Antimicrobial Research Centre and School of Biochemistry and Microbiology, University of Leeds, Leeds LS2 9JT, UK
Received 13 February 2004; returned 4 April 2004; revised 8 April 2004; accepted 14 April 2004
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Abstract |
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Methods: We determined PAE values for a variety of antibiotics against Staphylococcus aureus and Escherichia coli following exposure to 5 x MIC drug concentrations for 60 min in MuellerHinton Broth (MHB). The duration of the PAE was obtained by following the recovery of bacterial growth in antibiotic-free MHB measured either as colony forming units on MuellerHinton agar, or as culture absorbance (600 nm) in a microplate reader.
Results: For bacteriolytic agents there was poor correlation between the two methods for both S. aureus (R2=0.096) and E. coli (R2=0.5456). However, when PAEs for bacteriostatic agents and non-lytic bactericidal agents were compared, correlation between the two methods was high for both S. aureus (R2=0.7529) and E. coli (R2=0.7687).
Conclusions: The spectrophotometric microplate method for determining PAEs may be a suitable alternative to the classical method for those antibiotics that do not induce bacterial cell lysis.
Keywords: antibiotic action , S. aureus , E. coli
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Introduction |
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PAE periods are usually determined by a method, first established by McDonald and colleagues2,9 which involves exposure of organisms to 5 x antibiotic MIC for 60 min, removal of drug by dilution, or re-suspension of organisms in fresh medium, followed by viable count determinations to monitor resumption of bacterial growth.9 The PAE is then calculated using the standard formula PAE = TC, where T is the time required for the treated cells to increase 10-fold (1 log10 cfu/mL) after washout of drug and C is the time required for a non-treated control to increase 10-fold (1 log10 cfu/mL) after washout with fresh medium.9 PAE determination involving viable counting of organisms is labour-intensive and lengthy, and several new methods based on spectrophotometric techniques have been developed to measure more easily the resumption of bacterial growth after antibiotic exposure.6,1013 Spectrophotometric methods for PAE determination are particularly attractive because these measurements, when made in microplate format, could offer rapid, automated, high-throughput procedures for determination of PAEs.
However, spectrophotometric methods for determining PAEs have not been carried out with an extensive set of drugs and detailed comparison with the classical, viable count procedure, has not been conducted. Consequently, we devised a simple method for determining PAEs using a microplate reader and established its performance with a wide range of antibiotics active against Staphylococcus aureus and Escherichia coli.
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Materials and methods |
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The S. aureus strain used throughout this work was 8325-4 (i.e. NCTC 8325 cured of phages 11, 12 and 13).14 E. coli ATCC 25922 was obtained from the American Type Culture Collection (ATCC). E. coli 1411 (lacI3, lacZ118, proB, trp, nalA, rspL) and its derivative E. coli SM1411 (acrAB) which contains a acrAB: Tn903 Kanr insert have been described in an earlier publication from this laboratory.15
Antibiotics and growth media
Ciprofloxacin, linezolid, mupirocin, meropenem, tiamulin and mecillinam were gifts, respectively from Bayer AG (Leverkusen, Germany), Pharmacia and Upjohn Inc. (Kalamazoo, MI, USA), GlaxoSmithKline Pharmaceuticals (Harlow, Essex, UK), AstraZeneca Pharmaceuticals (Macclesfield, UK), Biochemie (Vienna, Austria) and Leo Pharmaceuticals (Ballerup, Copenhagen). Other antibiotics were purchased from SigmaAldrich (Poole, Dorset, UK). MuellerHinton broth (MHB) and agar (MHA) were purchased from Fisher (Loughborough, UK).
Determination of susceptibility of S. aureus and E. coli strains to antimicrobial agents
MICs were determined by broth microdilution in MHB using an inoculum of 106 colony forming units per mL for S. aureus 8325-4 and 104 cells per mL for E. coli strains in a final volume of 70 µL. Microtitre plates (384 wells) containing triplicate two-fold dilution series were incubated for 16 h at 37°C in a Spectramax 384 plus microtitre plate reader (Molecular Devices, Abingdon, Oxfordshire, UK), running SOFTmax PRO 3.1.1 software. Optical density readings (600 nm) were taken at 10 min intervals. Plates were shaken for 30 s before each reading. The MIC was taken as the lowest concentration of antibiotic that prevented growth in the triplicate wells. Susceptibility determinations were carried out on three separate occasions and the mean of these values recorded as the MIC.
PAE determination by viable counting
Bacteria were grown to the early logarithmic phase (2 x 108 cells/mL) in MHB. Cultures were then divided into two aliquots, one of which received test antibiotic at 5 x MIC, the other serving as a drug-free control. The cultures were incubated for a further 60 min and the bacteria harvested by centrifugation (5000 g, 5 min) in a Sigma 3K18 laboratory centrifuge (Philip Harris Scientific, Ashby Park, Leicestershire, UK) that had been pre-warmed to 37°C. Bacteria were resuspended in fresh pre-warmed sterile MHB and washed three times by centrifugation as above. Finally, the washed cell pellets were resuspended in fresh pre-warmed sterile MHB using volumes equivalent to the original culture volumes. These new cultures were then incubated at 37°C.
Samples were removed for viable counting before washing, immediately after washing and at hourly intervals thereafter. Samples were serially diluted in ice-cold PBS and plated onto MHA. Colonies were counted after incubation at 37°C for 16 h. The viable count for each sample was determined from the average number of colonies on at least three plates containing between 30 and 300 colonies. The duration (D) of the PAE was calculated according to McDonald et al.9 using D=TC where T is the time (h) required for the treated cell density to increase by 10-fold (by 1 log10 cfu/mL) and C is the time required for the non-treated control cell density to increase 10-fold (by 1 log10 cfu/mL).
PAE determination by optical density
Cultures were grown in MHB and exposed to antimicrobial agents at 5 x MIC for 60 min as described above. Samples (1 mL) were then removed from cultures containing antibiotics and drug-free controls and bacteria harvested by centrifugation at 16 000 g for 2 min in a microfuge. Supernatants were removed using a suction pump and the cell pellets resuspended in fresh pre-warmed MHB (1 mL) before the cells were again harvested by centrifugation. The washing and centrifugation steps were repeated a further two times. After washing, the bacteria were resuspended in fresh pre-warmed MHB (1 mL) and 70 µL of each culture was added to the wells of 384-well microtitre plates. Plates were then incubated as described for MIC determination with automated reading of culture turbidity every 10 min. PAE duration was calculated according to Odenholt-Tornqvist,16 i.e. the time taken for antibiotic-treated cultures to reach 50% of the ODmax of the control culture, minus the time taken for the control culture to reach the same point.
Correlation between viable counting and optical density techniques as methods for PAE determination was calculated as R2 values by the Pearson correlation calculation using GraphPad Prism (GraphPad Software Inc., San Diego, CA, USA) software in accordance with the manufacturer's instructions.
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Results and discussion |
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For E. coli, we used strain ATCC 25922, which has been widely used for PAE determinations based on viable count methods,2 together with strains 1411 and SM1411 (acrAB). For S. aureus, we used strain 8325-4 which has been previously used in our laboratory to determine PAEs for candidate anti-staphylococcal agents.5,17
The MIC values for a variety of antibiotics against these strains are shown in Table 1. Some of these antibiotics have no clinical application for the treatment of infections caused by E. coli or S. aureus, but were nevertheless included in our studies to explore the validity of spectrophotometric microplate procedures for measuring PAEs. We have already used E. coli strains 1411 and SM1411 (acrAB) for other studies on the mechanism of the PAE.18 In addition, the use of a mutant which is insertionally inactivated for acrAB more readily permits PAE studies in E. coli with antibiotics such as erythromycin and novobiocin that normally have poor activity against E. coli due to AcrAB-mediated efflux. The benefit of using an acrAB mutant for PAE determinations with certain antibiotics in E. coli is illustrated by the MIC data presented in Table 1.
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PAE data are often conflicting due to the use of various methods for determination of PAE and different conditions of exposure to the test antibiotic. In view of the importance of PAE as a pharmacodynamic predictor, it would be advantageous to develop a standard, rapid, method for PAE determination. Spectrophotometric methods have recently been used to determine PAEs. However, there has been no systematic evaluation of these methods and little comparison with the classical, viable-count-based, procedure of McDonald et al.9 In this paper, we have shown that PAE values, comparable to those obtained by the classical procedure, can be obtained with S. aureus and E. coli for non-lytic antibiotics using a rapid spectrophotometric method which involves a plate reader. Nevertheless, although the rapid method described here could have a useful application in the determination of PAEs for bacteriostatic and non-lytic bactericidal agents against these organisms, it appears that it will still be necessary to use the classical, viable-count-based, procedure for studies with lytic agents such as the ß-lactam antibiotics. Further work will be needed to assess the usefulness of the microplate method for determining PAEs of non-lytic agents against other Gram-positive and Gram-negative organisms, including clinical isolates.
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Acknowledgements |
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Footnotes |
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References |
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