Fecal pollution of water resources is an environmental problem of increasing importance. Identification of individual host sources of fecal Escherichia coli, such as humans, pets, production animals, and wild animals, is prerequisite to formulation of remediation plans. Ribotyping has been used to distinguish fecal E. coli of human origin from pooled fecal E. coli isolates of nonhuman origin. We have extended application of this technique to distinguishing fecal E. coli ribotype patterns from human and seven individual nonhuman hosts. Classification accuracy was best when the analysis was limited to three host sources. Application of this technique to identification of host sources of fecal coliforms in water could assist in formulation of pollution reduction plans.Fecal pollution of water resources is a problem of increasing worldwide concern (4, 15). Human population growth, inadequate sewage systems, and management of animal waste (especially related to concentrated animal feeding operations) are some of the issues associated with maintenance of supplies of clean water (17). Counts of commensal coliform bacteria have traditionally been used to indicate the potential presence of pathogenic microbes of intestinal origin (1). Total coliform and fecal coliform numbers (1) are useful for estimating fecal pollution levels but give no indication of the specific sources of microbial pollution, such as humans, production animals, pets, or migratory birds. Examples of methods which have been used as indicators of host sources include phage susceptibility (20) and the ratio of fecal coliforms to streptococci (5). Such indirect measurements are based on unstable parameters and may thereby lead to erroneous conclusions (11). More recently, DNA fingerprinting techniques such as ribotyping (11), pulsedfield gel electrophoresis (9), PCR of repetitive intergenic sequences (3), and 16S ribosomal DNA length heterogeneity PCR with terminal restriction fragment length polymorphism (2) have been described as promising for discriminating between fecal-origin bacteria from humans and animals. Multiple antibiotic resistance phenotype has been used to distinguish between human and nonhuman sources of Escherichia coli (7,10,11,19) and streptococci (6, 18), but genetic instability or changes in antibiotic use can alter the resistance profiles obtained.Ribotyping has been compared to multiple antibiotic resistance profiles, and both approaches were reportedly complementary in discriminating between human and nonhuman (collective) sources of fecal pollution (11). Ribotyping has since become a popular approach (personal communications) to the problem of differentiating between fecal E. coli pollution from humans and, in particular, that from animals and birds. We describe here the use of ribotyping for the identification of E. coli cultured from feces of humans, cattle, swine, horses, chickens, turkeys, dogs, and migratory geese. MATERIALS AND METHODSFecal E. coli. Table 1 presents the host sources of feces, the numbers of individuals samp...

Aims:  The goal of this study was to develop and test the efficacy of a PCR assay for the environmental detection of the nifH gene of Methanobrevibacter smithii, a methanogen found in human faeces and sewage. Methods and Results:  PCR primers for the nifH gene of M. smithii were designed, tested and used to detect the presence or absence of this organism in faecal and environmental samples. Specificity analysis showed that the Mnif primers amplified products only in M. smithii pure culture strains (100%), human faeces (29%), human sewage samples (93%) and sewage‐contaminated water samples (100%). No amplification was observed when primers were tested against 43 bacterial stock cultures, 204 animal faecal samples, 548 environmental bacterial isolates and water samples from a bovine waste lagoon and adjacent polluted creek. Sequencing of PCR products from sewers demonstrated that a 222‐bp product was the nifH gene of M. smithii. The minimal amount of total DNA required for the detection of M. smithii was 10 ng for human faeces, 10 ng for faecally contaminated water and 5 ng for sewage. Recreational water seeded with M. smithii established a lower detection limit of 13 cells ml−1. Conclusions:  The Mnif assay developed during this investigation showed successful detection of M. smithii in individual human faecal samples, sewage and sewage‐contaminated water but not in uncontaminated marine water or bovine‐contaminated waters. The Mnif assay appears to be a potentially useful method to detect sewage‐polluted coastal waters. Significance and Impact of the Study:  This study was the first to utilize methanogens as an indicator of sewage pollution. Mnif PCR detection of M. smithii was shown to be a rapid, inexpensive and reliable test for determining the presence or absence of sewage pollution in coastal recreational waters.

This report compares the performances of two popular genotypic methods used for tracking the sources of fecal pollution in water, ribotyping and repetitive extragenic palindromic-PCR (rep-PCR). The rep-PCR was more accurate, reproducible, and efficient in associating DNA fingerprints of fecal Escherichia coli with human and animal hosts of origin.

Microbial source tracking (MST) results, obtained using identical sample sets and pulsed field gel electrophoresis (PFGE), repetitive element PCR (rep-PCR) and ribotyping techniques were compared. These methods were performed by six investigators in analysis of duplicate, blind sets of water samples spiked with feces from five possible sources (sewage, human, dog, cow and seagull). Investigators were provided with samples of the fecal material used to inoculate the water samples for host origin database construction. All methods correctly identified the dominant source in the majority of the samples. Modifications of some of these methods correctly identified the dominant sources in over 90% of the samples; however, false positive rates were as high as 57%. The high false positive rates appeared to be indirectly proportional to the levels of stringency applied in pattern analysis. All the methods produced useful data but the results highlighted the need to modify and optimize these methods in order to minimize sources of error.

A bacterial primer set, known to produce a 542-bp amplicon specific for Bacteroides thetaiotaomicron, generated this product in PCR with 1 ng of extracted DNA from 92% of 25 human fecal samples, 100% of 20 sewage samples, and 16% of 31 dog fecal samples. The marker was not detected in 1 ng of fecal DNA from 61 cows, 35 horses, 44 pigs, 24 chickens, 29 turkeys, and 17 geese.

Aims:  The aims of this work were to develop a quantitative test, based on Bacteroides thetaiotaomicron, for human faecal pollution in water and to evaluate test performance. Methods and Results:  qPCR primers, based on the complete genomic sequence of B. thetaiotaomicron VPI 5482, were designed and tested. The single‐copy putative mannanase homologue, α‐1‐6 mannanase, was selected as the particular target and sequences within this gene chosen as the qPCR primers by Blast search for specificity to B. thetaiotaomicron. The average concentration of B. thetaiotaomicron in human faeces was 1·39 × 108 cells per gram faeces and the detection limit was 9·3 B. thetaiotaomicron copies per qPCR procedure. Comparison of B. thetaiotaomicron content in sewage vs pooled nonhuman faecal samples indicated that the current assay is specific for sewage. Conclusion:  The subject assay is potentially useful for quantification of sewage pollution in water. Significance and Impact of the Study:  Bacteroides‐associated markers, proposed for faecal source tracking, have exclusively been based on gene sequences related to generally classified and uncultured bacteria. However, genes associated with host‐microbe interaction have been suggested as more specific markers. The present assay targets such a gene of B. thetaiotaomicron which is considered to be a symbiont in the human gut.

Studies evaluating the relationship between microbes and human health at non-point source beaches are necessary for establishing criteria which would protect public health while minimizing economic burdens. The objective of this study was to evaluate water quality and daily cumulative health effects (gastrointestinal, skin, and respiratory illnesses) for bathers at a non-point source subtropical marine recreational beach in order to better understand the inter-relationships between these factors and hence improve monitoring and pollution prevention techniques. Daily composite samples were collected, during the Oceans and Human Health Beach Exposure Assessment and Characterization Health Epidemiologic Study conducted in Miami (Florida, USA) at a non-point source beach, and analyzed for several pathogens, microbial source tracking markers, indicator microbes, and environmental parameters. Analysis demonstrated that rainfall and tide were more influential, when compared to other environmental factors and source tracking markers, in determining the presence of both indicator microbes and pathogens. Antecedent rainfall and Fþ coliphage detection in water should be further assessed to confirm their possible association with skin and gastrointestinal (GI) illness outcomes, respectively. The results of this research illustrate the potential complexity of beach systems characterized by non-point sources, and how more novel and comprehensive approaches are needed to assess beach water quality for the purpose of protecting bather health.

A procedure for cloning Babesia bovis was developed. The procedure was used to establish and cultivate homogeneous populations of parasites and to isolate B. bovis from carrier animals. Three different clone lines of B. bovis based on in vitro growth rates were established.