Biﬁdobacteria in Feces and Environmental Waters (cid:1) †

Biﬁdobacteria have been recommended as potential indicators of human fecal pollution in surface waters even though very little is known about their presence in nonhuman fecal sources. The objective of this research was to shed light on the occurrence and molecular diversity of this fecal indicator group in different animals and environmental waters. Genus- and species-speciﬁc 16S rRNA gene PCR assays were used to study the presence of biﬁdobacteria among 269 fecal DNA extracts from 32 different animals. Twelve samples from three wastewater treatment plants and 34 water samples from two fecally impacted watersheds were also tested. The species-speciﬁc assays showed that Biﬁdobacterium adolescentis , B. biﬁdum , B. dentium , and B. catenulatum had the broadest host distribution (11.9 to 17.4%), whereas B. breve , B. infantis , and B. longum were detected in fewer than 3% of all fecal samples. Phylogenetic analysis of 356 biﬁdobacterial clones obtained from different animal feces showed that ca. 67% of all of the sequences clustered with cultured biﬁdobacteria, while the rest formed a supercluster with low sequence identity (i.e., <94%) to previously described Biﬁdobacterium spp. The B. pseudolongum subcluster (>97% similarity) contained 53 fecal sequences from seven different animal hosts, suggesting the cosmopolitan distribution of members of this clade. In contrast, two clades containing B. thermophilum and B. boum clustered exclusively with 37 and 18 pig fecal clones, respectively, suggesting host speciﬁcity. Using species-speciﬁc assays, biﬁdobacteria were detected in only two of the surface water DNA extracts, although other fecal anaerobic bacteria were detected in these waters. Overall, the results suggest that the use of biﬁdobacterial species as potential markers to monitor human fecal pollution in natural waters may be questionable. and (deer). We performed similar assays using serial dilutions of inﬂuent, return activated sludge, secondary aeration, efﬂuent, and environmental water DNA extracts that yielded positive signals with a given biﬁdobacterial assay. To determine the inhibition potential at a speciﬁc environmental DNA concentration, DNA extracts were used as a template in general and human-speciﬁc Bacteroides assays (4), as well as in C. coccoides PCR assays Detection limits were also established for seven of the nine biﬁdobacterial species markers using DNA extracts from pure cultures of B. adolescentis (DSM 20086), B. angulatum (DSM 20225), B. biﬁdum (DSM 20082), B. breve (DSM 20213), B. catenulatum (DSM 20103), B. gallicum (DSM 20093), and B. infantis (DSM 20088). Serial dilutions (10 (cid:2) 8 to 10 (cid:2) 16 g of DNA) from each pure culture were used as a template in the respective PCR assays. The potential cross-reactivity of the biﬁdobacteria species-speciﬁc assays was determined by using 1 ng of the aforementioned pure culture DNA extracts and nontarget species DNA extracts of additional species such as B. pseudolongum subsp. globosum (DSM 20092), B. ruminatum (DSM 6489), and B. suis (DSM 20211). Coverage of genus-speciﬁc assays was tested by using all 10 pure culture DNA extracts (1 ng) as a template in each of the three genus-speciﬁc biﬁdobacterial assays.