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Distinction between intact and antibiotic-inactivated bacteria by real-time PCR after treatment with propidium
Hideo Kobayashi1, Margret Oethinger, Marion J Tuohy
1Department of Anatomic Pathology, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, Ohio, USA.
Abstract:
One limitation to the use of the polymerase chain reaction (PCR) to identify orthopedic infections has been apparent false-positive 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.
Insights
Propidium monoazide (PMA) effectively distinguishes viable from antibiotic-killed bacteria in orthopedic infection diagnosis. This method significantly reduces false-positive polymerase chain reaction (PCR) results, improving diagnostic accuracy for patients with prior antibiotic exposure.
Area of Science:
- Molecular Biology
- Infectious Diseases
- Orthopedic Surgery
Background:
- Polymerase chain reaction (PCR) for orthopedic infections is limited by false-positive results from dead bacteria.
- Propidium monoazide (PMA) can differentiate viable from heat-inactivated bacteria.
Purpose of the Study:
- To evaluate PMA's efficacy in distinguishing viable from antibiotic-inactivated bacteria (vancomycin, gentamicin) relevant to clinical settings.
- To assess PMA's impact on PCR detection of bacteria inactivated by antibiotics with different mechanisms of action.
Main Methods:
- Staphylococcus aureus and S. epidermidis were inactivated using vancomycin and gentamicin.
- Bacteria were treated with PMA or left untreated prior to DNA extraction.
- PCR targeting 16S rDNA and tuf genes was performed, analyzing threshold cycle (C(t)) differences.
Main Results:
- PMA significantly inhibited PCR detection of antibiotic-inactivated bacteria at 24 hours (C(t) difference >3, p < 0.05) and 10 days (C(t) difference >4, p < 0.01).
- Vancomycin showed a stronger effect on C(t) values compared to gentamicin, reflecting distinct antibiotic mechanisms.
- Viable cells showed minimal C(t) difference (1-2) with or without PMA.
Conclusions:
- PMA effectively distinguishes between viable and antibiotic-killed bacteria, reducing false-positive PCR results in orthopedic infection diagnostics.
- This technique is particularly valuable for patients who have received antibiotics, such as those undergoing revision arthroplasty.
