Bifidobacterial diversity and the development of new microbial source tracking indicators

Abstract

Many studies suggest a close relationship between species of Bifidobacterium and their hosts. Thus, species such as B. adolescentis and B. thermacidophilum subsp. porcinum have been proposed as potential indicators of human and porcine fecal pollution. The diversity of bifidobacteria in wastewaters (human and animal) and slurries is analyzed using nested PCR followed by denaturant gradient gel electrophoresis (DGGE). The sewage samples showed similar DGGE patterns. The predominant bands were recognized as B. adolescentis, B. longum, and two unidentified species related to B. adolescentis. A single band detected in poultry samples was identified as B. saeculare. Bifidobacterial diversity was higher within porcine and bovine samples. The main bands in porcine samples were identified as B. minimum, an unknown species, and B. thermophilum/B. thermacidophilum subsp. porcinum. The latter species was also identified among the main bands in bovine samples together with B. pseudolongum and B. ruminantium. We then attempted to isolate the host-specific strains. DGGE bands were examined to develop specific probes to screen environmental samples by colony hybridization and further isolation of strains from positively hybridized colonies. Bifidobacterial strains that are host associated by DGGE bands to human and pig were successfully isolated from the environment: B. adolescentis from human sewage samples and the unidentified species related to pig from slurries and slaughterhouse wastewater. Neither the poultry-associated B. saeculare nor the ruminant-associated B. pseudolongum could be isolated with the current methodology, suggesting either a low prevalence in the samples or failure of the culture to grow in the media used.

Fig. 1.

Separation of the PCR product of the Bifidobacterium reference strains used in this study with genus-specific primers. Lanes: A, B. adolescentis; B, B. ruminantium; C, B. catenulatum; D, B. animalis subsp. lactis; E, B. minimum; F, B. merycicum; G, B. subtile; H, B. longum; I, B. longum subsp. infantis; J, B. saeculare; K, B. pullorum; L, B. pseudolongum subsp. pseudolongum; M, B. scardovii; N, B. breve; O, B. bifidum; P, B. pseudocatenulatum; Q, B. coryneforme; R, B. dentium; S, B. globosum; T, B. choerinum; U, B. angulatum; V, B. indicum; W, B. boum; X, B. thermophilum; Y, B. asteroides. The denaturant gradient range was 45% (urea and formamide) in the upper part of the gel and 55% at the bottom.

Fig. 2.

DGGE profiles and dendrogram of the Bifidobacterium population of wastewater of different sources. □, human; ■, poultry; , porcine; , bovine samples. Jaccard and UPGMA clustering.

Fig. 3.

DGGE profiles of the Bifidobacterium population of wastewater from different sources. Lanes: 1 and 2, ruminant; 3 to 6, pig; 7 to 10, human; 11 and 12, poultry.