Environmental Contamination and Persistence of Clostridioides difficile in Hospital Wastewater Systems.

Clostridioides difficile produces an environmentally resistant dormant spore morphotype that infected patients shed to the hospital environment. C. difficile spores persist in clinical reservoirs that are not targeted by hospital routine cleaning protocols. Transmissions and infections from these reservoirs present a hazard to patient safety. This study aimed to assess the impact of patients acutely suffering from C. difficile-associated diarrhea (CDAD) on C. difficile environmental contamination to identify potential reservoirs. Twenty-three hospital rooms accommodating CDAD inpatients with corresponding soiled workrooms of 14 different wards were studied in a German maximum-care hospital. Additionally, four rooms that never accommodated CDAD patients were examined as negative controls. Stagnant water and biofilms from sinks, toilets, and washer disinfector (WD) traps as well as swabs from cleaned bedpans and high-touch surfaces (HTSs) were sampled. For detection, a culture method was used with selective medium. A latex agglutination assay and a Tox A/B enzyme-linked immunosorbent assay were performed with suspect colonies. Stagnant water and biofilms in hospital traps (29%), WDs (34%), and HTSs (37%) were found to be reservoirs for large amounts of C. difficile during the stay of CDAD inpatients that decreased but could persist 13 ± 6 days after their discharge (13%, 14%, and 9.5%, respectively). Control rooms showed none or only slight contamination restricted to WDs. A short-term cleaning strategy was implemented that reduced C. difficile in stagnant water almost entirely. IMPORTANCE Wastewater pipes are microbial ecosystems. The potential risk of infection emanating from the wastewater for individuals is often neglected, since it is perceived to remain in the pipes. However, sewage systems start with siphons and are thus naturally connected to the outside world. Wastewater pathogens do not only flow unidirectionally to wastewater treatment plants but also retrogradely, e.g., through splashing water from siphons to the hospital environment. This study focused on the pathogen C. difficile, which can cause severe and sometimes fatal diarrheas. This study shows how patients suffering from such diarrheas contaminate the hospital environment with C. difficile and that contamination persists in siphon habitats after patient discharge. This might pose a health risk for hospitalized patients afterward. Since this pathogen's spore morphotype is very environmentally resistant and difficult to disinfect, we show a cleaning measure that can almost entirely eliminate C. difficile from siphons.

Legionella in drinking water: the detection method matters.

Legionella concentrations in drinking water have been regulated for decades and are evaluated with regard to their concentrations in drinking water plumbing systems (DWPS). The respective action levels differ at the international level. In Germany, the Federal Environment Agency (UBA) specifies the application of ISO 11731 for the detection of legionella in drinking water and gives a binding recommendation for the methods to be used for culturing and evaluation. Effective from 01 March 2019, the UBA recommendation was revised. The utilized culture media in the culture approach were altered, consequently affecting the spectrum of legionella colonies detected in drinking water. Using data from a routine legionella monitoring of a large laboratory, over a period of 6 years and 17,270 individual drinking water samples, allowed us to assess the impact of the alteration on the assessment of DWPS. By comparing the amount of action level exceedances before and after the method change, it could be demonstrated that exceedances are reported significantly more often under the new method. Consequently, the corresponding action level for evaluation of legionella contamination and the resulting risk to human health needs to be revised to avoid the misleading impression of increased health risk.

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.

Effect of portable HEPA filters on COVID-19 period prevalence: an observational quasi-interventional study in German kindergartens.

OBJECTIVES: The aim of the study was to evaluate the effect of high-efficiency particulate air (HEPA) filters on COVID-19 period prevalence in kindergartens. DESIGN: The observational study follows an intervention design with the intervention group using HEPA filters and the control group not. SETTING: The study was conducted in 32 (10 intervention, 22 control) kindergartens (daycare centres) in Rhineland Palatinate (Germany). PARTICIPANTS: Data of 2360 children (663 intervention, 1697 control) were reported by the kindergarten heads. Data were collected on institutional level without any identifying information on individuals. Thus, all children of all facilities were included; however, no demographic data were recorded. INTERVENTIONS: The study followed a quasi-interventional design, as no formal intervention was conducted. A charity foundation equipped kindergartens with HEPA filters. These kindergartens were enrolled as intervention group. The control group was recruited from the neighbouring communities and districts. OUTCOME MEASURES: The primary outcome measure was the number of COVID-19 cases reported by the kindergarten heads, converted into period prevalence rates per 1000 population. RESULTS: The mean COVID-19 period prevalence rates of the control and intervention groups were 186 (95% CI: 137.8 to 238.9) and 372 (95% CI: 226.6 to 517.6) per 1000 children, respectively. The one-sided Wilcoxon rank-sum test indicates a p value of 0.989; thus, the hypothesised preventive effect of HEPA filters could not be confirmed in the kindergarten setting. CONCLUSIONS: While HEPA filters can significantly reduce the viral load in room air, this does not lead to reduced COVID-19 prevalence in the selected kindergartens in Germany. It is known that contagion mainly occurs via direct face-to-face air exchange during play and that the contaminated air does not necessarily pass through the filter prior to air exchange between children. The use of HEPA filters may also lead to a sense of security, leading to reduced preventive behaviour.

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.

Prevalence of multidrug-resistant and extended-spectrum beta-lactamase-producing Escherichia coli in urban community wastewater.

Antibiotic resistance (ABR) and the spread of multidrug-resistant and extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli via wastewater to environmental compartments are of rapidly growing global health concern. Health care facilities, industries and slaughterhouses discharge high loads of ABR bacteria with their wastewater. However, the general community is often the biggest indirect discharger. Yet, research focusing explicitly on this important diffuse source is rather scarce raising questions about variations in the occurrence of ESBL-producing E. coli in wastewater from different communities and over time. Between April 2019 and March 2020, wastewater from three socio-spatially different districts in the Ruhr Metropolis, Germany, and the receiving wastewater treatment plant was sampled monthly and analysed for the occurrence of ESBL-producing E. coli via culture-based methods. Isolates were validated with matrix assisted laser desorption ionization time of flight mass spectrometry and antibiotic resistance profiles were analysed via microdilution. Results were interpreted using the European Committee on Antimicrobial Susceptibility Testing criteria. The German Commission for Hospital Hygiene and Infection Prevention criteria were used for multidrug-resistance categorization. Phenotypic ESBL-producing E. coli could be isolated from every wastewater sample demonstrating that the general community is an important indirect discharger. The socio-spatially disadvantaged area displayed higher absolute loads of ESBL-producing E. coli compared to the other two areas, as well as higher adjusted loads for domestic discharge and inhabitants, particularly during winter, indicating a higher ABR burden. Thirty-two isolates (28.6%) were characterized as multidrug-resistant Gram-negative bacteria (3MRGN). Resistance profiles varied only for those antibiotics, which can be administered in outpatient care. Resistance levels tended to be around 10% lower in the socio-spatially advantaged area. This study shows that spatial and seasonal influences regarding the occurrence of ESBL-producing E. coli in wastewater from socio-spatially different communities are identifiable.

Air filtration as a tool for the reduction of viral aerosols.

For testing the effectiveness of air purification devices in regard to the reduction of virus-containing aerosols, a test method involving test viruses has been lacking until now. The use of bacteriophages (phiX174 phages) is a method to test the efficiency of air purification devices under experimental conditions. Using air purifiers with a HEPA filter H14, a 4.6-6.1 Log reduction of test viruses can be achieved if bacteriophages are directly aerosolised into the air purifier, which corresponds to a reduction of 99.9974-99.9999%. Due to the complexity and individuality of air flow, an experimental approach was used in which all outside influences were minimised. The experimental setup was practical and chosen to project a scenario of direct transmission by an emitting source to a recipient. The experiments were performed with and without the air purifier at a distance of 0.75 m and 1.5 m each. Using the air purifier at a setting of 1000 m3/h, the concentration of the phiX174 phages in the air could be reduced by 2.86 Log (mean value). Nevertheless, the experiments without the air purifier showed a similar reduction rate of 2.61 Log (mean value) after 35 min. The concentration of phiX174 phages in the air could be additionally reduced up to 1 log step (maximum value) by the use of the air purifier in comparison to the experiments without. Distance was shown to be an important factor for risk reduction.

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.

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.

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.