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