Infectious rain? Evaluation of human pathogen concentrations in stormwater in separate sewer systems.

Separate sewer systems collect and discharge rainwater directly into surface water bodies. In residential areas covering moderate traffic load these are alternative drainage routes to avoid combined sewer overflow discharge and to keep rivers clean as required by the EU Water Framework Directive. This overflow's microbial quality, however, needs to be evaluated, since stormwater run-offs are potential pathways for pathogens into river systems. Between 2010 and 2016, two separate sewer systems in North Rhine-Westphalia (Germany) were investigated. The stormwater outflow was sampled during discharge events and microbiologically analysed. The results showed high concentrations of Escherichia coli (1,100-1,100,000 CFU/100 mL) and Clostridium perfringens (20-13,000 CFU/100 mL). Campylobacter and Salmonella were detected in 97% and 43% of the samples. Giardia cysts were more often detected (31.6%) than Cryptosporidium oocysts (10.5%). The sources of human pathogens in rainwater run-off are heterogeneous. While roads have already been declared as chemical polluters via rainwater run-off, our study detected supplementary pollution of mainly faecal microorganisms. Presumably, failed connections in the sewer system itself are important sources of human pathogens. We suggest treatment of stormwater run-offs before being discharged into the river system.

Antibiotic resistance among autochthonous aquatic environmental bacteria.

Antibiotics are widely used in both human and veterinary medicine and antibiotic-resistant bacteria cause problems in antibiotic therapy. The current study was conducted in the catchment area of the river Swist (Germany) and focuses on the resistance of environmental Rhodospirillaceae to antibiotics used in human medicine. The samples collected reflect different levels of human impact on the environment. In total, 614 isolates were tested for antibiotic susceptibility. About half of these isolates were susceptible to all substances tested. Oxacillin resistance was observed most frequently (41%). Resistant Rhodospirillaceae were detected in wastewater effluent from a municipal sewage treatment plant, as well as in non-polluted upper reaches. The highest multi-resistance level was detected in small tributaries and it surprisingly decreased with an increasing influence of municipal wastewater. It could be shown that the detected resistances were acquired rather than intrinsic. Besides natural occurrence of multi-resistance among non-sulphur purple bacteria, horizontal gene transfer and acquired cross-resistance against veterinary antibiotics are assumed to be important factors. To the authors' knowledge, this is the first study investigating the potential of Rhodospirillaceae as a reservoir for resistance to antibiotics used in human medicine. The consequence for resistance prevalence in human pathogens and for their antibiotic therapy needs evaluation.

Agroecosystem exploration for Antimicrobial Resistance in Ahmedabad, India: A Study Protocol.

Introduction: Antimicrobial resistance (AMR) has emerged as one of the leading threats to public health. AMR possesses a multidimensional challenge that has social, economic, and environmental dimensions that encompass the food production system, influencing human and animal health. The One Health approach highlights the inextricable linkage and interdependence between the health of people, animal, agriculture, and the environment. Antibiotic use in any of these areas can potentially impact the health of others. There is a dearth of evidence on AMR from the natural environment, such as the plant-based agriculture sector. Antibiotics, antibiotic-resistant bacteria (ARB), and related AMR genes (ARGs) are assumed to present in the natural environment and disseminate resistance to fresh produce/vegetables and thus to human health upon consumption. Therefore, this study aims to investigate the role of vegetables in the spread of AMR through an agroecosystem exploration in Ahmedabad, India. Protocol: The present study will be executed in Ahmedabad, located in Gujarat state in the Western part of India, by adopting a mixed-method approach. First, a systematic review will be conducted to document the prevalence of ARB and ARGs on fresh produce in South Asia. Second, agriculture farmland surveys will be used to collect the general farming practices and the data on common vegetables consumed raw by the households in Ahmedabad. Third, vegetable and soil samples will be collected from the selected agriculture farms and analyzed for the presence or absence of ARB and ARGs using standard microbiological and molecular methods. Discussion: The analysis will help to understand the spread of ARB/ARGs through the agroecosystem. This is anticipated to provide an insight into the current state of ARB/ARGs contamination of fresh produce/vegetables and will assist in identifying the relevant strategies for effectively controlling and preventing the spread of AMR.

Climate Change Impact Assessment of Food- and Waterborne Diseases.

The PubMed and ScienceDirect bibliographic databases were searched for the period of 1998-2009 to evaluate the impact of climatic and environmental determinants on food- and waterborne diseases. The authors assessed 1,642 short and concise sentences (key facts), which were extracted from 722 relevant articles and stored in a climate change knowledge base. Key facts pertaining to temperature, precipitation, water, and food for 6 selected pathogens were scrutinized, evaluated, and compiled according to exposure pathways. These key facts (corresponding to approximately 50,000 words) were mapped to 275 terminology terms identified in the literature, which generated 6,341 connections. These relationships were plotted on semantic network maps to examine the interconnections between variables. The risk of campylobacteriosis is associated with mean weekly temperatures, although this link is shown more strongly in the literature relating to salmonellosis. Irregular and severe rain events are associated with Cryptosporidium sp. outbreaks, while noncholera Vibrio sp. displays increased growth rates in coastal waters during hot summers. In contrast, for Norovirus and Listeria sp. the association with climatic variables was relatively weak, but much stronger for food determinants. Electronic data mining to assess the impact of climate change on food- and waterborne diseases assured a methodical appraisal of the field. This climate change knowledge base can support national climate change vulnerability, impact, and adaptation assessments and facilitate the management of future threats from infectious diseases. In the light of diminishing resources for public health this approach can help balance different climate change adaptation options.

Facultative pathogenic bacteria and antibiotic resistance genes in swine livestock manure and clinical wastewater: A molecular biology comparison.

Manure contains vast amounts of biological contaminants of veterinary origin. Only few studies analyse clinically critical resistance genes against reserve antibiotics in manure. In general, resistances against these high priority antibiotics involve a high potential health risk. Therefore, their spread in the soil as well as the aquatic environment has to be prevented. Manures of 29 different swine livestock were analysed. Abundances of facultative pathogenic bacteria including representatives of the clinically critical ESKAPE-pathogens (P. aeruginosa, K. pneumoniae, A. baumannii, E. faecium) and E. coli were investigated via qPCR. Antibiotic resistance genes against commonly used veterinary antibiotics (ermB, tetM, sul1) as well as various resistance genes against important (mecA, vanA) and reserve antibiotics (blaNDM, blaKPC3, mcr-1), which are identified by the WHO, were also obtained by qPCR analysis. The manures of all swine livestock contained facultative pathogenic bacteria and commonly known resistance genes against antibiotics used in veterinary therapies, but more important also a significant amount of clinically critical resistance genes against reserve antibiotics for human medicine. To illustrate the impact the occurrence of these clinically critical resistance genes, comparative measurements were taken of the total wastewater of a large tertiary care hospital (n = 8). Both manure as well as raw hospital wastewaters were contaminated with significant abundances of gene markers for facultative pathogens and with critical resistance genes of reserve antibiotics associated with genetic mobile elements for horizontal gene transfer. Hence, both compartments bear an exceptional potential risk for the dissemination of facultative pathogens and critical antibiotic resistance genes.

Dissemination of carbapenem resistant bacteria from hospital wastewater into the environment.

Infections with antibiotic resistant pathogens threaten lives and cause substantial costs. For effective interventions, knowledge of the transmission paths of resistant bacteria to humans is essential. In this study, carbapenem resistant bacteria were isolated from the wastewater of a maximum care hospital during a period of two years, starting in the patient rooms and following the sewer system to the effluent of the wastewater treatment plant (WWTP). The bacteria belonged to six different species and 44 different sequence types (STs). The most frequent STs, ST147 K. pneumoniae (blaNDM/blaOXA-48) and ST235 P. aeruginosa (blaVIM) strains, were present at nearly all sampling sites from the hospital to the WWTP effluent. After core genome multi-locus sequence typing (cgMLST), all ST147 K. pneumoniae strains presented a single epidemiological cluster. In contrast, ST235 P. aeruginosa formed five cgMLST clusters and the largest cluster contained the strain from the WWTP effluent, indicating without doubt, a direct dissemination of both high-risk clones into the environment. Thus, there are - at least two - possible transmission pathways to humans, (i) within the hospital by contact with the drains of the sanitary installations and (ii) by recreational or irrigation use of surface waters that have received WWTP effluent. In conclusion, remediation measures must be installed at both ends of the wastewater system, targeting the drains of the hospital as well as at the effluent of the WWTP.

Antibiotic-Resistant Bacteria in Clams-A Study on Mussels in the River Rhine.

Bacterial infections have been treated effectively by antibiotics since the discovery of penicillin in 1928. A worldwide increase in the use of antibiotics led to the emergence of antibiotic resistant strains in almost all bacterial pathogens, which complicates the treatment of infectious diseases. Antibiotic-resistant bacteria play an important role in increasing the risk associated with the usage of surface waters (e.g., irrigation, recreation) and the spread of the resistance genes. Many studies show that important pathogenic antibiotic-resistant bacteria can enter the environment by the discharge of sewage treatment plants and combined sewage overflow events. Mussels have successfully been used as bio-indicators of heavy metals, chemicals and parasites; they may also be efficient bio-indicators for viruses and bacteria. In this study an influence of the discharge of a sewage treatment plant could be shown in regard to the presence of E. coli in higher concentrations in the mussels downstream the treatment plant. Antibiotic-resistant bacteria, resistant against one or two classes of antibiotics and relevance for human health could be detected in the mussels at different sampling sites of the river Rhine. No multidrug-resistant bacteria could be isolated from the mussels, although they were found in samples of the surrounding water body.

Clinically relevant antibiotic-resistant bacteria in aquatic environments - An optimized culture-based approach.

The emergence of antibiotic-resistant clinically relevant facultative pathogenic bacteria in the environment has become one of the most important global health challenges. Antibiotic-resistant bacteria (ARB) have been found in surface waters and wastewater treatment plants. Drinking water guidelines and the EU bathing water directive 2006/7/EC include the surveillance of defined microbiological parameters on species level, while the monitoring of ARB is missing in all existing guidelines. However, standardized methods for the detection of ARB exist for clinical investigations of human materials only. They are based on cultivation on selective agar plates. These methods cannot be used directly for environmental samples, because of the high amount and diversity of bacterial background flora which interferes with the detection of human-relevant ARB. The aim of this study was to introduce a proposal for future normative standard operation procedures, with international relevance, for the culture-based detection of clinically-relevant antibiotic resistant bacteria in aquatic environmental samples like wastewater and surface water: gram-negative bacteria resistant against 3rd generation cephalosporins (ESBL) and against carbapenems (CARBA), gram-positive vancomycin-resistant enterococci (VRE) and methicillin-resistant Staphylococcus aureus (MRSA). The final adaptation of standardized cultivation methods included increasing the standard incubation temperature from 36 °C to 42 °C, which effectively inhibits the environmental background flora on agar plates while the desired target species survive. This enables the detection of target species in suitable sample volumes. Putative target colonies which belong to the remaining background flora had to be excluded by morphological and physiological differentiation. Therefore, a time and cost optimized testing scheme with good performance was developed, which allows an effective exclusion of non-target isolates in samples. Depending on the target species and sample type, sensitivity of up to 100% is achieved, and specificity ranges from 91.1% to 99.7%, while the positive predictive value, negative predicted value and accuracy rate are always >90%.

Bacteria isolated from hospital, municipal and slaughterhouse wastewaters show characteristic, different resistance profiles.

Multidrug-resistant bacteria cause difficult-to-treat infections and pose a risk for modern medicine. Sources of multidrug-resistant bacteria include hospital, municipal and slaughterhouse wastewaters. In this study, bacteria with resistance to 3rd generation cephalosporins were isolated from all three wastewater biotopes, including a maximum care hospital, municipal wastewaters collected separately from a city and small rural towns and the wastewaters of two pig and two poultry slaughterhouses. The resistance profiles of all isolates against clinically relevant antibiotics (including ß-lactams like carbapenems, the quinolone ciprofloxacin, colistin, and trimethoprim/sulfamethoxazole) were determined at the same laboratory. The bacteria were classified according to their risk to human health using clinical criteria, with an emphasis on producers of carbapenemases, since carbapenems are prescribed for hospitalized patients with infections with multi-drug resistant bacteria. The results showed that bacteria that pose the highest risk, i. e., bacteria resistant to all ß-lactams including carbapenems and ciprofloxacin, were mainly disseminated by hospitals and were present only in low amounts in municipal wastewater. The isolates from hospital wastewater also showed the highest rates of resistance against antibiotics used for treatment of carbapenemase producers and some isolates were susceptible to only one antibiotic substance. In accordance with these results, qPCR of resistance genes showed that 90% of the daily load of carbapenemase genes entering the municipal wastewater treatment plant was supplied by the clinically influenced wastewater, which constituted approximately 6% of the wastewater at this sampling point. Likewise, the signature of the clinical wastewater was still visible in the resistance profiles of the bacteria isolated at the entry into the wastewater treatment plant. Carbapenemase producers were not detected in slaughterhouse wastewater, but strains harboring the colistin resistance gene mcr-1 could be isolated. Resistances against orally available antibiotics like ciprofloxacin and trimethoprim/sulfamethoxazole were widespread in strains from all three wastewaters.

Reduction of micropollutants and bacteria in a constructed wetland for combined sewer overflow treatment after 7 and 10 years of operation.

Repeated investigations on constructed wetlands for the treatment of combined sewer overflows, also named bioretention filters or retention soil filters, are necessary to provide information on their long-term performance. In this study, a sampling campaign was conducted on micropollutants, indicator microorganisms and standard parameters ten years after such filters were in operation and three years after the first investigation; it revealed that the filters lost capacity to remove chemical substances with no or only slow biological degradability. This was the case e.g. for phosphate (decrease from 29 to 11%), diclofenac (67 to 34%) and TCPP (34% to negative reduction). They continued to remove easily degradable parameters such as COD (stable around 75%) stably. The indicator microorganisms Escherichia coli (1.1/0.8 log10), intestinal enterococci (1.3/0.8 log10) and somatic coliphages (0.6/1.0 log10) showed comparably low process variations given the difficulties in sampling and analysing microbial parameters representatively as well as given natural variations in microbial behaviour and growth. Additionally, for bisphenol A, we found a temperature-related difference of removal efficiencies: while in the cold months (winter), the removal was only 53% on average, it increased to 90% in the warm months (summer). As for the long-term prospective of retention soil filters, decision-makers need to identify the most important pollutants in a specific catchment area and adapt the filter design accordingly. If pollutants are targeted that lead to an exhausted filtration capacity, post treatment or the exchange of charged filter material is necessary. However, for easily biologically degradable substances, so far, there is no limit in their use.

Reduction of bacteria and somatic coliphages in constructed wetlands for the treatment of combined sewer overflow (retention soil filters).

Combined sewer overflows (CSOs) introduce numerous pathogens from fecal contamination, such as bacteria and viruses, into surface waters, thus endangering human health. In Germany, retention soil filters (RSFs) treat CSOs at sensitive discharge points and can contribute to reducing these hygienically relevant microorganisms. In this study, we evaluated the extent of how dry period, series connection and filter layer thickness influence the reduction efficiency of RSFs for Escherichia coli (E. coli), intestinal enterococci (I. E.) and somatic coliphages. To accomplish this, we had four pilot scale RSFs built on a test field at the wastewater treatment plant Aachen-Soers. While two filters were replicates, the other two filters were installed in a series connection. Moreover, one filter had a thinner filtration layer than the other three. Between April 2015 and December 2016, the RSFs were loaded in 37 trials with pre-conditioned CSO after dry periods ranging from 4 to 40 days. During 17 trials, samples for microbial analysis were taken and analyzed. The series connection of two filters showed that the removal increases when two systems with a filter layer of the same height are operated in series. Since the microorganisms are exposed twice to the environmental conditions on the filter surface and in the upper filter layers, there is a greater chance for abiotic adsorption increase. The same effect could be shown when filters with different depths were compared: the removal efficiency increases as filter thickness increases. This study provides new evidence that regardless of seasonal effects and dry period, RSFs can improve hygienic situation significantly.

Dissemination of multi-resistant Gram-negative bacteria into German wastewater and surface waters.

Carbapenem antibiotics constitute the mainstay therapy of nosocomial infections with extended spectrum beta-lactamase producing Gram-negative bacteria; however, resistance against these compounds is increasing. This study was designed to demonstrate that carbapenemase-producing bacteria are disseminated from hospitals into the environment. To this end, resistant bacteria were isolated from a clinical/urban and from a rural catchment system in Germany in 2016/17. The study followed the dissemination of resistant bacteria from the wastewater through the wastewater treatment plant (WWTP) into the receiving surface waters. The bacteria were cultivated on selective agar and characterized by antibiotic testing, real-time PCR targeting carbapenemase genes and typing. Bacteria with resistance to third generation cephalosporins were isolated from all sample sites. 134 isolates harboring carbapenemase genes encoding VIM, NDM and OXA-48 and 26 XDR (extensively drug-resistant) strains with susceptibility to only one or two antibiotics were isolated from the clinical/urban system. The rural system yielded eight carbapenemase producers and no XDR strains. In conclusion, clinical wastewaters were charged with a high proportion of multidrug resistant bacteria. Although most of these bacteria were eliminated during wastewater treatment, dissemination into surface waters is possible as single carbapenemase producers were still present in the effluent of the WWTP.

Modelling characteristics to predict Legionella contamination risk - Surveillance of drinking water plumbing systems and identification of risk areas.

For the surveillance of drinking water plumbing systems (DWPS) and the identification of risk factors, there is a need for an early estimation of the risk of Legionella contamination within a building, using efficient and assessable parameters to estimate hazards and to prioritize risks. The precision, accuracy and effectiveness of ways of estimating the risk of higher Legionella numbers (temperature, stagnation, pipe materials, etc.) have only rarely been empirically assessed in practice, although there is a broad consensus about the impact of these risk factors. We collected n = 807 drinking water samples from 9 buildings which had had Legionella spp. occurrences of >100 CFU/100mL within the last 12 months, and tested for Legionella spp., L. pneumophila, HPC 20°C and 36°C (culture-based). Each building was sampled for 6 months under standard operating conditions in the DWPS. We discovered high variability (up to 4 log(10) steps) in the presence of Legionella spp. (CFU/100 mL) within all buildings over a half year period as well as over the course of a day. Occurrences were significantly correlated with temperature, pipe length measures, and stagnation. Logistic regression modelling revealed three parameters (temperature after flushing until no significant changes in temperatures can be obtained, stagnation (low withdrawal, qualitatively assessed), pipe length proportion) to be the best predictors of Legionella contamination (>100 CFU/100 mL) at single outlets (precision = 66.7%; accuracy = 72.1%; F(0.5) score = 0.59).

Developing an easy-to-apply model for identifying relevant pathogen pathways into surface waters used for recreational purposes.

Swimming in inner-city surface waters is popular in the warm season, but can have negative consequences such as gastro-intestinal, ear and skin infections. The pathogens causing these infections commonly enter surface waters via several point source discharges such as the effluents from wastewater treatment plants and sewer overflows, as well as through diffuse non-point sources such as surface runoff. Nonetheless, the recreational use of surface waters is attractive for residents. In order to save financial and organizational resources, local authorities need to estimate the most relevant pathways of pathogens into surface waters. In particular, when detailed data on a local scale are missing, this is quite difficult to achieve. For this reason, we have developed an easy-to-apply model using the example of Escherichia coli and intestinal enterococci as a first approach to the local situation, where missing data can be replaced by data from literature. The model was developed based on a case study of a river arm monitored in western Germany and will be generalized for future applications. Although the limits of the EU Bathing Water Directive are already fulfilled during dry weather days, we showed that the effluent of wastewater treatment plants and overland flow had the most relevant impact on the microbial surface water quality. On rainy weather days, combined sewer overflows are responsible for the highest microbial pollution loads. The results obtained in this study can help decision makers to focus on reducing the relevant pathogen sources within a catchment area.

Application of flow cytometry and PMA-qPCR to distinguish between membrane intact and membrane compromised bacteria cells in an aquatic milieu.

The paper compares two methods of distinguishing between alive and dead cells by differentiation on the basis of their membrane structure: LIVE/DEAD flow cytometry and PMA-qPCR. LIVE/DEAD flow cytometry was established using the LIVE/DEAD(®) BacLight™ Bacterial Viability Kit with different ratios of Legionella pneumophila and Escherichia coli cells with intact and compromised membranes (heat treated). The PMA-qPCR method was tested and modified, and results were compared with those from LIVE/DEAD flow cytometry using L. pneumophila cells. Ratios of membrane intact to membrane compromised cells were well shown by LIVE/DEAD flow cytometry in all combinations. PMA-qPCR seems to work best in even mixed ratios (1:1) of intact and compromised cells. In other respects, we noticed an overestimation of intact cells in the samples which contained a high percentage of membrane compromised cells, and an underestimation of intact cells in samples with a small percentage of membrane compromised cells. However, looking at total counts instead of ratios, the results were within an order of magnitude. This implies that the use of PMA-qPCR is appropriate only for a qualitative analysis to monitor the success of a process such as disinfection. Furthermore, we were able to assess that both methods have advantages and disadvantages: LIVE/DEAD flow cytometry as applied in this study works well on some bacteria monocultures, but does not distinguish between bacteria species. The PMA-qPCR method allows the possibility of distinguishing between membrane intact cells and membrane compromised cells and can be used to screen for specific bacteria.

The impact of land use on microbial surface water pollution.

Our knowledge relating to water contamination from point and diffuse sources has increased in recent years and there have been many studies undertaken focusing on effluent from sewage plants or combined sewer overflows. However, there is still only a limited amount of microbial data on non-point sources leading to diffuse pollution of surface waters. In this study, the concentrations of several indicator micro-organisms and pathogens in the upper reaches of a river system were examined over a period of 16 months. In addition to bacteria, diffuse pollution caused by Giardia lamblia and Cryptosporidium spp. was analysed. A single land use type predestined to cause high concentrations of all microbial parameters could not be identified. The influence of different land use types varies between microbial species. The microbial concentration in river water cannot be explained by stable non-point effluent concentrations from different land use types. There is variation in the ranking of the potential of different land use types resulting in surface water contamination with regard to minimum, median and maximum effects. These differences between median and maximum impact indicate that small-scale events like spreading manure substantially influence the general contamination potential of a land use type and may cause increasing micro-organism concentrations in the river water by mobilisation during the next rainfall event.

Drinking water quality in household supply infrastructure--A survey of the current situation in Germany.

As a result of the amendment to the German Drinking Water Ordinance in 2001, local public health authorities are obliged to monitor the water supply in installations providing water for public use (Section 18 German Drinking Water Ordinance). With a systematic and nationwide survey of locally available data relating to hygienic drinking water quality and the existing drinking water infrastructure in buildings, the extent of microbial contamination of in-building distribution systems in Germany is intended to be assessed. To gain an overview of the microbial contamination of drinking water in public buildings all 419 local public health authorities in Germany were contacted in 2007. In a detailed study with a representative cooperation level of 5% of these local public health authorities, the available data relating to microbiological, chemical, physical and technical parameters gained from in-building distribution systems were collected. Drinking water parameters were combined with regard to the total number of analyses and the absolute number as well as the percentage of limit compliance failures (n=108,288). Limits exceeded were classified as the failure to comply with the German Drinking Water Ordinance, DVGW technical regulations and Federal Environment Agency recommended limits. The highest rates of samples exceeding these limits were found for the parameter Legionella sp. which contaminated 12.8% of all samples (n=22,786; limit: 100 CFU/100ml), followed by heterotrophic plate count at 36 degrees C (3.5%, n=10,928; limit: 100 CFU/1 ml) and Pseudomonas sp. (2.9%, n=3468; limit: 0 CFU/100ml). Legionella sp. and Pseudomonas sp. pose a direct health risk to immunosuppressed users. Additionally, for some chemical parameters, such as nickel, iron and lead, a potential risk for the health of consumers was detected. Further data analysis may reveal whether this contamination is related to stagnation where there is only sporadic use or whether other factors are involved in the process of microbial growth in installation systems.