Photoinactivation of Eight Health-Relevant Bacterial Species: Determining the Importance of the Exogenous Indirect Mechanism

It is presently unknown to what extent the endogenous direct, endogenous indirect, and exogenous indirect mechanisms contribute to bacterial photoinactivation in natural surface waters. In this study, we investigated the importance of the exogenous indirect mechanism by conducting photoinactivation experiments with eight health-relevant bacterial species (<i>Bacteroides thetaiotaomicron, Campylobacter jejuni</i>, <i>Enterococcus faecalis</i>, <i>Escherichia coli</i> K12, <i>E. coli</i> O157:H7, <i>Salmonella enterica</i> serovar Typhimurium LT2, <i>Staphylococcus aureus</i>, and <i>Streptococcus bovis</i>). We used three synthetic photosensitizers (methylene blue, rose bengal, and nitrite) and two model natural photosensitizers (Suwannee River natural organic matter and dissolved organic matter isolated from a wastewater treatment wetland) that generated singlet oxygen and hydroxyl radical. <i>B. thetaiotaomicron</i> had larger first order rate constants than all other organisms under all conditions tested. The presence of the synthetic photosensitizers generally enhanced photoinactivation of Gram-positive facultative anaerobes (<i>Ent. faecalis</i>, <i>Staph. aureus</i>, and <i>Strep. bovis</i>). Among Gram-negative bacteria, only methylene blue with <i>E. coli</i> K12 and rose bengal with <i>C. jejuni</i> showed an enhancing effect. The presence of model natural photosensitizers either reduced or did not affect photoinactivation rate constants. Our findings highlight the importance of the cellular membrane and photosensitizer properties in modulating the contribution of the exogenous indirect mechanism to the overall bacterial photoinactivation

Exogenous indirect photoinactivation of bacterial pathogens and indicators in water with natural and synthetic photosensitizers in simulated sunlight with reduced UVB

ABSTRACT Aims: To investigate the UVB-independent and exogenous indirect photoinactivation of eight human health-relevant bacterial species in the presence of photosensitizers. Methods and Results: Eight bacterial species were exposed to simulated sunlight with greatly reduced UVB light intensity in the presence of three synthetic photosensitizers and two natural photosensitizers. Inactivation curves were fit with shoulder-log linear or first order kinetic models, from which the presence of a shoulder and magnitude of inactivation rate constants were compared. 84% reduction in the UVB light intensity roughly matched a 72-95% reduction in the overall bacterial photoinactivation rate constants in sensitizer-free water. With the UVB light mostly reduced, the exogenous indirect mechanism contribution was evident for most bacteria and photosensitizers tested, although most prominently with the Gram-positive bacteria. Conclusions: Results confirm the importance of UVB light in bacterial photoinactivation and, with the reduction of the UVB light intensity, that the Gram-positive bacteria are more vulnerable to the exogenous indirect mechanism than Gram-negative bacteria. Significance and Impact of Study: UVB is the most important range of the sunlight spectrum for bacterial photoinactivation. In aquatic environments where photosensitizers are present and there is high UVB light attenuation, UVA and visible wavelengths can contribute to exogenous indirect photoinactivation

Quantitative PCR assays to detect whales, rockfish, and common murre environmental DNA in marine water samples of the Northeastern Pacific

© The Author(s), 2020. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Andruszkiewicz, E. A., Yamahara, K. M., Closek, C. J., & Boehm, A. B. Quantitative PCR assays to detect whales, rockfish, and common murre environmental DNA in marine water samples of the Northeastern Pacific. Plos One, 15(12), (2020): e0242689, doi:10.1371/journal.pone.0242689.Monitoring aquatic species by identification of environmental DNA (eDNA) is becoming more common. To obtain quantitative eDNA datasets for individual species, organism-specific quantitative PCR (qPCR) assays are required. Here, we present detailed methodology of qPCR assay design and testing, including in silico, in vitro, and in vivo testing, and comment on the challenges associated with assay design and performance. We use the presented methodology to design assays for three important marine organisms common in the California Current Ecosystem (CCE): humpback whale (Megaptera novaeangliae), shortbelly rockfish (Sebastes jordani), and common murre (Uria aalge). All three assays have excellent sensitivity and high efficiencies ranging from 92% to 99%. However, specificities of the assays varied from species-specific in the case of common murre, genus-specific for the shortbelly rockfish assay, and broadly whale-specific for the humpback whale assay, which cross-amplified with other two other whale species, including one in a different family. All assays detected their associated targets in complex environmental water samples.This work is a contribution to the Marine Biodiversity Observation Network (MBON). The MBON project was supported by NASA grant NNX14AP62A ‘National Marine Sanctuaries as Sentinel Sites for a Demonstration Marine Biodiversity Observation Network (MBON)’ funded under the National Ocean Partnership Program (NOPP RFP NOAA-NOS-IOOS-2014-2003803 in partnership between NOAA, BOEM, and NASA), and the U.S. Integrated Ocean Observing System (IOOS) Program Office

Enveloped and non-enveloped virus survival on microfiber towels

Background Handwashing is an important intervention which can reduce indirect disease transmission, however soap and water for handwashing purposes is not available in some low-resource regions. When handwashing with soap and water is not possible, individuals may use alternatives such as the Supertowel (a microfiber towel with an antimicrobial coating). Testing of viral inactivation as a result of antimicrobial treatment on the Supertowel, however, has been limited. The goal of this study is to provide information about the performance of the Supertowel’s antimicrobial treatment against viruses, which will help inform the use of the towels as handwashing alternatives. Methods We seeded the Supertowel and a regular microfiber towel with two bacteriophages (enveloped Phi6 and non-enveloped MS2) and monitored viral inactivation over time. Additionally, we assessed if temperature, humidity, whether the towel was initially wet or dry, or virus type had an effect on viral decay rate constants. Virus concentrations were measured repeatedly over 24 h. Results We found that neither towel type (whether the towel was a Supertowel or a regular microfiber towel) nor humidity were significant variables in our model of decay rate constants (P = 0.06 and P = 0.22, respectively). We found that the variables of temperature, whether towels were initially wet versus dry, and virus type were significantly different from 0, suggesting that these variables explained variance in the decay rate constant (P = 6.55 × 10−13, P = 0.001, and P < 2 × 10−16, respectively). Higher temperatures, dry towels, and enveloped viruses all resulted in increases in the decay rate constant. Conclusions Viruses seeded onto a Supertowel decay similar to viruses seeded onto a regular towel indicating that the virucidal potential of the Supertowel is minimal

Transfer of enteric viruses (adenovirus and coxsackievirus) and bacteriophage (MS2) from liquid to human skin

Indirect exposure to waterborne viruses increases the risk of infection, especially among children with frequent hand-to-mouth contacts. Here, we quantified the transfer of one bacteriophage (MS2) and two enteric viruses (adenovirus and coxsackievirus) from liquid to skin. MS2, a commonly used enteric virus surrogate, was used to compare virus transfer rates in a volunteer trial to those obtained using human cadaver skin and synthetic skin. MS2 transfer to volunteer skin was similar to transfer to cadaver skin but significantly different from transfer to synthetic skin. The transfer of MS2, adenovirus, and coxsackievirus to cadaver skin was modeled using measurements for viruses attaching to the skin (adsorbed) and viruses in liquid residual on skin (unadsorbed). We find virus transfer per surface area is a function of the concentration of virus in the liquid and the film thickness of liquid retained on the skin and is estimable using a linear model. Notably, the amount of MS2 adsorbed on the skin was on average 5 times higher than the amount of adenovirus and 4 times higher than the amount of coxsackievirus. Quantification of pathogenic virus retention to skin would thus be overestimated using MS2 adsorption data. This study provides models of virus transfer useful for risk assessments of water-related activities, demonstrates significant differences in the transfer of pathogenic virus and MS2, and suggests cadaver skin as an alternative testing system for studying interactions between viruses and skin.; IMPORTANCE; Enteric viruses (viruses that infect the gastrointestinal tract) are responsible for most water-transmitted diseases. They are shed in high concentrations in the feces of infected individuals, persist for an extended period of time in water, and are highly infective. Exposure to contaminated water directly (through ingestion) or indirectly (for example, through hand-water contacts followed by hand-to-mouth contacts) increases the risk of virus transmission. The work described herein provides a quantitative model for estimating human-pathogenic virus retention on skin following contact with contaminated water. The work will be important in refining the contribution of indirect transmission of virus to risks associated with water-related activities

Engineering Solutions to Improve the Removal of Fecal Indicator Bacteria by Bioinfiltration Systems during Intermittent Flow of Stormwater

Bioinfiltration systems facilitate the infiltration of urban stormwater into soil and reduce high flow events and flooding. Stormwater carries a myriad of pollutants including fecal indicator bacteria (FIB). Significant knowledge gaps exist about the ability of bioinfiltration systems to remove and retain FIB. The present study investigates the ability of model, simplified bioinfiltration systems containing quartz sand and iron oxide-coated quartz sand (IOCS) to remove two FIB (Enterococcus faecalis and Escherichia coli) suspended in synthetic stormwater with and without natural organic matter (NOM) as well as the potential for accumulated FIB to be remobilized during intermittent flow. The experiments were conducted in two phases: (1) the saturated columns packed with either sand or IOCS were contaminated by injecting stormwater with bacteria followed by injection of sterile stormwater and (2) the contaminated columns were subjected to intermittent infiltration of sterile stormwater preceded by a pause during which columns were either kept saturated or drained by gravity. During intermittent flow, fewer bacteria were released from the saturated column compared to the column drained by gravity: 12% of attached E. coli and 3% of attached Ent. faecalis were mobilized from the drained sand column compared to 3% of attached E. coli and 2% attached Ent. faecalis mobilized from the saturated sand column. Dry and wet cycles introduce moving air–water interfaces that can scour bacteria from grain surfaces. During intermittent flows, less than 0.2% of attached bacteria were mobilized from IOCS, which bound both bacteria irreversibly in the absence of NOM. Addition of NOM, however, increased bacterial mobilization from IOCS: 50% of attached E. coli and 8% of attached Ent. faecalis were released from IOCS columns during draining and rewetting. Results indicate that using geomedia such as IOCS that promote irreversible attachment of bacteria, and maintaining saturated condition, could minimize the mobilization of previous attached bacteria from bioinfiltration systems, although NOM may significantly decrease these benefits

Systematic review of marine environmental DNA metabarcoding studies: toward best practices for data usability and accessibility

The emerging field of environmental DNA (eDNA) research lacks universal guidelines for ensuring data produced are FAIR–findable, accessible, interoperable, and reusable–despite growing awareness of the importance of such practices. In order to better understand these data usability challenges, we systematically reviewed 60 peer reviewed articles conducting a specific subset of eDNA research: metabarcoding studies in marine environments. For each article, we characterized approximately 90 features across several categories: general article attributes and topics, methodological choices, types of metadata included, and availability and storage of sequence data. Analyzing these characteristics, we identified several barriers to data accessibility, including a lack of common context and vocabulary across the articles, missing metadata, supplementary information limitations, and a concentration of both sample collection and analysis in the United States. While some of these barriers require significant effort to address, we also found many instances where small choices made by authors and journals could have an outsized influence on the discoverability and reusability of data. Promisingly, articles also showed consistency and creativity in data storage choices as well as a strong trend toward open access publishing. Our analysis underscores the need to think critically about data accessibility and usability as marine eDNA metabarcoding studies, and eDNA projects more broadly, continue to proliferate

Enterococcus and Escherichia coli fecal source apportionment with microbial source tracking genetic markers - Is it feasible?

Fecal pollution is measured in surface waters using culture-based measurements of enterococci and Escherichia coli bacteria. Source apportionment of these two fecal indicator bacteria is an urgent need for prioritizing remediation efforts and quantifying health risks associated with source-specific pathogens. There are a number of quantitative real-time PCR (QPCR) assays that estimate concentrations of source-associated genetic markers; however, their concentrations are not necessarily amenable to source apportionment because the markers may differ in prevalence across sources. Here we mathematically derive and test, under ideal conditions, a method that utilizes the ratios of fecal source-associated genetic markers and culture and molecular measurements of general fecal indicators to apportion enterococci and E. coli. The source contribution is approximately equal to the ratio of the source-associated and the general fecal indicator concentrations in a water sample divided by their ratio in the source material, so long as cross-reactivity is negligible. We illustrate the utility of the ratio method using samples consisting of mixtures of various fecal pollution sources. The results from the ratio method correlated well with the actual source apportionment in artificial samples. However, aging of contamination can confound source allocation predictions. In particular, culturable enterococci and E. coli, the organisms presently regulated in the United States and much of the world, decay at different rates compared to source-associated markers and as a result cannot be apportioned using this method. However, limited data suggest a similar decay rate between source-associated and QPCR-measured Enterococcus and E. coli genetic markers, indicating that apportionment may be possible for these organisms; however further work is needed to confirm.Keywords: Water quality, Microbial source tracking, Fecal pollutio

Water quality criteria for an acidifying ocean: Challenges and opportunities for improvement

This is PMEL Contribution 4396. Acidification has sparked discussion about whether regulatory agencies should place coastal waters on the Clean Water Act 303(d) impaired water bodies list. Here we describe scientific challenges in assessing impairment with existing data, exploring use of both pH and biological criteria. Application of pH criteria is challenging because present coastal pH levels fall within the allowable criteria range, but the existing criteria allow for pH levels that are known to cause extensive biological damage. Moreover, some states express their water quality criteria as change from natural conditions, but the spatio-temporal distribution and quality of existing coastal pH data are insufficient to define natural condition. Biological criteria require that waters be of sufficient quality to support resident biological communities and are relevant because a number of biological communities have declined over the last several decades. However, the scientific challenge is differentiating those declines from natural population cycles and positively associating them with acidification-related water quality stress. We present two case studies, one for pteropods and one for oysters, which illustrate the opportunities, challenges and uncertainties associated with implementing biological criteria. The biggest challenge associated with these biological assessments is lack of co-location between long-term biological and chemical monitoring, which inhibits the ability to connect biological response with an acidification stressor. Developing new, ecologically relevant water quality criteria for acidification and augmenting coastal water monitoring at spatio-temporal scales appropriate to those criteria would enhance opportunities for effective use of water quality regulations.This is the publisher’s final pdf. The published article is copyrighted by Elsevier and can be found at: http://www.journals.elsevier.com/ocean-and-coastal-managementKeywords: Water quality criteria, 303(d), Acidification, Pteropod