Hollow-Fiber Ultrafiltration and PCR Detection of Human-Associated Genetic Markers from Various Types of Surface Water in Florida

In this study, the microbiological quality of household tap water samples fed from rainwater tanks was assessed by monitoring the numbers of Escherichia coli bacteria and enterococci from 24 households in Southeast Queensland (SEQ), Australia. Quantitative PCR (qPCR) was also used for the quantitative detection of zoonotic pathogens in water samples from rainwater tanks and connected household taps. The numbers of zoonotic pathogens were also estimated in fecal samples from possums and various species of birds by using qPCR, as possums and birds are considered to be the potential sources of fecal contamination in roofharvested rainwater (RHRW). Among the 24 households, 63% of rainwater tank and 58% of connected household tap water (CHTW) samples contained E. coli and exceeded Australian drinking water guidelines of <1 CFU E. coli per 100 ml water. Similarly, 92% of rainwater tanks and 83% of CHTW samples also contained enterococci. In all, 21%, 4%, and 13% of rainwater tank samples contained Campylobacter spp., Salmonella spp., and Giardia lamblia, respectively. Similarly, 21% of rainwater tank and 13% of CHTW samples contained Campylobacter spp. and G. lamblia, respectively. The number of E. coli (P ‫؍‬ 0.78), Enterococcus (P ‫؍‬ 0.64), Campylobacter (P ‫؍‬ 0.44), and G. lamblia (P ‫؍‬ 0.50) cells in rainwater tanks did not differ significantly from the numbers observed in the CHTW samples. Among the 40 possum fecal samples tested, Campylobacter spp., Cryptosporidium parvum, and G. lamblia were detected in 60%, 13%, and 30% of samples, respectively. Among the 38 bird fecal samples tested, Campylobacter spp., Salmonella spp., C. parvum, and G. lamblia were detected in 24%, 11%, 5%, and 13% of the samples, respectively. Household tap water samples fed from rainwater tanks tested in the study appeared to be highly variable. Regular cleaning of roofs and gutters, along with pruning of overhanging tree branches, might also prove effective in reducing animal fecal contamination of rainwater tanks.

Section: Discussionmentioning

confidence: 96%

Fecal Indicators and Zoonotic Pathogens in Household Drinking Water Taps Fed from Rainwater Tanks in Southeast Queensland, Australia

Ahmed

Hodgers

Sidhu

et al. 2012

“…The DEUF method, a modification of HFUF, has been used as a simple and portable technique for field concentration of bacteria, viruses, protozoa, and parasites from large water volumes (19,33,34). Two advantages to the DEUF method are that it is less likely to clog than other ultrafiltration methods and it is able to concentrate cells in the field followed by elution in the laboratory.…”

Section: Discussionmentioning

confidence: 99%

Ultrafiltration and Microarray for Detection of Microbial Source Tracking Marker and Pathogen Genes in Riverine and Marine Systems

Harwood

Weidhaas

2016

Self Cite

Pathogen identification and microbial source tracking (MST) to identify sources of fecal pollution improve evaluation of water quality. They contribute to improved assessment of human health risks and remediation of pollution sources. An MST microarray was used to simultaneously detect genes for multiple pathogens and indicators of fecal pollution in freshwater, marine water, sewage-contaminated freshwater and marine water, and treated wastewater. Dead-end ultrafiltration (DEUF) was used to concentrate organisms from water samples, yielding a recovery efficiency of >95% for Escherichia coli and human polyomavirus. Whole-genome amplification (WGA) increased gene copies from ultrafiltered samples and increased the sensitivity of the microarray. Viruses (adenovirus, bocavirus, hepatitis A virus, and human polyomaviruses) were detected in sewage-contaminated samples. Pathogens such as Legionella pneumophila, Shigella flexneri, and Campylobacter fetus were detected along with genes conferring resistance to aminoglycosides, beta-lactams, and tetracycline. Nonmetric dimensional analysis of MST marker genes grouped sewage-spiked freshwater and marine samples with sewage and apart from other fecal sources. The sensitivity (percent true positives) of the microarray probes for gene targets anticipated in sewage was 51 to 57% and was lower than the specificity (percent true negatives; 79 to 81%). A linear relationship between gene copies determined by quantitative PCR and microarray fluorescence was found, indicating the semiquantitative nature of the MST microarray. These results indicate that ultrafiltration coupled with WGA provides sufficient nucleic acids for detection of viruses, bacteria, protozoa, and antibiotic resistance genes by the microarray in applications ranging from beach monitoring to risk assessment. W aterborne pathogens pose a health risk to recreational water users (1), in drinking water systems (2), and in aquatic organisms such as shellfish that are consumed by humans (3). These waterborne pathogens include more than 40 different groups or genera, including viruses, bacteria, protozoa, cyanobacteria, and helminths (4). Additional waterborne pathogens will doubtless emerge over time due to increased proportions of sensitive populations, globalization of commerce, microbial evolution, and use of reclaimed water as drinking water (5). Many waterborne pathogens originate from fecal pollution in storm water runoff from agricultural and urban surfaces (6) or direct release of untreated sewage to surface water (7). Additional sources of waterborne fecal pathogens include wildlife and domesticated animals such as deer, dogs, raccoons, cats, and wild avian species (8). Still other waterborne pathogens, such as Vibrio spp., are autochthonous to aquatic environments (9).The microbiological safety of surface water has been assessed for over a century by enumeration of fecal indicator bacteria (FIB) (10). Other monitoring techniques such as microbial source tracking (MST) are advantageous compared to enumeration ...

“…A wide range of virus concentration methods have been developed and used to concentrate viruses from various types of environmental waters (25)(26)(27)(28)(29)(30)(31). Among these methods, an adsorption/elution-based protocol with negatively charged membrane has been shown to recover high concentration of viruses from small volume of water samples with minimal PCR inhibitory effects (27).…”

mentioning

confidence: 99%

Comparison of Concentration Methods for Quantitative Detection of Sewage-Associated Viral Markers in Environmental Waters

Ahmed

Harwood

Gyawali

et al. 2015

126

dPathogenic human viruses cause over half of gastroenteritis cases associated with recreational water use worldwide. They are relatively difficult to concentrate from environmental waters due to typically low concentrations and their small size. Although rapid enumeration of viruses by quantitative PCR (qPCR) has the potential to greatly improve water quality analysis and risk assessment, the upstream steps of capturing and recovering viruses from environmental water sources along with removing PCR inhibitors from extracted nucleic acids remain formidable barriers to routine use. Here, we compared the efficiency of virus recovery for three rapid methods of concentrating two microbial source tracking (MST) viral markers human adenoviruses (HAdVs) and polyomaviruses (HPyVs) from one liter tap water and river water samples on HA membranes (90 mm in diameter). Samples were spiked with raw sewage, and viral adsorption to membranes was promoted by acidification (method A) or addition of MgCl 2 (methods B and C). Viral nucleic acid was extracted directly from membranes (method A), or viruses were eluted with NaOH and concentrated by centrifugal ultrafiltration (methods B and C). No inhibition of qPCR was observed for samples processed by method A, but inhibition occurred in river samples processed by B and C. Recovery efficiencies of HAdVs and HPyVs were ϳ10-fold greater for method A (31 to 78%) than for methods B and C (2.4 to 12%). Further analysis of membranes from method B revealed that the majority of viruses were not eluted from the membrane, resulting in poor recovery. The modification of the originally published method A to include a larger diameter membrane and a nucleic acid extraction kit that could accommodate the membrane resulted in a rapid virus concentration method with good recovery and lack of inhibitory compounds. The frequently used strategy of viral absorption with added cations (Mg 2؉) and elution with acid were inefficient and more prone to inhibition, and will result in underestimation of the prevalence and concentrations of HAdVs and HPyVs markers in environmental waters. Discharges from sewage treatment plants (STPs), storm water drains, improperly designed septic systems, and fecal contamination from livestock and wildlife are known to degrade environmental water quality in terms of elevating fecal indicator bacteria and possibly pathogen concentrations (1-5). Fecal indicator bacteria such as Escherichia coli and Enterococcus spp. have been widely used as an indirect measure of microbial risk associated with environmental waters. However, identifying the health risks associated with enteric viruses and protozoa by monitoring fecal indicator bacteria has been questioned (5-8).The risk of infectious disease associated with recreational water use may well be of viral etiology (9). The transmission of these viruses occurs via the fecal-oral route, nasal mucosa or the conjunctiva and the infected individual may shed up to 10 11 viral particles/gram of feces (10). Some of these viruses are more res...