Post-antibiotic effect induced by an antibiotic combination: influence of altered susceptibility to individual components

Ronald C. Li* and Mei C. Tang

Department of Pharmacy, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong


* Corresponding author and current address. Clinical and Exploratory Pharmacology, International Clinical Investigations, Sanofi Aventis, 9 Great Valley Parkway, Malvern, PA 19355, USA. Tel: +1-610-889-6398; Fax: +1-610-889-6227; Email: ronald.li{at}sanofi-aventis.com

Received 8 October 2004; returned 10 December 2004; revised 21 December 2004; accepted 29 December 2004


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: The impact of altered susceptibility of an organism to the components of an antibiotic combination on post-antibiotic effect (PAE) was studied.

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 0.5–2x 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


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Antibiotic combinations are frequently used to provide empirical coverage for serious infections, and in critical conditions before confirmatory diagnosis and susceptibility data are available. However, with the rapidly emergent bacterial resistance and changing landscape of antibiotic resistance, the use of antibiotic combinations does not guarantee a satisfactory clinical response. To date, studies of antibiotic combinations concentrate mainly on overall bactericidal and/or inhibitory activities14 in the following areas: (i) interaction of newer antibiotic combinations for activity profiling, (ii) mechanistic investigation of an interaction, or (iii) interaction against organisms with special clinical interest, e.g. methicillin-resistant Staphylococcus aureus (MRSA). However, interaction pertaining to post-antibiotic effect (PAE) has only received minimal attention.57 The impact of resistance development at the level of individual components of a combination on PAE has yet to be considered.

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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Test organism

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

Mueller–Hinton 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 2–3 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.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
MIC

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.



View larger version (11K):
[in this window]
[in a new window]
 
Figure 1. The changes in bacterial density over time, showing the PAE and the extent of viable count reduction (a) after a 1 h exposure of the antibiotic-naive P. aeruginosa ATCC 27853 strain to piperacillin alone (filled circles), gentamicin alone (filled triangles) and the combination of the two antibiotics (filled squares), (b) following a 1 h exposure of the piperacillin/gentamicin combination to the antibiotic-naive P. aeruginosa control (filled circles) and its subpopulations resistant to 16 (filled triangles), 32 (filled diamonds) and 64 (filled squares) mg/L piperacillin, and (c) following a 1 h exposure of the piperacillin/gentamicin combination to the antibiotic-naive P. aeruginosa control (filled circles) and its subpopulations resistant to 1 (filled triangles), 2 (filled diamonds) and 4 (filled squares) mg/L gentamicin. For (a) to (c) the growth curve of the antibiotic-free control (open circles) is also shown. Timings of the antibiotic(s) addition, removal and the duration of exposure are indicated by vertical white arrows, horizontal white arrows and horizontal black arrows, respectively.

 
In the second study employing the subpopulations resistant to piperacillin over 16 to 64 mg/L, the PAEs expressed following the ß-lactam re-challenge ranged from –0.03 to –0.06 h and remained comparable to the –0.1 h for the antibiotic-naive control (Table 1) confirming the deficiency of ß-lactams to provoke a PAE on Gram-negative organisms regardless of ß-lactam resistance. However, when these subpopulations were tested against gentamicin, the PAE unexpectedly increased from 1.33 h for the antibiotic-naive control to 2.32–2.60 h, suggesting an inverse relationship between piperacillin susceptibility and gentamicin PAE. Interestingly, when these subpopulations were tested against the antibiotic combination (Figure 1b), PAEs in the range 2.47–2.62 h were obtained and were similar to that of the antibiotic-naive control when exposed to the combination, i.e. 2.46 h. Based on the data above, it could be concluded that gentamicin was the dominant antibiotic in the combination. Since the PAEs expressed by the combination on these subpopulations were devoid of any PAE by piperacillin, the sole contribution from the gentamicin component imposed the conclusion of an additive interaction on these subpopulations. Such a conclusion was in contrast to the synergistic interaction observed prior to the induction of piperacillin resistance. Although only a trend, it is also interesting to note that the combination appeared to produce a slightly longer PAE (Table 1) and a higher reduction in bacterial density, as the subpopulations became more resistant to piperacillin (Figure 1b).


View this table:
[in this window]
[in a new window]
 
Table 1. PAEs expressed by P. aeruginosa with different levels of susceptibility to piperacillin and gentamicin after 1 h of exposure to piperacillin (32 mg/L) and gentamicin (1 mg/L), alone and in combination

 
When the gentamicin-resistant subpopulations were tested, a progressively shorter PAE was recorded following the exposure to gentamicin from 1.33 h for the antibiotic-naive control to 0.86, 0.58 and –0.24 h, as the resistance level was incrementally increased from 0.5, 1 to 2x MIC, respectively. The same also applied to the combination when it was tested against these gentamicin subpopulations, i.e. the PAE for the antibiotic-naive control (2.46 h) decreased to 0.42 h with the most gentamicin-resistant subpopulation (Table 1 and Figure 1c). These data were, in fact, consistent with the conclusion that gentamicin was the dominant antibiotic. Unexpectedly, piperacillin produced a positive PAE reading on the gentamicin-resistant subpopulations, i.e. 0.48–0.53 h as compared with –0.10 h for the antibiotic-naive control (Table 1). In this case, the sum of PAEs produced individually by piperacillin and gentamicin was approximately equal to that of the combination on these gentamicin-resistant subpopulations, and thereby met the criteria of an additive interaction.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Study of the PAE has been limited mainly to single antibiotics, with antibiotic combinations receiving much less attention. A review of the literature revealed no systematic approach to examining the influence of altered susceptibility on the individual components of an antibiotic combination in relation to the disparity in PAE expressions. In view of the rapid development of resistance and the increasing use of antibiotic combinations, susceptibility profiling and its associated impact on all antimicrobial effects, including the PAE, cannot be ignored.

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. 2.47–2.62 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.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Sader, H. S., Huynh, H. K. & Jones, R. N. (2003). Contemporary in vitro synergy rates for aztreonam combined with newer fluoroquinolones and ß-lactams tested against gram-negative bacilli. Diagnostic Microbiology and Infectious Disease 47, 547–50.[CrossRef][ISI][Medline]

2 . Li, R. C., Schentag, J. J. & Nix, D. E. (1993). The fractional maximal effect method: a new way to characterize the effect of antibiotic combinations and other nonlinear pharmacodynamic interactions. Antimicrobial Agents Chemotherapy 37, 523–31.[Abstract]

3 . Sweeney, M. T. & Zurenko, G. E. (2003). In vitro activities of linezolid combined with other antimicrobial agents against staphylococci, enterococci, pneumococci, and selected gram-negative organisms. Antimicrobial Agents and Chemotherapy 47, 1902–6.[Abstract/Free Full Text]

4 . Jacqueline, C., Caillon, J., Le Mabecque, V. et al. (2003). In vitro activity of linezolid alone and in combination with gentamicin, vancomycin or rifampicin against methicillin-resistant Staphylococcus aureus by time-kill curve methods. Journal of Antimicrobial Chemotherapy 51, 857–64.[Abstract/Free Full Text]

5 . Hostacka, A. (1997). Comparison of postantibiotic effects of imipenem and netilmicin alone and in combination against Pseudomonas aeruginosa. Arzneimittelforschung 47, 965–7.[ISI][Medline]

6 . Totsuka, K., Shiseki, M., Kikuchi, K. et al. (1999). Combined effects of vancomycin and imipenem against methicillin-resistant Staphylococcus aureus (MRSA) in vitro and in vivo. Journal of Antimicrobial Chemotherapy 44, 455–60.[Abstract/Free Full Text]

7 . Hostacka, A. (1997). In vitro postantibiotic effect of imipenem and enoxacin alone and in combination against Pseudomonas aeruginosa. Pharmazie 52, 544–6.[ISI][Medline]

8 . National Committee for Clinical Laboratory Standards (1993). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically—Third Edition: Approved Standard M7-A3, M7-A3. NCCLS, Villanova, PA, USA.

9 . Fuursted, K. (1988). Comparative killing activity and postantibiotic effect of streptomycin combined with ampicillin, ciprofloxacin, imipenem, piperacillin or vancomycin against strains of Streptococcus faecalis and Streptococcus faecium. Chemotherapy 34, 229–34.[ISI][Medline]





This Article
Abstract
FREE Full Text (PDF)
All Versions of this Article:
55/4/583    most recent
dki035v1
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Disclaimer
Request Permissions
Google Scholar
Articles by Li, R. C.
Articles by Tang, M. C.
PubMed
PubMed Citation
Articles by Li, R. C.
Articles by Tang, M. C.