Optimization of Propidium Monoazide Quantitative PCR for Evaluating Performances of Bioaerosol Samplers for Sampling AirborneStaphylococcus aureus

Abstract

Airborne Staphylococcus aureus causes a significant proportion of nosocomial infections. The purpose of this study was to combine real-time quantitative polymerase chain reaction with the DNA-binding agent propidium monoazide (PMA-qPCR) to assess exposures to airborne S. aureus. In this work, we generated a S. aureus aerosol and used our assay to detect viable, airborne S. aureus in a study chamber. The biological collection efficiencies of three samplers (the AGI-30 impinger, BioSampler, and Nuclepore filter sampler) were evaluated using the S. aureus aerosols. The effects of storage in collection fluid on S. aureus sampled by the AGI-30 impinger and BioSampler were evaluated. Furthermore, air samples from an intensive care unit (ICU) and a gymnasium (GYM) were subsequently used to test the performance of a BioSampler combined with our PMA-qPCR technique. The BioSampler was more effective than the AGI-30 and Nuclepore filter samplers for preserving the culturability and viability of S. aureus aerosol samples. After sampling by impingement, the loss of viable S. aureus was minimized by treating the cells with PMA prior to storage at ¡20 C and analyzing the samples by qPCR within 3 weeks. In field applications, we noted that traditional culture assays tended to underestimate the viable concentrations of S. aureus by approximately one order of magnitude. Overall, combining qPCR with and without PMA staining may be useful for assessing exposure to airborne S. aureus. However, a complex set of parameters that may affect the efficiency of PMA-qPCR must be taken into account before applying PMA-qPCR to bioaerosol detection.

Altered susceptibility to air sampling stress by filtration is related to colistin resistance development inAcinetobacter baumannii

“…In our chamber study, A. baumannii aerosols collected by the Nuclepore filter were subjected to a combination of three stresses for filter sampling: aerosolization, impaction, and desiccation . The CR values of A. baumannii in the present study were very close to those reported for S. aureus . In addition, CRAB clearly demonstrated greater susceptibility than CSAB to the combination of these three stresses.…”

“…34 The CR values of A. baumannii in the present study were very close to those reported for S. aureus. 24 In addition, CRAB clearly demonstrated greater susceptibility than CSAB to the combination of these three stresses. The evolution of cells to become CRAB, in contrast to CSAB, resulted in the complete loss of LPS production.…”

“…In our current study, the filtration stress on bacteria was evaluated because filtration can cause mechanical and nonmechanical stresses such as desiccation and elution . These sampling stresses resulted from filtration causing greater stress to airborne bacteria than other stresses, including impaction and impingement . If the susceptibilities of antibiotic‐resistant bacteria to this sampling stress are different from those of antibiotic‐sensitive bacteria, the validity of the air sampling strategy for quantifying these harmful bacteria may need to be verified.…”

Aerobiological Stabilities of Different Species of Gram-Negative Bacteria, Including Well-Known Biothreat Simulants, in Single-Cell Particles and Cell Clusters of Different Compositions

“…It also could be interesting to study several strains of the same bacterial species to specifically address strain-specific aerobiological stability characteristics. The ambition for the future also involves adopting additional characterization methods, including flow cytometry in combination with membrane integrity and metabolic cell staining (20,45,46) and quantitative PCR in combination with ethidium or propidium monoazide live-dead discrimination techniques (47)(48)(49)(50), as well as the study of multiple strains of the same bacterial species to investigate strain-specific aerobiological stability characteristics.…”

Selection and identification of protective culture for controlling Staphylococcus aureus in fresh Domiati like cheese

“…The pellet of bacteria was washed twice with 1 ml TES buffer (25 mM TrisHCl, 0.1 M EDTA, 25% [wt/vol] saccharose [pH 8]), centrifuged at (5000 × g) for 10 min at 4°C, and then the supernatant was removed. Cell suspensions for propidium mono azide (PMA) staining, the tubes were stained with 10 mg/ml PMA for 5 min in dark place and then transferred to ice and exposed to a 500 W halogen light for 7 min Tseng et al (). Total DNA was extracted by using the Wizard DNA Purification Kit (Promega, Madison, WI).…”