Multiple lines of evidence to identify sewage as the cause of water quality impairment in an urbanized tropical watershed.

Indicator bacteria, which are conventionally used to evaluate recreational water quality, can originate from various non-human enteric and extra-enteric sources, hence they may not be indicative of human health risk nor do they provide information on the sources of contamination. In this study we utilized traditional (enterococci and Escherichia coli) and alternative (Clostridium perfringens) indicator bacteria, F+-specific coliphage, molecular markers for microorganisms associated with human sewage (human-associated Bacteroides and polyomaviruses), and microbial community analysis tools (16S rRNA gene fragment amplicon sequencing), to identify and evaluate human sewage-related impact in the Manoa watershed in Honolulu, Hawaii. Elevated concentrations of enterococci (geometric mean ranging from 1604 to 2575 CFU 100 mL-1) and C. perfringens (45-77 CFU 100 mL-1) indicated impairment of the urbanized section of the stream, while indicator bacteria concentrations decreased downstream in the tidally influenced Ala Wai Canal. The threshold values triggering water quality violation notifications in Hawaii were exceeded in 33.3-75.0% of samples collected at sites in the urbanized section of Manoa Stream, but were not exceeded in any of the samples collected at an upstream site located in a forested area. Correlation between indicator bacteria concentrations and rainfall amounts was weak to moderate but significant (E. coli R = 0.251, P = 0.009; enterococci R = 0.369, P < 0.001; C. perfringens R = 0.343, P < 0.001), while concentrations of human fecal-associated molecular markers were not significantly correlated with rainfall (human-associated Bacteroides, R = 0.131, P = 0.256; human-associated polyomaviruses, R = 0.213, P = 0.464). Presence of human sewage was confirmed by detection of human-associated Bacteroides and human polyomavirus in the urbanized section of Manoa Stream (83.3-100% and 41.7-66.7% positive samples respectively). It was further confirmed by microbial community analyses which suggested that an average 2.4-3.4% of the total bacterial population in this section was associated with sewage. Microbial community profiles were significantly influenced by rainfall (R2 = 0.4390, P < 0.001), pH (R2 = 0.3077, P = 0.006), salinity (R2 = 0.2614, P = 0.038), and conductivity (R2 = 0.2676, P = 0.031). Although microbial diversity fluctuated throughout the watershed, it was lower in the impaired section. Leaking sewer systems and illegal cross-connections are implicated in the impairment of the watershed, hence both the sewer and the storm water lines should be routinely inspected. Collectively, our data suggest that information derived from the analysis of microbial communities complements current marker-based microbial source tracking techniques and environmental monitoring programs.

Human-Associated Bacteroides spp. and Human Polyomaviruses as Microbial Source Tracking Markers in Hawaii.

Identification of sources of fecal contaminants is needed to (i) determine the health risk associated with recreational water use and (ii) implement appropriate management practices to mitigate this risk and protect the environment. This study evaluated human-associated Bacteroides spp. (HF183TaqMan) and human polyomavirus (HPyV) markers for host sensitivity and specificity using human and animal fecal samples collected in Hawaii. The decay rates of those markers and indicator bacteria were identified in marine and freshwater microcosms exposed and not exposed to sunlight, followed by field testing of the usability of the molecular markers. Both markers were strongly associated with sewage, although the cross-reactivity of the HF183TaqMan (also present in 82% of canine [n = 11], 30% of mongoose [n = 10], and 10% of feline [n = 10] samples) needs to be considered. Concentrations of HF183TaqMan in human fecal samples exceeded those in cross-reactive animals at least 1,000-fold. In the absence of sunlight, the decay rates of both markers were comparable to the die-off rates of enterococci in experimental freshwater and marine water microcosms. However, in sunlight, the decay rates of both markers were significantly lower than the decay rate of enterococci. While both markers have their individual limitations in terms of sensitivity and specificity, these limitations can be mitigated by using both markers simultaneously; ergo, this study supports the concurrent use of HF183TaqMan and HPyV markers for the detection of sewage contamination in coastal and inland waters in Hawaii. IMPORTANCE: This study represents an in-depth characterization of microbial source tracking (MST) markers in Hawaii. The distribution and concentrations of HF183TaqMan and HPyV markers in human and animal fecal samples and in wastewater, coupled with decay data obtained from sunlight-exposed and unexposed microcosms, support the concurrent application of HF183TaqMan and HPyV markers for sewage contamination detection in Hawaii waters. Both markers are more conservative and more specific markers of sewage than fecal indicator bacteria (enterococci and Escherichia coli). Analysis of HF183TaqMan (or newer derivatives) is recommended for inclusion in future epidemiological studies concerned with beach water quality, while better concentration techniques are needed for HPyV. Such epidemiological studies can be used to develop new recreational water quality criteria, which will provide direct information on the absence or presence of sewage contamination in water samples as well as reliable measurements of the risk of waterborne disease transmission to swimmers.

U.S. Recreational Water Quality Criteria: A Vision for the Future.

This manuscript evaluates the U.S. Recreational Water Quality Criteria (RWQC) of 2012, based upon discussions during a conference held 11-13 March 2013, in Honolulu, Hawaii. The RWQC of 2012 did not meet expectations among the research community because key recommended studies were not completed, new data to assess risks to bathers exposed to non-point sources of fecal indicator bacteria (FIB) were not developed, and the 2012 RWQC did not show marked improvements in strategies for assessing health risks for bathers using all types of recreational waters. The development of the 2012 RWQC was limited in scope because the epidemiologic studies at beach sites were restricted to beaches with point sources of pollution and water samples were monitored for only enterococci. The vision for the future is development of effective RWQC guidelines based on epidemiologic and quantitative microbial risk assessment (QMRA) studies for sewage specific markers, as well as human enteric pathogens so that health risks for bathers at all recreational waters can be determined. The 2012 RWQC introduced a program for states and tribes to develop site-specific water quality criteria, and in theory this approach can be used to address the limitations associated with the measurements of the traditional FIB.

Evidence for occurrence, persistence, and growth potential of Escherichia coli and enterococci in Hawaii's soil environments.

High densities of Escherichia coli and enterococci are common in freshwaters on Oahu and other Hawaiian Islands. Soil along stream banks has long been suspected as the likely source of these bacteria; however, the extent of their occurrence and distribution in a wide range of soils remained unknown until the current investigation. Soil samples representing the seven major soil associations were collected on the island of Oahu and analyzed for fecal coliforms, E. coli, and enterococci by the most probable number method. Fecal coliforms, E. coli, and enterococci were found in most of the samples analyzed; log mean densities (MPN ± SE g soil⁻¹) were 1.96 ± 0.18, n=61; 1.21 ± 0.17, n=57; and 2.99 ± 0.12, n=62, respectively. Representative, presumptive cultures of E. coli and enterococci collected from the various soils were identified and further speciated using the API scheme; at least six species of Enterococcus, including Enterococcus faecalis and Enterococcus faecium, were identified. In mesocosm studies, E. coli and enterococci increased by 100-fold in 4 days, after mixing sewage-spiked soil (one part) with autoclaved soil (nine parts). E. coli remained metabolically active in the soil and readily responded to nutrients, as evidenced by increased dehydrogenase activity. Collectively, these findings indicate that populations of E. coli and enterococci are part of the natural soil microflora, potentially influencing the quality of nearby water bodies.

The population structure of Escherichia coli isolated from subtropical and temperate soils.

While genotypically-distinct naturalized Escherichia coli strains have been shown to occur in riparian soils of Lake Michigan and Lake Superior watersheds, comparative analyses of E. coli populations in diverse soils across a range of geographic and climatic conditions have not been investigated. The main objectives of this study were to: (a) examine the population structure and genetic relatedness of E. coli isolates collected from different soil types on a tropical island (Hawaii), and (b) determine if E. coli populations from Hawaii and temperate soils (Indiana, Minnesota) shared similar genotypes that may be reflective of biome-related soil conditions. DNA fingerprint and multivariate statistical analyses were used to examine the population structure and genotypic characteristics of the E. coli isolates. About 33% (98 of 293) of the E. coli from different soil types and locations on the island of Oahu, Hawaii, had unique DNA fingerprints, indicating that these bacteria were relatively diverse; the Shannon diversity index for the population was 4.03. Nearly 60% (171 of 293) of the E. coli isolates from Hawaii clustered into two major groups and the rest, with two or more isolates, fell into one of 22 smaller groups, or individual lineages. Multivariate analysis of variance of 89, 21, and 106 unique E. coli DNA fingerprints for Hawaii, Indiana, and Minnesota soils, respectively, showed that isolates formed tight cohesive groups, clustering mainly by location. However, there were several instances of clonal isolates being shared between geographically different locations. Thus, while nearly identical E. coli strains were shared between disparate climatologically- and geographically-distinct locations, a vast majority of the soil E. coli strains were genotypically diverse and were likely derived from separate lineages. This supports the hypothesis that these bacteria are not unique and multiple genotypes can readily adapt to become part of the soil autochthonous microflora.

Temporal and spatial variability in culturable pathogenic Vibrio spp. in Lake Pontchartrain, Louisiana, following hurricanes Katrina and Rita.

We investigated the abundance, distribution, and virulence gene content of Vibrio cholerae, V. parahaemolyticus, and V. vulnificus in the waters of southern Lake Pontchartrain in Louisiana on four occasions from October 2005 to September 2006, using selective cultivation and molecular assays. The three targeted pathogenic vibrios were generally below the detection level in January 2006, when the water was cold (13°C), and most abundant in September 2006, when the lake water was warmest (30°C). The maximum values for these species were higher than reported previously for the lake by severalfold to orders of magnitude. The only variable consistently correlated with total vibrio abundance within a single sampling was distance from shore (P = 0.000). Multiple linear regression of the entire data set revealed that distance from shore, temperature, and turbidity together explained 82.1% of the variability in total vibrio CFU. The log-transformed mean abundance of V. vulnificus CFU in the lake was significantly correlated with temperature (P = 0.014), but not salinity (P = 0.625). Virulence-associated genes of V. cholerae (ctx) and V. parahaemolyticus (trh and tdh) were not detected in any isolates of these species (n = 128 and n = 20, respectively). In contrast, 16S rRNA typing of V. vulnificus (n = 298) revealed the presence of both environmental (type A) and clinical (type B) strains. The percentage of the B-type V. vulnificus was significantly higher in the lake in October 2005 (35.8% of the total) than at other sampling times (P ≤ 0.004), consistent with the view that these strains represent distinct ecotypes.

The coastal environment and human health: microbial indicators, pathogens, sentinels and reservoirs.

Innovative research relating oceans and human health is advancing our understanding of disease-causing organisms in coastal ecosystems. Novel techniques are elucidating the loading, transport and fate of pathogens in coastal ecosystems, and identifying sources of contamination. This research is facilitating improved risk assessments for seafood consumers and those who use the oceans for recreation. A number of challenges still remain and define future directions of research and public policy. Sample processing and molecular detection techniques need to be advanced to allow rapid and specific identification of microbes of public health concern from complex environmental samples. Water quality standards need to be updated to more accurately reflect health risks and to provide managers with improved tools for decision-making. Greater discrimination of virulent versus harmless microbes is needed to identify environmental reservoirs of pathogens and factors leading to human infections. Investigations must include examination of microbial community dynamics that may be important from a human health perspective. Further research is needed to evaluate the ecology of non-enteric water-transmitted diseases. Sentinels should also be established and monitored, providing early warning of dangers to ecosystem health. Taken together, this effort will provide more reliable information about public health risks associated with beaches and seafood consumption, and how human activities can affect their exposure to disease-causing organisms from the oceans.

Survey of methicillin-resistant Staphylococcus aureus (MRSA) carriage in healthy college students, Hawai'i.

Currently, the carriage rate for Community-Acquired Methicillin Resistant Staphylococcus aureus (CA-MRSA) is unknown in Hawai'i. This survey focuses on a healthy population of 95 college students and 5 faculty who completed a survey related to possible risk factors for colonization of Staphylococcus aureus and were sampled for S. aureus from their anterior nares. Thirty-three (33%) subjects were carrying Staphylococcus aureus and of those, 3 (3%) carried MRSA. There was no significant association between Staphylococcus aureus carriage and ethnicity, gender exposure to seawater, prior Staphylococcus aureus infections, recent antibiotic use, or pets. Additional testing of a larger group of healthy individuals would be beneficial in assessing factors associated with CA-MRSA and Methicillin-susceptible Staphylococcus aureus (MSSA) carriage in Hawai'i.