Phylogenetic Diversity and Molecular Detection of Bacteria in Gull Feces (cid:1)

In spite of increasing public health concerns about the potential risks associated with swimming in waters contaminated with waterfowl feces, little is known about the composition of the gut microbial community of aquatic birds. To address this, a gull 16S rRNA gene clone library was developed and analyzed to determine the identities of fecal bacteria. Analysis of 282 16S rRNA gene clones demonstrated that the gull gut bacterial community is mostly composed of populations closely related to Bacilli (37%), Clostridia (17%), Gammaproteobacteria (11%), and Bacteriodetes (1%). Interestingly, a considerable number of sequences (i.e., 26%) were closely related to Catellicoccus marimammalium , a gram-positive, catalase-negative bacterium. To determine the occurrence of C . marimammalium in waterfowl, species-speciﬁc 16S rRNA gene PCR and real-time assays were developed and used to test fecal DNA extracts from different bird ( n (cid:1) 13) and mammal ( n (cid:1) 26) species. The results showed that both assays were speciﬁc to gull fecal DNA and that C . marimammalium was present in gull fecal samples collected from the ﬁve locations in North America (California, Georgia, Ohio, Wisconsin, and Toronto, Canada) tested. Additionally, 48 DNA extracts from waters collected from six sites in southern California, Great Lakes in Michigan, Lake Erie in Ohio, and Lake Ontario in Canada presumed to be impacted with gull feces were positive by the C . marimammalium assay. Due to the widespread presence of this species in gulls and environmental waters contaminated with gull feces, targeting this bacterial species might be useful for detecting gull fecal contamination in waterfowl-impacted waters. assays, with SYBR green as the detection dye. The assays were performed with a 7900 HT Fast Real-Time Sequence Detector (Applied Biosystems). Reaction mixtures (20 (cid:3) l) contained 10 (cid:3) l 2x SYBR Premix Ex Taq , 0.4 (cid:3) l ROX reference dye (Takara Bio Inc., Shiga, Japan), 0.2 (cid:3) M (ﬁnal concentration) primers, and either 100 ng genomic DNA (fecal and water samples), a series of dilutions of plasmid DNA with a target insert with a log copy number of 8.8 to 1.8 (i.e., a copy number of 6.3 (cid:6) 10 8 to about 63) per reaction mixture, or a series of dilutions of gull fecal DNA ranging from 60 to 6 (cid:6) 10 (cid:2) 7 ng per reaction mixture. All reaction mixtures were prepared in triplicate in MicroAmp Optical 96-well reaction plates with MicroAmp Optical Caps (Applied Bio- systems). The ampliﬁcation protocol consisted of 50°C for 2 min, followed by 95°C for 2 min and then 40 cycles of 95°C for 5 s, 64°C for 15 s, and a 72°C extension for 10 s and an additional disassociation at 60°C for 15 s. Data were initially analyzed with Sequence Detector software (version 2.2.2) with a 0.2 threshold. Gene copies in fecal samples were calculated from standard curves based on the log transformation of a known concentration versus the thresh- old cycle ( C T ) . Comparison tests between gull species (laughing gull and ring-billed gull) were performed with Statistical Analysis Software (SAS Institute Inc., Cary, NC) by using PROC GLM with the contrast statement feature.