Improving clinical significance of PCR: Use of propidium monoazide to distinguish viable from dead <i>staphylococcus aureus</i> and <i>staphylococcus epidermidis</i>

Kobayashi, Hideo; Oethinger, Margret; Tuohy, Marion J.; Hall, Gerri S.; Bauer, Thomas W.

doi:10.1002/jor.20872

Cited by 71 publications

(66 citation statements)

References 20 publications

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“…To our knowledge, there have been two previous studies that investigated the effectiveness of PMA for quantifying S. aureus (Nocker et al 2006;Kobayashi et al 2009b). However, no optimal PMA treatment test for S. aureus has been developed.…”

Section: Resultsmentioning

confidence: 99%

“…PMA purchased from Biotium Inc. (Biotium, Hayward, CA, USA) was added at a final concentration of 50, 25, 10, or 2.5 mg/ml to sample tubes containing 300 ml of either viable (100% controlled viable sample) or heat-killed (0% controlled viable sample) cells (10 5 CFU/ml), as described in previous studies (Kobayashi et al 2009b(Kobayashi et al , 2010. For PMA staining, the tubes were placed in the dark for 5 min and then transferred to ice and exposed to a 500 W halogen light at a distance of 20 cm.…”

Section: Optimization Of the Pma Treatmentmentioning

confidence: 99%

“…C t levels are inversely proportional to the amount of the target gene in the sample. Therefore, the difference between the C t values obtained for untreated and PMAtreated MSSA by qPCR was calculated for each pair of samples, according to the method described in a previous study, as follows: DC t D C t with PMA ¡ C t without PMA (Kobayashi et al 2009b). A low DC t indicates the presence of amplifiable DNA from viable bacteria, whereas a high DC t indicates that the target DNA in the sample originated from bacteria with membrane damage (Miotto et al 2012).…”

Section: Optimization Of the Pma Treatmentmentioning

confidence: 99%

“…In contrast, PMA can only penetrate dead cells and has therefore been recommended as a highly selective dye to distinguish S. aureus that have been inactivated by heat or antibiotics from living bacteria (Kobayashi et al 2009b(Kobayashi et al , 2010. These results indicate that while EMA and PMA present similar mechanisms for discriminating between viable and dead bacteria, the penetration of these two dyes into bacteria could be species-dependent.…”

Section: Introductionmentioning

confidence: 96%

“…These results indicate that while EMA and PMA present similar mechanisms for discriminating between viable and dead bacteria, the penetration of these two dyes into bacteria could be species-dependent. Compared with EMA, PMA is a better reagent for rapidly detecting viable airborne S. aureus or MRSA (Kobayashi et al 2009b). …”

Section: Introductionmentioning

confidence: 99%

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

Tseng

Hsiao

Chang

et al. 2014

Aerosol Science and Technology

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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.

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Section: Resultsmentioning

confidence: 99%

Section: Optimization Of the Pma Treatmentmentioning

confidence: 99%

Section: Optimization Of the Pma Treatmentmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 96%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

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

Tseng

Hsiao

Chang

et al. 2014

Aerosol Science and Technology

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Distinction between intact and antibiotic‐inactivated bacteria by real‐time PCR after treatment with propidium monoazide

Kobayashi

Oethinger

Tuohy

et al. 2010

Journal Orthopaedic Research

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One limitation to the use of the polymerase chain reaction (PCR) to identify orthopedic infections has been apparent falsepositive results, possibly due to the detection of dead bacteria. We recently showed that the use of DNA-binding agent propidium monoazide (PMA) could distinguish viable from heat-inactivated bacteria, and, in this study, we investigated whether the same technique can be applied to bacteria killed by two antibiotics with distinctly different mechanisms of action, a test of greater clinical relevance than thermal inactivation. Staphylococcus aureus and S. epidermidis were inactivated by vancomycin and gentamicin and treated with PMA or left untreated before DNA extraction. The threshold cycle difference of antibiotic-treated bacteria with and without PMA pretreatment was investigated with PCR primers for the 16S rDNA and tuf genes. Our results indicated that PMA effectively inhibited detection by PCR of bacteria, which had been inactivated by either vancomycin or gentamicin. The effect was statistically significant at 24 h after treatment (C t difference consistently >3; p < 0.05) and after 10 days of treatment (C t difference >4; p < 0.01), when compared to viable cells (C t difference 1-2). Vancomycin had a stronger effect on the C t value than gentamicin, reflecting the different mechanism of action of each antibiotic. Techniques of this type may help reduce clinically false-positive PCR results caused by the detection of dead bacteria, and may be especially useful in patients who have received antibiotics, such as patients undergoing the second stage of a two-stage revision for infected arthroplasty. ß

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Diagnosis of Periprosthetic Joint Infection

Zmistowski¹,

Valle²,

Bauer³

et al. 2014

Journal Orthopaedic Research

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Improving clinical significance of PCR: Use of propidium monoazide to distinguish viable from dead staphylococcus aureus and staphylococcus epidermidis

Cited by 71 publications

References 20 publications

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

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

Distinction between intact and antibiotic‐inactivated bacteria by real‐time PCR after treatment with propidium monoazide

Diagnosis of Periprosthetic Joint Infection

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