# Presentation: 174887

## Evaluation of Bacteroides-Prevotella and Bifidobacterium Communities for Use in Fecal Source Tracking Studies

**D. L. Clough**, L. R. Fogarty, M. A. Voytek US Geological Survey, Reston, VA.

## ABSTRACT

- 		Current methods for detecting and for determining sources of fecal contamination rely on cultivation of fecal coliforms, *E. coli*, and/or enterococci. These groups of bacteria* *constitute a relatively small percentage of the fecal flora, and cultivation methods may bias the populations by selection, thus misrepresenting the source populations. Bacteroides-Prevotella and Bifidobacterium groups have recently been proposed as alternatives. Each of these species represent a much larger proportion of the fecal population than the fecal coliform and enterococci group. They can also be detected by molecular methods and therefore, could be more sensitive indicators and source determinants of fecal contamination. The variability in fecal bacterial communities within a fecal sample, amongst fecal samples from individuals from the same source species, or between different source species is poorly understood. In this study, terminal restriction fragment length polymorphism (T-RFLP) was used to evaluate differences in the Bacteroides-Prevotella and Bifidobacterium communities in fecal material from mammal and avian source species and from waters contaminated with fecal material. To determine spatial variation of bacterial populations multiple samples were collected from different geographical regions. DNA extracted from the samples was amplified by PCR using published primers for the Bacteroides-Prevotella and Bifidobacterium groups. T-RFLP fragments from different enzymes were analyzed and compared using principal component analysis to develop criteria to distinguish source groups. Results of this study indicate that differences in both the Bacteroides-Prevotella and Bifidobacterium populations occur between source species and that within a source species differences occur geographically. Both of these groups of bacteria could also be detected in fecal contaminated waters and correlated to the known source species of the contamination. These results suggest that this technique may be useful for detecting and for determining the source of fecal contamination.

## INTRODUCTION

- 		Currently sources of fecal pollution are inferred based on biochemical characterization or molecular fingerprinting of the cultivated indicator in a process often referred to as microbial source tracking (MST). The most commonly used MST methods include multiple antibiotic resistance, rep-PCR, pulse field gel electrophoresis, and ribotyping, all of which rely on cultivation of bacteria associated with fecal material such as fecal coliforms, *E. coli*, and/or enterococci. These groups of bacteria* *constitute a relatively small percentage of the fecal flora, and cultivation methods may bias the populations by selection, thus misrepresenting the source populations. In addition, these methods require a comprehensive library of isolate profiles from known sources to which isolates from an unknown source can then be compared.  Recently, other studies have proposed using members of the *Bacteroides* genera as indicators of fecal pollution (Bernhard and Field).

## Why Bacteroides-Prevotella or Bifidobacterium?

-  Members of these* *groups are strict anaerobes and do not naturally occur in environmental water supplies
-  Members of these groups represent a much larger proportion of the fecal population than the fecal coliforms or enterococci groups
-  With the use of molecular methods, cultivation is not necessary
-  Recent studies have identified potential source specific markers from these groups (Bernhard and Field)

## OBJECTIVES

-  Compare the Bacteroides-Prevotella and Bifidobacterium communities found in fecal samples from cows, deer, dogs, geese, horses, and humans.
-  Identify unique T-RFLP peaks to be used in fecal source tracking studies.

## METHODS

**DNA extraction:** DNA was extracted from the fecal samples
-  with the QIamp® DNA Stool Mini Kit (Qiagen, Valencia, CA).

**T-RFLP:** Extracted DNA was amplified using Bacteroides-Prevotella
-  specific primers and Bifidobacterium specific primers (Bernhard ref).
-  Amplified product was digested with restriction enzymes.

## METHODS

** **          **Target    **                  	              **Primers (Reference)****	**	          **Restriction Enzyme**
*Bacteroides-Prevotella          *Bac32F and Bac708R (Bernhard and Field April 2000)*           Aci*I, *Hae*III, *Msp*I
-      *Bifidobacterium*                         Lm26F and Lm3R (Satokari *et al*. 2001)	              *Aci*I, *Hae*III*, Hha*I

**Statistical analysis**: T-RFLP peak profiles were converted into binary form (peak height fluorescence _>_ 50 considered positive). These profiles were compared by calculating the Jaccard similarity coefficients for each and creating a UPGMA dendrogram by using BioNumerics software (version 2.5 Applied Maths; Kortrijk, Belgium). Jackknife analysis was then used to determine how accurately the similarity coefficients could predict the source group for each sample using average classification. With BioNumerics software, a discriminant analysis, with variance, was also performed to determine if there were common T-RFLP peaks associated with each source group. Samples were manually grouped based on source species and the best discriminating components were calculated.

## Jaccard cluster analyses of T-RFLP profiles of all all source groups

## Jaccard cluster analyses of T-RFLP profiles of the cow group colored in red to the left. Samples collected from three locations: WA, VA, IN.

## Average Correct Classification

## Samples within same source group are most similar to one another
Samples from the same location do not necessarily cluster together
No indication of seasonal differences in any source group
Slight correlation within the cow grouping based on whether the sample was from a beef or dairy cow
Ruminant groups cluster together

**Samples from the same location do not necessarily cluster together**
**No indication of seasonal differences in any source group**
**Slight correlation within the cow grouping based on whether the sample was from a beef or dairy cow**
**Ruminant groups cluster together**

- Potential Bacteroides-Prevotella Source Tracking Markers

## There was no peak exclusive to any one source group
Groups of peaks could be used as source markers in source tracking studies
Previously identified source markers were found in appropriate source samples

**Groups of peaks could be used as source markers in source tracking studies**
**Previously identified source markers were found in appropriate source samples**

- Potential Bacteroides-Prevotella Source Tracking Markers

## Preliminary Bifidobacterium Results

**Diversity within a single source species (cows) was analyzed**
**Samples grouped loosely by geographical origin of source**
**Bacteria survived at high levels in fecal-contaminated water under simulated environmental conditions for up to two weeks **

## Preliminary Bifidobacterium Results

## References

**Bernhard, A. E. and K. G. Field. **2000. A PCR assay to discriminate human and ruminant feces on the basis of host differences in *Bacteroides-Prevotella* genes encoding 16S rRNA. Appl. Environ. Microbiol.  **66:**4571-4574.
**Bernhard, A. E. and K. G. Field. **2000. Identification of nonpoint sources of fecal pollution in coastal waters by using host-specific 16S ribosomal DNA genetic markers from fecal anaerobes. Appl. Environ. Microbiol.  **66:**1587-1594.
**Kreader, C. A. **1995. Design and evaluation of *Bacteroides* DNA probes for the specific detection of human fecal pollution. Appl. Environ. Microbiol.  **61:**1171-1179.
**Satokari, R. M., E. E. Vaughan, A. D. L. Akkermans, M. Saarela, and W. M. de Vos***.* 2001. Bifidobacterial Diversity in Human Feces Detected by Genus-Specific PCR and Denaturing Gradient Gel Electrophoresis. *Appl. Environ. Microbiol. ****67:***504-512.