Assessment of Bioaerosol Sampling Techniques for Viable Acinetonacter baumannii by Propidium Monoazide Quantitative PCR

摘要

Background Acinetobacter baumannii is a Gram-negative coccobacillus, which is frequently associated with nosocomial infections worldwide. Since the introduction of antibiotics, A. baumannii has increased its resistance to broad-spectrum antibiotics and has thus become problematic in intensive care units (ICUs). Although A. baumannii is known to be transmitted by close contact with infected persons, some evidence suggests that airborne dispersal and transmission may also be important. Aims The purpose of this study is to establish the method by using PMA combined with real-time PCR (PMA-qPCR) for rapidly getting the concentration of airborne A. baumannii. Methods The Propidium Monoazide (PMA) is a nucleic acid dye which would be combined with Real-time PCR to determine the viability of bacteria by identifying the integrity of cell membrane. Results We established the method by using PMA-qPCR to detect A. baumannii in the air of our chamber. The PMA concentration used in this study is 10 μg/ml combined with a 500W light irradiation for 3 minutes. Based on the viability test, a linear relationship was observed between culture based method and PMA-qPCR (R=0.99). Consequently, PMA-qPCR has the potential to identify the viability of A. baumannii. The collection efficiency of bioaerosol sampler was evaluated by two parameters in this study; Culturability recovery (CR) and Viability recovery (VR). CR and VR were determined by the culture and PMA-qPCR method, respectively. Our results demonstrated Biosampler and AGI-30 have a batter performance in sampling airborne A. baumannii than the Nuclepore filter, no matter CR or VR was evaluated. Moreover, the stability of PMA binding samples were also evaluated when the samples were stored at refrigerated temperature. Once PMA is binding with A. baumannii DNA, the stability of the DNA bindings was observed to be stable up to 28 days. Conclusions Our study demonstrated PMA-qPCR may be a meaningful method for airborne A. baumannii aerosol detection.