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.

Human enteric viruses--potential indicators for enhanced monitoring of recreational water quality.

Recreational waters contaminated with human fecal pollution are a public health concern, and ensuring the safety of recreational waters for public use is a priority of both the Environmental Protection Agency (EPA) and the Centers for Disease Control and Prevention (CDC). Current recreational water standards rely on fecal indicator bacteria (FIB) levels as indicators of human disease risk. However present evidence indicates that levels of FIB do not always correspond to the presence of other potentially harmful organisms, such as viruses. Thus, enteric viruses are currently tested as water quality indicators, but have yet to be successfully implemented in routine monitoring of water quality. This study utilized enteric viruses as possible alternative indicators of water quality to examine 18 different fresh and offshore recreational waters on O'ahu, Hawai'i, by using newly established laboratory techniques including highly optimized PCR, real time PCR, and viral infectivity assays. All sample sites were detected positive for human enteric viruses by PCR including enterovirus, norovirus genogroups I and II, and male specific FRNA coliphage. A six time-point seasonal study of enteric virus presence indicated significant variation in virus detection between the rainy and dry seasons. Quantitative PCR detected the presence of norovirus genogroup II at levels at which disease risk may occur, and there was no correlation found between enteric virus presence and FIB counts. Under the present laboratory conditions, no infectious viruses were detected from the samples PCR-positive for enteric viruses. These data emphasize both the need for additional indicators for improved monitoring of water quality, and the feasibility of using enteric viruses as these indicators.

Leptospira infection prevalence in small mammal host populations on three Hawaiian islands.

We describe the geographic distribution and variation in host-pathogen specificity for Leptospira-infected small mammals collected concurrently from three Hawaiian Islands over a period of 14 years: 1990-2003. Four serogroups (Icterohaemorrhagiae, Ballum, Sejroe, and Australis) were identified from the 15,171 animals tested. Serogroup prevalence differed across host species and islands (P < 0.0001 for each), but not across years. The host associations and biogeographic patterns of Leptospira in Hawaii indicate a pathogen community shaped by ecological factors.

Preliminary characterization of the normal microbiota of the human vulva using cultivation-independent methods.

The objective of this study was to perform a preliminary characterization of the microbial populations of the normal human vulva. Genomic DNA was isolated from samples of the labia majora and labia minora from four healthy women, and sequences of bacterial 16S rRNA genes in each were determined. The sequences were compared with those of known bacterial species to classify the numerically abundant populations in these communities. Even among this limited number of individuals, the microbiota of the human vulva was found to be quite diverse. Each woman had a distinctive microbiota and no single species was common to all women. The microbiota of the labia majora and labia minora differed, although both had appreciable numbers of lactobacilli and strict anaerobes. A greater diversity of populations inhabited the labia majora compared with the labia minora. The results indicated that the microbiota of the vulva includes populations known to be commensals of the microbiota of the skin, colon and vagina, and is much more complex than previously thought, suggesting that more extensive investigations are warranted.

Caudal polymorphism and cephalic morphology among first-stage larvae of Parelaphostrongylus odocoilei (Protostrongylidae: Elaphostrongylinae) in Dall's sheep from the Mackenzie Mountains, Canada.

We demonstrate polymorphism in the structure of the tail among first-stage larvae of Parelaphostrongylus odocoilei (Protostrongylidae). Two distinct larvae, both with a characteristic dorsal spine, include (1) a morphotype with a kinked conical tail marked by 3 distinct transverse folds or joints and a symmetrical terminal tail spike and (2) a morphotype with a digitate terminal region lacking folds or joints and with an asymmetrical, subterminal tail spike. These divergent larval forms had been postulated as perhaps representing distinct species of elaphostrongyline nematodes. Application of a multilocus approach using ITS-2 sequences from the nuclear genome and COX-II sequences from the mitochondrial genome confirmed the identity of these larvae as P. odocoilei. Additionally, based on scanning electron microscopy (low-temperature field emission), the cephalic region of these larvae consisted of a cuticular triradiate stoma surrounded by 6 single circumoral papillae of the inner circle, 10 papillae of the outer circle (4 paired and 2 single), and 2 lateral amphids. Our's is the first demonstration of structural polymorphism among larval conspecifics in the Metastrongyloidea and Strongylida. The basis for this polymorphism remains undetermined, but such phenomena, if discovered to be more widespread, may contribute to continued confusion in discriminating among first-stage larvae for species, genera, and subfamilies within Protostrongylidae.