a Microbiology Service, General Specialty Hospital Virgen de las Nieves, Granada; b Department of Microbiology, University Hospital Virgen Macarena, Seville, Spain
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Abstract |
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Introduction |
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The laborious nature of this technique is its biggest drawback. For this reason, other techniques have been developed and applied to the study of PAE, such as bioluminescence,610 electrical impedance,11,12 bacterial morphology,13 infrared spectroscopy,14 radiometry,15 electrical counting,16,17 fluorometry18 and spectrophotometry. The spectrophotometric method involves either determining the time difference for a 5% decrease in transmittance, between the antibiotic-exposed and unexposed bacterial cultures,1921 or measuring the difference in time to reach a chosen point on the absorbance curve between the treated and control cultures.22,23
The objectives of this study were to develop a spectrophotometric method for determining PAE, and to validate it by comparison with the traditional method of viable counts on agar. With this spectrophotometric method, the PAE was calculated by measuring the separation of the spectrophotometric logarithmic growth curves of the control and treated cultures; the time of exposure of the cultures to the antimicrobial agent and the regrowth time, or the theoretical time that it takes for the exposed culture to recover from the bactericidal effect of the antibiotic and reach the initial inoculum, was subtracted from the separation time.
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Materials and methods |
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The strains used in this study were Escherichia coli ATCC 25922; Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 29213. The strains were cultured in MuellerHinton broth (Difco Laboratories, Detroit, MI, USA) to monitor the growth kinetics by spectrophotometry. MuellerHinton agar (Oxoid, Basingstoke, UK) was used for determination of viable counts.
Antimicrobial agents
The antimicrobial agents used were gentamicin (Instituto Llorente, Madrid, Spain) and ciprofloxacin (Bayer AG, Leverkusen, Germany) at 1 x, 2 x and 4 x MIC. The MIC was determined for each isolate using the macrodilution method.24
PAE determination
Two to three colonies of a 20 h growth on MuellerHinton agar of the organism to be studied were suspended in 50 mL prewarmed (37°C) MuellerHinton broth. The suspension was incubated overnight at 37°C, diluted 1/2500 in the same prewarmed medium and incubated in a waterbath at 37°C with agitation (50 rpm). The absorbance of the culture was monitored with a spectrophotometer (Spectronic 20, Milton Roy Company, PACISA, Madrid, Spain) using a wavelength of 450 nm and 19 mm diameter spectrophotometer tubes (Perkin-Elmer Hispania S.A., Granada, Spain), until an absorbance of 0.1 was reached (equivalent to 2.53.0 x 107 cfu/mL for E. coli and P. aeruginosa and to 1.82.0 x 107 cfu/mL for S. aureus). Equal volumes of the control culture and the cultures to be treated with the antibiotic were separated and a viability count was taken to determine the exposed bacterial inoculum (Ninic). The bacteriaantibiotic contact lasted 1 h (texpo), at the end of which drug activity was stopped by placing a 103 dilution of the bacterial suspension in drug-free prewarmed Mueller Hinton broth. In preliminary experiments this dilution was shown to be sufficient as the residual drug activity resulted in no significant deviation of the growth curve. Colony counts were taken at time zero (Ninic), after removal of the antibiotic (Nanti) and then at hourly intervals for 6 h for PAE determination by viable count. The control culture was also subjected to the 1/1000 dilution and growth rate was determined under identical conditions without antibiotic exposure. All the cultures were incubated at 37°C with agitation and the absorbance was measured every 30 min.
Calculation of the PAE
Once Ninic and Nanti were determined and the growth of the control and exposed cultures were spectrophotometrically monitored, we proceeded with the following steps. (i) Plotting, on semi-logarithmic paper, of the spectrophotometric growth curves of the control and post-exposure cultures, representing optical density (OD) along the y-axis and time along the x-axis (Figure 1). Customarily the first meaningful reading of OD can be taken for the control culture at 120 or 150 min after the initial time (tinic = 0). (ii) Determination of the generation time (tg) or duplication time. The tg is calculated by the spectrophotometric monitoring of the control culture and of a 1/8 dilution of the same culture. This dilution is separated in time by three generations (21, 22, 23); therefore, by dividing the separation time between both growth curves by 3, the generation time is obtained. (iii) The calculation of bactericidal effect (r):
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(iv) Graphical determination of the time separation of the spectrophotometric growth curves of the control culture and the post-exposure culture (tsep). (v) Calculation of the PAE according to the general formula:
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where tsep is the separation time of the spectrophotometric growth curves of the control culture and the post-exposure culture; texpo is the exposure time equivalent to 1 h duration and trecrt is the theoretical time that the treated culture takes for its viability count (Nanti) to match the initial count (Ninic); this depends on the succeeding generations (n) and the generation time (tg) of the bacteria.
Under constant conditions, after a long enough time, when cellcell interaction is small, growth measured by any method is expected to proceed according to:
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Where N0 and Nt are the counts at times zero and t, respectively. has been used for the specific growth rate.
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or
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thus
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At the same time, the growth kinetics were monitored by viable counts in agar, determining PAE with the standard formula of Craig & Gudmundsson,1 as the difference in time, between test (T) and control (C) cultures, for organisms to increase in number by a factor of 10:
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Colony counts were determined with MuellerHinton agar pour plates in the initial inoculum, after dilution to remove the antibiotic, and every hour for 6 h.
Statistical analysis
To compare both methods, Student's t-test at the 0.05 level of significance was used for matching data.
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Results |
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The MICs of gentamicin for E. coli ATCC 25922; P. aeruginosa ATCC 27853 and S. aureus ATCC 29213 were 0.5, 0.5 and 0.25 mg/L, respectively. The MIC of ciprofloxacin was 0.007 mg/L for E. coli ATCC 25922 and 0.25 mg/L for P. aeruginosa ATCC 27853 and S. aureus ATCC 29213.
Timekill and PAE experiments
Gentamicin demonstrated a marked bactericidal activity against the three standard strains when cultures in the logarithmic phase of growth with an inoculum of 35 x 107 cfu/mL were exposed to 4 x MIC. In the case of S. aureus a fall in the initial inoculum of 2.91 ± 0.12 log10 cfu/mL was detected. The PAE was not calculated in cases where a large decrease in viability was observed. No reductions in the inoculum exposed to 1 x and 2 x MIC were observed when this antibiotic was used against E. coli, evidence of bacteriostatic activity being observed at both concentrations.
The PAE induced by gentamicin on the three standard strains was concentration dependent, with a duration of between 0.52 ± 0.12 h against E. coli and 1 x MIC of gentamicin and 1.77 ± 0.14 h with S. aureus and 2 x MIC of gentamicin (Table).
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The duration of the PAE of ciprofloxacin was also dose dependent, being 0.14 ± 0.09 h and 0.41 ± 0.10 h against E. coli ; 0.88 ± 0.08 h and 1.27 ± 0.15 h against P. aeruginosa; and 0.55 ± 0.10 h and 0.65 ± 0.05 h against S. aureus, at 1 x and 2 x MIC, respectively (Table).
No statistically significant differences (P > 0.05) were detected in the duration of PAE between the spectrophotometric method developed by us and the traditional viability count in any of the combinations assayed. The results obtained with the latter method appear in the Table.
Figures 2, 3 and 4 show the growth curves monitored by spectrophotometry and by viability counting, for the control cultures and for those exposed to the antibiotic, once they had all been subjected to the same procedure of elimination of the antibiotic. As can be seen, the exponential growth phases of each culture run in parallel and the greater the concentration of the antimicrobial agent to which the bacterial culture has been exposed, the longer the time separation from the growth curve of the control culture.
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Discussion |
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A variety of techniques have been applied to the study of PAE in vitro, as well as models for its determination in vivo.
The spectrophotometric technique that we have developed enables results to be available in 24 h, for various antimicrobialmicroorganism combinations, easily and conveniently and with results that can be replicated. For every antimicrobial agentmicroorganism association and for every antimicrobial concentration tested, the results obtained are not significantly different from those obtained with the viable count technique.
Other authors have measured PAE using spectrophotometry and have compared it with the standard method. Bergan et al.26 observed discrepancies between turbidity and colony count with E. coligentamicin and E. coli ß-lactam antibiotics in contrast to the chloramphenicol and oxytetracycline combinations. However, with flucloxacillin and S. aureus, the cfu/mL and OD curves are essentially parallel.26 Rescott et al.21 demonstrated a considerable variation between the reference method and spectrophotometry using the Abbott MS-2 system to quantify the increase in OD of the cultures with tobramycinE. coli. With the MS-2 method, there is a threshold for growth detection at approximately 106107 cfu/mL. When the initial colony count was significantly below this threshold concentration, there was an apparent growth lag measured by MS-2. During this apparent growth lag, log-phase growth may be present. It is particularly important to stress that with the utilization of this spectrophotometric method, the observed lag in growth is dependent on the initial number of cfu/mL as well as on the growth rate of the organism during the monitoring period. With potent bactericidal drugs such as tobramycin, there was considerable variability in both the reference and MS-2 methods.21 These same authors found no differences between either method in the combinations of E. coli and ampicillin or ciprofloxacin. PAE durations for both the reference and MS-2 methods for E. coli ATCC 25922 exposed at 2 x MIC for 1 h were 0.0 and 26.0 min for ampicillin, 56.0 and 70.3 min for ciprofloxacin, 98.0 and 111.4 min for tobramycin.21 Li et al.19 established an excellent concordance between the reference method and the turbidimetric assay only with antibiotics that have a minimal or no PAE on the bacteria assayed. This assertion contrasts with our results and those indicated by Meng et al.27 and Rescott et al.,21 using the spectrophotometric technique.
Meng et al.27 developed a spectrophotometric method for determining PAE that was unaffected by the detection threshold of the OD of the culture. The PAE was quantified on the basis of the mean recovery time of the bacterial population exposed to the antimicrobial agent. To validate this technique, they studied the PAE of ciprofloxacin on E. coli ATCC 25922, comparing their method with the traditional one of viable count. They found no statistically significant difference between them. PAEs induced by a 1.5 h exposure of E. coli ATCC 25922 to ciprofloxacin at 2 x and 3 x MIC were 1.6 and 2.1 h, respectively.28
Odenholt-Tornqvist23 determined the PAE of meropenem for different strains of E. coli and P. aeruginosa with viable counts and then measured by continuous monitoring of OD in a BioScreen C apparatus. Again, no major difference was seen between the two methods. However, BioScreen C was found to present certain problems with the setting-up temperature and OD stability, and needs at least 1 h to reach the set-point temperature: the time lag is affected, but not the growth rate. Also there were perturbations of the measurements recorded at the beginning of the experiment.29
Consequently, there has been no basis for a uniform comparison of PAEs obtained from growth curves measured by colony counting and by OD. The limitation of sensitivity and changes in bacterial morphology are two major problems involved in the quantification of PAE using OD measurements for determination of bacteria growth.
The Spectronic 20 has an effective OD scale much too short for high precision assays. It follows that if the shorter wavelength (450 nm) is used, scattering will be greater and the absorbance measured more accurate.
We have established that the OD of a culture is directly proportional to the viable counts in agar at an interval that varies, depending on the organism being studied, between 4 x 106 and 3 x 108 cfu/mL with E. coli ATCC 25922; between 3 x 106 and 108 cfu/mL with P. aeruginosa ATCC 27853; and between 4 x 106 and 7 x 107 cfu/mL with S. aureus ATCC 29213. In our case, the sensitivity threshold of the spectrophotometer did not interfere with the calculation of PAE. This is because our method is based first on the linearity of the spectrophotometry ratio and viable counts in the range mentioned earlier, and secondly on the parallelism of the phases of logarithmic growth, measured by spectrophotometry, of the control cultures and of those exposed to the antibiotic. The change in size of the bacteria (e.g. with ciprofloxacinE. coli) is too small to influence the spectrophotometric growth curves. The photometric method measures cell mass, not cell number. Neither was the bactericidal activity affected by filamentation generated in E. coli after ciprofloxacin exposure because, in contrast to results with the ß-lactams, this morphological alteration did not appear until after drug removal.30
The duration of the PAE was concentration dependent for all the combinations studied. This would be related to the degree of bactericidal activity at the different concentrations. Therefore, increasing concentrations were associated with a larger tsep between the spectrophotometric growth curves of the control and the post-exposure cultures, due to a longer trecrt time being required for cellular recovery from drug-induced damage.
In conclusion, the spectrophotometric method that we have developed for measuring PAE is a less laborious and speedier alternative than the traditional one of viable counts. Only two counts are required, one initially and a second after the elimination of the antibiotic. An excellent correlation was demonstrated between the PAEs quantified by this system and by viable counts for gentamicin and ciprofloxacin with standard strains of E. coli, P. aeruginosa and S. aureus. The equipment needed is a standard item in most clinical microbiology laboratories. In addition, this method overcomes the lack of sensitivity of other photometric methods.
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Acknowledgments |
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Notes |
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References |
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Received 5 January 2000; returned 2 May 2000; revised 3 August 2000; accepted 3 November 2000