Monitoring coliphages to reduce waterborne infectious disease transmission in the One Water framework.

Coastal waters, surface waters, and groundwater are impacted by wastewater and stormwater discharges, as well as agricultural flows containing animal waste and nutrients. A One Water approach posits that components of the water system have overlapping and interactive impacts on other aspects of the system, for which a comprehensive approach to water management is needed to further inform public health decisions. Current frameworks for monitoring wastewater effluent and recreational surface waters include the measurement of fecal indicator bacteria. Although viral pathogens are likely to be transported further and can survive longer than bacterial pathogens, virus monitoring is not required for recreational waters. A scientific consensus is emerging that the use of bacterial indicators alone does not account for nor represent the health risks associated with viral pathogens due to the differences in the fate and transport of bacterial versus viral pathogens in wastewater treatment, surface water, and groundwater. Furthermore, it is likely that the public health risk associated with these waterborne pathogens is variable and diverse. For example, under drought conditions, effluents of urban water systems can comprise most of the dry weather flow in downstream waters, which are often used as sources of drinking water. This de facto reuse could increase viral risk for the end users of this water. A One Water approach will aid in protecting the health of the public from waterborne pathogens, regardless of where those pathogens entered the water system. In this review, we assert that monitoring for fecal indicator viruses can complement the monitoring of bacterial indicators, thereby improving public health protections. Bacteriophages have the strongest research foundation and correlation with viral pathogens along with some prediction power for risk to human health. Methods for detecting and quantifying coliphages are briefly summarized, as are challenges in the implementation of testing. Key knowledge gaps and research priorities are discussed so that the potential value and limitations of coliphage monitoring can be better addressed and understood.

Determining the effects of Class I landfill leachate on biological nutrient removal in wastewater treatment.

The disposal of landfill leachate is a chronic problem facing the municipal solid waste industry. The composition of landfill leachate is highly variable and often dependent on site-specific conditions. Due to the potentially disruptive impact on wastewater treatment processes, wastewater treatment plants (WWTP) are reluctant to accept landfill leachate for co-treatment. To improve the ability of WWTPs to screen the impact of landfill leachate and reduce landfill owners' cost of disposal, two bench scale methods were evaluated. First, six landfill leachates were screened with the specific oxygen uptake rate (SOUR) test, and second, the effect of leachate on the efficacy of activated sludge processes using lab scale sequencing batch reactors (SBRs) was determined with volumetric loading rates ranging from 5% to 20%. Results suggested that these tools can be used to estimate the impacts of leachate loading on biological processes. Both tools were able to identify loadings where biological activity was increased and inhibition of biological processes was minimized. The loading that maximized microbial activity was leachate specific and typically ranged from 5% to 10%. Taken together, these results suggest that improved landfill leachate screening and testing may improve outcomes at WWTPs by identifying a "Goldilocks" loading rate that increases biological activity. Nevertheless, our results also demonstrated that the effluent quality was degraded even at loading rates that increased biological activity. It is uncertain at this time if biological acclimation can remedy increased effluent nutrient mass loadings, suggesting further research is needed.

Evaluating the fate of bacterial indicators, viral indicators, and viruses in water resource recovery facilities.

A year-long sampling campaign at nine water resource recovery facilities (WRRFs) was conducted to assess the treatability and fate of bacterial indicators, viral indicators, and viruses. Influent concentrations of viral indicators (male-specific and somatic coliphages) and bacterial indicators (Escherichia coli and enterococci) remained relatively constant, typically varying by one order of magnitude over the course of the year. Annual average bacterial indicator reduction ranged from 4.0 to 6.7 logs, and annual average viral indicator reduction ranged from 1.6 to 5.4 logs. Bacterial and viral indicator reduction depended on the WRRF's treatment processes, and bacterial indicator reduction was greater than viral indicator reduction for many processes. Viral reduction (adenovirus 41, norovirus GI, and norovirus GII) was more similar to viral indicator reduction than bacterial indicator reduction. Overall, this work suggests that viral indicator reduction in WRRFs is variable and depends on specific unit processes. Moreover, for the same unit treatment process, viral indicator reduction and bacterial indicator reduction can vary. PRACTITIONER POINTS: A year-long sampling campaign was conducted at nine water resource recovery facilities (WRRFs). The treatability and fate of bacterial indicators, viral indicators, and viruses were assessed. Viral indicator reduction in WRRFs is variable and depends on specific unit processes. For the same unit treatment process, viral indicator reduction and bacterial indicator reduction can vary.

Reduction of invasive bacteria in ethanol fermentations using bacteriophages.

Invasive Lactobacillus bacteria inhibit ethanol fermentations and reduce final product yields. Due to the emergence of antibiotic resistant strains of Lactobacillus spp., alternative disinfection strategies are needed for ethanol fermentations. The feasibility of using the bacteriophage (phage) 8014-B2 to control Lactobacillus plantarum in ethanol fermentations by Saccharomyces cerevisiae was investigated. In 48 h media-based shake flask fermentations, phages achieved greater than 3-log inactivation of L. plantarum, protected final ethanol yields, and maintained yeast viability. The phage-based bacterial disinfection rates depended on both the initial phage and bacterial concentrations. Furthermore, a simple set of kinetic equations was used to model the yeast, bacteria, phage, reducing sugars, and ethanol concentrations over the course of 48 h, and the various kinetic parameters were determined. Taken together, these results demonstrate the applicability of phages to reduce L. plantarum contamination and to protect final product yields in media-based fermentations.

Modeling phage induced bacterial disinfection rates and the resulting design implications.

The phage induced disinfection rates of Escherichia coli K-12 MG1655 in the presence of coliphage Ec2 were determined under a wide range of phage and bacterial concentrations. These rates were elucidated to determine if phages could be used in water and wastewater treatment systems as a biological based disinfectant. Disinfection rates ranging from 0.13 ± 0.1 to 2.03 ± 0.1 h⁻¹ were observed for E. coli K12. A multiple linear regression model was used to explain the variance in the disinfection rates, and this model demonstrated an interaction effect between the initial phage and bacterial concentrations. Furthermore, the results were modeled with particle aggregation theory, which over predicted the disinfection rates at higher phage and bacterial concentrations, suggesting additional interactions. Finally, the observed and predicted disinfection rates were used to determine additional design parameters. The results suggested that a phage based disinfection process may be suitable for the inactivation of specific pathogens in plug flow reactors, such as the pathogens in hospital wastewater effluents and the bacteria responsible for foaming and sludge bulking in activated sludge processes.

A computational analysis of antisense off-targets in prokaryotic organisms.

The adoption of antisense gene silencing as a novel disinfectant for prokaryotic organisms is hindered by poor silencing efficiencies. Few studies have considered the effects of off-targets on silencing efficiencies, especially in prokaryotic organisms. In this computational study, a novel algorithm was developed that determined and sorted the number of off-targets as a function of alignment length in Escherichia coli K-12 MG1655 and Mycobacterium tuberculosis H37Rv. The mean number of off-targets per a single location was calculated to be 14.1 ± 13.3 and 36.1 ± 58.5 for the genomes of E. coli K-12 MG1655 and M. tuberculosis H37Rv, respectively. Furthermore, when the entire transcriptome was analyzed, it was found that there was no general gene location that could be targeted to minimize or maximize the number of off-targets. In an effort to determine the effects of off-targets on silencing efficiencies, previously published studies were used. Analyses with acpP, ino1, and marORAB revealed a statistically significant relationship between the number of short alignment length off-targets hybrids and the efficacy of the antisense gene silencing, suggesting that the minimization of off-targets may be beneficial for antisense gene silencing in prokaryotic organisms.

The long-term effects of phage concentration on the inhibition of planktonic bacterial cultures.

Since the early 1920s there has been an interest in using bacteriophages (phages) for the control of bacterial pathogens. While there are many factors that have limited the success of phage bio-control, one particular problem is the variability of outcomes between phages and bacteria. Specifically, there is a significant need for a better understanding of how initial phage concentrations affect long-term bacterial inhibition. In work reported herein three phages were isolated for Escherichia coli K12, Pseudomonas aeruginosa PAO1, as well as Bacillus cereus and bio-control experiments were performed with phage concentrations ranging from 10(5) to 10(8) plaque forming units per mL over the course of 72 h. For four of the nine phages isolated there was a linear relationship between inhibition and phage concentration, suggesting the effect of phage concentration is important at longer time scales. For three of the isolated phages, phage concentrations had no effect on bacterial inhibition suggesting that even at the lowest concentration the method of action was saturated and lower concentrations might still be effective. Additionally, a cocktail was created and was compared to the previously isolated phages. There was no statistical difference between the cocktail and the best performing phage highlighting the importance of selecting the appropriate phages for treatment. These results suggest that, for certain phages, there is a strong relationship between phage concentration and long-term bacterial growth inhibition and the initial phage concentration is an important indicator of the long-term outcome.