CHAPTER 1
Health
Evaluation of detection methods targeting host-specific Bacteroides Spp. as a microbial source tracking marker
T. Miura, Y.M. Chan, Y. Masago and T. Omura
Department of Civil and Environmental Engineering, Tohoku University. 6-6-06, Aoba, Sendai, 980-8579, Japan (E-mail: miura@water.civil.tohoku.ac.jp, stephycym@gmail.com, masago@water.civil.tohoku.ac.jp, omura@water.civil.tohoku.ac.jp)
Abstract Microbial source tracking is an increasingly used approach to determine host-specific contributions of fecal contamination to water bodies. Throughout the world, many studies have been done on fecal contamination in various kinds of water bodies; however, studies on fecal contamination in sediment are limited. In this study, detection methods for host-specific Bacteroides spp. previously developed were evaluated for their ability for applications to sediment, as well as water samples. In addition, fecal coliforms in sediment and its water column were quantified in order to compare their concentrations and to evaluate contribution of the sediment to the water in case of sediment's resuspension to the water. Bacteroides were detected in 22 out of 36 sediment samples (61%) and 1 out of 18 water samples (6%). The primer set for human-specific Bacteroides developed by Okabe et al. (2007) showed much higher positive ratio (47%, n = 17) in the sediment samples compared with that developed by Kildare et al. (14%, n = 5) (2007). The concentration of fecal coliforms in the sediment samples (CFU/100 g (dry weight)) was 12 to 663 times as high as that in the water samples (CFU/100 ml). These results imply that sediment is more appropriate target for microbial source tracking.
Keywords fecal coliforms, fecal contamination, host-specific Bacteroides, microbial source tracking, sediment
INTRODUCTION
In Southeast Asia, diarrheal diseases are responsible for as much as 18% of all deaths of children under 5 years of age (WHO, 2005). WHO (2004) also reported that 88% of diarrheal diseases is attributed to unsafe water supply and inadequate sanitation. Water bodies used as drinking water sources in developing countries are contaminated by human and animal feces (Moe et al., 1991; Vollaard et al., 2005) that may include waterborne pathogens partly due to the insufficient wastewater treatment systems. As many pathogens are host-specific, it is important to assess contribution of human feces to the fecal contamination in water.
Microbial source tracking is an increasingly used approach to determine host-specific contributions of fecal contamination to water bodies (Kildare et al., 2007) and appropriate measures against the fecal contamination can be discussed based on the result of the source tracking. Bacteroides, a group of anaerobic bacteria, has been applied for fecal source tracking in water (Reischer et al., 2006; Kildare et al., 2007; Savichtcheva et al., 2007) mainly due to its high abundance in the feces of warm-blooded animals (Fiksdal et al., 1985) and high host specificity (Bernhard and Field, 2000a and b).
Throughout the world, many studies have been done on fecal contamination in various kinds of water bodies, such as raw sewage, river water, seawater and tap water. However, although it is reported that sediment may provide more reliable information of long-term fecal contamination (Craig et al., 2002), studies on fecal contamination in sediment are limited, that warrants needs for the knowledge and techniques for detection and quantification of microbes in sediment.
In this study, detection methods for host-specific Bacteroides spp. previously developed were evaluated for their ability for applications to sediment samples, as well as water samples. In addition, fecal coliforms in sediment and its water column were quantified in order to compare their concentrations and to evaluate contribution of the sediment to the water in case the sediment is resuspended to the water. This evaluation experiment was done using sediment and water samples collected at the Takagi River estuary in Japan. Outcomes of this study would be applicable for fecal source tracking undertaken in other areas.
MATERIALS AND METHODS
Samples
Sediment and water samples were collected in the Takagi River estuary during the ebb tide monthly from November 2007 to May 2008. Locations of sample sites are shown in Figure 1: St.A, St.B and St.C were located at the bay, where oyster beds are placed (Figures 1 and 2); St. D was located at the river mouth; and St.E and St.F were located at the river downstream. There is a small dam to control the river flow at St.F (Figure 3).
About 100 mL of top layer sediment was collected using a Ekman-Birge type bottom sampler at St.A, St.B, St.C, St.D, St.E and St.F; about 20 L of surface seawater was collected at St.A, St.B, St.C and St.D, about 1 L of surface river water was collected at St.E and St.F, and about 1 L of treated wastewater were collected at the wastewater treatment plant (WWTP). All the samples were transported to the laboratory on ice in sterile containers and processed within a few hours of collection.
Sediment characterization
Water content and ignition loss of sediment samples were measured according to the standard protocols of Japan Sewage Works Association (1997). Briefly, sediment was dried in an oven at 105°C for 2 hr and water content (%) was calculated using the weight before and after the desiccation. Then the dried sediment was placed in an electric furnace at 600°C for 1 hr and weighed. The ignition loss (%) was calculated from these results. Particle size distribution of sediment samples was measured by Microtrac (9320HRA (X-100); NIKKISO, Tokyo).
DNA extraction and detection of Bacteroides
DNA in the sediment samples (from November 2007 to April 2008, 36 samples in total) was extracted from 0.5 g well-mixed wet sediment using ISOIL DNA extraction kit (NIPPON GENE, Tokyo), according to the protocols described by the manufacturer.
Bacteroides in the water samples (from February 2008 to April 2008, 18 samples in total) were concentrated and recovered, following the protocols of Okabe et al. (2007) with some modifications. Ten to 20 L of sea water, 1 L of river water and 1 L of treated waste water samples were passed through HA membrane filters (0.45 µm pore size; Millipore, Tokyo). The membranes were placed in phosphate buffered saline and captured cells were dispatched from the membranes by ultrasonication with Ultra S.Homogenizer (50 W, 20 kHz; Model VP-5S; TAITEC, Koshigaya). The solution containing Bacteroides was collected and centrifuged at 9000 · g at 4°C for 10 min. Supernatant was discarded and the pellet was resuspended in 1 mL of sterile Milli-Q water. Genome DNA...
