The purpose of this study was to examine host distribution patterns among fecal bacteria in the order Bacteroidales, with the goal of using endemic sequences as markers for fecal source identification in aquatic environments. We analyzed Bacteroidales 16S rRNA gene sequences from the feces of eight hosts: human, bovine, pig, horse, dog, cat, gull, and elk. Recovered sequences did not match database sequences, indicating high levels of uncultivated diversity. The analysis revealed both endemic and cosmopolitan distributions among the eight hosts. Ruminant, pig, and horse sequences tended to form host-or host group-specific clusters in a phylogenetic tree, while human, dog, cat, and gull sequences clustered together almost exclusively. Many of the human, dog, cat, and gull sequences fell within a large branch containing cultivated species from the genus Bacteroides. Most of the cultivated Bacteroides species had very close matches with multiple hosts and thus may not be useful targets for fecal source identification. A large branch containing cultivated members of the genus Prevotella included cloned sequences that were not closely related to cultivated Prevotella species. Most ruminant sequences formed clusters separate from the branches containing Bacteroides and Prevotella species. Host-specific sequences were identified for pigs and horses and were used to design PCR primers to identify pig and horse sources of fecal pollution in water. The primers successfully amplified fecal DNAs from their target hosts and did not amplify fecal DNAs from other species. Fecal bacteria endemic to the host species may result from evolution in different types of digestive systems.

Fecal indicator bacteria (FIB), commonly used to regulate sanitary water quality, cannot discriminate among sources of contamination. The use of alternative quantitative PCR (qPCR) methods for monitoring fecal contamination or microbial source tracking requires an understanding of relationships with cultivated FIB, as contamination ages under various conditions in the environment. In this study, the decay rates of three Bacteroidales 16S rRNA gene markers (AllBac for general contamination and qHF183 and BacHum for human-associated contamination) were compared with the decay rate of cultivated Escherichia coli in river water microcosms spiked with human wastewater. The following five sets of microcosms were monitored over 11 days: control, artificial sunlight, sediment exposure, reduced temperature, and no autochthonous predation. Decay was characterized by estimation of the time needed to produce a 2-log reduction (t 99 ). No treatmentassociated differences in the decay of the 4 targets were evident except with reduced predation, where E. coli, qHF183, and BacHum markers had lower levels of decay by day 3. However, there were substantial targetassociated differences. Decay curves for the AllBac marker indicated a larger persistent population than those of the other targets. Exposure to sunlight, sediment, and reduced predation resulted in more rapid decay of the human-associated markers relative to cultivable E. coli, but there were no differences in t 99 values among the 4 targets under control conditions or at reduced temperatures. Further evaluation of epidemiological relationships will be needed in order to relate the markers directly to health risk. These findings suggest that the tested human-associated markers can complement E. coli as indicators of the human impact on sanitary water quality under the constrained conditions described in this paper.Recreational water quality standards for freshwater are commonly based on the cultivable concentration of the fecal indicator bacterium (FIB) Escherichia coli (42). Epidemiological studies demonstrated a correlation between E. coli concentration and rates of gastrointestinal illness among swimmers (19,32,46), and water quality criteria based on E. coli have been established by the U.S. Environmental Protection Agency for the protection of human health (41). However, E. coli are found in many hosts, both human and nonhuman, that carry different cohorts of human-pathogenic microorganisms (13), and E. coli types have limited host specificity (4,16,17). E. coli also have been shown to reproduce in the environment under some conditions (7,12,23,37,49). These limitations of E. coli as an indicator of public health risk are well recognized (34) and may result in revision of U.S. recreational water quality criteria (44).In the pursuit of alternate assessment strategies, researchers in the field of microbial source tracking (MST) have developed and applied host-associated molecular markers of fecal contamination. Prominent among candidate MST protocols are those that det...

Avian feces contaminate waterways but contribute fewer human pathogens than human sources. Rapid identification and quantification of avian contamination would therefore be useful to prevent overestimation of human health risk. We used subtractive hybridization of PCR-amplified gull fecal 16S RNA genes to identify avian-specific fecal rRNA gene sequences. The subtracters were rRNA genes amplified from human, dog, cat, cow, and pig feces. Recovered sequences were related to Enterobacteriaceae (47%), Helicobacter (26%), Catellicoccus (11%), Fusobacterium (11%), and Campylobacter (5%). Three PCR assays, designated GFB, GFC, and GFD, were based on recovered sequence fragments. Quantitative PCR assays for GFC and GFD were developed using SYBR green. GFC detected down to 0.1 mg gull feces/100 ml (corresponding to 2 gull enterococci most probable number [MPN]/100 ml). GFD detected down to 0.1 mg chicken feces/100 ml (corresponding to 13 Escherichia coli MPN/100 ml). GFB and GFC were 97% and 94% specific to gulls, respectively. GFC cross-reacted with 35% of sheep samples but occurred at about 100,000 times lower concentrations in sheep. GFD was 100% avian specific and occurred in gulls, geese, chickens, and ducks. In the United States, Canada, and New Zealand, the three markers differed in their geographic distributions but were found across the range tested. These assays detected four important bird groups contributing to fecal contamination of waterways: gulls, geese, ducks, and chickens. Marker distributions across North America and in New Zealand suggest that they will have broad applicability in other parts of the world as well. Contamination from gulls, Canada geese, ducks, and other birds negatively impacts water quality (5, 16, 24, 33a, 49, 56). Their feces are sources of fecal coliforms, enterococci, and Escherichia coli, and their presence is correlated with elevated fecal indicator bacteria (FIB) and beach closures (2,22,23,38). Pathogenic E. coli and Campylobacter, Salmonella, Giardia, and Cryptosporidium spp. occur in bird feces (11,18,19,42) and can infect domestic poultry and humans (27, 41) and contaminate shellfish (1). Bird feces are also a source of antibiotic resistance genes (34,39,50). Recently, because of avian influenza, concerns have risen about pathogen movement due to bird migration (8,10,17,28,30).Although pathogens occur in bird feces, exposure to bird feces is considered less harmful to humans than exposure to other sources of fecal contaminants, especially that of humans (43, 51). For example, molecular evidence indicates that genotypes of certain parasites in birds, such as Giardia and Cryptosporidium, are host adapted and cannot cross-infect among different hosts (20, 57). The relative human health risks of bird and human fecal contamination will be more amenable to measurement once reliable methods are developed to distinguish them quantitatively. The ability to rapidly identify and quantify fecal contamination from birds will improve our ability to estimate human health risk from conta...

Assessment of health risk associated with fecal pollution requires a reliable fecal indicator and a rapid quantification method. We report the development of a Taq nuclease assay for enumeration of 16S rRNA genes of Bacteroidetes. Sensitivity and correlation with standard fecal indicators provide experimental evidence for application of the assay in monitoring fecal pollution.The enumeration of fecal indicators is the cornerstone of testing for fecal pollution in recreational, potable, and shellfish water. There is currently no single bacterial indicator used in all water systems. Total coliforms are the U.S. Environmental Protection Agency (EPA) standard indicators of pollution for drinking water (14), Escherichia coli and enterococci are approved for freshwater (12), enterococci are recommended for marine water (12), and fecal coliforms are used for shellfish waters (15). Traditional membrane filtration and most probable number (MPN) methods require 24 to 74 h for enumeration. Recent updates in methods include the Colilert-18 test (Idexx Laboratories, Westbrook, Maine), which has received U.S. EPA approval for use in ambient water testing (13). Colilert-18 uses a defined substrate medium to test colorimetrically for E. coli and total coliforms within 18 h. E. coli is considered a more specific fecal indicator than total or fecal coliforms, which are found in ambient water in the absence of fecal pollution (11). Epidemiological studies have established a correlation between standard fecal indicators and associated human health risks (5,7,12).Fecal members of the class Bacteroidetes have distinct advantages over coliforms and E. coli as fecal indicators. They are more abundant in the feces of warm-blooded animals than E. coli (8). They are likely to predict recent fecal contamination because they are obligate anaerobes and are unlikely to survive long outside the intestinal tract (1, 8). Enterococci and E. coli are facultative anaerobes, and they can proliferate in soil, sand, and sediments (6,10,16,17).Bernhard and Field (3, 4) developed 16S rRNA gene (rDNA) markers from Bacteroidetes to detect fecal pollution and to distinguish between human and ruminant sources by PCR. Markers for additional host sources have been recently developed (L. K. Dick, A. E. Bernhard, T. J. Brodeur, J. W. Santo Domingo, J. M. Simpson, S. P. Walters, and K. G. Field, unpublished data). PCR source identification is rapid, specific, and sensitive, and it does not require maintenance of databases or libraries of bacterial isolates.Here we report a quantitative Taq nuclease assay (TNA) (2, 9) for general fecal pollution using a Bacteroidetes 16S rDNA marker. The TNA was compared with the Colilert-18 system for accuracy, range, and limits of quantification in serial dilutions of primary sewage influent.A fluorogenic probe and primer set was designed for Bacteroidetes 16S rDNA by using the Primer Design function in the ARB software program (Ludwig and Strunk, Munich, Germany). The sequences were verified for use in a TNA with Primer Expres...

A long‐standing dilemma for soil microbial assays is how best to store soil samples between sampling and analysis. We studied the effects of sample handling and storage on methods used to determine soil microbial biomass, structure, and function. For this study, one forest soil (Gilpin), and two agricultural soils (Granby and Hoytville) were selected with five commonly used sample pretreatments: (i) fresh soil; (ii) air drying for 14 d followed by rewetting (65% water‐holding capacity) and incubation (25°C) for 14 d (D/R); (iii) 28 d at 4°C; (iv) 28 d at −20°C; and (v) 28 d at −80°C. Immediately after pretreatments, soils were analyzed for fatty acid methyl esters (FAMEs), total DNA (tDNA), seven enzyme activities, microbial biomass C, and respiration. Drying and rewetting significantly reduced microbial biomass, respiration, most enzyme activities, tDNA, and total FAME concentrations compared with fresh soil in all three soils. The percentage of fungal FAME markers and two enzyme assays were unaffected by 4°C storage in all soils, and microbial biomass C was unchanged in Hoytville and Gilpin soil at −20 and −80°C. Total DNA was unchanged in the Granby soil at −80°C, and in the Hoytville soil at both −20 and −80°C compared with fresh soil. Total FAME was reduced by all storage treatments in all three soils. We concluded that storage should be avoided whenever possible, particularly for extraction of FAME and total DNA, but that 4 or −20°C is the best storage method for FAME analysis, and −80°C is preferable for DNA analysis. Microbial biomass C and enzyme activities were least affected when stored at 4 or −20°C. The D/R treatment was the least desirable soil preparation method for microbial analyses, and we recommend that this pretreatment be avoided.