colistin and propidium-monoazide

Nucleic acid amplification-based methods are limited by their inability to discriminate between viable and dead cells. To overcome this drawback, propidium monoazide (PMA) combined with qPCR has been used to differentiate viable from nonviable cells in environmental samples. However, assessing bacterial physiology using PMA-qPCR remains a challenge due to its incapability of detecting metabolic activities, leading to overestimation of the viable bacteria population under an inactivation condition (e.g. antibiotic treatments). A recent advanced technique to amplify ribosomal RNA precursors (pre-rRNA) has been shown to detect viable cells because pre-rRNAs are intermediates in rRNA synthesis. This study investigated the effect of different types of antibiotics on the bacterial viability or viable but non-culturable (VBNC) state using both PMA-qPCR and pre-rRNA analyses with Pseudomonas aeruginosa. This study demonstrated that P. aeruginosa was more sensitive to colistin than it was to carbenicillin, gentamicin and levofloxacin. We could discriminate VBNCP. aeruginosa cells using PMA-qPCR when antibiotic pressure induced the VBNC state. Also, pre-rRNA was able to distinguish viable cells from colistin-inactivated bacteria cells, and it could detect the presence of VBNC and persister cells. Our results showed that these two molecular methods could successfully eliminate false-positive signals derived from antibiotics-inactivated cells.

Topics: Anti-Bacterial Agents; Azides; Colistin; Microbial Viability; Propidium; Pseudomonas aeruginosa; Real-Time Polymerase Chain Reaction

Acinetobacter baumannii represents a significant cause of nosocomial infections. Therefore, we combined real-time quantitative polymerase chain reaction (PCR) with the propidium monoazide (PMA-qPCR) to assess the feasibility of detecting viable, airborne A. baumannii. The biological collection efficiencies of three samplers for collecting airborne A. baumannii were evaluated by PMA-qPCR in a chamber study. After sampling, the effects of storage in collection fluid on A. baumannii were evaluated. The results showed that the culturable ratio of A. baumannii measured using the culture method was significantly correlated with the viable ratio measured using PMA-qPCR, but was not significantly correlated with the qPCR results. It was indicated that the AGI-30 impinger and the BioSampler were much more effective than the Nuclepore filter sampler for collecting airborne A. baumannii. The storage temperature was critical for aerosol samples, as the loss of viable A. baumannii was minimized when the PMA-bound DNA was stored at -20°C or if the collected cells were stored at 4°C and subsequently processed by PMA-qPCR within 1 month. The PMA-qPCR method was also to distinguish between colistin-sensitive and colistin-resistant A. baumannii, and no colistin-sensitive A. baumannii was detected by PMA-qPCR upon treatment of the BioSampler collection medium with 2 μg/ml colistin for 5 min.

Topics: Acinetobacter baumannii; Air Microbiology; Anti-Bacterial Agents; Azides; Colistin; Drug Resistance, Bacterial; Polymerase Chain Reaction; Propidium