title: Detection of live and antibiotic-killed bacteria by quantitative real-time PCR of specific fragments of rRNA.
abstract: 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.
authors.affiliation: Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland.
authors.forename: Steve
authors.initials: S
authors.lastname: Aellen
authors.forename: Yok-Ai
authors.initials: YA
authors.lastname: Que
authors.forename: Bertrand
authors.initials: B
authors.lastname: Guignard
authors.forename: Marisa
authors.initials: M
authors.lastname: Haenni
authors.forename: Philippe
authors.initials: P
authors.lastname: Moreillon
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D015169:Colony Count, Microbial; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D005798:Genes, Bacterial; D005819:Genetic Markers; D007700:Kinetics; D064704:Levofloxacin; D008826:Microbial Sensitivity Tests; D009154:Mutation; D015242:Ofloxacin / Q000494:pharmacology; D010406:Penicillins / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D012329:RNA, Bacterial / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D012335:RNA, Ribosomal / Q000032:analysis* / Q000737:chemistry; D012336:RNA, Ribosomal, 16S / Q000032:analysis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013291:Streptococcus / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development
chemical_list: D000900:Anti-Bacterial Agents; D004269:DNA, Bacterial; D005819:Genetic Markers; D010406:Penicillins; D012329:RNA, Bacterial; D012335:RNA, Ribosomal; D012336:RNA, Ribosomal, 16S; D064704:Levofloxacin; D015242:Ofloxacin