Roger S. Fujioka

Soil: the environmental source of Escherichia coli and Enterococci in Guam's streams

We have previously documented that faecal indicator bacteria (Escherichia coli, faecal coliform, enterococci) recommended by the U.S. Environmental Protection Agency (USEPA) to establish recreational water quality standards are naturally found in high concentrations in the surface and subsurface of soils in Hawaii. Rain, the source of all streams in Hawaii, washes the soil sources of faecal bacteria into all the streams of Hawaii, at concentrations which consistently exceed the USEPA recreational water quality standards. The objective of this study was to test the hypothesis that faecal bacteria are able to establish themselves in the soil environments of tropical islands by conducting the same study in Guam, a tropical pacific island with warmer temperatures and higher humidity than Hawaii. The same methods and study design used in Hawaii was used in Guam. The results of the study conducted in Guam revealed that all streams contain consistently high concentrations of faecal coliform, E. coli, and enterocci which exceeded the old USEPA recreational water quality standard of 200 faecal coliform/100 ml as well as the new water quality standards of 126 E. coli/100 ml or 33 enterococci/100 ml. These same faecal indicator bacteria were recovered in high concentrations in surface and subsurface (18–36 cm depth) soil samples in Guam. Limited coastal water analysis showed that most coastal marine waters contain low concentrations of faecal bacteria but coastal waters impacted by stream run‐off showed elevated levels of faecal bacteria. The results of this study support the hypothesis that environmental conditions in the tropical areas of the world can support the growth and establishment of populations of faecal bacteria in the soil. Thus, soil becomes an environmental, non‐faecal source of faecal indicator bacteria. These results indicate that USEPA water quality standards may not be directly applicable to tropical island environments.

Hematologic and blood chemistry values for Sarotherodon melanotheron and a red hybrid tilapia in freshwater and seawater

1. 1. The effects of salinity, sex and species (S. melanotheron and a red hybrid) on a selection of blood parameters were determined. 2. 2. The seawater (SW) adapted fish had higher (P < 0.05) PCV, HBG, RBCC, MCHC, TP, G, GLU, BUN, UA, ALT, AP, Na, K and Cl values compared to the freshwater (FW) adapted fish. 3. 3. Females had higher (P < 0.01) values than males for TP, A, G, CHOL, TRIGLY, Ca and P. 4. 4. The salinity x sex interaction had effects (P 0.05) salinity × sex × species 3-way interactions.

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.

Isolation and characterization of Bacteroides host strain HB-73 used to detect sewage specific phages in Hawaii.

Previous studies have shown that Escherichia coli and enterococci are unreliable indicators of fecal contamination in Hawaii because of their ability to multiply in environmental soils. In this study, the method of detecting Bacteroides phages as specific markers of sewage contamination in Hawaiis recreational waters was evaluated because these sewage specific phages cannot multiply under environmental conditions. Bacteroides hosts (GB-124, GA-17), were recovered from sewage samples in Europe and were reported to be effective in detecting phages from sewage samples obtained in certain geographical areas. However, GB-124 and GA-17 hosts were ineffective in detecting phages from sewage samples obtained in Hawaii. Bacteroides host HB-73 was isolated from a sewage sample in Hawaii, confirmed as a Bacteroides sp. and shown to recover phages from multiple sources of sewage produced in Hawaii at high concentrations (5.2-7.3 x 10(5) PFU/100 mL). These Bacteroides phages were considered as potential markers of sewage because they also survived for three days in fresh stream water and two days in marine water. Water samples from Hawaiis coastal swimming beaches and harbors, which were known to be contaminated with discharges from streams, were shown to contain moderate (20-187 CFU/100 mL) to elevated (173-816 CFU/100 mL) concentrations of enterococci. These same samples contained undetectable levels (<10 PFU/100 mL) of F+ coliphage and Bacteroides phages and provided evidence to suggest that these enterococci may not necessarily be associated with the presence of raw sewage. These results support previous conclusions that discharges from streams are the major sources of enterococci in coastal waters of Hawaii and the most likely source of these enterococci is from environmental soil rather than from sewage.

Use of the nitrocellulose-enzyme immunosorbent assay for rapid, sensitive and quantitative detection of human enteroviruses

A modified enzyme immunosorbent assay (EIA) employing nitrocellulose (NC) membrane as a high-capacity solid phase was successfully employed for the sensitive and rapid detection of human enteric viruses, poliovirus and Coxsackievirus B-5. The sensitivity of the NC-EIA ranged from 7 to 70 pg of viral antigen diluted in phosphate-buffered saline. When virus was added to crude supernatants of mollusc tissue homogenates prepared by the standard procedure for the recovery of viruses in molluscs, the sensitivity was reduced by approximately 10-fold. The sensitivity of the NC-EIA for the detection of viral antigens was 10- to 100-fold higher than conventional EIA using polystyrene microtiter plates as solid phase supports. This simple, rapid and sensitive assay using minimal amounts of antibodies should prove to be a useful and practical diagnostic tool to augment infectivity assays currently employed by various virus monitoring procedures. The method also may be applicable for the detection of difficult to grow and/or non-cultivatable enteric viruses which may be present in sewage-contaminated environments.

Recovery, survival and dissemination of human enteric viruses in ocean waters receiving sewage in Hawaii

A commercial model of a portable virus concentrator operating on the principle of the membrane virus adsorption-elution technique was used to examine the recovery and distribution of human enteropathogenic viruses in Hawaiian waters receiving sewage wastes. Although operating at an efficiency of 20%, a variety of human enteric viruses was isolated, including polioviruses, Coxsackievirus and Echoviruses. Positive virus isolations were consistently made and at high concentrations at sampling stations closest to the sewage outfall. The frequency of virus isolations decreased with increasing distance from the discharge site. viruses were recovered in all directions from the outfall and even as far as 3.2 km east but not 6.4 km away from the discharge site.Tidal conditions to some degree were found to influence the dissemination of viruses. However, since sampling was generally performed at the surface of the water (0.3 to 0.6 m depth), the influence of winds and surface currents was more difficult to assess. Although a positive correlation was found between the presence of high concentrations of indicator coliforms and viruses in waters closest to the discharge site, this relationship was not seen at sampling stations farther away. At these latter stations, positive virus isolations with corresponding low to negligible coliform counts were made which reaffirmed previous published observations that human enteric viruses are relatively more stable in ocean water than coliform bacteria. Furthermore, the instability of the coliform bacteria consequently affected the fecal coliform/fecal streptococci ratios which thus invalidated the use of such ratios to indicate contamination by human wastes.

Recycling of water for irrigation: Persistence of enteroviruses in sewage effluent and natural waters receiving the effluent

A virological analyses of a sewage treatment plant which provided chlorinated, activated sludge treated sewage effluent to irrigate a complete two-year crop of sugarcane was made. The raw, the activated sludge treated and the chlorinated sewage effluent, as well as streams and a harbor receiving sewage effluents were concentrated by either the polymer two-phase, PE-60, Al(OH)3, protamine sulfate, or cellulose membrane method and assayed for human enteric viruses. Viruses were recovered from 100% (11/11) of the raw sewages tested at concentrations ranging from 27 to 19 000 PFU l−1 while 76% (13/17) of the activated sludge treated effluent was positive at concentrations ranging from 7 to 5222 PFU l−1. After chlorination, 58% (31/53) of the samples was positive for virus at concentrations ranging from 2 to 750 PFU l−1. Human enteroviruses were also isolated from shallow flowing streams at distances up to 3 mi (5 km) from the closest known sewage effluent discharge point and from a harbor approximately 0.5 mi (0.8 km) from the point of sewage discharge entering the harbor. The viruses most often isolated were echovirus 7, coxsackievirus B-4, B-5 and poliovirus 1, 2, and 3. These results indicate that although activated sludge treatment plus chlorination remove approximately 90% of the virus from the raw sewage, the final treated sewage effluent, which is normally discharged into a stream and in this experimental study to irrigate sugarcane, still contains a significant concentration of infectious viruses. Furthermore, the recovery of enteroviruses from waterways at points distant from the sewage treatment plants indicates that sewage-borne viruses persist in natural water environment. The significance of enteric viruses in waters accessible to the public and used for irrigation purposes remains to be determined.

Human enteric viruses in sewage and their discharge into the ocean

Twenty-four hourly as well as 12 two-hour composite samples of Honolulus untreated sewage which is discharged into the ocean were assayed for human enteric viruses. Both sampling methods yielded similar results with peaks of virus discharge occurring between 11 to 14 h and again between 20 to 23 h. Over a 24 h period, 8.5 × 1010 PFU of virus based on the 24 hourly samples and 7.5 x 1010 PFU based on the 12 two-hour composite samples were determined to be discharged into the ocean via the sewage ocean outfall. Four-hour composite samples of sewage from two selected communities were also analyzed for human enteric viruses. Higher concentrations of viruses were consistently recovered from the sewage draining Kuhio Park Terrace community than from Nuuanu community and support the hypothesis that a community (Kuhio Park Terrace) characterized by a relatively high percentage of children, high density and low socio-economic conditions will excrete higher amounts of enteric viruses than a community (Nuuanu) with contrasting characteristics.

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

ABSTRACT 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.

Comparative disinfection of poliovirus by bromine chloride and chlorine in a model contact chamber

Abstract The effectiveness of BrCl vs chlorine as a disinfectant of poliovirus was evaluated using a model contact chamber capable of automatically treating water at the rate of 4 gal min− to simulate conditions at an operating wastewater treatment plant. The results show that in clean tap water both BrCl and chlorine are effective in inactivating poliovirus. However, in the presence of NH4Cl or 10% sewage effluent, BrCl was demonstrated to be superior to chlorine as a disinfectant of poliovirus.