mb 401 research presentation final
TRANSCRIPT
Developing molecular techniques for determining chlamydial recombinants
Beau Bradford
Chlamydiae – Obligate intracellular, gram negative, cocci bacteria– Developmental cycle occurs in membrane-bound vacuole
termed “inclusion”– Chlamydia trachomatis• Human pathogen of urogenital and ocular epithelial
mucosa• Inclusion are fusogenic when cell is multiply infected• Readily undergo recombination during infection
– Chlamydia caviae • Guinea pig pathogen• Non-fusogenic “grapevine” inclusions • No current methods for recombination with other
Chlamydia species
Rationale• Recombination has been observed between C.
trachomatis serovars and other Chlamydiae spp.
• No current methods of recombination for C. caviae with C. trachomatis
• Can C. trachomatis L2 undergo recombination with C. caviae?
Can L2 and C. caviae recombine?
C. caviae ofloxacin/R parent L2 rifampicin/R parentGrown in Rif & Ofl
Doubly-resistant offspring are determined to be C. caviae or L2 by MOMP
Evaluating Candidates
• PCR for gyrA and rpoB• Purify • Restriction digest• Send for sequencing
Digest products
gyrA results10 11 26 27
Candidate # MOMP type Restriction type
10 L2 L2
11 caviae caviae
26 caviae caviae
27 L2 L2
Single point mutations conveying resistance
rpoB results
56 58 61 67
Candidate #
MOMP type
Restriction type
56 caviae caviae
58 caviae caviae
61 caviae caviae
67 L2 L2
Conclusions
• Despite documented ability of C. trachomatis to undergo recombination there is no evidence of recombination with C. caviae
• Recombinant candidates developed antibiotic resistance mutations independently
Acknowledgements
• Dr. Rockey• Bob Suchland• Erik Cram• Zoe Seigel • Emaan Khan• Stormy Scharzenberger