In vitro activity of the aerosolized agents colistin and tobramycin and five intravenous agents against Pseudomonas aeruginosa isolated from cystic fibrosis patients in southwestern Germany

Tanja Schülin,*

Department of Microbiology and Hygiene, University of Freiburg, Freiburg, Germany


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Three hundred and eighty-five mucoid and non-mucoid strains of Pseudomonas aeruginosa isolated from 192 sputa from 57 adult cystic fibrosis patients were studied. Susceptibility testing using an agar dilution technique was performed for ceftazidime, ciprofloxacin, fosfomycin, meropenem and piperacillin, and the aerosolized agents colistin and tobramycin. Meropenem, ceftazidime and piperacillin were the most potent agents (susceptibility 86.2%, 84.2% and 84%, respectively). Tobramycin and colistin susceptibility rates were lower, but in light of higher intrabronchial concentrations of these drugs a greater percentage of strains might still be clinically susceptible. Only 46.2% and 41.8% of isolates were susceptible to ciprofloxacin and fosfomycin, respectively.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Pseudomonas aeruginosa is the most common colonizing pathogen in lungs of adult cystic fibrosis (CF) patients. Chronic colonization and infection with this pathogen causes lung tissue damage and shortens the life span considerably. Colonization can be delayed by inhaling antibiotics such as colistin or tobramycin.1 In most cases, therapy for infection consists of a combination of two agents given intravenously, but the therapeutic benefit of nebulized agents such as tobramycin and colistin has also been described. Treatment with many courses of antibiotics can lead to dominance of subpopulations with different susceptibility patterns in the strains harboured by the patient, and therapy should therefore be guided by actual susceptibility data. Susceptibility data for colistin have rarely been reported for P. aeruginosa strains from CF patients. Therefore, the in vitro susceptibility of 385 P. aeruginosa strains against two aerosolized and five iv agents was studied.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Strains

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 Mueller–Hinton (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 1–5 x 104 cfu/spot was inoculated on antibiotic-containing MH-II plates (concentration range 0.25–128 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 16–20 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.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The percentages of strains susceptible, intermediate and resistant to each agent for non-mucoid and mucoid isolates are presented in Table 1Go. For all antibiotics tested, except tobramycin, the mucoid isolates were more susceptible than the non-mucoid strains. The most potent agents for all strains were meropenem (86.2% susceptible) and ceftazidime (84.2% susceptible). A total of 83.9% of the strains were susceptible to piperacillin; 41.8% and 43.1% of strains were susceptible to the aerosolized agents colistin and tobramycin, respectively; and 46.2% of strains were susceptible to ciprofloxacin. MIC distributions of all agents are shown in Table 2Go.


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Table 1. Percentages of susceptibilities of 229 non-mucoid and 156 mucoid isolates of P. aeruginosa to seven antibiotics
 

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Table 2. MIC distribution of 229 non-mucoid and 156 mucoid strains of P. aeruginosa to seven antibiotics
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Antibiotic therapy plays a major role in the management of chronic P. aeruginosa infection in CF patients. Many patients harbour different strains of P. aeruginosa in their lungs, which often have different susceptibility patterns. Antibiotic-resistant subpopulations of P. aeruginosa may become dominant during therapy. Therefore, susceptibility testing for all morphotypes of P. aeruginosa in each sputum is important to select the agents most likely to be effective. For these reasons, isolates from subsequent sputa from the same patient were investigated as well as the first-visit isolates, even though this may have biased the selection towards more resistant isolates from sicker patients. Data were not available on individual antibiotic treatment, so correlation between therapy and resistant subpopulations was not possible.

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,8–10 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.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The author was supported by Grünenthal GmbH, Aachen, Germany.


    Notes
 
* Correspondence address. UMC St Radboud, University of Nijmegen, Department of Clinical Microbiology, MMB 440, PO Box 9101, 6500 HB Nijmegen, The Netherlands. Tel: +31-24-361-9560; Fax: +31-24-3540216; E-mail: T.Schulin{at}mmb.azn.nl Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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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, 831–8.[Abstract]

6 . 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, 749–67.[Abstract/Free Full Text]

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, 1190–6.[ISI][Medline]

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Received 29 June 2001; returned 15 August 2001; revised 26 October 2001; accepted 2 November 2001