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 antibioticinduced 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 log 10 CFU/ml of viability over 48 h of penicillin treatment. In comparison, the Tol1 mutant lost <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 (>4 log 10 CFU/ml and <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.

Direct sampling of cystic fibrosis lungs indicates that DNA-based analyses of upper-airway specimens can misrepresent lung microbiota

“…This scenario seems unlikely. The fact that DNA-based methods readily detect bacteria killed by antibiotics and other mechanisms (29,30) also makes it unlikely that abundant species were missed because of antibiotic killing.…”

Detection of Periprosthetic Infections With Use of Ribosomal RNA-Based Polymerase Chain Reaction

“…rRNA is also a more robust RNA species and will survive the extraction process at a considerably higher concentration than will mRNA 19 . Interestingly, the biodegradation kinetic profile of rRNAs has been shown to be similar to that of mRNAs, suggesting that rRNAs may serve well as a cell viability marker 20 . Since rRNAs are highly conserved among bacterial species, rRNA-based detection targeting the conserved sequences in the rRNA should provide universal coverage for all orthopaedically relevant bacterial species.…”

Estimation of Viable Salmonella Cell Numbers in Meat and Meat Product Using Real-Time PCR

“…The method gave results similar to the conventional colony counting method, but was not fully accurate. For some samples, it estimated viable cells over 10 times higher than of the conventional method 22) . Our method is thought to be reliable for quantitative detection of the target cell in a sample, but it needs an incubation procedure and more PCR reagents than a conventional real-time PCR procedure.…”

“…And the detection limit of it is as low as 10 1 CFU/mL 7) . On the other hand, Aellen et al 22) reported a method for viable target cell number by quantitative real-time PCR of specific fragments of rRNA. The method gave results similar to the conventional colony counting method, but was not fully accurate.…”

“…Some investigators have studied quantitative methods for viable bacterial cells with real-time PCR 21), 22) . Rudi et al 21) used a stain, ethidium monoazide, which covalently binds to DNA from viable target cells only and such bound DNA cannot be amplified by PCR.…”