Sunny C. Jiang

Human adenoviruses and coliphages in urban runoff-impacted coastal waters of southern California

ABSTRACT A nested-PCR method was used to detect the occurrence of human adenovirus in coastal waters of Southern California. Twenty- to forty-liter water samples were collected from 12 beach locations from Malibu to the border of Mexico between February and March 1999. All sampling sites were located at mouths of major rivers and creeks. Two ultrafiltration concentration methods, tangential flow filtration (TFF) and vortex flow filtration (VFF), were compared using six environmental samples. Human adenoviruses were detected in 4 of the 12 samples tested after nucleic acid extraction of VFF concentrates. The most probable number of adenoviral genomes ranged from 880 to 7,500 per liter of water. Coliphages were detected at all sites, with the concentration varying from 5.3 to 3332 PFU/liter of water. F-specific coliphages were found at 5 of the 12 sites, with the concentration ranging from 5.5 to 300 PFU/liter. The presence of human adenovirus was not significantly correlated with the concentration of coliphage (r = 0.32) but was significantly correlated (r = 0.99) with F-specific coliphage. The bacterial indicators (total coliforms, fecal coliforms, and enterococci) were found to exceed California recreational water quality daily limits at 5 of the 12 sites. However, this excess of bacterial indicators did not correlate with the presence of human adenoviruses in coastal waters. The results of this study call for both a reevaluation of our current recreational water quality standards to reflect the viral quality of recreational waters and monitoring of recreational waters for human viruses on a regular basis.

Quantification of Enterococci and Human Adenoviruses in Environmental Samples by Real-Time PCR

ABSTRACT Pathogenic bacteria and enteric viruses can be introduced into the environment via human waste discharge. Methods for rapid detection and quantification of human viruses and fecal indicator bacteria in water are urgently needed to prevent human exposure to pathogens through drinking and recreational waters. Here we describe the development of two real-time PCR methods to detect and quantify human adenoviruses and enterococci in environmental waters. For real-time quantification of enterococci, a set of primers and a probe targeting the 23S rRNA gene were used. The standard curve generated using Enterococcus faecalis genomic DNA was linear over a 7-log-dilution series. Serial dilutions of E. faecalis suspensions resulted in a lower limit of detection (LLD) of 5 CFU/reaction. To develop real-time PCR for adenoviruses, degenerate primers and a Taqman probe targeting a 163-bp region of the adenovirus hexon gene were designed to specifically amplify 14 different serotypes of human adenoviruses, including enteric adenovirus serotype 40 and 41. The standard curve generated was linear over a 5-log-dilution series, and the LLD was 100 PFU/reaction using serial dilutions of purified adenoviral particles of serotype 40. Both methods were optimized to be applicable to environmental samples. The real-time PCR methods showed a greater sensitivity in detection of adenoviruses in sewage samples than the viral plaque assay and in detection of enterococci in coastal waters than the bacterial culture method. However, enterococcus real-time PCR overestimated the number of bacteria in chlorinated sewage in comparison with the bacterial culture method. Overall, the ability via real-time PCR to detect enterococci and adenoviruses rapidly and quantitatively in the various environmental samples represents a considerable advancement and a great potential for environmental applications.

Taking the “Waste” Out of “Wastewater” for Human Water Security and Ecosystem Sustainability

Humans create vast quantities of wastewater through inefficiencies and poor management of water systems. The wasting of water poses sustainability challenges, depletes energy reserves, and undermines human water security and ecosystem health. Here we review emerging approaches for reusing wastewater and minimizing its generation. These complementary options make the most of scarce freshwater resources, serve the varying water needs of both developed and developing countries, and confer a variety of environmental benefits. Their widespread adoption will require changing how freshwater is sourced, used, managed, and priced.

Molecular analysis of Vibrio cholerae O1, O139, non-O1, and non-O139 strains: clonal relationships between clinical and environmental isolates

ABSTRACT A total of 26 strains of Vibrio cholerae, including members of the O1, O139, and non-O1, non-O139 serogroups from both clinical and environmental sources, were examined for the presence of genes encoding cholera toxin (ctxA), zonula occludens toxin (zot), accessory cholera enterotoxin (ace), hemolysin (hlyA), NAG-specific heat-stable toxin (st), toxin-coregulated pilus (tcpA), and outer membrane protein (ompU), for genomic organization, and for the presence of the regulatory protein genes tcpI andtoxR in order to determine relationships between epidemic serotypes and sources of isolation. While 22 of the 26 strains were hemolytic on 5% sheep blood nutrient agar, all strains were PCR positive for hlyA, the hemolysin gene. When multiplex PCR was used, all serogroup O1 and O139 strains were positive fortcpA, ompU, and tcpI. All O1 and O139 strains except one O1 strain and one O139 strain were positive for the ctxA, zot, and ace genes. Also, O1 strain VO3 was negative for the zot gene. All of the non-O1, non-O139 strains were negative for the ctxA,zot, ace, tcpA, andtcpI genes, and all of the non-O1, non-O139 strains except strain VO26 were negative for ompU. All of the strains except non-O1, non-O139 strain VO22 were PCR positive for the gene encoding the central regulatory protein, toxR. All V. cholerae strains were negative for the NAG-specificst gene. Of the nine non-ctx-producing strains of V. cholerae, only one, non-O1, non-O139 strain VO24, caused fluid accumulation in the rabbit ileal loop assay. The other eight strains, including an O1 strain, an O139 strain, and six non-O1, non-O139 strains, regardless of the source of isolation, caused fluid accumulation after two to five serial passages through the rabbit gut. Culture filtrates of all non-cholera-toxigenic strains grown in AKI media also caused fluid accumulation, suggesting that a new toxin was produced in AKI medium by these strains. Studies of clonality performed by using enterobacterial repetitive intergenic consensus sequence PCR, Box element PCR, amplified fragment length polymorphism (AFLP), and pulsed-field gel electrophoresis (PFGE) collectively indicated that theV. cholerae O1 and O139 strains had a clonal origin, whereas the non-O1, non-O139 strains belonged to different clones. The clinical isolates closely resembled environmental isolates in their genomic patterns. Overall, there was an excellent correlation among the results of the PCR, AFLP, and PFGE analyses, and individual strains derived from clinical and environmental sources produced similar fingerprint patterns. From the results of this study, we concluded that the non-cholera-toxin-producing strains of V. cholerae, whether of clinical or environmental origin, possess the ability to produce a new secretogenic toxin that is entirely different from the toxin produced by toxigenic V. cholerae O1 and O139 strains. We also concluded that the aquatic environment is a reservoir for V. cholerae O1, O139, non-O1, and non-O139 serogroup strains.

Predictability of Vibrio cholerae in Chesapeake Bay

ABSTRACT Vibrio cholerae is autochthonous to natural waters and can pose a health risk when it is consumed via untreated water or contaminated shellfish. The correlation between the occurrence of V. cholerae in Chesapeake Bay and environmental factors was investigated over a 3-year period. Water and plankton samples were collected monthly from five shore sampling sites in northern Chesapeake Bay (January 1998 to February 2000) and from research cruise stations on a north-south transect (summers of 1999 and 2000). Enrichment was used to detect culturable V. cholerae, and 21.1% (n = 427) of the samples were positive. As determined by serology tests, the isolates, did not belong to serogroup O1 or O139 associated with cholera epidemics. A direct fluorescent-antibody assay was used to detect V. cholerae O1, and 23.8% (n = 412) of the samples were positive. V. cholerae was more frequently detected during the warmer months and in northern Chesapeake Bay, where the salinity is lower. Statistical models successfully predicted the presence of V. cholerae as a function of water temperature and salinity. Temperatures above 19°C and salinities between 2 and 14 ppt yielded at least a fourfold increase in the number of detectable V. cholerae. The results suggest that salinity variation in Chesapeake Bay or other parameters associated with Susquehanna River inflow contribute to the variability in the occurrence of V. cholerae and that salinity is a useful indicator. Under scenarios of global climate change, increased climate variability, accompanied by higher stream flow rates and warmer temperatures, could favor conditions that increase the occurrence of V. cholerae in Chesapeake Bay.

Real-Time PCR Quantification of Human Adenoviruses in Urban Rivers Indicates Genome Prevalence but Low Infectivity

ABSTRACT Real-time PCR was applied to quantify the abundance of human adenoviruses in two southern California urban rivers, the San Gabriel and Los Angeles. A total of 114 river samples from five different locations were collected over a 1-year period and analyzed for human adenoviruses, along with fecal indicator bacteria and coliphages. Adenoviruses were detected by real-time PCR in ∼16% of the samples, with concentrations ranging from 102 to 104 genomes per liter. However, a plaque assay using two human tissue culture cell lines, HEK-293A and A549, yielded negative results, suggesting that adenoviruses detected by real-time PCR are likely noninfectious. Enterovirus genome was detected in ∼7% of the samples by reverse transcription-PCR. Analysis by Spearmans rho rank order correlation showed significant correlations between fecal indicator bacteria and indicator virus (total coliform, fecal coliform, enterococcus, and coliphage values). However, no significant correlations were found between human adenoviruses quantified by real-time PCR and culturable coliphages or fecal indicator bacteria. Kruskal-Wallis chi-square analysis showed significant seasonal variability of all fecal indicator bacteria and coliphages, while no significant variability was observed for adenoviruses or enteroviruses. This study presents the first quantitative measurement of human adenovirus genomes in urban rivers and their statistical relationship to fecal indicator bacteria and coliphages. The uncoupling between high-number genome copies of adenoviruses detected by real-time PCR and the absence of infectivity detected by tissue culture suggests that genome-based detection methods are inadequate for direct assessment of human health risk.

Significance of Lysogeny in the Marine Environment: Studies with Isolates and a Model of Lysogenic Phage Production

A bstractThe importance of lysogeny in marine microbial populations is just beginning to be understood. To determine the abundance of lysogens in bacterial populations, we studied the occurrence of lysogenic bacteria among bacterial isolates from a variety of marine environments. More than 116 bacteria isolated on artificial seawater nutrient agar plates were tested for the presence of inducible prophage by mitomycin C and UV radiation. Induction was determined as a decrease in culture absorbance at 600 nm, after treatment with inducing agents. Samples in which optical density decreased or remained the same after induction were further examined by transmission electron microscopy, for the presence of virus-like particles. More than 40% of the bacterial isolates contained inducible prophage, as determined by mitomycin C induction. A higher percentage of lysogenic bacteria was found in isolates from oligotrophic environments, compared to coastal or estuarine environments. These studies suggest that lysogenic bacteria are important components in marine microbial populations. However, a mathematical model based on viral and bacterial abundance and production rates suggests that, under normal conditions, lysogenic viral production contributes less than 0.02% of total viral production. Therefore, lysogens in the marine environment may serve as a source of viruses and only contribute significantly to viral production during natural induction events.

PCR detection of pathogenic viruses in southern California urban rivers

Aims:  To investigate human viral contamination in urban rivers and its impact on coastal waters of southern California, USA.

Evidence for groundwater and surface marine water contamination by waste disposal wells in the Florida keys

Abstract One of the methods for domestic wastewater disposal in the Florida Keys is injection of partially treated wastewater into the highly porous limestone bedrock using waste disposal or injection wells. In an effort to understand the transport and fate of wastewater, we utilized bacteriophages as tracers in a 12.2-m deep, simulated injection well in Key Largo and an active (27.4 m deep) class V disposal well in the Middle Keys. The latter is the currently permitted type of injection well used for multi-unit domestic waste disposal in the Keys. Surface waters and groundwater at several sites were monitored for 5 days after seeding of the injection wells. In both environments, viral tracers appeared after short periods of time in groundwater (8 h after injection) and surface marine waters (10 h and 53 h for Key Largo and the Middle Keys, respectively). Estimated rates of tracer movement were greatest in Key Largo (2.5–35 m/h), where tidal pumping was implicated in tracer movement. In the Middle Keys, the pattern of movement showed little evidence of tidal pumping, and migration rates were slower (0.12–2 m/h). Collectively these results indicate that wastewater injected into the subsurface can make its way rapidly to surface marine waters, where it may contribute to water quality deterioration.

Isolation and characterization of an atrazine-degrading bacterium from industrial wastewater in China

Aims: To isolate and characterize atrazine‐degrading bacteria in order to identify suitable candidates for potential use in bioremediation of atrazine contamination.