Faecal pollution in coastal or fresh waters leads to human disease and economic losses such as closure of commercial shellfish harvesting and recreational and bathing areas. Recent incidents include the isolation of human enteric viruses and bacteria such as norovirus, hepatitis A virus, and Salmonella from coastal waters and shellfish, which were implicated in shellfish-borne outbreaks after oyster consumption (Potasman et al., 2002; Martinez-Urtaza et al., 2004). In light of this risk to health and safety, it is important to identify the source of faecal contamination to better facilitate resource management and remediation.
Faecal contamination of water resources is currently evaluated by employing culturing methods to detect and enumerate living facultative-anaerobic bacteria, such as Escherichia coli, enterococci, or faecal coliforms. Samples are normally obtained from shellfish or directly from bathing waters (Directives 2006/113/CE; 2006/7/CE). The species traditionally used as faecal indicators, however, have limitations owing to several factors, including (1) their short survival time in an open-water environment, (2) their ability to proliferate in soil, sand or sediments absent in any point-source faecal contamination, (3) the low levels of correlation with the actual presence of pathogens, (4) the underestimation of true bacterial presence through omission of noncultivable bacteria, (5) their inability to track the source of faecal contamination because coliforms and enterococci are common to all mammalian hosts (Roszak & Colwell, 1987; Pommepuy et al., 1996; Gordon & Cowling, 2003; Wheeler et al., 2003; Hörman et al., 2004; Savichtcheva & Okabe, 2006). In order to overcome these shortcomings, alternative methods and indicators must be developed. Potential alternative indicators of faecal contamination could be anaerobic bacteria such as Bacteroides and Bifidobacterium that are more abundant in the faeces of warm-blooded animals than E. coli (Fiksdal et al., 1985; Suau et al., 1999). Importantly, these species have been shown to exhibit host-specific adaptation on the genetic level (Dick et al., 2005). While these bacteria are fastidious to enumerate with conventional culture techniques, they can nonetheless be easily detected using current molecular methods. Because uncultivated bacteria represent 70–80% of the total human microbiota, culture-independent methods of analysis based on 16S rRNA gene have been developed (Suau et al., 1999; Eckburg et al., 2005). These studies showed that the most highly represented bacterial groups in human stools were the Clostridium leptum and the Clostridium coccoides groups of the Firmicutes followed by the Bacteroides/Prevotella group and the Bifidobacterium genus (Harmsen et al., 2002; Lay et al., 2005a). Studies involving domestic animal microbiota are less numerous and are mainly focused on the phylogenetic diversity of the intestinal bacterial community in pigs, cattle and chicken (Lan et al., 2002; Leser et al., 2002; Ozutsumi et al., 2005). Recently, specific quantitative PCR (qPCR) approaches were used to estimate a limited number of bacterial species or groups of faecal microbiota (Matsuki et al., 2004; Seurinck et al., 2005; Reischer et al., 2006).
The work presented here seeks to establish a more comprehensive dataset in comparing human and farm animal microbiota. To this end, we developed and optimized a qPCR-based approach, which was subsequently applied to analyse faecal samples collected from humans and farm animals. Using such molecular techniques, we overcome the limits of traditional faecal indicators, including culturing methods, which consistently underestimate faecal population. The development and application of our qPCR systems quantifies faecal bacteria groups in human and animal faecal samples and provides essential information concerning potential alternative faecal indicators and host-specific bacterial groups.