Towards risk assessment for antibiotic resistant pathogens in recycled water: a systematic review and summary of research needs.

Risk assessment is critical for identifying target concentrations of antibiotic resistant pathogens necessary for mitigating potential harmful exposures associated with water reuse. However, there is currently limited available data characterizing the concentrations of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in recycled water to support robust efforts at risk assessment. The objective of this systematic review was to identify and synthesize the existing literature documenting the presence and abundance of ARB and ARGs in recycled water. In addition, this review identifies best practices and explores monitoring targets for studying ARB and ARGs in recycled water to guide future work and identifies key research needs aimed at better supporting quantitative microbial risk assessment focused on recycled water and antibiotic resistance. Future efforts to collect data about ARB and ARG prevalence in recycled water should report concentration data per unit volume. Sample metadata should also be provided, including a description of treatment approach, a description of planned water uses (e.g., potable, irrigation), methods for conveyance to the point of use, and available physicochemical water quality data. Additional research is needed aimed at identifying recommended ARB and ARG monitoring targets and for developing approaches to incorporate metagenomic data into risk assessment.

Microbial Chain Elongation and Subsequent Fermentation of Elongated Carboxylates as H2-Producing Processes for Sustained Reductive Dechlorination of Chlorinated Ethenes.

In situ anaerobic groundwater bioremediation of trichloroethene (TCE) to nontoxic ethene is contingent on organohalide-respiring Dehalococcoidia, the most common strictly hydrogenotrophic Dehalococcoides mccartyi (D. mccartyi). The H2 requirement for D. mccartyi is fulfilled by adding various organic substrates (e.g., lactate, emulsified vegetable oil, and glucose/molasses), which require fermenting microorganisms to convert them to H2. The net flux of H2 is a crucial controlling parameter in the efficacy of bioremediation. H2 consumption by competing microorganisms (e.g., methanogens and homoacetogens) can diminish the rates of reductive dechlorination or stall the process altogether. Furthermore, some fermentation pathways do not produce H2 or having H2 as a product is not always thermodynamically favorable under environmental conditions. Here, we report on a novel application of microbial chain elongation as a H2-producing process for reductive dechlorination. In soil microcosms bioaugmented with dechlorinating and chain-elongating enrichment cultures, near stoichiometric conversion of TCE (0.07 ± 0.01, 0.60 ± 0.03, and 1.50 ± 0.20 mmol L-1 added sequentially) to ethene was achieved when initially stimulated by chain elongation of acetate and ethanol. Chain elongation initiated reductive dechlorination by liberating H2 in the conversion of acetate and ethanol to butyrate and caproate. Syntrophic fermentation of butyrate, a chain-elongation product, to H2 and acetate further sustained the reductive dechlorination activity. Methanogenesis was limited during TCE dechlorination in soil microcosms and absent in transfer cultures fed with chain-elongation substrates. This study provides critical fundamental knowledge toward the feasibility of chlorinated solvent bioremediation based on microbial chain elongation.

Legionnaires' disease in dental offices: Quantifying aerosol risks to dental workers and patients.

Legionella pneumophila is an opportunistic bacterial respiratory pathogen that is one of the leading causes of drinking water outbreaks in the United States. Dental offices pose a potential risk for inhalation or aspiration of L. pneumophila due to the high surface area to volume ratio of dental unit water lines-a feature that is conducive to biofilm growth. This is coupled with the use of high-pressure water devices (e.g., ultrasonic scalers) that produce fine aerosols within the breathing zone. Prior research confirms that L. pneumophila occurs in dental unit water lines, but the associated human health risks have not been assessed. We aimed to: (1) synthesize the evidence for transmission and management of Legionnaires' disease in dental offices; (2) create a quantitative modeling framework for predicting associated L. pneumophila infection risk; and (3) highlight influential parameters and research gaps requiring further study. We reviewed outbreaks, management guidance, and exposure studies and used these data to parameterize a quantitative microbial risk assessment (QMRA) model for L. pneumophila in dental applications. Probabilities of infection for dental hygienists and patients were assessed on a per-exposure and annual basis. We also assessed the impact of varying ventilation rates and the use of personal protective equipment (PPE). Following an instrument purge (i.e., flush) and with a ventilation rate of 1.2 air changes per hour, the median per-exposure probability of infection for dental hygienists and patients exceeded a 1-in-10,000 infection risk benchmark. Per-exposure risks for workers during a purge and annual risks for workers wearing N95 masks did not exceed the benchmark. Increasing air change rates in the treatment room from 1.2 to 10 would achieve an ∼85% risk reduction, while utilization of N95 respirators would reduce risks by ∼95%. The concentration of L. pneumophila in dental unit water lines was a dominant parameter in the model and driver of risk. Future risk assessment efforts and refinement of microbiological control protocols would benefit from expanded occurrence datasets for L. pneumophila in dental applications.

Antibiotic Resistance and Sewage-Associated Marker Genes in Untreated Sewage and a River Characterized During Baseflow and Stormflow.

Since sewage is a hotspot for antibiotic resistance genes (ARGs), the identification of ARGs in environmental waters impacted by sewage, and their correlation to fecal indicators, is necessary to implement management strategies. In this study, sewage treatment plant (STP) influent samples were collected and analyzed using quantitative polymerase chain reaction (qPCR) to investigate the abundance and correlations between sewage-associated markers (i.e., Bacteroides HF183, Lachnospiraceae Lachno3, crAssphage) and ARGs indicating resistance to nine antibiotics (belonging to aminoglycosides, beta-lactams, sulfonamides, macrolides, and tetracyclines). All ARGs, except bla VIM, and sewage-associated marker genes were always detected in untreated sewage, and ermF and sul1 were detected in the greatest abundances. intl1 was also highly abundant in untreated sewage samples. Significant correlations were identified between sewage-associated marker genes, ARGs and the intl1 in untreated sewage (τ = 0.488, p = 0.0125). Of the three sewage-associated marker genes, the BIO-ENV procedure identified that HF183 alone best maximized correlations to ARGs and intl1 (τ = 0.590). Additionally, grab samples were collected from peri-urban and urban sites along the Brisbane River system during base and stormflow conditions, and analyzed for Escherichia coli, ARGs, the intl1, and sewage-associated marker genes using quantitative polymerase chain reaction (qPCR). Significant correlations were identified between E. coli, ARGs, and intl1 (τ = 0.0893, p = 0.0032), as well as with sewage-associated marker genes in water samples from the Brisbane River system (τ = 0.3229, p = 0.0001). Of the sewage-associated marker genes and E. coli, the BIO-ENV procedure identified that crAssphage alone maximized correlations with ARGs and intl1 in river samples (τ = 0.4148). Significant differences in E. coli, ARGs, intl1, and sewage-associated marker genes, and by flow condition (i.e., base vs. storm), and site types (peri-urban vs. urban) combined were identified (R = 0.3668, p = 0.0001), where percent dissimilarities between the multi-factorial groups ranged between 20.8 and 11.2%. Results from this study suggest increased levels of certain ARGs and sewage-associated marker genes in stormflow river water samples compared to base flow conditions. E. coli, HF183 and crAssphage may serve as potential indicators of sewage-derived ARGs under stormflow conditions, and this merits further investigation. Data presented in this study will be valuable to water quality managers to understand the links between sewage pollution and ARGs in urban environments.

The occurrence and ecology of microbial chain elongation of carboxylates in soils.

Chain elongation is a growth-dependent anaerobic metabolism that combines acetate and ethanol into butyrate, hexanoate, and octanoate. While the model microorganism for chain elongation, Clostridium kluyveri, was isolated from a saturated soil sample in the 1940s, chain elongation has remained unexplored in soil environments. During soil fermentative events, simple carboxylates and alcohols can transiently accumulate up to low mM concentrations, suggesting in situ possibility of microbial chain elongation. Here, we examined the occurrence and microbial ecology of chain elongation in four soil types in microcosms and enrichments amended with chain elongation substrates. All soils showed evidence of chain elongation activity with several days of incubation at high (100 mM) and environmentally relevant (2.5 mM) concentrations of acetate and ethanol. Three soils showed substantial activity in soil microcosms with high substrate concentrations, converting 58% or more of the added carbon as acetate and ethanol to butyrate, butanol, and hexanoate. Semi-batch enrichment yielded hexanoate and octanoate as the most elongated products and microbial communities predominated by C. kluyveri and other Firmicutes genera not known to undergo chain elongation. Collectively, these results strongly suggest a niche for chain elongation in anaerobic soils that should not be overlooked in soil microbial ecology studies.

Identification of reliable marker genes for the detection of canine fecal contamination in sub-tropical Australia.

Animal fecal contamination in aquatic environments is a major source of zoonotic diseases in humans. While concerns are focused on livestock, companion animals such as dogs can also be a source of a wide range of zoonotic pathogens. Therefore, detection of dog or canine fecal contamination in aquatic environments is important for mitigating risks. In this study, host-sensitivity and specificity of four canine fecal-associated marker genes were evaluated by analyzing 30 canine and 240 non-canine fecal samples. The application of these markers was also tested in water from an urban river under dry weather conditions. The host sensitivity values of the Bacteroides BacCan-UCD, DogBact, DF113 and DF418 were 1.00, 0.90, 0.83, and 0.90, respectively. The host specificity value of the BacCan-UCD, DogBact, DF113 and DF418 were 0.87, 0.98, 0.83, and 0.41, respectively. The mean concentrations of DF418 were highest (7.82 ± 1.13 log10 gene copies (GC)/g of feces) followed by BacCan-UCD (7.61 ± 1.06 log10 GC/g) and DogBact (7.15 ± 0.92 log10 GC/g). The mean concentration of DF113 (5.80 ± 1.25 log10 GC/g) was 1.5 to 2.5 orders of magnitude lower than the other marker genes. The DogBact marker gene was not detected in any other animal feces other than a small number of untreated sewage samples. The BacCan-UCD marker gene cross-reacted with cat, chicken, and pig fecal samples, while the DF113 marker gene cross-reacted with cat, chicken, cattle fecal and untreated sewage samples. The DF418 marker gene was detected in all sewage and animal feces and deemed not suitable for canine fecal contamination tracking in sub-tropical Australia. Canine fecal contamination was infrequently detected in environmental water samples. Based on the results obtained in this study, we recommend that at least two canine feces-associated marker genes should be used in field studies.