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Appl Environ Microbiol. May 2004; 70(5): 3171–3175.
PMCID: PMC404414

Multiplex PCR with 16S rRNA Gene-Targeted Primers of Bifidobacterium spp. To Identify Sources of Fecal Pollution

Abstract

Bifidobacteria are one of the most common bacterial types found in the intestines of humans and other animals and may be used as indicators of human fecal pollution. The presence of nine human-related Bifidobacterium species was analyzed in human and animal wastewater samples of different origins by using species-specific primers based on 16S rRNA sequences. Only B. adolescentis and B. dentium were found exclusively in human sewage. A multiplex PCR approach with strain-specific primers was developed. The method showed a sensitivity threshold of 10 cells/ml. This new molecular method could provide useful information for the characterization of fecal pollution sources.

Determining the origin of fecal pollution is important in a variety of situations: for the protection of water supplies, in epidemiological studies, and, from a legal perspective, in the identification of perpetrators of environmental pollution (12). Traditional microbial indicators have been used for many years to predict the presence of fecal pollution in water. However, it is well established that most of these organisms are not restricted to humans but exist also in the intestines of many other warm-blooded animals. New microbial indicators have therefore been proposed that may provide information about both the presence and the origin of fecal pollution (1, 3, 6, 8, 11, 13, 16, 18, 20, 22, 25, 28, 29). Bifidobacteria are one of the most common bacterial types found in the intestines of humans and other animals. They have been examined as potential candidates for use as indicators of human fecal pollution (1, 21, 25, 29). The distribution of Bifidobacterium species is highly variable: some are associated exclusively with humans and others are exclusive to other animals (4, 27). One molecular approach to species identification in ecological and taxonomic studies is the analysis of the 16S rRNA molecule and its gene as a target (5, 14). Sequence-specific primers or probes based on its sequence have been effective in the detection and identification of the genus (15) or individual species of Bifidobacterium in mixed populations, which are difficult and sometimes impossible by phenotypic characterization (24, 26, 30, 31). A set of 16S rRNA target primers has been developed for the examination of Bifidobacterium distribution in human feces (20), targeting nine Bifidobacterium species: B. adolescentis, B. angulatum, B. bifidum, B. breve, B. catenulatum, B. dentium, B. gallicum, B. infantis, and B. longum.

The aim of this study was to measure the presence of human Bifidobacterium spp. in municipal sewage (samples mainly of human origin) and in slaughterhouse wastewater (samples mainly of animal origin). A specific multiplex PCR for the detection of a few Bifidobacterium species associated exclusively with humans is proposed.

Bacterial type strains used in this study were B. adolescentis DSM 20083T, B. angulatum DSM 20098T, B. bifidum DSM 20456T, B. breve DSM 20213T, B. catenulatum DSM 20103T, B. dentium DSM 20084T, B. gallicum DSM 20093T, B. infantis DSM 20088T, and B. longum DSM 20219T. Bifidobacterium strains were grown anaerobically at 37°C on Columbia blood agar (Difco, Detroit, Mich.) supplemented with 5 g of glucose/liter and 5 g of l-cysteine-HCl/liter before sterilization and reinforced clostridial medium (Oxoid, Basingstoke, Hampshire, England). The incubation period was between 48 and 72 h, as previously described (23). DNA extraction from bifidobacteria and environmental samples was performed as previously described (23). Sewage samples were centrifuged at 250 × g for 15 min to remove major particulate debris. Total DNA was extracted from an aliquot of 200 μl of the supernatant by using the QIAamp DNA blood minikit (Qiagen GmbH) according to the manufacturer's instructions. The PCR amplification of the 16S rRNA gene for the genus Bifidobacterium was performed with the Bifidobacterium genus-specific primers lm26 and lm3 and according to the amplification program previously described (15).

Most bifidobacterial media (2, 19, 22) do not distinguish the origin of the isolates. Additionally, a culture-based method may be limited by the anaerobic physiology of the bacteria. The use of molecular rather than culture-based detection methods could overcome the problems associated with growing strict anaerobes (23). In order to confirm the exclusive presence of human-related Bifidobacterium spp. in wastewater samples, the set of nine specific primers reported above was used for species-specific PCR amplification with raw municipal sewage and slaughterhouse wastewater samples. The procedure consisted of two amplification steps. First, a PCR was performed using the Bifidobacterium genus-specific primers lm26 and lm3 (15). Then, PCR products were used for PCR with sets of primers specific for Bifidobacterium spp. The Bifidobacterium spp. related to humans detected in sewage samples are shown in Table Table1.1. All the samples from human and animal sources showed positive amplification of the 16S rRNA by the Bifidobacterium genus-specific primers. The second PCR with the species-specific primers revealed that all the human sewage samples were positive for B. adolescentis. Eleven human sewage samples were positive for B. dentium and B. catenulatum. B. bifidum and B. longum were found in eight and seven human sewage samples, respectively. Half of these samples were positive for B. infantis and B. breve, while B. angulatum was found in only five of these human sewage samples. Finally, B. gallicum was not found in any human sewage sample. Only B. adolescentis, B. dentium, and B. longum were absent in all animal wastewater samples. One bovine wastewater sample was positive for B. infantis, and two wastewater samples were positive for B. catenulatum. Three bovine wastewater samples were positive for B. angulatum, and three were positive for B. breve. Finally, B. bifidum was found in four of the bovine wastewater samples. Consequently, the specific association of B. adolescentis and B. dentium with human sewage could be used to identify the origin of fecal pollution in water. This result agrees with independent studies by different authors who proposed B. adolescentis (17) and B. dentium (23) as human-specific species. Both studies suggested these species for distinguishing human fecal pollution, although some false positives were observed in certain cases (17).

TABLE 1.
Distribution of the human-related Bifidobacterium species in municipal sewage and animal wastewater samples of different origins by 16S rRNA gene amplification with species-specific primers (24)

The species-specific primers for the selected Bifidobacterium species from the results of the previous analysis were used for the definition of a specific “ADO-DEN multiplex PCR.” The procedure consisted of two amplification steps. First, a PCR was performed using the Bifidobacterium genus primers lm26 and lm3 (15). Then, PCR products were used for ADO-DEN multiplex PCR with the species-specific sets of primers (Table (Table2).2). The PCR mixture for the first PCR was as described above. The ADO-DEN multiplex PCR was performed in 25-μl volumes composed of 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2, 0.1% Triton X-100, each deoxynucleoside triphosphate at a 200 μM concentration, the species-specific primers for B. adolescentis at an 0.25 μM concentration (21), the species-specific primer for B. dentium at an 0.50 μM concentration, 1 μl of the extracted bacterial DNA, and 0.9 U of Taq DNA polymerase (Eppendorf, Hamburg, Germany). The PCR was carried out in a Perkin-Elmer thermal cycler (Perkin-Elmer, Norwalk, Conn.). The following amplification program was used: one cycle consisting of 94°C for 5 min; followed by 35 cycles consisting of 94°C for 20 s, 55°C for 20 s, and 72°C for 30 s; and finally one cycle at 72°C for 5 min. The amplification products were subjected to gel electrophoresis in a 3% agarose gel, followed by ethidium bromide staining. In order to establish the threshold of the B. adolescentis and B. dentium detection by ADO-DEN multiplex PCR, a set of 10-fold dilutions was made from pure cultures of both species (B. adolescentis DSM 20083T and B. dentium DSM 20084T), which had a concentration of around 108 CFU/ml. DNA extraction by the QIAamp DNA blood minikit (catalog no. 51104) was performed from each dilution, and the 16S rRNA was amplified using two-step PCR amplification as described above. Figure Figure11 shows the PCR products obtained from these 10-fold dilutions by the ADO-DEN multiplex PCR. The limit of detection for the new method was 10 CFU/ml.

FIG. 1.
Agarose gel electrophoresis patterns of the ADO-DEN multiplex PCR products obtained from 10-fold dilutions of mixtures of pure cultures of B. adolescentis DSM 20083T and B. dentium DSM 20084T to determine the threshold of the method. Lanes: M, DNA marker ...
TABLE 2.
Sequences of the specific primers for B. adolescentis (Bi-ADO) and B. dentium (Bi-DEN) used in the ADO-DEN multiplex PCR (21)

A preliminary evaluation of the specificity of the defined ADO-DEN multiplex PCR was performed by analyzing four experimental mixtures of human and animal wastewater samples (90:10, vol/vol) and four experimental mixtures of animal and human wastewater samples (90:10, vol/vol). Later, serially diluted mixtures of animal and human wastewater samples were analyzed to determine the lowest proportion of human pollution that could be detected by the ADO-DEN multiplex PCR. The tested mixtures of animal (A) and human (H) wastewaters (vol/vol) were 100H:0A (control for human sewage), 90H:10A, 10H:90A, 5H:95A, 2.5H:97.5A, 1H:99A, and 0H:100A (control for animal sewage). The DNA extraction and the 16S rRNA genus-specific amplifications were performed as described above. Then, the species-specific second PCR was performed for each mixture. DNAs extracted and amplified from pure cultures of B. adolescentis DSM 20083T and B. dentium DSM 20084T were used as positive controls in all the ADO-DEN multiplex PCR assays. The experimental mixtures of human sewage and animal wastewater samples showed positive amplification for both species when the ADO-DEN multiplex PCR was used until the proportions of human sewage and animal wastewater reached 1 and 99%, respectively (Fig. (Fig.22).

FIG. 2.
Agarose gel electrophoresis patterns of the ADO-DEN multiplex PCR products obtained from experimental mixtures of wastewater samples to evaluate the lowest proportion of human pollution that can be detected. Lanes: M, DNA marker ([var phi]X174); A to ...

A total of 44 wastewater samples from different origins were collected. Twenty-two samples of raw sewage were collected from five municipal sewage treatment plants. The main characteristics of the sewage treatment plants are shown in Table Table3.3. Twenty-two samples of animal wastewater were taken from the drains of pig, cattle, and poultry slaughterhouses. All samples were transported at 4°C to the laboratory according to standardized protocols (7). Samples were analyzed immediately or kept at −80°C prior to analysis. The enumeration of fecal coliforms, enterococci, and spores of sulfite-reducing anaerobes (clostridia) was performed for all samples. The enumeration of fecal coliforms and enterococci was performed by membrane filtration on membranes with a pore size of 0.45 μm (Millipore, Molsheim, France). Later, they were transferred onto m-FC agar plates (Difco) and cultured for the enumeration of fecal coliforms (10) and onto m-Enterococcus agar plates (Difco) for the enumeration of enterococci. Then, they were incubated at 44.5°C for 24 h and 37°C for 48 h, respectively. Later, the membranes for enumeration of enterococci were transferred to bile esculin agar (Difco) for 1 h at 37°C to confirm the enterococcal colonies on the basis of hydrolysis of esculin, which turns them black. Counts of black colonies were then performed for each of the analyzed membranes to confirm enterococcal enumerations (9). Spores of sulfite-reducing anaerobes (clostridia) were enumerated by heat shock of samples at 80°C for 10 min as previously described (9). Later, 10-fold dilutions were made in one-quarter Ringer solution, and 1 ml of each dilution was inoculated in 50 ml of liquid sulfite polymyxin sulfadiazine agar (Scharlau, Barcelona, Spain). Inoculated tubes were shaken to homogenize the solution, and the medium was then allowed to solidify. Tubes were then incubated at 44°C for 24 h. The enumeration of fecal coliforms, enterococci, and spores of sulfite-reducing anaerobes (clostridia) is shown in Table Table4.4. The concentration of fecal coliforms was around 7 log CFU/100 ml in most of the samples, except for one sewage sample from treatment plant 4. The concentration of enterococci was usually 1 log value below that of the fecal coliforms. The ratio of fecal coliforms to enterococci was calculated for all samples by using the average concentration for each sampling site. The concentration of spores of sulfite-reducing anaerobes was around 4 to 5 log CFU/100 ml in all samples. No differences were observed between human and animal wastewater samples in the concentration of fecal coliforms, enterococci, or clostridia (Table (Table4).4). Consequently, none of these parameters nor the fecal coliform/enterococcus ratio can discriminate the origin of the studied wastewater; however, the molecular approach developed here has allowed the discrimination of wastewater origin in most cases.

TABLE 3.
Characteristics of urban sewage treatment plants sampled in this study
TABLE 4.
Results of ADO-DEN multiplex PCR and microbial indicators in human and animal wastewater samples

The detection of B. adolescentis and B. dentium in human sewage and animal wastewater is also shown in Table Table4.4. All of the human sewage samples showed positive amplification in the ADO-DEN multiplex PCR. Animal wastewater samples did not show positive amplification in the ADO-DEN multiplex PCR, with the exception of three samples, one from poultry and two from cattle. In the first case (slaughterhouse 3, pig) both species showed positive amplification. The wastewater network system was then analyzed, and it was found that sewage from the employees' toilet was pouring to feed into the drain sampled. In the second case (slaughterhouse 6, poultry), only B. adolescentis was positive and the possibility that it was of avian origin could not be discarded, as B. adolescentis is also related to some species of avian bacteria. Further studies should consider analyzing the presence of this species.

In conclusion, a simple and specific molecular procedure to discriminate between human and animal fecal pollution in waters with high concentrations of bifidobacteria has been described. The ADO-DEN multiplex PCR is based on the detection of the 16S rRNA gene of B. dentium and B. adolescentis by species-specific primers and does not require the prior use of a selective culture medium. At present, there is no information on the persistence of these Bifidobacterium species in water over time. Consequently, the use of this new approach in “aged” waters (i.e., water with old pollution) should be further analyzed when data on the persistence of these species become available. This new molecular method could provide information for the characterization of fecal pollution sources.

Acknowledgments

This research was supported by the European project TOFPSW EVK1-2000-22080.

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