RESULTS

CONCLUSIONS:1. Continuous subculturing of M. tuberculosis produces a remarkable decrease in the citotoxicity against a THP-1 macrophage monolayer.

2. It induces also genetic changes measured as movement of the IS6110 elements in the chromosome

3. Changes in virulence are possibly related to genomic deletions of the chromosome, although at this stage we are confirming this findings by PCR and Southern Blot.

4. The attenuation of M. tuberculosis isolates will serve for analysis of the immune resistance against this organism.

INTRODUCTIONTuberculosis is caused by the bacterium Mycobacterium tuberculosis, considered as a re-emergent infectious disease and a major public health problem affecting one third of earth’s population. One of the main difficulties for the control of tuberculosis, is the lack of appropriate therapeutical options. The BCG vaccine, derived from an M. bovis strain, has a very variable efficacy in humans; despite of its antigenic similarity with M. tuberculosis it’s not exactly the same microorganism. In vitro serial passage has been used to induce changes in virulence of different microorganisms, including M. bovis for the production of BCG. The present study has the objective of analyzing changes in the genetic composition of Mycobacterium tuberculosis H37Rv and a Beijing strain, DR-689, after 200 serial passages.

METHODSStrains and culture conditions. Mycobacterium tuberculosis H37Rv and M. tuberculosis DR-689 (which contains a deletion in the plcA-plcB-plcC locus) were subcultured in M7H9 broth, with and without 0.5% ox bile, in 50 ml Erlenmeyer flasks and incubated at 37ºC with shaking at 150 rpm until observing visible growth (about 7 days) for a total of 200 passages.

Cloning of individual colonies.- In order to avoid genetic heterogeneity, bacterial clones were prepared. From the cultures in suspension, a loopful was separated on agar plates. After bacterial growth was obtained, an individual colony was selected and cultured in 7H9 broth. The procedure was repeated two more times.

Mycobacterial genomic DNA extraction. DNA was extracted using the CTAB-NaCl technique with some modifications.

Cytotoxic activity.- The cytotoxicity assays were done according to the technique previously reported by Castro- Garza et al. (2007).

RFLP analysis for IS6110. Southern blotting with labelled IS6110 DNA was performed as previously described using an international protocol (van Embden et al., 1993). Briefly, M. tuberculosis DNA was digested with PvuII, electrophoresed, and hybridized with a labelled 245 bp PCR-generated probe as previously described.

Genomic mass sequencing.- High throughput sequencing was carried out by using the Illumina MiSeqmachine using a V2 cartrdige of 25x2 PE cycles. Readings gave a 25-46x genome coverage; they were assembled using SPAdes Genome Assmbler (v 3.5.0). The metric analysis was done using QUAST (v3.1). The obtained contigs were aligned against the M. tuberculosis H37Rv reference sequence NC_000962.3 using Sequencher 5.3 (Gene Codes). The deletions observed in the passaged isolates were confirmed to exist in the parental (non subculured) isolates genomic sequence.

"Genomic sequence analysis of Mycobacterium tuberculosis H37Rv and M. tuberculosis DR-689

subjected to 200 continous subcultures"

L. Vera-Cabrera*, C.A. Molina-Torres, Rocio Ortiz-Lopez, Ramon Barcenas, Guillermo I. Guerrero-Ramirez, J. Castro-Garza, and J. Ocampo-Candiani. Servicio de Dermatologia, Universidad Autonoma de Nuevo Leon,

Monterrey, Mexico, Centro de Investigación Biomédica del Noreste. IMSS, Monterrey, México.

M. bovis BCG

DR689 subcultured 200x

DR689 subcultured

200x+ bile

DR689 Parental isolate

Fig. x. RFLP analysis by Southern blotting for IS6110. Lanes: 1, Ladder 1 Kb; 2, M. tuberculosis H37Rv without subculturing; 3, M. tuberculosis H37Rv subcultured 200 times in Middlebrook 7H9; 4, M. tuberculosis H37Rv subcultured 200 times in Middlebrook 7H9 plus ox bile; 5, M- tuberculosis Dr-689 without subculturing; 6, M. tuberculosis DR-689 subcultured 200 times in Middlebrook 7H9; 7, M. tuberculosis DR-689 subcultured 200 times in Middlebrook 7H9 plus ox bile

Fig. X. Cytotoxic activity of M. tuberculosis isolates on the THP-1 macrophage monolayer. Culture plate wells were seeded with 4 x105 macrophages and infected with the bacilli at an m.o.i. of 1 : 10.

Fig. X. Microscopic features of THP-1 macrophage´s monolayers infected with M. tuberculosis strains using a m.o.i. of 10 after one (left), four (center) and seven (right) days post-infection.

RESULTS

3120650-3163824

4032502-4059083

2146083-2195923

1779348-1982259

733269-846904

Acknoledgements: This work was supported by CONACYT GRANT 155892

P192

*Corresponding author