a School of Pharmacy and c School of Biology and Biochemistry, The Queen's University of Belfast, Medical Biology Centre, Belfast BT9 7BL b School of Life and Health Sciences, Aston University, Birmingham B4 7ET, UK
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Abstract |
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Introduction |
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Materials and methods |
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Azlocillin and tobramycin were prepared in water from Securopen powder (Bayer plc, Newbury, UK) and Nebcin solution (Eli Lilly & Co. Ltd, Basingstoke, UK), respectively.
Pseudomonas aeruginosa isolates and MIC determinations
Eight isolates from the sputum of CF patients were identified as distinct strains by pyocin typing and serotyping, as described previously.5 MICs were determined by broth microdilution in Iso-Sensitest broth (ISB; Oxoid, Basingstoke, UK) with a final inoculum of 105106 cfu/mL, and incubation at 37°C for 24 h.
Development of resistance by in-vitro serial subculture
The procedure for studying resistance development consisted of subculturing and regrowing bacteria that survived in the presence of half of the MIC of the treatment antibiotic, and reassessing the MIC.4 Controls in drug-free media were run in parallel.
Chequerboard broth microdilution method for serial subculture of antibiotic combinations
Eight doubling dilutions (based on initial MICs) of azlocillin and tobramycin in ISB were prepared. Each dilution (50 µL) of each antibiotic was placed in wells of a microtitre plate to give 64 drug combinations. The MICs of each antibiotic alone and in the combination (taken as the highest dilution of both antibiotics which failed to show visible growth) were determined after incubation at 37°C for 24 h.
A subculture (0.1 mL) was taken from the well containing one-half the MIC of both antibiotics, transferred to 9.9 mL of drug-free ISB and incubated at 37°C for 24 h. The MIC of the individual antibiotics was determined for this culture by the broth microdilution method. An additional sample of this culture was used to inoculate the next microtitre plate, and the process of treating with the combination of antibiotics, subculturing and re-growing was continued for at least eight treatments. Controls in drug-free media were included.
Measurement and characterization of b-lactamases
ß-Lactamase levels were quantitatively determined based on the rate of degradation of nitrocefin (Glaxo research 87/312, Oxoid) and the pI of the enzymes characterized by isoelectric focusing, as described previously.4
Statistical treatment of results
Changes in MIC following serial subculture were tested for significance using the Wilcoxon Signed Rank Test.
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Results |
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Discussion |
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Resistance to the azlocillin component of the combination was delayed considerably in four isolates, and could not be induced in another four isolates after 16 subcultures. In two of the isolates tested (Nos 1 and 3), levels of ß-lactamase remained low and fairly constant for up to eight subcultures, and corresponded to a low MIC of azlocillin in the combination. In the subsequent three to four subcultures, the ß-lactamase activity and the MIC of azlocillin gradually increased. The pI of the extracted enzymes changed also and may be due to increased production of existing enzymes or expression of aditional enzymes. The results suggest that resistance to the azlocillin component of the combination was due to selection of resistant mutants with ß-lactamase activity and that this selection process was delayed by the presence of an additional antimicrobial agent even at subinhibitory concentrations. Resistance mechanisms in P. aeruginosa have recently been reviewed, and in the case of ß-lactam antibiotics, the mechanism is usually mediated by chromosomal ß-lactamases.7 Stably resistant mutants containing Class I ß-lactamases are present in most P. aeruginosa isolates,8 and this is consistent with the low level of ß-lactamase activity detected in the isolates before the serial subculture experiments. In the third isolate (No. 2), the increase in ß-lactamase activity was of a lower order and there was no change in pI of the extracted enzymes.
The principle of combining two antibiotics to prevent emergence of resistance is well established, and clinical evidence for this happening in P. aeruginosa infections has been reviewed by Barriere.9 This investigation describes a method to study resistance development to antibiotic combinations. Further investigation of the ability of antibiotic combinations to delay development of resistance in P. aeruginosamay be increasingly necessary as more antibiotics become ineffective when used as monotherapy.
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Acknowledgments |
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Notes |
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References |
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2 . Farrell, W., Wilks, M. & Drasar, F. A. (1979). Synergy between aminoglycosides and semi-synthetic penicillins against gentamicin-resistant Gram-negative rods. Journal of Antimicrobial Chemotherapy 5, 239.[ISI][Medline]
3 . Moellering, R. C., Allsop, A., Bush, K., Quinn, J., Baquero, F. & Philipps, I. (1998). Antibiotic resistance: lessons for the future. Clinical Infectious Diseases 27, Suppl. 1, S13540.[ISI][Medline]
4 . Wu, Y. L., Scott, E. M., Li Wan Po, A. & Tariq, V. N. (1999). Development of resistance and cross-resistance in Pseudomonas aeruginosa exposed to subinhibitory antibiotic concentrations. APMIS 107, in press.
5 . Steen, H. J., Scott., E. M., Stevenson, M. I., Black, A. E., Redmond, A. O. B. & Collier, P. S. (1989). Clinical and pharmacokinetic aspects of ciprofloxacin in the treatment of acute exacerbations of Pseudomonasinfection in cystic fibrosis patients. Journal of Antimicrobial Chemotherapy 24, 78795.[Abstract]
6 . Giwercman, B., Lambert, P. A., Rosdahl, V. T., Shand, G. H. & Høiby, N. (1990). Rapid emergence of resistance in Pseudomonas aeruginosa in cystic fibrosis patients due to in-vivo selection of stable partially derepressed ß-lactamase producing strains.Journal of Antimicrobial Chemotherapy 26, 24759.[Abstract]
7 . Hancock, R. E. W. (1998). Resistance mechanisms in Pseudomonas aeruginosa and other nonfermentative Gram-negative bacteria. Clinical Infectious Diseases 27, Suppl. 1, S939.[ISI][Medline]
8 . Eliopoulos, G. M. (1988). Induction of ß-lactamases. Journal of Antimicrobial Chemotherapy 22, Suppl. A, 3744.[ISI][Medline]
9 . Barriere, S. L. (1992). Bacterial resistance to ß-lactams, and its prevention with combination antimicrobial therapy. Pharmacotherapy 12, 397402.[ISI][Medline]
Received 27 October 1998; returned 24 February 1999; revised 7 April 1999; accepted 19 April 1999