The potential utility of MST indicators depends on the same criteria as for faecal indicators in general. The ideal indicator organism should fulfil the following criteria (Auckenthaler et al., 2003):
- The indicator should be present if pathogens are present
- The indicator should persist in the environment as long as pathogens persist - The indicator should be more resistant than pathogens to treatment procedure - Detection of the indicator should be fast and easy
- The indicator should be found in higher concentrations than pathogens
In addition to these criteria, the MST indicator used to discriminate between human and animal faecal contamination should be restricted to either human or animal faeces. A target fulfilling all these criteria is difficult to find. Based on a search of the literature, the following MST indicators (which successfully identified human or animal sources of
contamination in faecal samples and surface water) were chosen for further research in Switzerland. Sorbitol-fermenting bifidobacteria, R. coprophilus and phages infecting different Bacteroides host strains were selected for assessment. Methods available from the literature and new approaches were tested.
Sorbitol-fermenting bifidobacteria
Bifidobacteria have potential both as general faecal indicators and MST indicators. Sorbitol-fermenting bifidobacteria were shown to be human-specific, whereas strains grown on trypticase phytone yeast broth (TPYB) at 45°C were mainly derived from animal faeces (Sinton 1998). In 1983, sorbitol-fermenting bifidobacteria were shown to be specific indicators for human faecal contamination, and human bifid sorbitol agar (HBSA) was developed as selective medium for these bacteria (Mara and Oragui, 1983). Sorbitol- fermenting bifidobacteria are present in high concentrations in human faeces and wastewater. In comparison with E. coli, concentrations of sorbitol-fermenting bifidobacteria were higher in human faeces and comparable in raw and treated sewage (Mara and Oragui, 1983). Environmental water presents a different picture. Gram-positive, anaerobic bifidobacteria are not able to grow in water, and they do not survive for a long time in the environment. Jagals et al. (Jagals et al., 1995) demonstrated an increase in the faecal coliform-to-bifidobacteria ratio with distance from sources of human faecal contamination. Although sorbitol-fermenting bifidobacteria were found in animal faeces and mainly in faeces from pigs, they were successfully applied to indicate human faecal contamination in sewage and surface water (Blanch et al., 2006; Long et al., 2005; Mara and Oragui, 1983). Molecular methods for detecting B. dentium and B. adolescentis performed well in indicating human faecal contamination (Bonjoch et al., 2004; Nebra et al., 2003). Both culture-based detection of sorbitol-fermenting bifidobacteria and
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molecular methods for detecting B. dentium and B. adolescentis were included in a comparative study assessing several MST parameters in Europe (Blanch et al., 2006). Sorbitol-fermenting bifidobacteria were found to be potential useful MST indicators, but molecular approaches were not (Blanch et al., 2006). Therefore, the culture-based method was selected for use of these bacteria as an indicator for recent human faecal pollution.
Rhodococcus coprophilus
Rhodococcus coprophilus is an aerobic, Gram-positive bacterium able to form a fungus-
like mycelium. It occurs naturally in the intestines of herbivores. First described and classified by Rowbotham and Cross (1977a and b), R. coprophilus was one of the first bacteria used in MST. Due to the fact that it was never isolated from human faeces, it is a promising indicator for animal faecal contamination. The bacterium is present in faeces from a broad spectrum of different animal species, including cattle, sheep, pigs, horses, ducks, geese and hens, and has also been found in water contaminated with animal faeces (Jagals et al., 1995; Mara and Oragui, 1981; Oragui and Mara, 1983; Savill et al., 2001). When R. coprophilus-contaminated grass and hay is ingested by herbivores, the bacterium survives passage through the digestive system, and recontaminates voided dung (Savill et
al., 2001). Both culture-based and molecular methods for detecting R. coprophilus have
been described (Mara and Oragui, 1981; Savill et al., 2001). The culture-based detection procedure is time-consuming, requiring more than 21 days of incubation (Mara and Oragui, 1981; Oragui and Mara, 1983; Jagals et al., 1995). In addition, complete inhibition of contaminating bacteria was not achieved on selective media (Mara and Oragui, 1981). Sinton et al. (Sinton et al., 1998) described the long survival of R. coprophilus in environmental waters and therefore concluded that the organism cannot be used to indicate recent pollution. In order to identify faecal contamination of spring water, the long
persistence of the target microorganism in water may be beneficial. Although it is important to note that results cannot give any indication about the time of faecal pollution, they are useful in determining animal-derived faecal contamination. In the present study, culture-based and molecular methods that are available in the literature as well as a new molecular approach were used to detect R. coprophilus.
Phages of Bacteroides host strains
Bacteroides phages infect defined host strains of Gram-negative, anaerobic bacteria Bacteroides spp., which are naturally found in the intestinal tract. For use as an indicator
organism, detection of the bacteriophages is superior to host detection because phages are more persistent in the environment (Savichtcheva and Okabe, 2006). Prior to this thesis, bacteriophages of the host strains B. fragilis (HSP40), B. thetaotaomicron GA-17 and
B. ovatus GB-124 were shown to indicate human faecal pollution (Blanch et al., 2006;
Ebdon et al., 2007; Payan et al., 2005; Scott et al., 2002). The restriction of these organisms to human faeces makes them a promising indicator for human contamination. Using the host strain B. thetaotaomicron GA-17 for detecting bacteriophages was considered to be a very reliable approach in a comparative study (Blanch et al., 2006). Bacteriophages of the host strain GB-124, not included in the comparative study by Blanch et al. (2006), were never detected in animal samples but were abundant in human wastewater and in surface water (Ebdon et al., 2007; Payan et al., 2005). However,
Bacteroides bacteriophages were shown to be restricted to geographic area. Consequently,
the specificity and sensitivity of bacteriophage methods must be tested before use. The
Bacteroides host strains GA-17 and GB-124 were selected for assessment in Switzerland.
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