTIC Network's primer set and bioinformatics pipeline to evaluate the presence of variants in partial genome data. Based on the wastewater data from November 2021 to February 2022, the Delta variant was dominant until mid-December in Helsinki, Finland's capital, and thereafter in late December 2022 Omicron became the most common variant. At the same time, the Omicron variant of SARS-CoV-2 outcompeted the previous Delta variant in Finland in new COVID-19 cases. The SARS-CoV-2 variant findings from wastewater are in agreement with the variant information obtained from the patient samples when visually comparing trends in the sewerage network area. This indicates that the sequencing of wastewater is an effective way to monitor temporal and spatial trends of SARS-CoV-2 variants at the population level.

Developing wastewater-based surveillance schemes for multiple pathogens: The WastPan project in Finland.

Wastewater comprises multiple pathogens and offers a potential for wastewater-based surveillance (WBS) to track the prevalence of communicable diseases. The Finnish WastPan project aimed to establish wastewater-based pandemic preparedness for multiple pathogens (viruses, bacteria, parasites, fungi), including antimicrobial resistance (AMR). This article outlines WastPan's experiences in this project, including the criteria for target selection, sampling locations, frequency, analysis methods, and results communication. Target selection relied on epidemiological and microbiological evidence and practical feasibility. Within the WastPan framework, wastewater samples were collected between 2021 and 2023 from 10 wastewater treatment plants (WWTPs) covering 40 % of Finland's population. WWTP selection was validated for reported cases of Extended Spectrum Beta-lactamase-producing bacterial pathogens (Escherichia coli and Klebsiella pneumoniae) from the National Infectious Disease Register. The workflow included 24-h composite influent samples, with one fraction for culture-based analysis (bacteria and fungi) and the rest of the sample was reserved for molecular analysis (viruses, bacteria, antibiotic resistance genes, and parasites). The reproducibility of the monitoring workflow was assessed for SARS-CoV-2 through inter-laboratory comparisons using the N2 and N1 assays. Identical protocols were applied to same-day samples, yielding similar positivity trends in the two laboratories, but the N2 assay achieved a significantly higher detection rate (Laboratory 1: 91.5 %; Laboratory 2: 87.4 %) than the N1 assay (76.6 %) monitored only in Laboratory 2 (McNemar, p < 0.001 Lab 1, = 0.006 Lab 2). This result indicates that the selection of monitoring primers and assays may impact monitoring sensitivity in WBS. Overall, the current study recommends that the selection of sampling frequencies and population coverage of the monitoring should be based on pathogen-specific epidemiological characteristics. For example, pathogens that are stable over time may need less frequent annual sampling, while those that are occurring across regions may require reduced sample coverage. Here, WastPan successfully piloted WBS for monitoring multiple pathogens, highlighting the significance of one-litre community composite wastewater samples for assessing community health. The infrastructure established for COVID-19 WBS is valuable for monitoring various pathogens. The prioritization of the monitoring targets optimizes resource utilization. In the future legislative support in target selection, coverage determination, and sustained funding for WBS is recomended.

Composition of active bacterial communities and presence of opportunistic pathogens in disinfected and non-disinfected drinking water distribution systems in Finland.

Many factors, including microbiome structure and activity in the drinking water distribution system (DWDS), affect the colonization potential of opportunistic pathogens. The present study aims to describe the dynamics of active bacterial communities in DWDS and identify the factors that shape the community structures and activity in the selected DWDSs. Large-volume drinking water and hot water, biofilm, and water meter deposit samples were collected from five DWDSs. Total nucleic acids were extracted, and RNA was further purified and transcribed into its cDNA from a total of 181 water and biofilm samples originating from the DWDS of two surface water supplies (disinfected with UV and chlorine), two artificially recharged groundwater supplies (non-disinfected), and a groundwater supply (disinfected with UV and chlorine). In chlorinated DWDSs, concentrations of <0.02-0.97 mg/l free chlorine were measured. Bacterial communities in the RNA and DNA fractions were analysed using Illumina MiSeq sequencing with primer pair 341F-785R targeted to the 16S rRNA gene. The sequence libraries were analysed using QIIME pipeline, Program R, and MicrobiomeAnalyst. Not all bacterial cells were active based on their 16S rRNA content, and species richness was lower in the RNA fraction (Chao1 mean value 490) than in the DNA fraction (710). Species richness was higher in the two DWDSs distributing non-disinfected artificial groundwater (Chao1 mean values of 990 and 1 000) as compared to the two disinfected DWDSs using surface water (Chao1 mean values 190 and 460) and disinfected DWDS using ground water as source water (170). The difference in community structures between non-disinfected and disinfected water was clear in the beta-diversity analysis. Distance from the waterworks also affected the beta diversity of community structures, especially in disinfected distribution systems. The two most abundant bacteria in the active part of the community (RNA) and total bacterial community (DNA) belonged to the classes Alphaproteobacteria (RNA 28 %, DNA 44 %) and Gammaproteobacteria (RNA 32 %, DNA 30 %). The third most abundant and active bacteria class was Vampirovibrionia (RNA 15 %), whereas in the total community it was Paceibacteria (DNA 11 %). Class Nitrospiria was more abundant and active in both cold and hot water in DWDS that used chloramine disinfection compared to non-chlorinated or chlorine-using DWDSs. Thirty-eight operational taxonomic units (OTU) of Legionella, 30 of Mycobacterium, and 10 of Pseudomonas were detected among the sequences. The (RT)-qPCR confirmed the presence of opportunistic pathogens in the DWDSs studied as Legionella spp. was detected in 85 % (mean value 4.5 × 104 gene copies/100 ml), Mycobacterium spp. in 95 % (mean value 8.3 × 106 gene copies/100 ml), and Pseudomonas spp. in 78 % (mean value 1.6 × 105 gene copies/100 ml) of the water and biofilm samples. Sampling point inside the system (distance from the waterworks and cold/hot system) affected the active bacterial community composition. Chloramine as a chlorination method resulted in a recognizable community composition, with high abundance of bacteria that benefit from the excess presence of nitrogen. The results presented here confirm that each DWDS is unique and that opportunistic pathogens are present even in conditions when water quality is considered excellent.

Detection of fecal bacteria and source tracking identifiers in environmental waters using rRNA-based RT-qPCR and rDNA-based qPCR assays.

In this study, we evaluated the use of RT-qPCR assays targeting rRNA gene sequences for the detection of fecal bacteria in water samples. We challenged the RT-qPCR assays against RNA extracted from sewage effluent (n = 14), surface water (n = 30), and treated source water (n = 15) samples. Additionally, we applied the same assays using DNA as the qPCR template. The targeted fecal bacteria were present in most of the samples tested, although in several cases, the detection frequency increased when RNA was used as the template. For example, the majority of samples that tested positive for E. coli and Campylobacter spp. in surface waters, and for human-specific Bacteroidales, E. coli, and Enterococcus spp. in treated source waters were only detected when rRNA was used as the original template. The difference in detection frequency using rRNA or rDNA (rRNA gene) was sample- and assay-dependent, suggesting that the abundance of active and nonactive populations differed between samples. Statistical analyses for each population exhibiting multiple quantifiable results showed that the rRNA copy numbers were significantly higher than the rDNA counterparts (p < 0.05). Moreover, the detection frequency of rRNA-based assays were in better agreement with the culture-based results of E. coli, intestinal enterococci, and thermotolerant Campylobacter spp. in surface waters than that of rDNA-based assays, suggesting that rRNA signals were associated to active bacterial populations. Our data show that using rRNA-based approaches significantly increases detection sensitivity for common fecal bacteria in environmental waters. These findings have important implications for microbial water quality monitoring and public health risk assessments.

Manual faucets induce more biofilms than electronic faucets.

Electronic faucets (types E1 and E2) and manual (M) faucets were studied for microbial quality, i.e., biomass and pathogenic microbes of biofilms in the faucet aerator, the water, and the outer surface of faucet in a hospital in Finland. Heterotrophic plate count content reflecting culturable microbial biomass and adenosine triphosphate content representing viable microbial biomass were smaller in the biofilms of E1-type electronic faucets than E2-type electronic faucets or M faucets. The likely explanation is the mixing point of cold and hot water (E1 and M: in the faucet; E2: in a separate box 50 cm before the actual faucet part). The highest amounts of Legionella (serogroups 2-15 of Legionella pneumophila) in a water sample (5000 cfu/L) and in biofilm samples (May-June 2008 sampling: 240 cfu/mL; November 2008: 1100 cfu/mL) were found in one E1-type faucet, which was lacking a back pressure valve due to faulty installation. This study reveals that certain types of electronic faucets seem to promote hospital hygiene, as they were associated with less microbial growth in biofilms in the faucet aerator, than some other types of electronic faucets or manual faucets, likely owing to the mixing point of cold and hot water. However, the faucet type had no direct effect on the presence of Legionella spp. Also correct installation is crucial.

Occurrence of thermotolerant Campylobacter spp. and adenoviruses in Finnish bathing waters and purified sewage effluents.

A total of 50 Finnish bathing water samples and 34 sewage effluent samples originating from 17 locations were studied in the summers of 2006 and 2007. Campylobacter were present in 58% and adenoviruses in 12% of all bathing water samples; 53% of all sewage effluent samples were positive for Campylobacter spp. and 59% for adenoviruses. C. jejuni was the most common Campylobacter species found and human adenovirus serotype 41 was the most common identified adenovirus type. Bathing water temperature displayed a significant negative relationship with the occurrence of Campylobacter. One location had identical pulsed-field gel electrophoresis patterns of C. coli isolates in the bathing water and in sewage effluent, suggesting that sewage effluent was the source of C. coli at this bathing site. The counts of faecal indicator bacteria were not able to predict the presence of Campylobacter spp. or adenoviruses in the bathing waters. Thus the observed common presence of these pathogens in Finnish sewage effluents and bathing waters may represent a public health risk. The low water temperature in Finland may enhance the prevalence of Campylobacter in bathing waters. More attention needs to be paid to minimizing the concentrations of intestinal pathogens in bathing waters.

Microbial reduction in wastewater treatment using Fe(3+) and Al(3+) coagulants and PAA disinfectant.

Wastewater is an important source of pathogenic enteric microorganisms in surface water and a major contaminating agent of drinking water. Although primary and secondary wastewater treatments reduce the numbers of microorganisms in wastewater, significant numbers of microbes can still be present in the effluent. The aim of this study was to test the feasibility of tertiary treatment for municipal wastewater treatment plants (WWTPs) using PIX (FeCl3) or PAX (AlCl3) coagulants and peracetic acid (PAA) the disinfectant to reduce microbial load in effluent. Our study showed that both PIX and PAX efficiently reduced microbial numbers. PAA disinfection greatly reduced the numbers of culturable indicator microorganisms (Escherichia coli, intestinal enterococci, F-specific RNA coliphages and somatic DNA coliphages). In addition, pathogenic microorganisms, thermotolerant Campylobacter, Salmonella and norovirus GI, were successfully reduced using the tertiary treatments. In contrast, clostridia, Legionella, rotavirus, norovirus GII and adenovirus showed better resistance against PAA compared to the other microorganisms. However, interpretation of polymerase chain reaction (PCR) analysis results will need further studies to clarify the infectivity of the pathogenic microbes. In conclusion, PIX and PAX flocculants followed by PAA disinfectant can be used as a tertiary treatment for municipal WWTP effluents to reduce the numbers of indicator and pathogenic microorganisms.

Clostridium difficile contamination of public tap water distribution system during a waterborne outbreak in Finland.

AIMS: In November through December 2007, the drinking water distribution system in the town of Nokia, Finland, was contaminated with treated sewage effluent that resulted in a large gastroenteritis outbreak in the community. The aim of the present study was to investigate if the contaminated water in this outbreak was also a potential source of Clostridium difficile infections. METHODS: Samples from the contaminated tap water and treated sewage effluent were collected. Stool samples from a portion of patients that fell ill during the outbreak were examined for C. difficile. PCR ribotyping was performed on toxin positive C. difficile isolates and the genetic profiles of the water and patient isolates were compared. RESULTS: Twelve toxin-positive C. difficile isolates were found in water samples: five from contaminated tap water and seven from treated sewage effluent. Among these, four and five distinct PCR ribotype profiles were identified, respectively. Four PCR ribotype profiles were found among nine human faecal C. difficile isolates. Two isolates, one from tap water and one from a patient, had an indistinguishable PCR ribotype profile. CONCLUSIONS: Our findings demonstrate for the first time C. difficile contamination of a tap water distribution system and waterborne transmission of toxigenic C. difficile seems possible.