The primers Bfgi2_Int_F and Bfgi2_Int_R (Table 4) were designed directed outwards across the proposed attL and attR sites. Using these primers, amplification of product should only occur if a circularized form of Bfgi2 is present in the cell. The size (2.25 Kb) sequence of the resulting PCR product confirmed the presence of the circular intermediate (Fig. 6 panel B, Lane 3). Attempts to show plaque formation using NCTC9343 as

Cabozantinib supplier an indicator strain did not produce any visible plaques. This could be due to the phenomenon of limited host range for the bacteriophage. However, given that Bfgi2 circular intermediate was detected it is tempting to speculate that it is, or is a derivative of an active phage and such phage could be transmitted to a non-lysogenized strain of B. fragilis, bringing with

it a copy of a C10 protease. C10 protease genes are present in clinical isolates of B. fragilis and in the healthy human faecal microbiota In addition to the 3 genome strains, a panel of 5 clinical isolates of B. fragilis from ZD1839 several human infection sites (Table 7) were tested by allele-specific PCR for the C10 protease genes they harbour. The results indicated that this panel of strains have a complement of bfp genes more similar to NCTC9343 than to 638R (Table 1). The distribution of bfp genes in the clinical isolates is not identical, and none of the 5 isolates carried all four bfp genes. The bfp1-4 genes were detected in 3, 5, 1 and 0 clinical isolates respectively. The bfp4 gene was not be detected in any of these clinical strains, while bfp1 was not detected in two strains (NCTC 10584 and NCTC 11295). In contrast, bfp2 was encoded by all strains. In B. fragilis strain YCH46, there is a CTnERL-type conjugative transposon 353 bp distance from the bfi1A-bfp1-bfi1B gene cluster. However, this conjugative transposon is not present

in either of the other two sequenced B. fragilis genomes, 638R and NCTC 9343. The bfp3 gene was only detected in one clinical isolate (NCTC 9344), with a concomitant detection from of the Bfgi2 insertion. In all cases a 595 bp fragment was successfully amplified using the primer pair Bfgi2_attB_F and Bfgi2_attB_R (not shown), indicating the presence of a free integration site for Bfgi2 in all strains. It should be noted that for NCTC 9344 and 638R, there was a lower product yield and although not quantitative this is likely due to the integration of Bfgi2 in a sub-population of the cells. Table 7 Bacterial strains used in this study B. fragilis strain Source of isolate Reference 638R Clinical isolate, human [57] YCH46a Bacteraemia, human [19] NCTC9343 Appendix abscess, human [58] NCTC9344 Septic operation wound, human [59] NCTC10581 Empyema fluid, human [60] NCTC10584 Pus, human [58] NCTC11295 Pus from fistula, human [61] NCTC11625 Post-operative wound infection, human [62] a. Analysis of genome sequence only.