Didier Raoult, Unité des Rickettsies, Faculté de Médecine, 27, Boulevard Jean Moulin, 13385 Marseille Cedex 5, France
Received 3 February 2004; returned 8 April 2004; revised 13 May 2004; accepted 14 May 2004
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Abstract |
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Methods: Antibiotic susceptibilities for 24 bacterial strains of various species were tested by real-time quantitative PCR assay and by conventional methods. Quantification of DNA copies of either the 16S RNA genes or rpoB were recorded over time in the presence or absence of antibiotics to determine the bacterial growth kinetics and the optimal testing time.
Results: Molecular results for antibiotic susceptibility or resistance were in accordance with those obtained using a standard macrodilution broth assay. The method was reproducible, sensitive and rapid (2 h for Gram-negative bacilli and 4 h for Gram-positive cocci). Moreover, this assay was also able to determine the antibiotic susceptibilities of fastidious bacteria, such as mycobacteria, within 5 days.
Conclusions: These results demonstrate that molecular detection of bacteria could be more rapid than phenotypic methods for antibiotic susceptibility testing.
Keywords: quantitative PCR , antibiotic resistance , MICs
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Introduction |
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The mathematical descriptions of PCR and bacterial growth are very similar, with an initial exponential rate of growth. Growth kinetics of bacteria may be determined more accurately by enumeration of DNA copies over time. PCR is faster and more specific than bacterial culture; using short cycle times, and assuming a good PCR efficiency, DNA doubles 40 times faster than bacteria.3
Recent advances in molecular biology have led to the development of genotypic assays suitable for antibiotic susceptibility testing.4,5
Here, we describe a universal method for measuring the inhibitory effects of antimicrobial agents on common bacterial pathogens using universal primers and quantification of DNA copies using a LightCycler.
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Methods |
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LightCycler PCR assay
Total genomic DNA was extracted from aliquots using a MagnaPure LC instrument (Roche Molecular Biochemicals, Mannheim, Germany) as described by the manufacturer. Genomic DNAs were stored at 4°C until their use as templates in PCR assays. PCR was performed with a LightCycler (Roche Biochemicals, Mannheim, Germany) using primers for 16S rDNA or rpoB. Those for 16S rDNA were: for Pseudomonas aeruginosa, 5'-TCAGTCACACTGGAACTGAG-3' and 5'-GTAATTCCGAGGAACGCTTG-3'; for staphylococci, 5'-CGGTACCTAATCAGAAAG-3' and 5'-TTTCCAGTTTCCAATGAC-3'; for streptococci, 5'-CTCTAGAGATAGAGTTTTAC-3' and 5'-CGACTCGTTGTACCAACCA-3'; and for mycobacteria, 5'-GAATTACTGGGCGTAAAGAG-3' and 5'-GCCGTAGCTAACGCATTAAG-3'. Primers for rpoB were: for Enterobacteriaceae, 5'-GCCAGCTGTCTCAGTTTATG-3' and 5'-ACATACGCGACCGTAGTG-3'; and for Haemophilus influenzae, 5'-ACAAGTGGTTGTGCCTTCTG-3' and 5'-TGTCATAAGTTGGATCGACAC-3'.
The PCR mixture had a final volume of 20 µL containing 2 µL of DNA master SYBR Green (DNA Master SYBR Green I Kit; Roche Diagnostics), 2.4 µL of 3 mM MgCl2, 1 µL (10 pmol) of each primer (primers were selected according to the tested bacteria), 11.6 µL of distilled water, and 2 µL of extracted DNA. Each PCR included sterile distilled water as a negative control. The amplification conditions were: an initial denaturation step at 95°C for 2 min, followed by 30 cycles of denaturation at 95°C for 15 s, annealing at 54°C for 20 s and extension at 68°C for 1 min, with fluorescence acquisition in single mode. The number of DNA copies obtained after incubation of bacteria with or without antibiotic was determined using standard curves for each bacterial species, and plotted against time to obtain the growth kinetics of the bacteria. Antibacterial activity was defined as the absence of growth with antibiotic as compared with the growth control. Conversely, resistance to an antibiotic was defined as an increase in the number of DNA copies during the time of incubation.
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Results |
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Melting curves obtained with standard concentrations of the tested bacteria were always reproducible and specific for the bacteria studied. Indeed, a specific peak fusion temperature was obtained for each bacteria species and was found to be at the same temperature in each experiment (Table 1). DNA sequencing of PCR products confirmed the identification of bacteria (data not shown).
Initially, we determined the kinetics of growth for all the bacteria tested in the absence of antibiotics. Exponential phase growth ranged from t=2 h to t=8 h for Gram-positive bacteria (Figure 1a), and from t=1 h to t=4 h for Gram-negative bacteria (Figure 1b). During exponential phase, the number of DNA copies increased by 3 log10 as compared with the beginning of the experiment with a standard 0.5 McFarland inoculum. For mycobacteria the exponential phase was during days 37.
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In the second part of the study, we determined the number of DNA copies obtained when bacteria were grown in the presence of breakpoint-equivalent concentrations of antibiotics. This number remained similar to the number of DNA copies at the beginning of the experiment if the tested strain was susceptible to the antibiotic tested. Conversely, if the strain was resistant to the antibiotic tested, the number of DNA copies increased similarly to the growth control without antibiotic. We determined the optimal time for the evaluation of antibiotic activity against each species tested. The incubation time necessary to provide results of antibiotic susceptibility was 4 h for Gram-positive cocci (Figure 1a and Table 1) and H. influenzae and 2 h for Gram-negative bacilli (Figure 1b and Table 1). For mycobacteria, the real-time PCR method gave susceptibility results in only 5 days, as compared with 1015 days for the conventional assay.
For all 24 strains tested, the susceptibility results obtained with the LightCycler assay were in accordance with results obtained using conventional methods.
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Discussion |
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Rapid return of susceptibility results is also the case for automated systems, with MICs for Enterobacteriaceae being obtained within 7 h9 and MICs for Gram-positive bacteria in 617 h.10 For mycobacteria, the result of antibiotic susceptibility testing was obtained in 5 days, which is considerably faster than conventional assays (1015 days). In this report, we have not tested the ability of our method to reliably detect bacteria with inducible resistance mechanisms, although we believe that molecular biological methods combined with growth curves may help in these situations.
At the present time, the method we have described is not entirely automated; it takes about 2 h to perform the assay, with a previous incubation step of 24 h for bacteria in the presence of antibiotic. However, automization of molecular biological methods in the future could lead to the development of multiple real-time PCR for the determination of susceptibility to many antibiotics. Although there were large differences between the MICs for the susceptible and resistant strains tested in this study, our preliminary results demonstrate that molecular detection of bacteria could be a more rapid method for determining antibiotic susceptibility. Presently, the major drawback of this method, as compared with conventional assays, is cost, but this differential is likely to decrease in the future as the cost of reagents falls (for example, Taq polymerase will be free of patent restrictions in the future) and as greater emphasis is placed on automation, miniaturization and computerization in the clinical microbiology laboratory.
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Acknowledgements |
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Footnotes |
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References |
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