Isolation of conditional expression mutants in Mycobacterium tuberculosis by transposon mutagenesis.

In Mycobacterium tuberculosis identification of essential genes has been hampered by the scarcity of suitable genetic tools for genome wide screenings. We constructed two Himar1 transposon derivatives in which the Streptomyces pristinamycin I-inducible ptr promoter was inserted at one transposon end in outward orientation. These transposons, Tn-pip/pptr (which harbours the promoter and its repressor pip gene) and Tn-pptr (which depends on a host expressing the pip gene), were inserted in the thermosensitive mycobacteriophage phAE87. After transduction into M. tuberculosis H37Rv, hygromycin resistant clones were selected in the presence of pristinamycin, screened for inducer dependent growth, and the transposon insertion point mapped by sequencing. Out of 3530 Hyg(R) mutants tested, we obtained 14 (0.4%) single insertion conditional mutants. In three (leuA, mazE6, rne) pptr was located upstream of genes whose function had been assessed by experimental evidence, whereas in seven the transposon targeted genes (ftsK, glf, infB, metC, pyrD, secY, and tuf) whose function had been assigned by similarity with homologous genes and four ORFs of unknown function (Rv0883c, Rv1478, Rv2050 and Rv2204c). These results validate our mutagenesis system and provide previously unavailable conditional expression mutants in genes of known, putative and unknown functions for genetic and physiological studies.

Outcome of males with high anorectal malformations treated with laparoscopic-assisted anorectal pull-through: preliminary results of a comparative study with the open approach in a single institution.

OBJECTIVE: The aim of this study was to analyze the outcome of males with HARM treated with a laparoscopic-assisted anorectal pull-through compared with the open posterior sagittal approach in a single institution. METHODS: This study includes 32 patients: 17 (9 with a rectoprostatic fistula [RPF] and 8 with a rectovesical fistula [RVF]) who underwent laparoscopic-assisted anorectal pull-through from October 2001 onward and 15 (8 with an RPF and 7 with an RVF) treated by posterior sagittal approach before that date. Patients were reviewed retrospectively but were operated on by the authors and had longitudinal follow-up. Parameters analyzed included associated anomalies, sacral ratio (SR) index, age at surgery, operative time, complications, presence of voluntary bowel movements, constipation, and soiling. A good outcome was determined by absent or grade 1 soiling and a poor outcome result by soiling grades 2 and 3. RESULTS: Mean age at surgery was 22 and 37.5 months for patients with RPF and RVF, respectively, in the laparoscopic group and 29.2 and 25.7 months in the open group. Operative time was significantly shorter (P < .0036) for the laparoscopic RVF repair compared with the open approach. In patients with RPF, 50% in the laparoscopic (L) and 37.5% in the open (O) approach had an SR below 0.6. Fifty percent of all patients with RVF had an SR below 0.6, making groups comparable in terms of evaluating bowel function. Four patients were excluded in the analysis of functional results. Voluntary bowel movements with previous defecatory sensation were present in 83.l3% (5/6) in L vs 87.5% (7/8) in O patients with RPF and 62.5% (5/8) L vs 50% (3/6) in O patients with RVF. Grade 1 soiling was present in 50% (3/6) vs 62.5% (5/8) of patients with RPF and 37.5% (3/8) vs 16% (1/6) of patients with RVF in the L and O groups, respectively. Soiling grade 2 or 3 was present in 50% (3/6) vs 12.5% (1/8) of patients with RPF and 37.5% (3/8) vs 50% (3/6) of patients with RVF in the L and O groups, respectively. The risk of poor outcome was 61% in the group with SR lower than 0.6 vs 13% in the group with a higher ratio. By stratifying the groups according to type of surgery or anatomical type, these results were maintained. CONCLUSION: The laparoscopic approach is a reasonable surgical option for the management of HARM. Laparoscopic approach was less time consuming in patients with RVF without impairing functional results.