Department of Pharmacy, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong
Received 8 October 2004; returned 10 December 2004; revised 21 December 2004; accepted 29 December 2004
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Abstract |
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Methods: The baseline PAEs expressed by Pseudomonas aeruginosa ATCC 27853 were recorded following 1 h of exposure to piperacillin and gentamicin, alone and in combination. Similar PAE assessments were made after resistance to the individual antibiotics was induced over 0.52x of their respective MIC.
Results: Before any induction, the PAE produced by piperacillin alone was negligible and that by the combination was synergistic. After piperacillin resistance was induced, PAE exhibited by the ß-lactam remained negligible, and comparable PAEs were observed for gentamicin and the combination, suggesting an additive interaction with a dominant effect from gentamicin. When resistance was induced against gentamicin, progressively shorter PAE was expressed by the aminoglycoside alone and the combination at increasing levels of resistance. In addition, a measurable PAE was unexpectedly observed for piperacillin, whereas the interaction also became additive.
Conclusions: In summary, the PAE expressed by the test combination was highly dependent on the status of gentamicin resistance. The resistance profile exhibited by the organism against individual antibiotics of the combination showed an impact on the type of interaction expressed.
Keywords: PAEs , gentamicin , piperacillin , interaction , Pseudomonas aeruginosa
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Introduction |
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In this study, gentamicin and piperacillin, alone and in combination, were utilized to test against a standard strain of Pseudomonas aeruginosa, before and after induction of resistance to each of the two antibiotics. The selection of this combination was based on the known mechanism of interaction, i.e. enhancing the uptake of aminoglycoside following the disruption of cell wall by the ß-lactam.2 One key study objective was to generate some baseline data in a systematic manner that would help stimulate further discussions.
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Materials and methods |
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Lyophilized P. aeruginosa ATCC 27853 purchased from Difco Laboratories (Detroit, MI, USA) was used. After initial isolation, an organism from a single colony was maintained on agar slants at 4 °C before use.
Culture media
MuellerHinton broth supplemented with 25 mg of Ca2+ and 12.5 mg of Mg2+ per litre (MHB-S) was used throughout. Nutrient agar was employed for the colony count assay via a pour plate technique. Both culture media were purchased from Difco Laboratories and were sterilized per the manufacturer's instructions.
Antimicrobial agents
Gentamicin and piperacillin were purchased from Sigma Chemical Co. (St Louis, MO, USA). Aqueous stock solutions were aseptically prepared and stored frozen at 20 °C before use.
MIC
MICs were measured by the macrodilution technique8 after 18 h of incubation at 37 °C.
Induction of resistance
Induction of resistance against the two study antibiotics was performed on the original P. aeruginosa ATCC 27853 strain. Resistant subpopulations for the individual antibiotics were selected over the range of 0.5, 1 and 2x their respective initial MICs. This was achieved by 10 daily passages of the organism in a sequential manner, i.e. the organism harvested from the last passage was submitted to the next higher concentration for another 10 daily passages. At the end, subpopulations of P. aeruginosa ATCC 27853 resistant to 16, 32 and 64 mg/L of piperacillin, and 1, 2 and 4 mg/L of gentamicin were obtained.
Bacterial cultures
On the day of the PAE experiment, culture containing the test organism harvested immediately after the last daily passage at the desired resistance level was negated of its antibiotic content by saline wash (3x). The organism was then diluted with MHB-S and allowed to grow for 23 h at 37 °C. The actively growing culture was subsequently adjusted to achieve a turbidity equivalent to that of a 0.5 McFarland standard by diluting with MHB-S. The stability of resistance of the test organism used in the PAE study was confirmed by visible growth following overnight incubation at 37 °C, when re-exposing it to the same antibiotic concentration at which resistance was developed.
PAE assessments
To start the experiment, 0.1 mL of the adjusted culture at a turbidity equivalent to that of a 0.5 McFarland standard was introduced to 9.9 mL of antibiotic-containing MHB-S to yield a total volume of 10 mL. The antibiotic-naive control strain and organisms at each of the three levels of resistance for the two antibiotics were exposed to 32 mg/L of piperacillin, 1 mg/L of gentamicin and their combination for 1 h. These concentrations were established in our preliminary studies to avoid excessive reduction in viable bacterial density in relation to the 200 cfu/mL limit of quantification. At the end of the 1 h exposure, the antibiotic(s) was removed by 3x washing using sterile 0.9% saline and centrifugation at 1200 g for 10 min. Throughout the experiments, cultures were kept at 37 °C using a calibrated water bath. Samples (0.1 mL) were withdrawn every 45 min and were submitted to the pour plate assay until steady growth was observed following antibiotic removal. PAE was determined as the difference between the time required for the viable count in the test culture to increase by one log unit from that recorded immediately after antibiotic removal and the time required after the same procedure for the antibiotic-free culture.
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Results |
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The MICs of piperacillin and gentamicin against the original antibiotic-naive P. aeruginosa ATCC 27853 strain were 32 and 2 mg/L, respectively.
PAE
Following 1 h of exposure, the PAE estimates for the P. aeruginosa ATCC 27853 strain (Figure 1a) were 0.1 h for piperacillin and 1.33 h for gentamicin. Simultaneous exposure to the combination resulted in a PAE of 2.46 h. Since the PAE produced by the combination was longer than the sum of those by the individual antibiotics alone, the combination was synergistic. The same conclusion applied as far as the extent of bacterial killing was concerned.
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Discussion |
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Results here showed a vast difference in the PAE responses to individual antibiotics, as well as combinations, as the resistance status towards the individual components of a combination varied. Resistance to piperacillin did not affect the general observation that ß-lactams do not produce a PAE on Gram-negative organisms. However, resistance development to piperacillin caused a prolongation of PAE generated by gentamicin. In fact, this observation deviates from the proposed mechanism that is widely used to explain the synergy brought by the combination, i.e. an increase in cellular aminoglycoside uptake as a result of the cell wall damage inflicted by the ß-lactam. Also unexpectedly, gentamicin resistance caused a prolongation of piperacillin PAE from 0.1 h to > 0.5 h. In view of the fact that these two antibiotics target distinctly different microbial receptors, reasons as to why resistance development to one component affected the PAE of another component of the combination is complex and remains unclear.
For the antibiotic-naive control organism, the piperacillin/gentamicin combination was clearly synergistic in terms of both bactericidal activity and PAE. However, this conclusion did not hold when resistance was developed to either component of the combination. Taking the case of the piperacillin-resistant subpopulations, by contrasting the PAEs produced by the combination in the antibiotic-naive control, i.e. 2.46 h, and those in the piperacillin-resistant subpopulations, i.e. 2.472.62 h, a wrong conclusion of synergy could be easily reached if PAE measurements were not performed on the two antibiotics individually. Indeed, this is an important point to consider when designing future PAE interaction studies. As shown in this study, when either piperacillin- or gentamicin-resistant subpopulations were tested, the interaction type would shift from synergistic to additive.
Present data suggest that resistance development against the ß-lactam component of the combination showed a relatively lower impact on PAE in comparison with the aminoglycoside component. This observation is in agreement with previous reports that combinations of ß-lactam/aminoglycoside would produce synergy on PAE only in aminoglycoside-susceptible strains of Enterococcus faecalis and faecium, but not in the resistant strains.9 Unfortunately, that study did not include the examination of ß-lactam susceptibility. Since the negative impact on PAE caused by resistance development against the gentamicin component was more dominant, aminoglycoside resistance should be weighed more heavily when judgment is to be made on selecting antibiotic combinations for use. In addition, present PAE data showed that the interaction type is largely affected by the resistance profile of the test organism against the components of a combination. Therefore, defining an interaction without considering the organism's resistance profile can be misleading. This work represents only the first steps in the study of the inter-relationship between resistance profiling and PAE expression of an antibiotic combination. More studies are needed to improve the understanding of some of the unanticipated responses observed here.
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References |
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