Microbial Source Tracking Studies Research Articles

Evidence for a human‐specific Escherichia coli clone

Escherichia coli is a widespread commensal of the vertebrate intestinal tract. Until recently, no strong association between a particular clone and a given host species has been found. However, members of the B2 subgroup VIII clone with an O81 serotype appear to be human host specific. To determine the degree of host specificity exhibited by this clone, a PCR-based assay was used to screen 723 faecal and clinical isolates from humans, and 904 faecal isolates from animals. This clone was not detected among the animal isolates, but was discovered in people living in Africa, Europe and South America. The clone is rarely isolated from people suffering from intestinal or extraintestinal disease and is avirulent in a mouse model of extraintestinal infection. Fine-scale epidemiological analysis suggests that this clone is competitively dominant relative to other members of the B2 phylogenetic group and that it has increased in frequency over the past 20 years. This clone appears to be a good candidate for use as a probiotic, and may be suitable as an indicator of human faecal contamination in microbial source tracking studies.

The effect of in-stream activities on the Njoro River, Kenya. Part II: Microbial water quality

The influence of periodic in-stream activities of people and livestock on the microbial water quality of the Njoro River in Kenya was monitored at two disturbed pools (Turkana Flats and Njoro Bridge) at the middle reaches. A total of 96 sets of samples were obtained from the two pools in six weeks during dry weather (January–April) in 2006. On each sampling day, two trips were made before and during in-stream activities and on each trip, two sets of samples were collected upstream and downstream of activities. This schedule was repeated four times each for Wednesday, Saturday and Sunday. Samples were processed for heterotrophic plate count bacteria (HPC), total coliform (TC), presumptive Escherichia coli and presumptive Enterococci. Additional samples were analysed for total suspended solids (TSS), turbidity, BOD 5 and ammonium-N. The microbial water quality deteriorated significant ( p < 0.05) downstream during activities at both pools. A similar trend was observed with the chemical indicators (TSS, turbidity, BOD 5 and ammonium-N). The two groups of indicators demonstrated high capacity for site segregation based on pollution levels. Pollution levels for specific days were not significantly different ( p > 0.05). This was incompatible with the variability of in-stream activities with specific days. The pooled data was explained largely by three significant principal components – recent pollution (PC1), metabolic activity (PC2) and residual pollution (PC3). It was concluded that the empirical site parity/disparity in the levels of microbial and non-microbial indicators reflected the diurnal periodicity of in-stream activities and the concomitant pollution they caused. However, microbial source tracking studies are required to distinguish faecal sources. In the meantime, measures should be undertaken to regulate in-stream activities along the stream and minimize the movement of livestock in the catchment.

Enterococci Concentrations in Diverse Coastal Environments Exhibit Extreme Variability

Fecal indicator bacteria (FIB) concentrations in a single grab sample of water are used to notify the public about the safety of swimming in coastal waters. If concentrations are over a single-sample standard, waters are closed or placed under an advisory. Previous work has shown that notification errors occur often because FIB vary more quickly than monitoring results can be obtained (typically 24 h). Rapid detection technologies (such as quantitative polymerase chain reaction) that allow FIB quantification in hours have been suggested as a solution to notification errors. In the present study, I explore variability of enterococci (ENT) over time scales less than a day that might affect interpretation of FIB concentrations from a single grab sample, even if obtained rapidly. Five new data sets of ENT collected at 10 and 1 min periodicities for 24 and 1 h, respectively, are presented. Data sets are collected in diverse marine environments from a turbulent surf zone to a quiescent bay. ENT vary with solar and tidal cycles, as has been observed in previous studies. Over short time scales, ENT are extremely variable in each environment even the quiescent bay. Changes in ENT concentrations between consecutive samples (1 or 10 min apart) greater than the single-sample standard (104 most probable number per 100 mL) are not unusual. Variability, defined as the change in concentration between consecutive samples, is not distinct between environments. ENT change by 60% on average between consecutive samples, and by as much as 700%. Spectral analyses reveal no spectral peaks, but power-law decline of spectral density with frequency. Power-law exponents are close to 1 suggesting ENT time series share properties with 1/f noise and are fractal in nature. Since fractal time series have no characteristic time scale associated with them, it is not obvious how the fractal nature of ENT can be exploited for adaptive sampling or management. Policy makers, as well as scientists designing field campaigns for microbial source tracking and epidemiology studies, are cautioned that a single sample of water reveals little about the true water quality at a beach. Multiple samples must be taken to gain a snapshot into the patchy structure of microbial water quality and associated human health risk.

Performance, Design, and Analysis in Microbial Source Tracking Studies

Microbial source tracking (MST) includes a group of methodologies that are aimed at identifying, and in some cases quantifying, the dominant source(s) of fecal contamination in resource waters, including drinking, ground, recreational, and wildlife habitat waters. MST methods can be grouped into two major types. Library-dependent methods are culture based and rely on isolate-by-isolate typing of bacteria cultured from various fecal sources and from water samples These isolates are matched to their corresponding source categories by direct subtype matching (41, 70) or by statistical means (23, 37, 40, 41, 80, 83, 102). In contrast, library-independent methods frequently are based on sample-level detection of a specific, host-associated genetic marker in a DNA extract by PCR (6, 11, 26, 88). Analyses of certain chemicals associated with sewage, including fecal sterols (29, 30, 47), optical brighteners (29, 30, 68), and host mitochondrial DNA (67), have also been utilized for what can be more broadly termed fecal source tracking; however, in this review we compare the performance of only fecal source tracking studies in which the target(s) is microbial. Mounting pressure to determine the origin of nonpoint source fecal pollution, as exemplified by the U.S. Environmental Protection Agency's total maximum daily load program, has led to a steady increase in manuscript submissions and grant applications that include MST approaches. At the same time, resource managers concerned with water quality and regulatory pressures struggle with the choice of methodology in the face of requirements for immediate application. Although there has been significant progress in the MST field over the last 10 years, variability among performance measurements and validation approaches in laboratory and field studies has led to a body of literature that is very difficult to interpret, both for scientists and for end users (99). In this review, we first consider the development and validation of MST methods in a historical context to describe the lessons learned in early studies. Next, uniform performance characteristics are introduced to allow comparison of method performance across MST studies (Tables ​(Tables11 and ​and2),2), and this is followed by a discussion of considerations for field study design and implementation. TABLE 1. Performance statistics for tests in which results were based on isolate-by-isolate classification into the various known-source categoriesa TABLE 2. Performance statistics for tests in which MST methods were tested with reference samples to determine the ability or failure to detect the sole source of fecal contamination

Diversity and Distribution of Escherichia coli Genotypes and Antibiotic Resistance Phenotypes in Feces of Humans, Cattle, and Horses

Escherichia coli is the most completely characterized prokaryotic model organism and one of the dominant indicator organisms for food and water quality testing, yet comparatively little is known about the structure of E. coli populations in their various hosts. The diversities of E. coli populations isolated from the feces of three host species (human, cow, and horse) were compared by two subtyping methods: ribotyping (using HindIII) and antibiotic resistance analysis (ARA). The sampling effort required to obtain a representative sample differed by host species, as E. coli diversity was consistently greatest in horses, followed by cattle, and was lowest in humans. The diversity of antibiotic resistance patterns isolated from individuals was consistently greater than the diversity of ribotypes. E. coli populations in individuals sampled monthly, over a 7- to 8-month period, were highly variable in terms of both ribotypes and ARA phenotypes. In contrast, E. coli populations in cattle and humans were stable over an 8-h period. Following the cessation of antibiotic therapy, the E. coli population in the feces of one human experienced a rapid and substantial shift, from a multiply antibiotic-resistant phenotype associated with a particular ribotype to a relatively antibiotic-susceptible phenotype associated with a different ribotype. The high genetic diversity of E. coli populations, differences in diversity among hosts, and temporal variability all indicate complex population dynamics that influence the usefulness of E. coli as a water quality indicator and its use in microbial source tracking studies.

Evaluation of antibiotic resistance analysis and ribotyping for identification of faecal pollution sources in an urban watershed

The accuracy of ribotyping and antibiotic resistance analysis (ARA) for prediction of sources of faecal bacterial pollution in an urban southern California watershed was determined using blinded proficiency samples. Antibiotic resistance patterns and HindIII ribotypes of Escherichia coli (n = 997), and antibiotic resistance patterns of Enterococcus spp. (n = 3657) were used to construct libraries from sewage samples and from faeces of seagulls, dogs, cats, horses and humans within the watershed. The three libraries were analysed to determine the accuracy of host source prediction. The internal accuracy of the libraries (average rate of correct classification, ARCC) with six source categories was 44% for E. coli ARA, 69% for E. coli ribotyping and 48% for Enterococcus ARA. Each library's predictive ability towards isolates that were not part of the library was determined using a blinded proficiency panel of 97 E. coli and 99 Enterococcus isolates. Twenty-eight per cent (by ARA) and 27% (by ribotyping) of the E. coli proficiency isolates were assigned to the correct source category. Sixteen per cent were assigned to the same source category by both methods, and 6% were assigned to the correct category. Addition of 2480 E. coli isolates to the ARA library did not improve the ARCC or proficiency accuracy. In contrast, 45% of Enterococcus proficiency isolates were correctly identified by ARA. None of the methods performed well enough on the proficiency panel to be judged ready for application to environmental samples. Most microbial source tracking (MST) studies published have demonstrated library accuracy solely by the internal ARCC measurement. Low rates of correct classification for E. coli proficiency isolates compared with the ARCCs of the libraries indicate that testing of bacteria from samples that are not represented in the library, such as blinded proficiency samples, is necessary to accurately measure predictive ability. The library-based MST methods used in this study may not be suited for determination of the source(s) of faecal pollution in large, urban watersheds.

Persistence and Differential Survival of Fecal Indicator Bacteria in Subtropical Waters and Sediments

Fecal coliforms and enterococci are indicator organisms used worldwide to monitor water quality. These bacteria are used in microbial source tracking (MST) studies, which attempt to assess the contribution of various host species to fecal pollution in water. Ideally, all strains of a given indicator organism (IO) would experience equal persistence (maintenance of culturable populations) in water; however, some strains may have comparatively extended persistence outside the host, while others may persist very poorly in environmental waters. Assessment of the relative contribution of host species to fecal pollution would be confounded by differential persistence of strains. Here, freshwater and saltwater mesocosms, including sediments, were inoculated with dog feces, sewage, or contaminated soil and were incubated under conditions that included natural stressors such as microbial predators, radiation, and temperature fluctuations. Persistence of IOs was measured by decay rates (change in culturable counts over time). Decay rates were influenced by IO, inoculum, water type, sediment versus water column location, and Escherichia coli strain. Fecal coliform decay rates were significantly lower than those of enterococci in freshwater but were not significantly different in saltwater. IO persistence according to mesocosm treatment followed the trend: contaminated soil > wastewater > dog feces. E. coli ribotyping demonstrated that certain strains were more persistent than others in freshwater mesocosms, and the distribution of ribotypes sampled from mesocosm waters was dissimilar from the distribution in fecal material. These results have implications for the accuracy of MST methods, modeling of microbial populations in water, and efficacy of regulatory standards for protection of water quality.