Article: article from journal or magazin.
Detection of live and antibiotic-killed bacteria by quantitative real-time PCR of specific fragments of rRNA.
Antimicrobial Agents and Chemotherapy
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 > or =4 log(10) CFU/ml of viability over 48 h of penicillin treatment. In comparison, the Tol1 mutant lost < or =1 log(10) 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 (> or =4 log(10) CFU/ml and < or =1 log(10) 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.
Anti-Bacterial Agents/pharmacology, Colony Count, Microbial, DNA, Bacterial/genetics, DNA, Bacterial/isolation & purification, Genes, Bacterial, Genetic Markers, Kinetics, Microbial Sensitivity Tests, Mutation, Ofloxacin/pharmacology, Penicillins/pharmacology, Polymerase Chain Reaction, RNA, Bacterial/analysis, RNA, Bacterial/genetics, RNA, Ribosomal/analysis, RNA, Ribosomal/chemistry, RNA, Ribosomal, 16S/analysis, RNA, Ribosomal, 16S/chemistry, Streptococcus/drug effects, Streptococcus/genetics
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