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

Abstract

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 ...

Frequent Detection and Genetic Diversity of Human Bocavirus in Urban Sewage Samples

“…Indeed, Hamza and collaborators described the detection of HBoV in 41 % of river water samples (Hamza et al 2009). Also, Li and coworkers recently detected HBoV along with other enteric viruses in sewage-contaminated fresh and marine waters (Li et al 2016). Therefore, a potential role of water in the transmission of HBoV should not be neglected.…”

Current Status of Marker Genes of Bacteroides and Related Taxa for Identifying Sewage Pollution in Environmental Waters

“…Li and colleagues [134] developed a custom microarray targeting pathogens, MST markers, and antibiotic resistance genes. In a subsequent study, the microarray was used to detect multiple pathogenic genes and MST markers in fresh and marine water and as well as sewage and treated effluent seeded environmental water [135]. One drawback of this approach is that it does not provide quantitative data.…”

Detection ofCampylobacterspp. in water by dead‐end ultrafiltration and application at farm level

“…However, the variation in detection results at low spiking levels indicates that a level of around 10 CFU 60 l À1 is close to the detection limit of the method, which was also shown by the statistical analysis where LOD 95 was 15 CFU 60 l À1 with the confidence interval 6Á6-33 CFU 60 l À1 . Although DEUF has previously been used for Campylobacter in water (Li et al, 2016), to our knowledge we are the first to report the detection limit of DEUF for Campylobacter in water. The detection limit identified in this study agrees with previous work on other organisms, which has demonstrated that viable spores of Bacillus artrophaeus at levels of <10 CFU l À1 can be concentrated and recovered from >100 l tap water (Kearns et al, 2008) and that high recovery rates (>95%) can be obtained for Escherichia coli and human polyomavirus using DEUF (Li et al, 2016).…”

“…Although DEUF has previously been used for Campylobacter in water (Li et al, 2016), to our knowledge we are the first to report the detection limit of DEUF for Campylobacter in water. The detection limit identified in this study agrees with previous work on other organisms, which has demonstrated that viable spores of Bacillus artrophaeus at levels of <10 CFU l À1 can be concentrated and recovered from >100 l tap water (Kearns et al, 2008) and that high recovery rates (>95%) can be obtained for Escherichia coli and human polyomavirus using DEUF (Li et al, 2016). Also, Humrighouse et al (2015) determined the method detection limit for a tangential flow ultrafiltration-based automated waterborne pathogen concentrator using the same filter type as in this study.…”