The bacterial diversity of the openings of the urogenital and anal tracts of the adult female tammar wallaby, Macropus eugenii, was determined in order to ascertain whether the physical proximity of the openings of these tracts within the cloaca affected the two populations of bacteria. Terminal restriction fragment length polymorphism (T-RFLP) analyses of 42 wallabies identified 81 different terminal fragments, indicative of diverse and complex microbiomes at these anatomical locations. Subsequent amplified rDNA restriction analysis (ARDRA) identified 72 phylotypes from the urogenital tract and 50 from the anal tract. Twenty-two of these phylotypes were common to both tracts. Phylogenetic analysis of sequenced 16S rDNA showed that 83 % of the phylotypes were unidentified species based on the premise that any sequence possessing ,97 % homology to a known bacterial species or phylotype was novel. Thus, despite the close proximity of the openings of the urogenital and anal tracts within the cloaca, the two sites retained a diverse range of distinct bacteria, with only a small percentage of overlapping species.
INTRODUCTIONMetatherian (marsupial) mammals offer a unique biological opportunity to investigate the relationship between the microflora of the urogenital and anal tracts. In contrast to eutherian (placental) mammals, which possess separate openings of the digestive and urogenital systems, most marsupials possess a common posterior chamber for these systems -the cloaca (Tyndale-Briscoe, 2005; Fig. 1). This structural feature might imply that bacteria from one tract have easy access to the other. To date, the bacteria associated with these two sites have not been studied in marsupials, although in humans it has been noted that the bacteria of the urogenital tract may vary and can be influenced by bacteria growing in the anal tract and thus occasionally can cause urogenital disease (Reid et al., 2001;Tannock, 1999).The tammar wallaby, Macropus eugenii, is regarded as a model marsupial (Tyndale-Briscoe, 2005) with more than 30 years of fundamental research into its basic biology. Despite this, little is known about the bacterial populations of this animal, all published studies having used culture-based methods that are known to be biased in favour of a small percentage of the total microbiome (Hume, 1999; Lentle et al., 2006;Old & Deane, 1998;Yadav et al., 1972).In this study we used molecular-based methods targeting the 16S rDNA gene to document the bacteria at the openings of the urogenital and anal tracts of 42 female tammar wallabies and undertook a comprehensive comparison of the major phylotypes and their diversity. This is believed to be the first extensive report of the bacterial species within the openings of these two tracts in any marsupial. The results showed that despite the close anatomical proximity of the openings of these tracts, each opening possessed a diverse and complex bacterial population comprising mainly unidentified species.Abbreviations: ARDRA, amplified rDNA restriction analysis; T-...