Lauren M. Sassoubre

Occurrence and Persistence of Bacterial Pathogens and Indicator Organisms in Beach Sand along the California Coast

ABSTRACT This report documents the presence of fecal indicators and bacterial pathogens in sand at 53 California marine beaches using both culture-dependent and -independent (PCR and quantitative PCR [QPCR]) methods. Fecal indicator bacteria were widespread in California beach sand, with Escherichia coli and enterococci detected at 68% and 94% of the beaches surveyed, respectively. Somatic coliphages and a Bacteroidales human-specific fecal marker were detected at 43% and 13% of the beaches, respectively. Dry sand samples from almost 30% of the beaches contained at least one of the following pathogens: Salmonella spp., Campylobacter spp., Staphylococcus aureus, and methicillin-resistant Staphylococcus aureus (MRSA), which were detected at 15%, 13%, 14%, and 3% of tested beaches, respectively. Fecal indicators and pathogens were poorly correlated to one another and to land cover. Sands were dry at the time of collection, and those with relatively high moisture tended to have higher concentrations or a more frequent occurrence of both indicators and pathogens. Using culture-dependent assays, fecal indicators decayed faster than pathogens in microcosm experiments using unaltered beach sand seeded with sewage and assessed by culture-dependent assays. The following order of persistence was observed (listed from most to least persistent): Campylobacter > Salmonella > somatic coliphages > enterococci > E. coli > F+ phages. In contrast, pathogens decayed faster than fecal indicators in culture-independent assays: enterococci > Bacteroidales human-specific marker > Salmonella > Campylobacter. Microcosm experiments demonstrated that both indicators and pathogens were mobilized by wetting with seawater. Decay rates measured by QPCR were lower than those measured with culture-dependent methods. Enterococcal persistence and possible growth were observed for wetted microcosms relative to unwetted controls.

Conducting Nanosponge Electroporation for Affordable and High- Efficiency Disinfection of Bacteria and Viruses in Water

High-efficiency, affordable, and low energy water disinfection methods are in great need to prevent diarrheal illness, which is one of the top five leading causes of death over the world. Traditional water disinfection methods have drawbacks including carcinogenic disinfection byproducts formation, energy and time intensiveness, and pathogen recovery. Here, we report an innovative method that achieves high-efficiency water disinfection by introducing nanomaterial-assisted electroporation implemented by a conducting nanosponge filtration device. The use of one-dimensional (1D) nanomaterials allows electroporation to occur at only several volts, which is 2 to 3 orders of magnitude lower than that in traditional electroporation applications. The disinfection mechanism of electroporation prevents harmful byproduct formation and ensures a fast treatment speed of 15,000 L/(h·m(2)), which is equal to a contact time of 1 s. The conducting nanosponge made from low-cost polyurethane sponge coated with carbon nanotubes and silver nanowires ensures the devices affordability. This method achieves more than 6 log (99.9999%) removal of four model bacteria, including Escherichia coli, Salmonella enterica Typhimirium, Enterococcus faecalis, and Bacillus subtilis, and more than 2 log (99%) removal of one model virus, bacteriophage MS2, with a low energy consumption of only 100 J/L.

Performance of viruses and bacteriophages for fecal source determination in a multi-laboratory, comparative study.

An inter-laboratory study of the accuracy of microbial source tracking (MST) methods was conducted using challenge fecal and sewage samples that were spiked into artificial freshwater and provided as unknowns (blind test samples) to the laboratories. The results of the Source Identification Protocol Project (SIPP) are presented in a series of papers that cover 41 MST methods. This contribution details the results of the virus and bacteriophage methods targeting human fecal or sewage contamination. Human viruses used as source identifiers included adenoviruses (HAdV), enteroviruses (EV), norovirus Groups I and II (NoVI and NoVII), and polyomaviruses (HPyVs). Bacteriophages were also employed, including somatic coliphages and F-specific RNA bacteriophages (FRNAPH) as general indicators of fecal contamination. Bacteriophage methods targeting human fecal sources included genotyping of FRNAPH isolates and plaque formation on bacterial hosts Enterococcus faecium MB-55, Bacteroides HB-73 and Bacteroides GB-124. The use of small sample volumes (≤50 ml) resulted in relatively insensitive theoretical limits of detection (10-50 gene copies or plaques × 50 ml(-1)) which, coupled with low virus concentrations in samples, resulted in high false-negative rates, low sensitivity, and low negative predictive values. On the other hand, the specificity of the human virus methods was generally close to 100% and positive predictive values were ∼40-70% with the exception of NoVs, which were not detected. The bacteriophage methods were generally much less specific toward human sewage than virus methods, although FRNAPH II genotyping was relatively successful, with 18% sensitivity and 85% specificity. While the specificity of the human virus methods engenders great confidence in a positive result, better concentration methods and larger sample volumes must be utilized for greater accuracy of negative results, i.e. the prediction that a human contamination source is absent.

Characterization of fecal concentrations in human and other animal sources by physical, culture-based, and quantitative real-time PCR methods

The characteristics of fecal sources, and the ways in which they are measured, can profoundly influence the interpretation of which sources are contaminating a body of water. Although feces from various hosts are known to differ in mass and composition, it is not well understood how those differences compare across fecal sources and how differences depend on characterization methods. This study investigated how nine different fecal characterization methods provide different measures of fecal concentration in water, and how results varied across twelve different fecal pollution sources. Sources investigated included chicken, cow, deer, dog, goose, gull, horse, human, pig, pigeon, septage and sewage. A composite fecal slurry was prepared for each source by mixing feces from 6 to 22 individual samples with artificial freshwater. Fecal concentrations were estimated by physical (wet fecal mass added and total DNA mass extracted), culture-based (Escherichia coli and enterococci by membrane filtration and defined substrate), and quantitative real-time PCR (Bacteroidales, E. coli, and enterococci) characterization methods. The characteristics of each composite fecal slurry and the relationships between physical, culture-based and qPCR-based characteristics varied within and among different fecal sources. An in silico exercise was performed to assess how different characterization methods can impact identification of the dominant fecal pollution source in a mixed source sample. A comparison of simulated 10:90 mixtures based on enterococci by defined substrate predicted a source reversal in 27% of all possible combinations, while mixtures based on E. coli membrane filtration resulted in a reversal 29% of the time. This potential for disagreement in minor or dominant source identification based on different methods of measurement represents an important challenge for water quality managers and researchers.

A Coupled Modeling and Molecular Biology Approach to Microbial Source Tracking at Cowell Beach, Santa Cruz, CA, United States

Consistently high levels of bacterial indicators of fecal pollution rank Cowell Beach as the most polluted beach in California. High levels of fecal indicator bacteria (FIB), E. coli and enterococci, are measured throughout the summer, resulting in beach advisories with social and economic consequences. The source of FIB, however, is unknown. Speculations have been made that the wrack accumulating on the beach is a major source of FIB to the surf zone. The present study uses spatial and temporal sampling coupled with process-modeling to investigate potential FIB sources and the relative contributions of those sources. Temporal sampling showed consistently high FIB concentrations in the surf zone, sand, and wrack at Cowell Beach, and ruled out the storm drain, the river, the harbor, and the adjacent wharf as the sources of the high concentrations observed in the surf zone. Spatial sampling confirmed that the source of FIB to the beach is terrestrial rather than marine. Modeling results showed two dominant FIB sources to the surf zone: sand for enterococci and groundwater for E. coli. FIB from wrack represented a minor contribution to bacterial levels in the water. Molecular source tracking methods indicate the FIB at the beach is of human and bird origin. The microbial source tracking (MST) approach presented here provides a framework for future efforts.

Mechanisms for Photoinactivation of Enterococcus faecalis in Seawater

ABSTRACT Field studies in fresh and marine waters consistently show diel fluctuations in concentrations of enterococci, indicators of water quality. We investigated sunlight inactivation of Enterococcus faecalis to gain insight into photoinactivation mechanisms and cellular responses to photostress. E. faecalis bacteria were exposed to natural sunlight in clear, filtered seawater under both oxic and anoxic conditions to test the relative importance of oxygen-mediated and non-oxygen-mediated photoinactivation mechanisms. Multiple methods were used to assess changes in bacterial concentration, including cultivation, quantitative PCR (qPCR), propidium monoazide (PMA)-qPCR, LIVE/DEAD staining using propidium iodide (PI), and cellular activity, including ATP concentrations and expression of the superoxide dismutase-encoding gene, sodA. Photoinactivation, based on numbers of cultivable cells, was faster in oxic than in anoxic microcosms exposed to sunlight, suggesting that oxygen-mediated photoinactivation dominated. There was little change in qPCR signal over the course of the experiment, demonstrating that the nucleic acid targets were not damaged to a significant extent. The PMA-qPCR signal was also fairly stable, consistent with the observation that the fraction of PI-permeable cells was constant. Thus, damage to the membrane was minimal. Microbial ATP concentrations decreased in all microcosms, particularly the sunlit oxic microcosms. The increase in relative expression of the sodA gene in the sunlit oxic microcosms suggests that cells were actively responding to oxidative stress. Dark repair was not observed. This research furthers our understanding of photoinactivation mechanisms and the conditions under which diel fluctuations in enterococci can be expected in natural and engineered systems.

Diversity and transport of microorganisms in intertidal sands of the California coast

ABSTRACT Forced by tides and waves, large volumes of seawater are flushed through the beach daily. Organic material and nutrients in seawater are remineralized and cycled as they pass through the beach. Microorganisms are responsible for most of the biogeochemical cycling in the beach; however, few studies have characterized their diversity in intertidal sands, and little work has characterized the extent to which microbes are transported between different compartments of the beach. The present study uses next-generation massively parallel sequencing to characterize the microbial community present at 49 beaches along the coast of California. In addition, we characterize the transport of microorganisms within intertidal sands using laboratory column experiments. We identified extensive diversity in the beach sands. Nearly 1,000 unique taxa were identified in sands from 10 or more unique beaches, suggesting the existence of a group of “cosmopolitan” sand microorganisms. A biogeographical analysis identified a taxon-distance relationship among the beaches. In addition, sands with similar grain size, organic carbon content, exposed to a similar wave climate, and having the same degree of anthropogenic influence tended to have similar microbial communities. Column experiments identified microbes readily mobilized by seawater infiltrating through unsaturated intertidal sands. The ease with which microbes were mobilized suggests that intertidal sands may represent a reservoir of bacteria that seed the beach aquifer where they may partake in biogeochemical cycling.

Solar Inactivation of Enterococci and Escherichia coli in Natural Waters: Effects of Water Absorbance and Depth

The decay of sewage-sourced Escherichia coli and enterococci was measured at multiple depths in a freshwater marsh, a brackish water lagoon, and a marine site, all located in California. The marine site had very clear water, while the waters from the marsh and lagoon contained colored dissolved organic matter that not only blocked light but also produced reactive oxygen species. First order decay rate constants of both enterococci and E. coli were between 1 and 2 d(-1) under low light conditions and as high as 6 d(-1) under high light conditions. First order decay rate constants were well correlated to the daily average UVB light intensity corrected for light screening incorporating water absorbance and depth, suggesting endogenous photoinactivation is a major pathway for bacterial decay. Additional laboratory experiments demonstrated the presence of colored dissolved organic matter in marsh water enhanced photoinactivation of a laboratory strain of Enterococcus faecalis, but depressed photoinactivation of sewage-sourced enterococci and E. coli after correcting for UVB light screening, suggesting that although the exogenous indirect photoinactivation mechanism may be active against Ent. faecalis, it is not for the sewage-source organisms. A simple linear regression model based on UVB light intensity appears to be a useful tool for predicting inactivation rate constants in natural waters of any depth and absorbance.

Impacts of Beach Wrack Removal via Grooming on Surf Zone Water Quality

Fecal indicator bacteria (FIB) are used to assess the microbial water quality of recreational waters. Increasingly, nonfecal sources of FIB have been implicated as causes of poor microbial water quality in the coastal environment. These sources are challenging to quantify and difficult to remediate. The present study investigates one nonfecal FIB source, beach wrack (decaying aquatic plants), and its impacts on water quality along the Central California coast. The prevalence of FIB on wrack was studied using a multibeach survey, collecting wrack throughout Central California. The impacts of beach grooming, to remove wrack, were investigated at Cowell Beach in Santa Cruz, California using a long-term survey (two summers, one with and one without grooming) and a 48 h survey during the first ever intensive grooming event. FIB were prevalent on wrack but highly variable spatially and temporally along the nine beaches sampled in Central California. Beach grooming was generally associated with either no change or a slight increase in coastal FIB concentrations and increases in surf zone turbidity and silicate, phosphate, and dissolved inorganic nitrogen concentrations. The findings suggest that beach grooming for wrack removal is not justified as a microbial pollution remediation strategy.

Temporal Stability of the Microbial Community in Sewage-Polluted Seawater Exposed to Natural Sunlight Cycles and Marine Microbiota

ABSTRACT Billions of gallons of untreated wastewater enter the coastal ocean each year. Once sewage microorganisms are in the marine environment, they are exposed to environmental stressors, such as sunlight and predation. Previous research has investigated the fate of individual sewage microorganisms in seawater but not the entire sewage microbial community. The present study used next-generation sequencing (NGS) to examine how the microbial community in sewage-impacted seawater changes over 48 h when exposed to natural sunlight cycles and marine microbiota. We compared the results from microcosms composed of unfiltered seawater (containing naturally occurring marine microbiota) and filtered seawater (containing no marine microbiota) to investigate the effect of marine microbiota. We also compared the results from microcosms that were exposed to natural sunlight cycles with those from microcosms kept in the dark to investigate the effect of sunlight. The microbial community composition and the relative abundance of operational taxonomic units (OTUs) changed over 48 h in all microcosms. Exposure to sunlight had a significant effect on both community composition and OTU abundance. The effect of marine microbiota, however, was minimal. The proportion of sewage-derived microorganisms present in the microcosms decreased rapidly within 48 h, and the decrease was the most pronounced in the presence of both sunlight and marine microbiota, where the proportion decreased from 85% to 3% of the total microbial community. The results from this study demonstrate the strong effect that sunlight has on microbial community composition, as measured by NGS, and the importance of considering temporal effects in future applications of NGS to identify microbial pollution sources.