Department of Microbiology and Hygiene, University of Freiburg, Freiburg, Germany
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Abstract |
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Introduction |
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Materials and methods |
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Three hundred and eighty-five P. aeruginosa strains were collected between August 1998 and December 1999, isolated from 192 sputa from 57 CF patients at the local CF outpatient clinic at Freiburg University Hospital. Two hundred and twenty-nine isolates were non-mucoid and 156 were mucoid.
MIC determination
Agar dilution was performed on MuellerHinton (MH) II agar (Oxoid Ltd, Basingstoke, UK) according to NCCLS guidelines. Briefly, frozen strains were subcultured twice on Colombia blood agar plates and inocula were prepared from overnight cultures in MH broth. A final inoculum of 15 x 104 cfu/spot was inoculated on antibiotic-containing MH-II plates (concentration range 0.25128 mg/L). Glucose-6-phosphate 25 mg/L (Sigma Chemicals, Munich, Germany) was supplemented to the agar when testing fosfomycin. Staphylococcus aureus ATCC 23923 and Escherichia coli ATCC 29522 were used as controls. The plates were incubated for 1620 h at 35°C and MICs were read visually. All strains showed sufficient growth after that time on antibiotic-free media. MICs were interpreted according to the latest NCCLS breakpoints (breakpoints according to the Societé Française de Microbiologie for fosfomycin and to German DIN breakpoints for colistin).
Antibiotics tested
The following agents were tested and were sourced as follows: ceftazidime (Glaxo Wellcome, Hamburg, Germany), ciprofloxacin (Bayer AG, Wuppertal, Germany), fosfomycin (InfectoPharm, Heppenheim, Germany) and meropenem (Grünenthal GmbH, Aachen, Germany). Tobramycin was used from the clinical preparation and piperacillin and colistin base were purchased from Sigma Chemicals.
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Results |
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Discussion |
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Our results demonstrate that non-mucoid strains are more resistant to antibiotics in vitro than mucoid strains (except for tobramycin). Other in vitro investigations have noted similar results,2 and it has been speculated that resistance to antibiotics is inversely correlated to alginate production.
Meropenem and ceftazidime are used frequently in the management of CF patients at the local CF clinic. Resistant strains were rare, and in the population investigated those agents seem to be a good choice. The percentage of susceptible strains compares favourably with other investigations.3 A high percentage of strains was also susceptible to piperacillin. This is in contrast to other studies, perhaps because piperacillin was not used frequently in the local CF clinic at the time of the investigation, and the selective pressure was therefore small.
Aerosolized antibiotics such as tobramycin and colistin may postpone colonization with P. aeruginosa.1,4 Given to patients chronically infected with P. aeruginosa they may also improve lung function5 and reduce the incidence of exacerbations.6 However, interpretation of susceptibility testing results for those agents is hampered by several factors. First, NCCLS breakpoints for colistin have not been updated recently. Other authorities have recently published guidelines: the German DIN (S < 0.5 mg/L, R > 4 mg/L), the BSAC (S < 4 mg/L, R > 8 mg/L) and the Societé Française de Microbiologie (S < 2 mg/L, R > 2 mg/L). The German DIN breakpoints were used in this study. Secondly, the existing breakpoints for tobramycin and colistin were evaluated for intravenous use of these agents, and may not be applicable when those agents are used locally. Thirdly, high concentrations in sputum have been reported, and efforts to determine a breakpoint for resistance to aerosolized tobramycin by correlating MICs and clinical efficacy have been made, but so far, results are inconclusive.7 Colistin concentrations in sputum are difficult to determine, since it is not colistin itself that is inhaled, but rather the methanesulphonate prodrug. No clinical studies have been undertaken to correlate the local concentration to MICs.
Breakpoints ranging from 16 to 128 mg/L for tobramycin in sputum have been proposed, but are not proven. Implementing a breakpoint of 128 mg/L to this collection of strains, the percentage of susceptible strains would rise to 87.7%. Similarly, for colistin, only 10.9% of isolates had an MIC > 2 mg/L. No controlled clinical studies have been undertaken where intrabronchial concentrations of colistin were measured and correlated to clinical efficacy. Since it was shown in one study that aerosolized colistin is still efficacious after inhalation, one could speculate that intrabronchial concentrations might also be active against strains with MICs above the breakpoint.
The MIC distribution pattern in our collection shows two populations of MICs of tobramycin and colistin. This may indicate an emerging resistance problem with use of the respective agents. In studies using aerosolized tobramycin in CF patients, a trend towards more resistant strains was seen in the tobramycin-treated group compared with the placebo-treated patients.7 In several recent studies,810 MICs of colistin have been determined for P. aeruginosa, including strains from CF patients. MICs were generally low and the results were very comparable to the results in the present study. In one small study, resistance development was seen using inhalative colistin therapy, but receded after withdrawal of the drug.11 In another study, synergy against P. aeruginosa from CF patients was shown when colistin was combined with ciprofloxacin.12 These data, together with the clinical efficacy described, indicate that colistin may be a useful drug against P. aeruginosa in this setting.
Ciprofloxacin is a widely used agent in CF patients. In this study, only 43.7% of all strains were susceptible. In light of increasing resistance, fosfomycin could be used for combination therapy. The combination of fosfomycin with meropenem or fluoroquinolones has been shown to be synergic for P. aeruginosa.13 In the collection studied, 38% of strains had fosfomycin MICs < 64 mg/L.
Susceptibility testing plays a crucial part in managing chronic P. aeruginosa infection in CF patients, and is also important in detecting trends towards resistance to frequently used agents. As use of aerosolized agents in CF is increasing, breakpoints need to be re-determined for agents such as colistin and tobramycin in this setting to predict clinical efficacy.
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Acknowledgements |
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Notes |
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References |
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2
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Shawar, R. M., MacLeod, D. L., Garber, R. L., Burns, J. L., Stapp, J. R., Clausen, C. R. et al. (1999). Activities of tobramycin and six other antibiotics against Pseudomonas aeruginosa isolates from patients with cystic fibrosis. Antimicrobial Agents and Chemotherapy 43, 287780.
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Christenson, J. C., Korgenski, E. K. & Daly, J. A. (2000). In vitro activity of meropenem, imipenem, cefepime and ceftazidime against Pseudomonas aeruginosa isolates from cystic fibrosis patients. Journal of Antimicrobial Chemotherapy 45, 899901.
4 . Littlewood, J. M., Miller, M. G., Ghoneim, A. T. & Ramsden, C. H. (1985). Nebulised colomycin for early pseudomonas colonisation in cystic fibrosis. Lancet i, 865.
5 . Jensen, T., Pedersen, S. S., Garne, S., Heilmann, C., Hoiby, N. & Koch, C. (1987). Colistin inhalation therapy in cystic fibrosis patients with chronic Pseudomonas aeruginosa lung infection. Journal of Antimicrobial Chemotherapy 19, 8318.[Abstract]
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.
Doring, G., Conway, S. P., Heijerman, H. G., Hodson, M. E., Hoiby, N., Smyth, A. et al. (2000). Antibiotic therapy against Pseudomonas aeruginosa in cystic fibrosis: a European consensus. European Respiratory Journal 16, 74967.
7 . Burns, J. L., Van Dalfsen, J. M., Shawar, R. M., Otto, K. L., Garber, R. L., Quan, J. M. et al. (1999). Effect of chronic intermittent administration of inhaled tobramycin on respiratory microbial flora in patients with cystic fibrosis. Journal of Infectious Diseases 179, 11906.[ISI][Medline]
8 . Catchpole, C. R., Andrews, J. M., Brenwald, N. & Wise, R. (1997). A reassessment of the in-vitro activity of colistin sulphomethate sodium. Journal of Antimicrobial Chemotherapy 39, 25560.[Abstract]
9
.
Gales, A. C., Reis, A. O. & Jones, R. N. (2001). Contemporary assessment of antimicrobial susceptibility testing methods for polymyxin B and colistin: review of available interpretative criteria and quality control guidelines. Journal of Clinical Microbiology 39, 18390.
10
.
Li, J., Turnidge, J., Milne, R., Nation, R. L. & Coulthard, K. (2001). In vitro pharmacodynamic properties of colistin and colistin methanesulfonate against Pseudomonas aeruginosa isolates from patients with cystic fibrosis. Antimicrobial Agents and Chemotherapy 45, 7815.
11 . Tamm, M., Eich, C., Frei, R., Gilgen, S., Breitenbucher, A. & Mordasini, C. (2000). Inhaled colistin in cystic fibrosis. Schweizer Medizinische Wochenschrift 130, 136672.[ISI]
12 . Richards, R. M. & Xing, D. K. (1993). Investigation of synergism between combinations of ciprofloxacin, polymyxin, sulphadiazine and p-aminobenzoic acid. Journal of Pharmacy and Pharmacology 45, 1715.[ISI][Medline]
13 . Tessier, F. & Quentin, C. (1997). In vitro activity of fosfomycin combined with ceftazidime, imipenem, amikacin, and ciprofloxacin against Pseudomonas aeruginosa. European Journal of Clinical Microbiology and Infectious Diseases 16, 15962.[ISI][Medline]
14 . National Committee for Clinical Laboratory Standards. (2000). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow AerobicallyFifth Edition: Approved Standard M7-A5. NCCLS, Wayne, PA.
15 . Comité de lantibiogramme de la societé francaise de microbiologie. (2001). Communique 20002001, 30.
16 . Deutsches Institut für Normung e. V. (2000). Methoden zur Empfindlichkeitsprüfung von bakteriellen Krankheitserregern (ausser Mykobakterien) gegen Chemotherapeutika. DIN. 58 940. Berlin, Beuth-Verlag.
Received 29 June 2001; returned 15 August 2001; revised 26 October 2001; accepted 2 November 2001