Molecular approaches to microbiological monitoring: fecal source detection

Molecular methods are useful both to monitor natural communities of bacteria, and to track specific bacterial markers in complex environments. Length-heterogeneity polymerase chain reaction (LH-PCR) and terminal restriction fragment length polymorphism (T-RFLP) of 16S rDNAs discriminate among 16S rRNA genes based on length polymorphisms of their PCR products. With these methods, we developed an alternative indicator that distinguishes the source of fecal pollution in water. We amplify 16S rRNA gene fragments from the fecal anaerobic genus Bacteroides with specific primers. Because Bacteroides normally resides in gut habitats, its presence in water indicates fecal pollution. Molecular detection circumvents the complexities of growing anaerobic bacteria. We identified Bacteroides LH-PCR and T-RFLP ribosomal DNA markers unique to either ruminant or human feces. The same unique fecal markers were recovered from polluted natural waters. We cloned and sequenced the unique markers; marker sequences were used to design specific PCR primers that reliably distinguish human from ruminant sources of fecal contamination. Primers for more species are under development. This approach is more sensitive than fecal coliform assays, is comparable in complexity to standard food safety and public health diagnostic tests, and lends itself to automation and high-throughput. Thus molecular genetic markers for fecal anaerobic bacteria hold promise for monitoring bacterial pollution and water quality.