, 2009). In our study, tet(40) was located in tandem with tet(O). Sequence homology search showed that the ARGs we identified in this study

were of diverse bacterial origin, including nonpathogenic species such as Bifidobacterium longum, as well as opportunistic pathogens such as Streptococcus suis and Staphylococcus pseudintermedius. Because the potential for gene transfer in the human gut is very high due to the dense microbial population (Kazimierczak & Scott, 2007), it is worth addressing in the future to what extent these bacteria serve as donors, disseminating the ARGs to other bacteria, especially the incoming pathogenic bacteria. The GSI-IX fosmid-based method has some potential disadvantages in ARG screening. Genes on smaller plasmids (< 30 kb) might not be represented in the metagenomic library. Moreover, only ARGs that are properly expressed in E. coli with their own promoters will be identified. However, the fosmid-based

method also has advantages. The larger insert size increases the likelihood PF-02341066 mouse of cloning complete ARGs. In fact, nearly one-third of resistant fosmid clones could not be subcloned, even after several trials. This could be because different vectors were used for cloning (pCC2FOS) and subcloning (pUC118 or pHSG298) or because some resistant determinants are out of the range of length chosen for subcloning (1–5 kb). Our further work will focus on whole-length sequencing to elucidate the resistance mechanisms conferred by the clones that failed to be subcloned.

It is worth noting that although the human subjects we used in this study were not exposed to antibiotic treatment for at least SDHB 6 months prior to sampling, we cannot exclude their antibiotic consumption history. As antibiotic-resistant strains can persist in the human host environment in the absence of selective pressure for a long time (Jernberg et al., 2010), the ARGs we identified cannot be considered intrinsic; they are probably the results of selective pressure conferred by antibiotics that the gut microbes previously encountered and somehow managed to maintain in the gut. In summary, we constructed a metagenomic library from four human gut microbiota and screened for ARGs, uncovering diverse new genes, including a new kanamycin resistance gene fusion. This work helps us to further understand the ARG reservoir of the human gut microbiota, and we believe that other new ARGs will be mined from human gut in the near future. However, to what degree these ARGs in our gut are linked to the potential emergence and dissemination of antimicrobial resistance genes in human pathogens is unclear. This work was supported in part by the National Basic Research Program of China (973 Program grants 2007CB513002 and 2009CB522605). Please note: Wiley-Blackwell is not responsible for the content or functionality of any supporting materials supplied by the authors.