Bifidobacteria in Feces and Environmental Waters

Bifidobacteria have been recommended as potential indicators of human fecal pollution in surface waters even though very little is known about their presence in nonhuman fecal sources. The objective of this research was to shed light on the occurrence and molecular diversity of this fecal indicator group in different animals and environmental waters. Genus- and species-specific 16S rRNA gene PCR assays were used to study the presence of bifidobacteria among 269 fecal DNA extracts from 32 different animals. Twelve samples from three wastewater treatment plants and 34 water samples from two fecally impacted watersheds were also tested. The species-specific assays showed that Bifidobacterium adolescentis, B. bifidum, B. dentium, and B. catenulatum had the broadest host distribution (11.9 to 17.4%), whereas B. breve, B. infantis, and B. longum were detected in fewer than 3% of all fecal samples. Phylogenetic analysis of 356 bifidobacterial clones obtained from different animal feces showed that ca. 67% of all of the sequences clustered with cultured bifidobacteria, while the rest formed a supercluster with low sequence identity (i.e., <94%) to previously described Bifidobacterium spp. The B. pseudolongum subcluster (>97% similarity) contained 53 fecal sequences from seven different animal hosts, suggesting the cosmopolitan distribution of members of this clade. In contrast, two clades containing B. thermophilum and B. boum clustered exclusively with 37 and 18 pig fecal clones, respectively, suggesting host specificity. Using species-specific assays, bifidobacteria were detected in only two of the surface water DNA extracts, although other fecal anaerobic bacteria were detected in these waters. Overall, the results suggest that the use of bifidobacterial species as potential markers to monitor human fecal pollution in natural waters may be questionable.     

Detection of Human-Derived Fecal Pollution in Environmental Waters by Use of a PCR-Based Human Polyomavirus Assay

Regulatory agencies mandate the use of fecal coliforms, Escherichia coli or Enterococcus spp., as microbial indicators of recreational water quality. These indicators of fecal pollution do not identify the specific sources of pollution and at times underestimate health risks associated with recreational water use. This study proposes the use of human polyomaviruses (HPyVs), which are widespread among human populations, as indicators of human fecal pollution. A method was developed to concentrate and extract HPyV DNA from environmental water samples and then to amplify it by nested PCR. HPyVs were detected in as little as 1 μl of sewage and were not amplified from dairy cow or pig wastes. Environmental water samples were screened for the presence of HPyVs and two additional markers of human fecal pollution: the Enterococcus faecium esp gene and the 16S rRNA gene of human-associated Bacteroides. The presence of human-specific indicators of fecal pollution was compared to fecal coliform and Enterococcus concentrations. HPyVs were detected in 19 of 20 (95%) samples containing the E. faecium esp gene and Bacteroides human markers. Weak or no correlation was observed between the presence/absence of human-associated indicators and counts of indicator bacteria. The sensitivity, specificity, and correlation with other human-associated markers suggest that the HPyV assay could be a useful predictor of human fecal pollution in environmental waters and an important component of the microbial-source-tracking “toolbox.”     

Histo-Blood Group Antigen-Like Substances of Human Enteric Bacteria as Specific Adsorbents for Human Noroviruses

Histo-blood group antigens (HBGAs) have been suggested to be receptors or coreceptors for human noroviruses (HuNoVs) expressed on the intestinal epithelium. We isolated an enteric bacterium strain (SENG-6), closely related to Enterobacter cloacae, bearing HBGA-like substances from a fecal sample of a healthy individual by using a biopanning technique with anti-HBGA antibodies. The binding capacities of four genotypes of norovirus-like particles (NoVLPs) to Enterobacter sp. SENG-6 cells were confirmed by enzyme-linked immunosorbent assay (ELISA). Transmission electron microscopy demonstrated that NoVLPs bound mainly to extracellular polymeric substances (EPS) of Enterobacter sp. SENG-6, where the HBGA-like substances were localized. EPS that contained HBGA-like substances extracted from Enterobacter sp. SENG-6 was shown by enzyme-linked immunosorbent assay (ELISA) to be capable of binding to NoVLPs of a GI.1 wild-type strain (8fIIa) and a GII.6 strain that can recognize A antigen but not to an NoVLP GI.1 mutant strain (W375A) that loses the ability to bind to A antigen. Enzymatic cleavage of terminal N-acetyl-galactosamine residues in the bacterial EPS weakened bacterial EPS binding to the GI.1 wild-type strain (8fIIa). These results indicate that A-like substances in the bacterial EPS play a key role in binding to NoVLPs. Since the specific binding of HuNoVs to HBGA-positive enteric bacteria is likely to affect the transmission and infection processes of HuNoVs in their hosts and in the environment, further studies of human enteric bacteria and their binding capacity to HuNoVs will provide a new scientific platform for understanding interactions between two types of microbes that were previously regarded as biologically unrelated.     

诺瓦克病毒研究进展

诺瓦克病毒(Noroviruses,NVs)是1972年在美国首次发现的新生病毒,到1995年国内才有报道。该病毒是严重危害人类健康的重要食源性病毒,可导致不同年龄阶段人群的急性病毒性腹泻。因为至今没有发现合适的细胞系和动物模型用于NVs的体外增殖,所以对于该病毒的研究相对滞后。随着分子生物学及其他学科的不断发展,不同类群NVs全基因组测序完成并且病毒核衣壳蛋白分别获得真核和原核系统体外表达,从而对该病毒特性产生了一些新的认识和观点。论文从NVs的基因组结构与功能、蛋白质组成与功能、检测方法应用与发展和流行病学积累四个方面,系统介绍了该病毒国内外的研究现状以及其中存在的问题;其中,其蛋白质研究及其检测方法成为NVs的热点。论文还对NVs分子进化、检测技术和病毒体外增殖模式等提出不同的看法和建议。     

Environmental Factors Affecting the Occurrence of Mycobacteria in Brook Waters

To evaluate the impact of environmental factors on the occurrence of environmental mycobacteria, viable counts of mycobacteria were measured in samples of brook water collected from 53 drainage areas located in a linear belt crossing Finland at 63° north latitude. The numbers of mycobacteria were correlated with characteristics of the drainage area, climatic parameters, chemical and physical characteristics of the water, and counts of other heterotrophic bacteria in the water. The numbers of mycobacteria in the water ranged from 10 to 2,200 CFU/liter. The counts correlated positively (P < 0.001) with the presence of peatlands, precipitation data, chemical oxygen demand, water color, and concentrations of Fe, Al, Cu, Co, and Cr. The mycobacterial counts correlated negatively (P < 0.001) with water pH, whereas other heterotrophic bacterial counts lacked any correlation with pH. A linear regression model with four independent variables (i.e., peatlands in the drainage area, chemical oxygen demand, concentration of potassium, and pH) explained 83% of the variation in mycobacterial counts in brook waters. Our results suggest that acidification may enhance the growth of environmental mycobacteria.     

Detection of Giardia in Environmental Waters by Immuno-PCR Amplification Methods

Genomic DNA was extracted either directly from Giardia muris cysts seeded into environmental surface waters or from cysts isolated by immunomagnetic beads (IMB). A 0.171-kbp segment of the giardin gene was PCR-amplified following “direct extraction” of Giardia DNA from seeded Cahaba river water concentrate with moderate turbidity (780 JTU's), but DNA purified from seeded Colorado river water concentrates with high turbidity (2 × 10⁵ JTUs) failed to amplify. However, if the cysts were first separated by the IMB approach from seeded Cahaba or Colorado river waters, and the DNA released by a freeze-boil Chelex?100 treatment, detection of G. muris by PCR amplification could be achieved at a sensitivity of 3 × 10⁰ or 3 × 10¹ cysts/ml, respectively. If, however, the G. muris cysts used to seed even moderately turbid river waters (780 JTUs) were formalin treated (which is conventionally used for microscopic examination), neither direct extraction nor IMB purification methods yielded amplifiable DNA. Use of immunomagnetic beads to separate Giardia cysts from complex matrices of environmental surface waters followed by DNA release and PCR amplification of the target giardin gene improved the reliability of detection of this pathogen with the required sensitivity. Received: 23 April 1997 / Accepted: 4 August 1997     

Human Health Risks of Exposure to Estuary Waters

The convergence of data strongly suggests the presence of an estuary-related toxin (pfiesteria piscicida or similar organism) that produces adverse human health effects. A diversity of methodological approaches is necessary to answer important preliminary questions about the clinical syndrome, its immediate public health risk, prioritize ongoing scientific studies and direct future research. These include intensive case and multiple-case studies; case-control studies; and epidemiological cohort studies that are conducted in parallel with basic science research. Findings to date indicate the estuary-related toxin is neurotoxic to persons with high levels of exposure. The most pronounced effects are neurocognitive and include problems with complex attention, memory, and visual contrast sensitivity. The disturbances are detectable with standard neuropsychological procedures. Although the most debilitating effects are reversible, the possibility of a sensitization phenomenon exists. The Estuary Clinical Syndrome joins other well-documented outbreaks of human illness for which the specific toxin agent remains to be identified. The methodological approaches for identifying and investigating this newly identified syndrome in the absence of a known toxin may provide a model for the early investigation of other emerging toxins and related illnesses.     

Visions of Nature and Environmental Sustainability: Shellfish Harvesting in the Dutch Wadden Sea

The concept of sustainability has several, sometimes contrasting, meanings that may generate confusion, misunderstanding, and conflict concerning conservation and restoration practices. It is therefore desirable to clarify the concept of sustainability, thereby potentially contributing to mutual understanding, especially when social conflicts arise. This article discusses a recently published typology of three conceptions of sustainability that range from economic to ecocentric valuations of nature. We argue that the typology is incomplete because it does not include the arcadian approaches. For this reason, we introduce a “tripolar model” for conceptions of sustainability, applying it to the debate on shellfish harvesting in the Dutch Wadden Sea. We conclude that the particular visions or conceptions of sustainability held by relevant actors may have an impact on strategies for conservation.     

Human Exposure to Dioxin-Like Compounds in Fish and Shellfish Consumed in South Korea

Dietary intake is the most important source of exposure to dioxins for the general population. This pathway contributes more than 90% of the daily intake for the general population of Korea. The objective of this study was to assess current exposure to dioxin-like compounds in fish and shellfish consumed by the general population in Korea. Residues of polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs), and dioxin-like, non-, and mono-substituted polychlorinated biphenyls (co-planar PCBs) were quantified in 32 fish and shellfish collected from domestic fisheries markets. The contributions of individual DL-PCB congeners to the total 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) equivalents (TEQ) were greater than 50%. Concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents based on the TCDD equivalency factors (TEFs) developed by the World Health Organization (TEQ_WHO) were compared to guidelines suggested for the protection of human health by the World Health Organization (WHO). The greatest TEQ concentration was observed in herring, followed by that in dried anchovy and Sailfin sandfish. The exposure to dioxin-like compounds from current fish consumption patterns was estimated to be 72 pg TEQ_WHO/day, which is equal to 1.2 pg TEQ_WHO/kg, bw/d, a value that is less than the current tolerable daily intake (TDI) guideline in Korea, which is 4 pg TEQ_WHO/kg, bw/d. The relatively great exposure was determined to be due to greater fish consumption rate in Korea, rather than greater concentrations of residues in food.     

Environmental Factors Influencing Isolation of Enteroviruses from Polluted Surface Waters

The influence of water quality upon the concentration of virus on location was assessed in field studies conducted in the Houston ship channel, Galveston Bay, and Houston waste treatment plants. Clarification of polluted surface waters was accomplished with minimal loss of virus. Virus from clarified sewage effluents and saline waters was then adsorbed and concentrated on textile and membrane filter surfaces. Direct measurements of virus from large volumes of polluted surface waters under existing field conditions were then made using the virus concentrator equipment.     

Shigella dysenteriae Type 1-Specific Bacteriophage from Environmental Waters in Bangladesh

Shigella dysenteriae type 1 is the causative agent of the most severe form of bacillary dysentery, which occurs as epidemics in many developing countries. We isolated a bacteriophage from surface water samples from Bangladesh that specifically lyses strains of S. dysenteriae type 1. This phage, designated SF-9, belongs to the Podoviridae family and has a 41-kb double-stranded DNA genome. Further screening of water samples for the prevalence of the phage revealed 9 of 71 (12.6%) water samples which were positive for the phage. These water samples were also positive in PCR assays for one or more S. dysenteriae type 1-specific genes, including ipaBCD and stx1, and live S. dysenteriae type 1 was isolated from three phage-positive samples. The results of this study suggest that phage SF-9 may have epidemiological applications in tracing the presence of S. dysenteriae type 1 in environmental waters.     

Detection of Infectious Adenoviruses in Environmental Waters by Fluorescence-Activated Cell Sorting Assay

Methods for rapid detection and quantification of infectious viruses in the environment are urgently needed for public health protection. A fluorescence-activated cell-sorting (FACS) assay was developed to detect infectious adenoviruses (Ads) based on the expression of viral protein during replication in cells. The assay was first developed using recombinant Ad serotype 5 (rAd5) with the E1A gene replaced by a green fluorescent protein (GFP) gene. Cells infected with rAd5 express GFP, which is captured and quantified by FACS. The results showed that rAd5 can be detected at concentrations of 1 to 10⁴ PFU per assay within 3 days, demonstrating a linear correlation between the viral concentration and the number of GFP-positive cells with an r² value of >0.9. Following the same concept, FACS assays using fluorescently labeled antibodies specific to the E1A and hexon proteins, respectively, were developed. Assays targeting hexon showed greater sensitivity than assays targeting E1A. The results demonstrated that as little as 1 PFU Ads was detected by FACS within 3 days based on hexon protein, with an r² value greater than 0.9 over a 4-log concentration range. Application of this method to environmental samples indicated positive detection of infectious Ads in 50% of primary sewage samples and 33% of secondary treated sewage samples, but none were found in 12 seawater samples. The infectious Ads ranged in quantity between 10 and 165 PFU/100 ml of sewage samples. The results indicate that the FACS assay is a rapid quantification tool for detecting infectious Ads in environmental samples and also represents a considerable advancement for rapid environmental monitoring of infectious viruses.Waterborne viral infection is one of the most important causes of human morbidity in the world. There are hundreds of different types of human viruses present in human sewage, which, if improperly treated, may become the source of contamination in drinking and recreational waters (6, 12, 19). Furthermore, as water scarcity intensifies in the nation, so has consideration of wastewater reuse as a valid and essential alternative for resolving water shortages (31).Currently, routine viral monitoring is not required for drinking or recreational waters, nor is it required for wastewater that is discharged into the environment. This lack of a monitoring effort is due largely to the lack of methods that can rapidly and sensitively detect infectious viruses in environmental samples. In the past 20 years, tremendous progress has been made in detection of viruses in the environment based on molecular technology (32, 33, 35). PCR and quantitative real-time PCR (qPCR) methods have improved both the speed and sensitivity of viral detection compared with detection by the traditional tissue culture method (2, 11, 17, 18). However, they provide little information on viral infectivity, which is crucial for human health risk assessment (22-24, 35). Our previous work using a real-time PCR assay to detect human adenoviruses (Ads) in sewage could not differentiate the infectious viruses in the secondary treated sewage from those killed by chlorination disinfection (15). In this research, we pursued an innovative approach to detecting infectious viruses in water using fluorescence-activated cell sorting (FACS). This method is rapid and sensitive, with an established record in microbiological research (29, 34, 39).FACS is a specialized type of flow cytometry which provides a method for counting and sorting a heterogeneous mixture of biological cells into two or more kinds, one cell at a time, based upon the specific light-scattering and fluorescent characteristics of each cell (4, 25, 34, 38). It is a useful method since it provides fast and quantitative recording of fluorescent signals from individual cells (14, 16, 34, 47). The FACS viral assay is based on the expression of viral protein inside the recipient cell during viral replication (16). Specific antibody labeled with fluorescence is bound to the target viral protein, which results in fluorescence emission from infected cells. Viral particles outside the cell will not be captured, because the size of virus is below the detection limit of flow cytometry. Therefore, detection of cells, which can be captured with fluorescently labeled viral antibody, is a definitive indication of the presence of infectious virus.This research used human Ads as the target for development of the FACS method. The rationale for this choice is as follows. (i) Ads are important human pathogens that may be transmitted by water consumption and water spray (aerosols) (26, 32). The health hazard associated with exposure to Ads has been demonstrated by epidemiological data and clinical research (1, 7, 9, 35, 40, 43). (ii) Ads are among the most prevalent human viruses identified in human sewage and are frequently detected in marine waters and the Great Lakes (17, 32, 33, 35). (iii) Ads are more resistant to UV disinfection than any other bacteria or viruses (3, 5, 10, 24, 41, 42, 44). Thus, they may survive wastewater treatment as increasing numbers of wastewater treatment facilities switch from chlorination to UV to avoid disinfection by-products. (iv) Some serotypes of Ads, including enteric Ad 40 and 41, are fastidious. They are difficult to detect by plaque assay, and a routine assay of infectivity takes 7 to 14 days (8, 20).In this study, recombinant Ad serotype 5 (rAd5) with the E1A gene (the first transcribed gene after infection) replaced by a green fluorescent protein (GFP) gene was first used to test for sensitivity and speed of the assay. Two other viral proteins were then used as targets for development of FACS assays using Ad serotype 2 (Ad2) and Ad41. This study demonstrated the feasibility, sensitivity, and reliability of the assay for detection of infectious Ads in environmental samples.