Potassium ferrate's disinfecting ability: a study on human adenovirus, Giardia duodenalis, and microbial indicators under varying pH and water temperature conditions.

Ferrate (Fe(VI): HFeO4- /FeO42-), a potent oxidant, has been investigated as an alternative chemical disinfectant in water treatment due to its reduced production of disinfection by-products. In this study, we assessed the disinfecting ability of potassium ferrate against a variety of microorganisms, including waterborne pathogens, under varying pH and water temperature conditions. We presented CT values, a metric of ferrate concentrations (C) and contact time (T), to quantify microbial inactivation rates. Among the tested microorganisms, human adenovirus was the least resistant to ferrate, followed by waterborne bacteria such as Escherichia coli and Vibrio cholerae, and finally, the protozoan parasite Giardia duodenalis. We further investigated the impact of two pH values (7 and 8) and two temperatures (5 and 25 °C) on microbial inactivation rates, observing that inactivation rates increased with lower pH and higher temperature. In addition to showcasing ferrate's capacity to effectively inactivate a range of the tested microorganisms, we offer a ferrate CT table to facilitate the comparison of the effectiveness of various disinfection methods.

Ciprofloxacin- and azithromycin-resistant bacteria in a wastewater treatment plant.

The occurrence of antibiotic-resistant bacteria (ARB) in wastewater treatment plants (WWTPs) has become an occupational and environmental concern. WWTPs are engineered systems that treat wastewater to meet public health standards before release into the environment. The residuals, as either effluent or solids, are then discharged or beneficially recycled into the environment. Since these wastes contain a diverse array of microorganisms, some of which are resistant to commonly used antibiotics, there is a potential for these organisms to spread in the environment via residual recycling and effluent discharge. Human infections with ARB are increasing, and it is not well known how the interaction between humans and the environment plays a role in this process. WWTP workers, who are on the front lines, may come into direct contact with materials containing these microbes. This study aimed to determine the number of ARB present in both air and sewage sludges in a WWTP using nonselective media supplemented with two antibiotics (ciprofloxacin and azithromycin). The densities of total heterotrophic bacteria, ciprofloxacin-resistant bacteria, and azithromycin-resistant bacteria were 7.82 × 105 - 4.7 × 109, 7.87 × 103 - 1.05 × 108, and 2.27 × 105 - 1.16 × 109 CFU/g, respectively. The prevalence [(concentration on medium with antibiotics/concentration on medium without antibiotics) × 100] of ciprofloxacin-resistant bacteria in treated sludge was twice as low as in digested sludge and approximately three times lower than in raw sludge. For azithromycin, the prevalence of resistant bacteria in treated sludge was about the same in digested and nearly twice lower than in raw sludge. Despite a marked reduction in the mean prevalence of resistant bacteria in dewatered treated sludge for both antibiotics, these differences were not significant. The highest prevalence of antibiotic resistance was observed for azithromycin. Similarly, the prevalence of airborne azithromycin-resistant bacteria inside the belt filter press room (BFPR) was nearly seven times higher than the prevalence of airborne ciprofloxacin-resistant bacteria. These concentrations of ARB were not negligible and may represent an exposure pathway for some workers in WWTPs.

Comparison of two culture methods for the enumeration of Legionella pneumophila from potable water samples.

Legionella infections have steadily increased in the United States over the last 20 years, and most of these infections have been attributed to contaminated water. The gold standard for confirmation of Legionella presence in water is culturing with Buffered Charcoal Yeast Extract (BCYE) agar. Following many modifications, this method is still time-consuming, expensive, and can take longer than 10 days for full confirmation. The Legiolert is a newer and simpler culture product that is claimed to be able to quantify Legionella pneumophila in 7 days with high sensitivity and specificity and does not need further confirmation for the presence of L. pneumophila. This study compared the culturability of L. pneumophila occurring in a simulated home plumbing system using both Legiolert and BCYE agar methods. Out of 185 water samples, Legiolert and BCYE method detected L. pneumophila in 83 and 85% of the samples, respectively. The two methods were determined to be statistically equivalent for culturability of L. pneumophila, though the detected levels by Legiolert were slightly higher than the BCYE method. The molecular confirmation of positive (n = 254) and negative wells (n = 82) with Legiolert also showed a high specificity of 96.5% (i.e., 3.5% false positives (9/254) and 0% false negatives (0/82)).

Survey of US wastewater for carbapenem-resistant Enterobacteriaceae.

A survey for antibiotic-resistant (AR) Escherichia coli in wastewater was undertaken by collecting samples from primary clarifiers and secondary effluents from seven geographically dispersed US wastewater treatment plants (WWTPs). Samples were collected at each WWTP in cool and summer months and cultured using selective media. The resulting isolates were characterized for resistance to imipenem, ciprofloxacin, cefotaxime, and ceftazidime, presence of carbapenemase and extended-spectrum beta-lactamase (ESBL) genes, and phylogroups and sequence types (STs). In total, 322 AR E. coli isolates were identified, of which 65 were imipenem-resistant. Of the 65 carbapenem-resistant E. coli (CREC) isolates, 62% were positive for more than one and 31% were positive for two or more of carbapenemase and ESBL genes targeted. The most commonly detected carbapenemase gene was blaVIM (n = 36), followed by blaKPC (n = 2). A widespread dispersal of carbapenem-resistant STs and other clinically significant AR STs observed in the present study suggested the plausible release of these strains into the environment. The occurrence of CREC in wastewater is a potential concern because this matrix may serve as a reservoir for gene exchange and thereby increase the risk of AR bacteria (including CR) being disseminated into the environment and thence back to humans.

Determining pathogen and indicator levels in class B municipal organic residuals used for land application.

Biosolids are nutrient-rich organic residuals that are currently used to amend soils for food production. Treatment requirements to inactivate pathogens for production of Class A biosolids are energy intensive. One less energy intensive alternative is to treat biosolids to Class B standards, but it could result in higher pathogen loads. Quantitative microbial risk assessments models have been developed on land application of Class B biosolids but contain many uncertainties because of limited data on specific pathogen densities and the use of fecal indicator organisms as accurate surrogates of pathogen loads. To address this gap, a 12-mo study of the levels and relationships between , , and human adenovirus (HAdV) with fecal coliform, somatic, and F-RNA coliphage levels in Class B biosolids from nine wastewater treatment plants throughout the United States was conducted. Results revealed that fecal coliform, somatic, and F-RNA coliphage densities were consistent throughout the year. More important, results revealed that HAdV ( = 2.5 × 10 genome copies dry g) and ( = 4.14 × 10 cysts dry g) were in all biosolids samples regardless of treatment processes, location, or season. oocysts were also detected (38% positive; range: 0-1.9 × 10 oocysts dry g), albeit sporadically. Positive correlations among three fecal indicator organisms and HAdV, but not protozoa, were also observed. Overall, this study reveals that high concentrations of enteric pathogens (e.g., , , and HAdV) are present in biosolids throughout the United States. Microbial densities found can further assist management and policymakers in establishing more accurate risk assessment models associated with land application of Class B biosolids.

Assessing residential activity in a home plumbing system simulator: monitoring the occurrence and relationship of major opportunistic pathogens and phagocytic amoebas.

Opportunistic premise plumbing pathogens (OPPPs) have been detected in buildings' plumbing systems causing waterborne disease outbreaks in the United States. In this study, we monitored the occurrence of OPPPs along with free-living amoeba (FLA) and investigated the effects of residential activities in a simulated home plumbing system (HPS). Water samples were collected from various locations in the HPS and analyzed for three major OPPPs: Legionella pneumophila, nontuberculous mycobacterial species (e.g., Mycobacterium avium, M. intracellulare, and M. abscessus), and Pseudomonas aeruginosa along with two groups of amoebas (Acanthamoeba and Vermamoeba vermiformis). A metagenomic approach was also used to further characterize the microbial communities. Results show that the microbial community is highly diverse with evidence of spatial and temporal structuring influenced by environmental conditions. L. pneumophila was the most prevalent pathogen (86% of samples), followed by M. intracellulare (66%) and P. aeruginosa (21%). Interestingly, M. avium and M. abscessus were not detected in any samples. The data revealed a relatively low prevalence of Acanthamoeba spp. (4%), while V. vermiformis was widely detected (81%) across all the sampling locations within the HPS. Locations with a high concentration of L. pneumophila and M. intracellulare coincided with the highest detection of V. vermiformis, suggesting the potential growth of both populations within FLA and additional protection in drinking water. After a period of stagnation lasting at least 2-weeks, the concentrations of OPPPs and amoeba immediately increased and then decreased gradually back to the baseline. Furthermore, monitoring the microbial population after drainage of the hot water tank and partial drainage of the entire HPS demonstrated no significant mitigation of the selected OPPPs. This study demonstrates that these organisms can adjust to their environment during such events and may survive in biofilms and/or grow within FLA, protecting them from stressors in the supplied water.

A comparison of methods to detect low levels of Salmonella enterica in surface waters to support antimicrobial resistance surveillance efforts performed in multiple laboratories.

Developing effective and sensitive detection methods for antimicrobial resistant Salmonella enterica from surface water is a goal of the National Antimicrobial Resistance Monitoring System (NARMS). There are no specified methods for recovery of S. enterica in surface waters in the U.S. A multi-laboratory evaluation of four methods - bulk water enrichment (BW), vertical Modified Moore Swab (VMMS), modified Standard Method 9260.B2 (SM), and dead-end ultrafiltration (DEUF) - was undertaken to recover S. enterica from surface water. In Phase 1, one-liter volumes of water were collected from the same site on five different dates. Water was shipped and analyzed at four different laboratory locations (A, B, C, and D) for recovery of 1) inoculated fluorescent S. Typhimurium strain (ca. 30 CFU/L) and 2) Salmonella present in the water sampled. At each location, BW, VMMS, or SM recovery was performed on five separate 1 L water samples. Twenty 1 L water samples were subjected to each recovery method, and overall, sixty 1 L samples were assayed for Salmonella. Inoculated, fluorescent Salmonella Typhimurium and environmental Salmonella spp. were recovered from 65 % (39/60) and 45 % (27/60) of water samples, respectively. BW, VMMS, and SM recovered fluorescent S. Typhimurium from 60 %, 60 %, and 75 % of inoculated samples, respectively. Analysis by Chi-squared test determined laboratory location had a significant (p < 0.05) effect on fluorescent S. Typhimurium recovery compared to method or date of water collection. In Phase 2, recovery of inoculated fluorescent S. Typhimurium from 1 L samples by SM and DEUF was compared at laboratory locations B and D. SM and DEUF recovered fluorescent S. Typhimurium from 100 % (20/20) and 95 % (19/20) of inoculated water samples, respectively; laboratory location (p > 0.05) did not affect Salmonella recovery. Uniform laboratory methodology and training should be prioritized in conducting Salmonella recovery from surface water in laboratories.

Development of a sensitive detection method for stressed E. coli O157:H7 in source and finished drinking water by culture-qPCR.

A sensitive and specific method that also demonstrates viability is of interest for detection of E. coli O157:H7 in drinking water. A combination of culture and qPCR was investigated. Two triplex qPCRs, one from a commercial source and another designed for this study were optimized from 5 different assays to be run on a single qPCR plate. The qPCR assays were specific for 33 E. coli O157:H7 strains tested and detected 500 cells spiked in a background of 10(8) nontarget bacterial cells. The qPCR detection was combined with an enrichment process using Presence Absence (P/A) broth to detect chlorine and starvation stressed cells. qPCR analysis performed post-enrichment allowed the detection of 3-4 cells/L as indicated by a sharp increase in fluorescence (lowering of Ct values) from pre-enrichment levels, demonstrating a 5-6 log increase in the number of cells. When six vulnerable untreated surface water samples were examined, only one was positive for viable E. coli O157:H7 cells. These results suggest that the culture-PCR procedure can be used for rapid detection of E. coli O157:H7 in drinking water.

Closed Genome and Plasmid Sequences of Legionella pneumophila AW-13-4, Isolated from a Hot Water Loop System of a Large Occupational Building.

Unused water in unoccupied buildings can become stagnant, with reductions in temperature and levels of disinfectant resulting in increased microbial growth. We report the closed and complete genome and plasmid of Legionella pneumophila strain AW-13-4 (serogroup 1), which was isolated from a hot water loop system of a large building.

Understanding Microbial Loads in Wastewater Treatment Works as Source Water for Water Reuse.

Facing challenges in water demands and population size, particularly in the water-scarce regions in the United States, the reuse of treated municipal wastewater has become a viable potential to relieve the ever-increasing demands of providing water for (non-)potable use. The objectives of this study were to assess microbial quality of reclaimed water and to investigate treatability of microorganisms during different treatment processes. Raw and final treated effluent samples from three participating utilities were collected monthly for 16 months and analyzed for various microbial pathogens and fecal indicator organisms. Results revealed that the detectable levels of microbial pathogens tested were observed in the treated effluent samples from all participating utilities. Log10 reduction values (LRVs) of Cryptosporidium oocysts and Giardia cysts were at least two orders of magnitude lower than those of human adenovirus and all fecal indicator organisms except for aerobic endospores, which showed the lowest LRVs. The relatively higher LRV of the indicator organisms such as bacteriophages suggested that these microorganisms are not good candidates of viral indicators of human adenovirus during wastewater treatment processes. Overall, this study will assist municipalities considering the use of wastewater effluent as another source of drinking water by providing important data on the prevalence, occurrence, and reduction of waterborne pathogens in wastewater. More importantly, the results from this study will aid in building a richer microbial occurrence database that can be used towards evaluating reuse guidelines and disinfection practices for water reuse practices.

Chlorine disinfection of blended municipal wastewater effluents.

Blending is used in the wastewater industry to manage wet-weather events. Wastewater is treated through primary clarification, with flows in excess of the hydraulic capacity of the secondary system being directed to effluent disinfection. Before disinfection, the primary clarified effluent is "blended" with effluents that have been treated through the secondary system. The combined or "blended" effluents are then disinfected before being discharged to receiving waters. This study evaluated the effectiveness of chlorine to disinfect blended effluents. Experiments were conducted at bench-scale on primary and secondary effluents and three ratios of primary to secondary effluent (1:9, 3:7, and 5:5) from three publicly owned treatment works. Results from this study found that blending 10% or more primary effluent with secondary reduces the efficacy of chlorine disinfection, and coliphage survived chlorine disinfection better than bacterial indicator organisms. A simple empirical model for predicting indicator organism densities following chlorine disinfection was developed using data from this research.

Legionellosis and Recent Advances in Technologies for Legionella Control in Premise Plumbing Systems: A Review.

This review discusses Legionella, among the most prolific and publicly well-known waterborne pathogens, and advances in potential treatment technologies. The number of cases associated with Legionella continues to rise, as does its public awareness. Currently, cases associated with premise plumbing account for the largest number of legionellosis cases in the United States. So, while it is important to understand Legionella as such, it is also important to investigate how to treat drinking water in premise plumbing for Legionella and other waterborne pathogens. While there are currently several methods recognized as potential means of inactivating waterborne pathogens, several shortcomings continue to plague its implementation. These methods are generally of two types. Firstly, there are chemical treatments such as chlorine, chlorine dioxide, monochloramine, ozone, and copper-silver ionization. Secondly, there are physical treatments such as thermal inactivation and media filtration. Their shortcomings range from being labor-intensive and costly to having negative health effects if not properly operated. Recently developed technologies including ultraviolet (UV) irradiation using light emitting diodes (LEDs) and innovative carbon nanotube (CNT) filters can better control waterborne pathogens by allowing for the simultaneous use of different treatment measures in plumbing systems.

Draft Genome Sequences of Seven Legionella pneumophila Isolates from a Hot Water System of a Large Building.

Public health data show that a significant fraction of the nation's waterborne disease outbreaks are attributable to premise plumbing. We report the draft genome sequences of seven Legionella pneumophila serogroup 1 isolates from hot water lines of a large building. Genomic analysis identified the isolates as belonging to sequence type 1.

Efficacy of inactivation of human enteroviruses by dual-wavelength germicidal ultraviolet (UV-C) light emitting diodes (LEDs).

The efficacy of germicidal ultraviolet (UV-C) light emitting diodes (LEDs) was evaluated for inactivating human enteroviruses included on the United States Environmental Protection Agency (EPA)'s Contaminant Candidate List (CCL). A UV-C LED device, emitting at peaks of 260 nm and 280 nm and the combination of 260∣280 nm together, was used to measure and compare potential synergistic effects of dual wavelengths for disinfecting viral organisms. The 260 nm LED proved to be the most effective at inactivating the CCL enteroviruses tested. To obtain 2-log10 inactivation credit for the 260 nm LED, the fluences (UV doses) required are approximately 8 mJ/cm2 for coxsackievirus A10 and poliovirus 1, 10 mJ/cm2 for enterovirus 70, and 13 mJ/cm2 for echovirus 30. No synergistic effect was detected when evaluating the log inactivation of enteroviruses irradiated by the dual-wavelength UV-C LEDs.

Applicability of integrated cell culture reverse transcriptase quantitative PCR (ICC-RTqPCR) for the simultaneous detection of the four human enteric enterovirus species in disinfection studies.

A newly developed integrated cell culture reverse transcriptase quantitative PCR (ICC-RTqPCR) method and its applicability in UV disinfection studies is described. This method utilizes a cell culture system coupled with four RTqPCR assays to detect four species of human enterovirus (e.g., Enterovirus A, Enterovirus B, Enterovirus C and Enterovirus D). Evaluation of the RTqPCR assays was conducted with coxsackievirus A10, echovirus 30, poliovirus 1 and enterovirus 70 and resulted in 100% specificity for the tested assays. A comparison of ICC-RTqPCR between the individual enteroviruses and a mixture of all four viruses resulted in an approximate 1:1 correlation, demonstrating a lack of competition during incubation in cell culture and RTqPCR. The simultaneous detection of multiple human enterovirus species within mixed cultures is relevant to many applications, including virus disinfection studies. This high-throughput, multiplex approach costs less in money and time. By helping with data collection, this approach will lead to more statistically sound data sets that directly compare the inactivation rates of enteroviruses tested.

Evaluating UV-C LED disinfection performance and investigating potential dual-wavelength synergy.

A dual-wavelength UV-C LED unit, emitting at peaks of 260 nm, 280 nm, and the combination of 260|280 nm together was evaluated for its inactivation efficacy and energy efficiency at disinfecting Escherichia coli, MS2 coliphage, human adenovirus type 2 (HAdV2), and Bacillus pumilus spores, compared to conventional low-pressure and medium-pressure UV mercury vapor lamps. The dual-wavelength unit was also used to measure potential synergistic effects of multiple wavelengths on bacterial and viral inactivation and DNA and RNA damage. All five UV sources demonstrated similar inactivation of E. coli. For MS2, the 260 nm LED was most effective. For HAdV2 and B. pumilus, the MP UV lamp was most effective. When measuring electrical energy per order of reduction, the LP UV lamp was most efficient for inactivating E. coli and MS2; the LP UV and MP UV mercury lamps were equally efficient for HAdV2 and B. pumilus spores. Among the UV-C LEDs, there was no statistical difference in electrical efficiency for inactivating MS2, HAdV2, and B. pumilus spores. The 260 nm and 260|280 nm LEDs had a statistical energy advantage for E. coli inactivation. For UV-C LEDs to match the electrical efficiency per order of log reduction of conventional LP UV sources, they must reach efficiencies of 25-39% or be improved on by smart reactor design. No dual wavelength synergies were detected for bacterial and viral inactivation nor for DNA and RNA damage.

The widespread occurrence of the enterohemolysin gene ehlyA among environmental strains of Escherichia coli.

The putative virulence factor enterohemolysin, encoded by the ehlyA gene, has been closely associated with the pathogenic enterohemorrhagic Escherichia coli (EHEC) group. Escherichia coli isolates from effluents from seven geographically dispersed municipal wastewater treatment plants were screened for the presence of enterohemolysin. A total of 338 E. coli isolates were found to express the ehlyA gene. However, none of the isolates contained the toxin-encoding genes (stxA or stxB) associated with EHEC. Two of the 338 isolates possessed the virulence factor intimin, encoded by the eae gene. These findings suggest that the ehlyA gene may be widely distributed among non-EHEC isolates in the environment.

Applicability of UV resistant Bacillus pumilus endospores as a human adenovirus surrogate for evaluating the effectiveness of virus inactivation in low-pressure UV treatment systems.

Recent studies have demonstrated the potential to use Bacillus pumilus endospores as a surrogate for human adenovirus (HAdV) in UV disinfection studies. The use of endospores has been limited by observations of batch-to-batch variation in UV sensitivity. This study reports on a propagation method that utilizes a commercially available medium to produce UV tolerant B. pumilus endospores with a consistent UV sensitivity. It is further demonstrated that the endospores of B. pumilus strain (ATCC 27142), produced using this protocol (half strength Columbia broth, 5 days incubation, with 0.1mM MnSO4), display a UV dose-response that is similar to that of HAdV. Endospore stocks could be stored in ethanol for up to two months at 4 °C without a significant change in UV sensitivity. Synergistic endospore damage was observed by pre-heat treatment of water samples followed by UV irradiation. UV tolerant B. pumilus endospores are a potential surrogate of HAdV for UV treatment performance tests in water utilities which do not have in-house research virology laboratories.

Detection of intrinsic vancomycin resistant enterococci in animal and human feces.

Fecal samples from animal species and humans were analyzed by quantitative culture for enterococci and vancomycin resistant enterococci (VRE). Each host species carried enterococci which exhibited intrinsic intermediate resistance to vancomycin and sensitivity to teicoplanin (Van C phenotype). The carriage rate in humans was 9%. Carriage rates varied among animal species with the highest percentages being found in deer, duck, goose, horse and turkey.

Occurrence of antibiotic-resistant uropathogenic Escherichia coli clonal group A in wastewater effluents.

Isolates of Escherichia coli belonging to clonal group A (CGA), a recently described disseminated cause of drug-resistant urinary tract infections in humans, were present in four of seven sewage effluents collected from geographically dispersed areas of the United States. All 15 CGA isolates (1% of the 1,484 isolates analyzed) exhibited resistance to trimethoprim-sulfamethoxazole (TMP-SMZ), accounting for 19.5% of the 77 TMP-SMZ-resistant isolates. Antimicrobial resistance patterns, virulence traits, O:H serotypes, and phylogenetic groupings were compared for CGA and selected non-CGA isolates. The CGA isolates exhibited a wider diversity of resistance profiles and somatic antigens than that found in most previous characterizations of this clonal group. This is the first report of recovery from outside a human host of E. coli CGA isolates with virulence factor and antibiotic resistance profiles typical of CGA isolates from a human source. The occurrence of "human-type" CGA in wastewater effluents demonstrates a potential mode for the dissemination of this clonal group in the environment, with possible secondary transmission to new human or animal hosts.