Nicholas J. Ashbolt

Microbial contamination of drinking water and disease outcomes in developing regions

Abstract Drinking water is a major source of microbial pathogens in developing regions, although poor sanitation and food sources are integral to enteric pathogen exposure. Gastrointestinal disease outcomes are also more severe, due to under-nutrition and lack of intervention strategies in these regions. Poor water quality, sanitation and hygiene account for some 1.7 million deaths a year world-wide (3.1% of all deaths and 3.7% of all DALY’s), mainly through infectious diarrhoea. Nine out of 10 such deaths are in children and virtually all of the deaths are in developing countries. Major enteric pathogens in these children include: rotavirus, Campylobacter jejuni, enterotoxigenic Escherichia coli, Shigella spp. and Vibrio cholerae O1, and possibly enteropathogenic E. coli, Aeromonas spp. V. cholerae O139, enterotoxigenic Bacteroides fragilis, Clostridium difficile and Cryptosporidium parvum. All except the latter are easily control by chlorination of water, but recontamination of treated water is a huge problem. Emerging environmental pathogens, such as Helicobacter pylori and Burkholderia pseudomallei, may well be of significance in some regions. In adults, much less is understood of various sequellae such as myocarditis, diabetes, reactive arthritis and cancers some months–years after initial infections. So in addition to the traditional pathogens (helminths, Entamoeba histolytica, Giardia lamblia hepatitis A and E) various enteroviruses, C. jejuni and H. pylori are emerging issues in adults.

Fate and transport of surface water pathogens in watersheds

Pathogens present in animal fecal deposits excreted to land undergo a poorly defined process of dispersion, transport or attenuation, and inactivation. The transport of pathogens overland in surface runoff is clearly responsible for event-related increases in the concentrations of in-stream waterborne pathogens in many watersheds. However, there are significant knowledge gaps concerning the precise mechanisms of pathogen transport. This article reviews the fate and transport of pathogens in watersheds supplying drinking water, from their deposition in feces and septic seepages on land to their dispersion in major tributaries. Pathogens considered representative of those associated with waterborne disease included enteric viruses derived from human fecal contamination, bacterial pathogens represented by Escherichia coli O157:H7, and the protozoan pathogens Cryptosporidium and Giardia. References to suitable model and index organisms for these pathogens are described. The key processes determining the fate and transport of pathogens within watersheds are discussed in the context of changing agricultural practices, climate, and scale factors. A generic conceptual model for watershed processes is described in light of the knowledge gaps identified from this review. Future areas for fundamental research were identified and included: (1) inactivation kinetics of pathogens in soil and fecal matrices; (2) characterization of the particle sizes with which pathogens are transported; (3) characterization of pathogen properties and watershed-specific features that affect terrestrial transport and attenuation; and (4) the inactivation and sedimentation of pathogens during their initial introduction to the aquatic environment. Such information is critical to advance the assessment of pathogen total maximum daily loads (TMDL), determining management priorities and appropriate control points, as well as integrating pathogens within the broader watershed hydrologic models.

Estimated human health risks from exposure to recreational waters impacted by human and non-human sources of faecal contamination

This work was conducted to determine whether estimated risks following exposure to recreational waters impacted by gull, chicken, pig, or cattle faecal contamination are substantially different than those associated with waters impacted by human sources such as treated wastewater. Previously published Quantitative Microbial Risk Assessment (QMRA) methods were employed and extended to meet these objectives. Health outcomes used in the analyses were infection from reference waterborne pathogens via ingestion during recreation and subsequent gastrointestinal (GI) illness. Illness risks from these pathogens were calculated for exposure to faecally contaminated recreational water at the U.S. regulatory limits of 35 cfu 100 mL(-1) enterococci and 126 cfu 100 mL(-1)Escherichia coli. The probabilities of GI illness were calculated using pathogen dose-response relationships from the literature and Monte Carlo simulations. Three scenarios were simulated, representing a range of feasible interpretations of the available data. The primary findings are that: 1) GI illness risks associated with exposure to recreational waters impacted by fresh cattle faeces may not be substantially different from waters impacted by human sources; and 2) the risks associated with exposure to recreational waters impacted by fresh gull, chicken, or pig faeces appear substantially lower than waters impacted by human sources. These results suggest that careful consideration may be needed in the future for the management of recreational waters not impacted by human sources.

Relationships between indicators, pathogens and water quality in an estuarine system

This study examined water and sediment samples for a range of indicator and pathogenic microorganisms from six sites in an urban estuary, Sydney, Australia. Water quality was affected by rainfall and sewage overflows which were associated with significant increases in the concentration of faecal coliforms, faecal streptococci, Clostridium perfringens spores, F-RNA bacteriophage, Aeromonas spp., Giardia and Cryptosporidium spp. However, in sediments, only faecal coliform concentrations were significantly increased by rainfall, although sewage overflow again resulted in increased concentrations of faecal coliforms, faecal streptococci, C. perfringens spores and Aeromonas. Isolation of Salmonella appeared to coincide with wet weather events and occasionally identical serotypes were detected in sediments at several locations within the estuary. However, isolations of enteric virus were sporadic and did not appear to be exclusively related to wet weather events. C. perfringens was identified as the most useful indicator of faecal pollution and was the only indicator significantly correlated to the presence of pathogenic Giardia (r = 0.41, p < 0.05) and the opportunistic bacterial genus Aeromonas (r = 0.39, p < 0.05). F-RNA bacteriophage was not significantly correlated with any of the pathogens examined.

Human Health Risk Assessment (HHRA) for Environmental Development and Transfer of Antibiotic Resistance

Background: Only recently has the environment been clearly implicated in the risk of antibiotic resistance to clinical outcome, but to date there have been few documented approaches to formally assess these risks. Objective: We examined possible approaches and sought to identify research needs to enable human health risk assessments (HHRA) that focus on the role of the environment in the failure of antibiotic treatment caused by antibiotic-resistant pathogens. Methods: The authors participated in a workshop held 4–8 March 2012 in Québec, Canada, to define the scope and objectives of an environmental assessment of antibiotic-resistance risks to human health. We focused on key elements of environmental-resistance-development “hot spots,” exposure assessment (unrelated to food), and dose response to characterize risks that may improve antibiotic-resistance management options. Discussion: Various novel aspects to traditional risk assessments were identified to enable an assessment of environmental antibiotic resistance. These include a) accounting for an added selective pressure on the environmental resistome that, over time, allows for development of antibiotic-resistant bacteria (ARB); b) identifying and describing rates of horizontal gene transfer (HGT) in the relevant environmental “hot spot” compartments; and c) modifying traditional dose–response approaches to address doses of ARB for various health outcomes and pathways. Conclusions: We propose that environmental aspects of antibiotic-resistance development be included in the processes of any HHRA addressing ARB. Because of limited available data, a multicriteria decision analysis approach would be a useful way to undertake an HHRA of environmental antibiotic resistance that informs risk managers. Citation: Ashbolt NJ, Amézquita A, Backhaus T, Borriello P, Brandt KK, Collignon P, Coors A, Finley R, Gaze WH, Heberer T, Lawrence JR, Larsson DG, McEwen SA, Ryan JJ, Schönfeld J, Silley P, Snape JR, Van den Eede C, Topp E. 2013. Human health risk assessment (HHRA) for environmental development and transfer of antibiotic resistance. Environ Health Perspect 121:993–1001; http://dx.doi.org/10.1289/ehp.1206316

The role of biofilms and protozoa in Legionella pathogenesis: implications for drinking water.

Current models to study Legionella pathogenesis include the use of primary macrophages and monocyte cell lines, various free‐living protozoan species and murine models of pneumonia. However, there are very few studies of Legionella spp. pathogenesis aimed at associating the role of biofilm colonization and parasitization of biofilm microbiota and release of virulent bacterial cell/vacuoles in drinking water distribution systems. Moreover, the implications of these environmental niches for drinking water exposure to pathogenic legionellae are poorly understood. This review summarizes the known mechanisms of Legionella spp. proliferation within Acanthamoeba and mammalian cells and advocates the use of the amoeba model to study Legionella pathogenicity because of their close association with Legionella spp. in the aquatic environment. The putative role of biofilms and amoebae in the proliferation, development and dissemination of potentially pathogenic Legionella spp. is also discussed. Elucidating the mechanisms of Legionella pathogenicity development in our drinking water systems will aid in elimination strategies and procedural designs for drinking water systems and in controlling exposure to Legionella spp. and similar pathogens.

A sea change ahead for recreational water quality criteria

The United States Environmental Protection Agency is committed to developing new recreational water quality criteria for coastal waters by 2012 to provide increased protection to swimmers. We review the uncertainties and shortcomings of the current recreational water quality criteria, describe critical research needs for the development of new criteria, as well as recommend a path forward for new criteria development. We believe that among the most needed research needs are the completion of epidemiology studies in tropical waters and in waters adversely impacted by urban runoff and animal feces, as well as studies aimed to validate the use of models for indicator and pathogen concentration and health risk predictions.

Do free-living amoebae in treated drinking water systems present an emerging health risk?

There is an expanding body of evidence that free-living amoebae (FLA) increase both the numbers and virulence of water-based, human-pathogenic, amoeba-resisting microorganisms (ARM). Legionella spp., Mycobacterium spp., and other opportunistic human pathogens are known to be both ARM and also the etiologic agents of potentially fatal human lung infections. However, comparatively little is known about the FLA that may facilitate ARM growth in drinking water. This review examines the available literature on FLA in treated drinking water systems; in total 26 studies from 18 different countries. FLA were reported to breakthrough the water treatment barrier and enter distribution systems, in addition to the expected post-treatment system ingress. Once in the distribution system there is evidence of FLA colonization and regrowth especially in reservoirs and in-premise plumbing storage tanks. At the point of use the average FLA detection rate was 45% but highly variable (n = 16, σ = 31) due to both differences in both assay methods and the type of water systems examined. This review reveals that FLA are consistently detected in treated drinking water systems around the world and present a yet unquantified emerging health risk. However, more research is urgently required before accurate risks assessments can be undertaken to assess the impacts on human health, in households and institutions, due to exposure to FLA facilitated pathogenic ARM.

The use of a ribosomal RNA targeted oligonucleotide probe for fluorescent labelling of viable Cryptosporidium parvum oocysts

A fluorescence in situ hybridization (FISH) technique has been developed for the fluorescent labelling of Cryptosporidium parvum oocysts in water samples. The FISH technique employs a fluorescently labelled oligonucleotide probe (Cry1 probe) targeting a specific sequence in the 18S ribosomal RNA (rRNA) of C. parvum. Hybridization with the Cry1 probe resulted in fluorescence of sporozoites within oocysts that were capable of excystation, while oocysts that were dead prior to fixation did not fluoresce. Correlation of the FISH method with viability as measured by in vitro excystation was statistically highly significant, with a calculated correlation coefficient of 0·998. Examination of sequence data for Cryptosporidium spp. other than C. parvum suggests that the Cry1 probe is C. parvum‐specific. In addition, 19 isolates of C.parvum were tested, and all fluoresced after hybridization with the Cry1 probe. Conversely, isolates of C.baileyi and C. muris were tested and found not to fluoresce after hybridization with the Cry1 probe. The fluorescence of FISH‐stained oocysts was not bright enough to enable detection of oocysts in environmental water concentrates containing autofluorescent algae and mineral particles. However, in combination with immunofluorescence staining, FISH enabled species‐specific detection and viability determination of C. parvum oocysts in water samples.

Assessing Pathogen Risk to Swimmers at Non-Sewage Impacted Recreational Beaches

The risk of gastrointestinal illness to swimmers from fresh sewage and non-sewage fecal sources at recreational beaches was predicted using quantitative microbial risk assessment (QMRA). The QMRA estimated the probability of illness for accidental ingestion of recreational water with a specific concentration of fecal indicator bacteria, here the geometric mean enterococci limit of 35 cfu 100 mL(-1), from either a mixture of sources or an individual source. Using seagulls as an example non-sewage fecal source, the predicted median probability of illness was less than the illness benchmark of 0.01. When the fecal source was changed to poorly treated sewage, a relativity small difference between the median probability of illness and the illness benchmark was predicted. For waters impacted by a mixture of seagull and sewage waste, the dominant source of fecal indicator was not always the predicted dominant source of risk.