Medical Microbiology, Aberdeen Royal Infirmary, Foresterhill, Aberdeen AB25 2ZN, UK
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Abstract |
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Introduction |
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This study is an investigation of the following pharmacodynamic parameters: post-antibiotic effect (PAE), post-antibiotic sub-MIC effect (PA-SME) and control-related effective regrowth time (CERT). These parameters were studied in relation to morphology and kill induced in ten strains of Enterobacteriaceae by ceftibuten. PAE measures the delayed regrowth of bacteria after antibiotic exposure. 1 The emphasis placed on PAE is questionable as, increasingly, it is recognized that this parameter is dependent on the methods used to quantify bacterial cells. CERT, which is a measure of both PAE and kill, is independent of these methods. It has, therefore, been suggested that less emphasis should be placed on PAE data and that CERT should be used in future pharmacodynamic studies. 2 So far, this suggestion has not found favour, relatively few CERT studies having appeared in the literature. PA-SME is also considered by some to be a more important pharmacodynamic measurement than PAE, reflecting more closely the in-vivo state of slowly declining antibiotic concentrations with long periods of persistently sub-inhibitory concentrations.
If PAE is to remain pre-eminent in the realm of pharmacodynamics, careful consideration must be given to the methodology employed in enumerating bacterial cells. Methods which have been employed in the past include the `reference' viable counting method, 1 spectrophotometry, 3 electronic particle counting, 4 direct microscopy, 5,6 bioluminescence assay of bacterial adenosine triphosphate (ATP) 7,8 and impedance monitoring. 9,10 Traditional methods of bacterial enumeration employ viable counting, which relies on the growth of colony-forming units on a solid agar surface. The suitability of this method has been questioned when enumerating aberrant morphological forms 7,8 induced by antibiotic exposure, as viable counting underestimates numbers of aberrant morphological forms, leading to an underestimation of the PAE. 8 Previously, we have used the alternative techniques of the bioluminescence assay of ATP and impedance monitoring. We have found no significant differences in PAE measured by viable counting alone and in combination with impedance (IMP/VC) and also no significant differences by bioluminescence alone, and in combination with impedance (IMP/BIOL). 8 We have found significant differences in PAE measured by IMP/VC and IMP/ BIOL. 8,11 This current study aims to further investigate PAE, PA-SME and CERT in relation to the methods used as well as in relation to post-exposure morphology and kill.
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Materials and methods |
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The bacteria used in this study were a mixture of control strains and recent clinical isolates sensitive to ceftibuten and are listed in Table I. Strains CTX 224, CTX 603 and BLR 02 are multiply resistant, exhibiting extended spectrum ß-lactamase (ESBL) activity due to an SHV type enzyme. 12 I 112 and T 767 are clinical isolates which we have studied in the past. 11 MuellerHinton broth was used throughout the study. Ceftibuten was provided by Schering-Plough Ltd (Welwyn Garden City, UK).
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MICs were determined by the microdilution method according to National Committee for Clinical Laboratory Standards (NCCLS) guidelines. 13 The standard inoculum of 5 x 105 cfu/mL was used in addition to an inoculum of 1 x 108 cfu/mL.
Kill curves
Kill curves were carried out in duplicate by exposing test cultures to ceftibuten and establishing bacterial numbers by three methods. An overnight broth was diluted 1:100 and exposed to 0.1, 1 and 10 x MIC. An unexposed control was obtained by diluting an overnight broth 1:1000. Counts at times 0, 1, 2, 3, 4, 5, 6 and 24 h were determined by bioluminescence and viable counting. Counts by direct microscopy were determined up to 6 h after exposure to 0, 1, 10 and 100 x MIC.
Determination of PAE and CERT
Inocula of c. 107 cfu/mL were exposed to ceftibuten (0.1100 x MIC) for 2 h, after which antibiotic was eliminated by 1:1000 dilution in test medium. Regrowth of these and a control culture was followed by impedance 9 (eight replicates) in combination with viable counting and bioluminescence (both of which were carried out in duplicate). 7,8 PAE was calculated as the difference in time between antibiotic-exposed and unexposed cultures to reach 107 cfu/mL (allowing for differences in their respective inocula). 8 CERT was calculated as the difference in time after antibiotic exposure and elimination for test and control cultures to grow to their pre-exposure inocula plus 1 log1011
Determination of PA-SME
The primary method used is based on that of Odenholt-Tornqvist et al. 14 and was carried out on selected test isolates as detailed in Table II. An inoculum of approximately 107 cfu/mL was exposed to 10 x MIC ceftibuten for 2 h, after which the cultures were diluted to achieve sub-MIC concentrations of 0.1, 0.2 and 0.3 x MIC. After the 2 h exposure, bioassays were performed to confirm the ceftibuten concentration. Regrowth was followed by impedance (eight replicates) in combination with viable counting (carried out in duplicate). Unexposed controls were run in parallel and PA-SME was calculated in the same way as PAE.
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Determination of predominant morphological forms
Inocula of c. 107 cfu/mL were exposed to ceftibuten (0.1100 x MIC) for 2 h, after which the cultures were observed by interference contrast microscopy and the predominant morphological forms present were recorded.
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Results |
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Figure 1 presents mean kill-curve data for all ten test isolates. By viable counting (Figure 1a) the cultures exposed to 0 and 0.1 x MIC showed no kill although growth was slower in the culture exposed to 0.1 x MIC. The 100 x MIC culture showed kill over the 24 h test period whereas all of the other cultures demonstrated kill over 6 h followed by concentration-dependent regrowth during the 6 24 h period. Direct microscopy (Figure 1c) gave similar results, with similar kill patterns after exposure to 1, 10 and 100 x MIC over the 6 h test period. Kill curves by bioluminescence (Figure 1b) gave similar patterns for the 0 and 0.1 x MIC exposed cultures, but not for the cultures exposed to ceftibuten concentrations of 1 x MIC or above. During the first 2 h no kill was demonstrated: in fact, growth of over 1 log10 was observed.
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Discussion |
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Aberrant morphological forms, such as filaments, are clinically beneficial in that they undergo phagocytosis more easily than bacilli, 17 but they are problematic in the laboratory. In common with other investigators, we have found that, after antibiotic exposure, different cell enumeration methods quantify aberrant morphological forms to varying degrees.8,19,20 This is exemplified in Figure 2, which shows the concentration-dependent nature of the differing abilities of viable counting and bioluminscence to quantify cells after ceftibuten exposure. Counts determined by bioluminscence were greater than those by viable counting, probably because although one filament may contain as many as 20 genomes, it will be represented by only 1 cfu by viable counting. 21 Upon cell division one filament will split into separate cells, giving the impression of a rapid increase in cell numbers. In contrast, the decrease in counts by bioluminscence was not as pronounced as that by viable counting because levels of intracellular ATP were measured directly. Bioluminescence may better represent aberrant morphological forms than viable counting but it also has disadvantages. A constant ATP content of bacterial cells is assumed, but after antibiotic exposure, an enlarged bacillus may contain more ATP than a healthy bacillus. Also, if ATP from intact dead cells is measured, counts determined by BIOL will be falsely inflated, giving the impression of less kill than has actually taken place. This may account for the difference between the kill curves determined by bioluminescence and those determined by viable counting and direct microscopy.
The concentration-dependent nature of the differences in post-exposure counts (Figure 2) was most marked up to 4 x MIC after a plateau effect was seen. This correlates with the morphological responses shown by the test cultures in response to ceftibuten exposure. It was only above 4 x MIC that all of the cultures showed a predominance of filaments (Table III). The largest differences in counts after ceftibuten exposure and the longest PAE and CERT values were found in cultures made up of filaments. PAE and CERT may represent the time required for filamentous forms to resynthesize new PBPs, or the time during which antibiotic which has accumulated in the periplasmic space continues to inhibit newly formed PBPs. They may also represent the time during which the cells regenerate active enzyme molecules after the bound antibiotic has dissociated from the target site. The rate of synthesis varies for different bacteria 22 and this could account for corresponding variations in the duration of both the PAE and CERT.
Impedance monitoring brings with it the associated benefits of an automated system. The only disadvantage is that the initial enumeration of bacterial cells must be performed by an additional method and this value used as a baseline. PAE and CERT values determined by IMP/VC and IMP/BIOL are presented in Table III. Differing abilities of viable counting and bioluminscence to enumerate aberrant morphological forms translated into highly significant differences between PAE values determined by IMP/VC and IMP/BIOL (Student's t-test, t = 8.67). Negative PAE values determined by IMP/VC are clearly artefactual and a function of the methodology. In contrast there were no significant differences between the CERT values (t = 1.61). As discussed previously, 10 viable counting of aberrant morphological forms probably yields falsely low counts, which leads to an inflation of the bactericidal activity and an underestimation of PAE when calculated by viable counting, both alone and in combination with impedance monitoring.
Different strains of the same species do not respond in the same way to a specific antibiotic as demonstrated by the large standard deviation values in Table III. To illustrate this further, the CERT values for the three isolates of E. coli are presented in Figure 4. Differences in these values are most marked at 100 x MIC with a range of 0.254.11 h. As PAE is method dependent but CERT is not, CERT values would yield a more meaningful pharmacodynamic parameter to compare the results of different research groups.
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In conclusion, concentration-dependent differences in post-antibiotic exposure counts by bioluminescence and viable counting were found after exposure of the test Enterobacteriaceae to ceftibuten. These may form a direct relationship with morphological forms present in each culture. PAE, PA-SME and kill were found to be method dependent whereas CERT was not. Generally neither PAE nor CERT was concentration dependent above 1 x MIC, although different strains of E. coliand K. pneumoniaeresponded to ceftibuten differently with respect to PAE and CERT. The results of the PA-SME studies, which may be the most clinically relevant pharmacodynamic parameter, confirm the appropriateness of the current once or twice a day dosing schedules despite the lack of PAE.
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Acknowledgments |
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Notes |
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References |
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Received 4 March 1998; returned 27 April 1998; revised 11 May 1998; accepted 6 August 1998