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Antimicrobial Agents and Chemotherapy, June 2006, p. 1913-1920, Vol. 50, No. 6
0066-4804/06/$08.00+0     doi:10.1128/AAC.00869-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Detection of Live and Antibiotic-Killed Bacteria by Quantitative Real-Time PCR of Specific Fragments of rRNA

Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland,¹ Department of Fundamental Microbiology, University of Lausanne, Lausanne, Switzerland²

Received 11 July 2005/ Returned for modification 11 October 2005/ Accepted 10 March 2006

Assessing bacterial viability by molecular markers might help accelerate the measurement of antibiotic-induced killing. This study investigated whether rRNA could be suitable for this purpose. Cultures of penicillin-susceptible and penicillin-tolerant (Tol1 mutant) Streptococcus gordonii were exposed to mechanistically different penicillin and levofloxacin. Bacterial survival was assessed by viable counts and compared to quantitative real-time PCR amplification of either the 16S rRNA genes or the 16S rRNA, following reverse transcription. Penicillin-susceptible S. gordonii lost 4 log₁₀ CFU/ml of viability over 48 h of penicillin treatment. In comparison, the Tol1 mutant lost 1 log₁₀ CFU/ml. Amplification of a 427-bp fragment of 16S rRNA genes yielded amplicons that increased proportionally to viable counts during bacterial growth but did not decrease during drug-induced killing. In contrast, the same 427-bp fragment amplified from 16S rRNA paralleled both bacterial growth and drug-induced killing. It also differentiated between penicillin-induced killing of the parent and the Tol1 mutant (4 log₁₀ CFU/ml and 1 log₁₀ CFU/ml, respectively) and detected killing by mechanistically unrelated levofloxacin. Since large fragments of polynucleotides might be degraded faster than smaller fragments, the experiments were repeated by amplifying a 119-bp region internal to the original 427-bp fragment. The amount of 119-bp amplicons increased proportionally to viability during growth but remained stable during drug treatment. Thus, 16S rRNA was a marker of antibiotic-induced killing, but the size of the amplified fragment was critical for differentiation between live and dead bacteria.