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Development of resistance in Pseudomonas aeruginosa obtained from patients with cystic fibrosis at different times
ORIGINAL ARTICLE Development of resistance in Pseudomonas aeruginosa obtained from patients with cystic fibrosis at different times F. B. Spencker1, L. Staber1, T. Lietz2, R. Schille2 and A. C. Rodloff1 Institute for Medical Microbiology and Epidemiology of Infectious Diseases, and 2Department of Pediatrics, University of Leipzig, Leipzig, Germany 1
Objective Determination of the extent of changes in quantitative resistance in Pseudo-
monas aeruginosa isolates from patients with cystic fibrosis over a period of approximately 2 years. Methods Three hundred and ninety nine isolates of P. aeruginosa collected from 34 pediatric patients in the period between April 1994 and April 1996 were investigated. During the 2 years the children were treated with a combination of a betalactam and an aminoglycoside, approximately every 3 months. In between they received ciprofloxacin orally, when required. The minimal inhibitory concentrations (MICs) of 38 clones of P. aeruginosa defined by different patterns in macrorestriction analysis (pulse field gel electrophoresis, PFGE) were established for 12 antibiotics: gentamicin, amikacin, tobramycin, ciprofloxacin, levofloxacin, moxifloxacin, trovafloxacin, imipenem, meropenem, ceftazidime, cefepime, and piperacillin by means of broth microdilution tests according to DIN 58940. Results Twenty-four of the 38 clones developed increased MIC values during the time of observation especially for aminoglycosides and quinolones. Comparatively less affected were ceftazidime, imipenem and meropenem. An association between the number of the intravenous treatment courses and the increase of the MIC values could not be verified. Conclusions A trend towards an increase of the MICs against antipseudomonal agents was observed over a limited period of time. It is necessary to prevent this development possibly by employing suitable combinations of antibiotics and the introduction of new substances. Keywords Pseudomonas aeruginosa, antibiotic resistance, minimal inhibitory concentrations (MICs), cystic fibrosis Accepted 30 April 2002
Clin Microbiol Infect 2003; 9: 370–379
INTRODUCTION Cystic fibrosis (CF) is an autosomal recessive, multisystem disease with Pseudomonas aeruginosa as the major prognostic factor in chronic respiratory infection [1]. Once colonization is Corresponding author and reprints request: Prof Dr med F.B. Spencker, Institute of Medical Microbiology and Epidemiology of Infectious Diseases, University of Leipzig, 04103 Leipzig, Liebigstr. 24, Germany Tel: þ49 341 9715204 Fax: þ49 341 9715209 E-mail: [email protected]
established the eradication of this pathogen is almost impossible owing to the nature of cystic fibrosis itself and the appearance of mucoid phenotypes of P. aeruginosa forming microcolonies on the mucosal surfaces which are surrounded by an abundant mass of the microbial exopolysaccharide alginate [2,3]. This unique bacterial product may present a polyanionic barrier to antimicrobial agents so that their action is prevented. As a result of the repeated use of antimicrobial agents in the management of cystic fibrosis, there may be a subsequent emergence of resistant
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>500
SUBJECTS AND METHODS
2.7732 (0.59; L3.03) 6.6659 (0.93; 17.46) 0.6942 (0.28; 1.75) 0.2355 (0.09; 0.64) 0.5781 (0.2; 1.67) 1.7281 (0.52; 5.78) 0.2731 (0.08; 0.91) 1.0565 (0.47; 2.38) 0.2152 (0.07; 0.67) 1.667 (0.6; 4.66) 1.9997 (0.76; 5.26) 3.7062 (0.8; 17.18)
P. aeruginosa [8]. The objective of this study was to investigate the gradual development of in vitro antibiotic resistance over a period of 2 years in P. aeruginosa isolates from CF patients and to determine changes in relation to the way the antimicrobial agents used were applied.
1
GM (standard deviation) (mg/L)
Spencker et al Development of resistance of pseudomonas aeruginosa in CF 371
1
64 2
>32
2 9
1
5 9
1
3 2 3
6
1 1 1
2
32 16 >8
1 6
2 2 8
8 13 3 1 3 9 2 2 8 5 2 2 2 8 1 11 11
6
2
13
8 2
1 3
GEN AMK TOB CIP LEV MOX TROVA IPM MEM CAZ CPM PIP
1
4 1
1 9 3
4 15 8 2 4 4 3
21 5 12 5 12 7 13 7
13 1 6 1 8 7 3 13 3 11 10 3 2
14 19 3
8 1 0.063 0.031
Table 1 Patient demographics
0.125
0.25
0.5
2
4
>4
Bacteriologic methods Sputum samples or deep-throat swab specimens were collected before and after the antibiotic treatment. Isolated strains were assessed by morphologic features, including pigment production, and identified as P. aeruginosa by a positive oxidase reaction and appropriate results with the API-20 NE system (Bio-Me´ rieux, Nu¨ rtingen, Germany). The strains were stored in special vials (Microbank, Mast, Liverpool, UK) at 208. The P. aeruginosa strains were analyzed for different pyocin types by the spotting method as described by Fyfe et al. [4] and pulse field gel electrophoresis (PFGE) patterns by contourclamped homogeneous electric field electrophoresis as described earlier [5]. Genomic DNA was subjected to the rare-cutting restriction enzyme SpeI. Twenty-two patients were chronically colonized with strains with the same PFGE pattern, seven were transiently colonized with two or more different types and five were permanently colonized with two different types in the observed period (details have been published elsewhere) [5]. As a result of the variable number of colonizing strains we investigated 38 P. aeruginosa clones with
2
>64
500
Isolates of P. aeruginosa, 399 in number, collected from 34 children, who regularly attended the CF center at the Department of Pediatrics of the University of Leipzig between April 1994 and April 1996, were investigated. Most of the patients were hospitalized either for regular 2-week courses or for the treatment of acute exacerbations as a rule with an intravenous combination of ceftazidime and gentamicin. Altogether there were 83 intravenous treatment courses in the period of investigation, for details see Figure 1. When deemed necessary the children received oral ciprofloxacin in between and nebulized aminoglycosides for inhalation. The patients were followed up in the outpatient clinic of the above mentioned department.
1
Patients
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GEN
AMK
n-muc (n¼122) 5.6746 11.5075 (0.97; 33.25) (4.1; 32.3) Muc (n¼277) 3.1677 7.4038 (0.78; 12.79) (2.99; 18.36) P-value 0.0001 0.0001
TOB
CIP
LEV
MOX
TROVA
IPM
MEM
CAZ
CPM
PIP
0.9493 (0.32; 2.78) 0.6811 (1.5; 0.31) 0.006
0.2997 (0.09; 0.97) 0.3237 (0.1; 1.07) 0.697
0.6859 (0.19; 2.5) 0.7945 (0.22; 2.83) 0.278
2.4181 (0.76; 7.67) 2.4473 (6.84; 0.88) 0.803
0.3116 (0.07; 1.43) 0.386 (0.08; 1.81) 0.185
0.7364 (0.27; 1.99) 1.1275 (2.89; 0.44) 0.0001
0.1977 (0.06; 0.65) 0.2157 (0.72; 0.06) 0.268
1.3979 (0.34; 5.73) 1.4681 (0.55; 3.93) 0.090
2.3655 (0.73; 7.68) 2.4968 (0.97; 6.44) 0.842
3.3368 (0.49; 8.32) 3.7062 (0.86; 18.8) 0.119
n-muc, non-mucoid phenotype; muc, mucoid phenotype; P-value, Mann–Whitney U-test; GEN, Gentamicin; AMK, Amikacin; TOB, Tobramycin; CIP, Ciprofloxacin; LEV, Levofloxacin; MOX, Moxifloxacin; TROVA, Trovafloxacin; IPM, Imipenem; MEM, Meropenem; CAZ, Ceftazidim; CPM, Cefepim; PIP, Piperacillin.
Table 3 MICs (mg/L), geometric mean, standard deviation in brackets, of 38 P. aeruginosa clones at the end of observation in relation to the duration of colonization in April 96.
G1 G2 G3 G4 P-value
GEN
AMK
TOB
CIP
LEV
MOX
TROVA
IPM
1.68 (4.8; 0.6) 1.68 (7.3; 0.4) 1.99 (4.4; 0.9) 27.97 (328.9; 2.4) 0.0056
5.66 (11.3; 2.8) 5.19 (25.9; 1.0) 5.79 (14.8; 2.3) 19.34 (51.8; 7.2) 0.033
0.71 (1.4; 0.4) 0.71 (2.3; 0.2) 0.57 (1.2; 0.3) 1.76 (6.3; 0.5) 0.071
0.18 (0.3; 0.1) 0.23 (0.8; 0.1) 0.16 (0.8; 0.0) 0.53 (2.9; 0.1) 0.262
0.42 (1.0; 0.2) 0.65 (2.5; 0.2) 0.38 (1.7; 0.1) 1.29 (5. 6; 0.3) 0.256
1.41 (2.8; 0.7) 1.41 (8.1; 0.2) 1.45 (5.8; 0.4) 3.99 (9.6; 1.7) 0.178
0.21 (0.5; 0.1) 0.29 (1.3; 0.1) 0.21 (1.2; 0.0) 0.78 (4.2; 0.2) 0.218
1.42 (2.1; 0.71 (2.6; 0.69 (1.9; 1.07 (3.5; 0.3
0.9) 0.2) 0.2) 0.3)
MEM
CAZ
CPM
PIP
0.18 (0.6; 0.1) 0.18 (0.4; 0.1) 0.17 (0.6; 0.1) 0.21 (1.1; 0.0) 0.835
1.68 (3.27; 0.86) 1.54 (2.58; 0.92) 1.32 (4.99; 0.35) 1.99 (10.61; 0.38) 0.65
1.41 (2.1; 0.9) 1.99 (4.6; 0.9) 1.59 (5.8; 0.4) 5.14 (15.2; 1.7) 0.056
2.38 (14.7; 4.76 (11.8; 2.76 (16.9; 3.75 (26.3; 0.623
0.4) 1.9) 0.5) 0.5)
GEN, Gentamicin; AMK, Amikacin; TOB, Tobramycin; CIP, Ciprofloxacin; LEV, Levofloxacin; MOX, Moxifloxacin; TROVA, Trovafloxacin; IPM, Imipenem; MEM, Meropenem; CAZ, Ceftazidim; CPM, Cefepim; PIP, Piperacillin. Group 1 (G1) 0.5 years, n¼4; group 2 (G2): 0.5 – 2 years, n¼8; group 3 (G3): >2– 4 years, n¼14; group 4 (G4): >4 years, n¼12)
372 Clinical Microbiology and Infection, Volume 9 Number 5, May 2003
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Table 2 Comparison of geometric mean (GM) values of the MICs (mg/L), standard deviation in brackets, between phenotypic non mucoid and mucoid P. aeruginosa isolate
Spencker et al Development of resistance of pseudomonas aeruginosa in CF 373
Figure 1 Antibiotics and combinations of antibiotics used in 83 episodes of i.v. therapy.
Figure 2 MICs of ceftazidime and moxifloxacin against Pseudomonas aeruginosa clones with 38 PFGE-patterns at the beginning of the investigation (t ¼ 0; 38 isolates), after 1 year (t ¼ 1 year; 31 isolates) and after 2 years (t ¼ 2 years; 19 isolates).
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374 Clinical Microbiology and Infection, Volume 9 Number 5, May 2003
Figure 3 MICs of ciprofloxacin and levofloxacin against Pseudomonas aeruginosa clones with 38 PFGE patterns at the beginning of the investigation (t ¼ 0; 38 isolates), after 1 year (t ¼ 1 year; 31 isolates) and after 2 years (t ¼ 2 years; 19 isolates).
different PFGE patterns. Two to 31 isolates per PFGE-pattern were isolated. Minimal inhibitory concentration (MIC) The MIC is defined as the lowest concentration of an antimicrobial agent that prevents visible growth of bacteria [6]. The MICs of 38 strains were established for 12 antibiotics: gentamicin (Serva, Heidelberg, Germany), amikacin (Bristol-Myers Squibb, Munich, Germany), tobramycin (Sigma, Munich, Germany), ciprofloxacin (Bayer, Leuerkusen, Germany), levofloxacin (Daiichi, Aventis, Bad Soden, Germany), moxifloxacin (Bayer), trovafloxacin (Pfizer, Karlsruhe, Germany), imipenem (Merck Sharp & Dohme, Haar, Germany), meropenem (AstraZeneca, Wedel, Germany), ceftazidime (GlaxoSmithKline, Munich, Germany), cefepime (Bristol) and piperacillin
(Lederle, Mu¨ nster, Germany) by means of broth microdilution tests according to DIN 58940 [6]. The interpretative categories with regard to the breakpoints were fixed [6] as follows: gentamicin, tobramycin 1 mg/mL (s), 2–4 mg/ mL (i), 8 mg/mL (r); amikacin 4 mg/mL (s), 8–16 mg/mL (i), 32 mg/mL (r); ciprofloxacin, levofloxacin, moxifloxacin, trovafloxacin 1 mg/mL (s), 2 mg/mL (i), 4 mg/ mL (r); imipenem ¼ 2 mg/mL (s), 4 mg/mL (i), ¼ 8 mg/ mL (r); meropenem ¼ 2 mg/mL (s), 4–8 mg/mL (i), ¼ 16 mg/mL (r); ceftazidime, cefepime ¼ 4 mg/mL (s), 8–16 mg/ mL (i), ¼ 32 mg/mL (r); piperacillin ¼ 4 mg/mL (s), 8–32 mg/mL(i), ¼ 64 mg/mL (r).
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Spencker et al Development of resistance of pseudomonas aeruginosa in CF 375
Figure 4 MICs of gentamicin and tobramycin against Pseudomonas aeruginosa clones with 38 PFGE patterns at the beginning of the investigation (t ¼ 0; 38 isolates), after 1 year (t ¼ 1 year; 31 isolates) and after 2 years (t ¼ 2 years; 19 isolates).
RESULTS Table 1 shows the susceptibility of the 38 P. aeruginosa clones at the beginning of the observation. When the geometric means were compared, tobramycin showed about four times lower MICs than gentamicin and about nine times lower MICs than amikacin. Among the quinolones, ciprofloxacin and trovafloxacin possessed the lowest MIC values. They were two times lower than levofloxacin and seven times lower than moxifloxacin. Cefepime was somewhat more active than ceftazidime. The geometric mean MIC of meropenem showed the lowest value, being about five times lower than that of imipenem. The number of samples with the typical mucoid colony morphology (n ¼ 277) was greater than those without alginate production (n ¼ 122).
The mucoid isolates were, except for the aminoglycosides, less susceptible to the anti-pseudomonal agents, but significance was only achieved by imipenem (Table 2). The mucoid isolates were significantly more susceptible to gentamicin, tobramycin, and amikacin (the Mann– Whitney U-test). The mean time the patients were colonized with P. aeruginosa was 3.8 years in April 1996. Four patients were colonized for less than 0.5 years, eight patients for 0.5–2 years, 14 patients for 2– 4 years and eight patients for more than 4 years. The length of colonization correlated with increasing MIC values, especially for aminoglycosides and quinolones, a statistical significance was reached for gentamicin and amikacin (Kruskal– Wallis one-way test). Isolates from patients colonized for more than 4 years accounted for this
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376 Clinical Microbiology and Infection, Volume 9 Number 5, May 2003
Figure 5 MICs of meropenem and imipenem against Pseudomonas aeruginosa clones with 38 PFGE patterns at the beginning of the investigation (t ¼ 0; 38 isolates), after 1 year (t ¼ 1 year; 31 isolates) and after 2 years (t ¼ 2 years; 19 isolates).
statistical significance (Mann–Whitney-Test, a ¼ 0.05, P <0.0083) (Table 3). During the study period 24 clones showed an increase of MIC values that resulted in alterations of the qualitative susceptibility (from susceptible to intermediate, from susceptible to resistant and from intermediate to resistant), at least against one of the investigated antibiotics. The extent of the development of resistance differed for the investigated antibiotics. Ceftazidime, imipenem and meropenem were less affected than the other substances. Figures 2–5 show, in eight representative examples, the gradual development of resistance during the observed time. The MICs (geometric mean) did not change the qualitative interpretative categories except for levofloxacin (sensitive to intermediate) and moxifloxacin (intermediate to resistant). There were no correlations between the
number of parenteral antibiotic treatment courses or the number of ciprofloxacin applications, respectively, and the development of resistance in 14 clones from 12 patients with at least four parenteral antibiotic courses, and ten clones from nine patients with at least four ciprofloxacin applications (Chi2 Test), respectively (Table 4). The proportion of strains resistant to gentamicin and amikacin increased in proportion to the amount of inhaled gentamicin (Chi2 Test, gentamicin P ¼ 0.0229; amikacin P ¼ 0.01; tobramycin P >0.05, Table 5). For parenteral treatment the patients received an intravenous combination, as a rule consisting of ceftazidime (150 mg/kg/day) and gentamicin (9 mg/kg/day). In between the children were treated with long-term nebulized gentamicin (80 mg/day) as a rule and, when necessary, oral ciprofloxacin for 2 weeks (40 mg/kg/ day).
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Spencker et al Development of resistance of pseudomonas aeruginosa in CF 377
Table 4 Proportion (%) of sensitive, intermediate and resistant P. aeruginosa strains with 14 different PFGE patterns (¼ 14 isolates pro cycle) in relation to the number of parenteral antibiotic courses Number of i.v. therapys 1 sensitive intermediate resistant 2 sensitive intermediate resistant 3 sensitive intermediate resistant 4 sensitive intermediate resistant
GEN
AMK
TOB
CIP
LEV
MOX
TROVA
IPM
MEM
CAZ
CPM
PIP
14.3% 71.4% 14.3%
28.6% 57.1% 14.3%
85.7% 7.1% 7.1%
85.7% 7.1% 7.1%
71.4% 14.3% 14.3%
35.7% 14.3% 50%
85.7% 0% 14.3%
92.9% 0% 7.1%
100% 0% 0%
78.6% 14.3% 7.1%
78.6% 14.3% 7.1%
42.9% 50% 7.1%
14.3% 50% 35.7%
21.4% 57.2% 21.4%
57.1% 35.8% 7.1%
71.4% 21.5% 7.1%
64.3% 14.3% 21.4%
21.4% 28.6% 50%
71.4% 7.2% 21.4%
64.3% 28.6% 7.1%
92.9% 7.1% 0%
71.4% 28.6% 0%
64.3% 21.4% 14.3%
50% 35.7% 14.3%
14.3% 57.1% 28.6%
21.4% 57.2% 21.4%
71.4% 21.5% 7.1%
71.4 21.5% 7.1%
64.3% 14.3% 21.4%
28.6% 35.7% 35.7%
71.4% 7.2% 21.4%
57.1% 28.6% 14.3%
92.9% 7.1% 0%
71.4% 21.5% 7.1%
57.1% 35.8% 7.1%
50% 35.7% 14.3%
7.1% 57.2% 35.7%
7.1% 78.6% 14.3%
71.4% 28.6% 0%
92.9% 0% 7.1%
57.1% 28.6% 14.3%
28.6% 7.1% 64.3%
85.7% 0% 14.3%
85.7% 0% 14.3%
92.9% 7.1% 0%
78.6% 14.3% 7.1%
57.1% 35.8% 0%
50% 42.9% 7.1%
GEN, Gentamicin; AMK, Amikacin; TOB, Tobramycin; CIP, Ciprofloxacin; LEV, Levofloxacin; MOX, Moxifloxacin; TROVA, Trovafloxacin; IPM, Imipenem; MEM, Meropenem; CAZ, Ceftazidim; CPM, Cefepim; PIP, Piperacillin.
DISCUSSION P. aeruginosa strains isolated from patients with cystic fibrosis over time are often multiply resistant. Emergence of resistance in strains isolated from CF patients has been reported earlier [8]. Current epidemiological data on the prevalence of antibiotic resistant P. aeruginosa infecting CF patients in European countries other than Italy [9] and Denmark [7] are rare. Of the aminoglycosides, the P. aeruginosa isolates investigated here were most susceptible to tobramycin, followed by gentamicin and amikacin. Shawar et al. [10] reported that tobramycin has good activity, even against multiply resistant P. aeruginosa strains. Among the quinolones ciprofloxacin showed the highest activity, followed by trovafloxacin, levofloxacin and moxifloxacin. Other authors reported similar results [11,12]. In P. aeruginosa strains isolated from CF the effluxsystem MexC-MexD-OprJ and MexE-MexF-OprN are the dominant mechanisms of resistance [13] to fluoroquinolones. According to previous studies [14], the carbapenems have excellent activities, and meropenem is about five times more active than imipenem. The higher activity of meropenem could be related to the lower induction of chromosomal beta-lactamase, its ability to cross the outer membrane through the OprD-Porin and an additional channel, and the additional target protein PBP3 [15–17].
With increasing time of colonization with P. aeruginosa, the MICs increased, especially for aminoglycosides and the quinolones. It seemed that the susceptibility of the clones remained relatively constant during the first years of colonization. In our study resistant P. aeruginosa isolates were predominantly found in patients who were colonized for more than 2 years. A correlation between the number of courses of parenteral antibiotic therapy and the development of resistance could not be verified. Carmeli et al. [18] suggested that treatment with ceftazidime is associated with a relatively low risk of subsequent emergence of resistance in P. aeruginosa, as compared to other substances such as imipenem. This is in accordance with our results. The low extent of development of resistance to the carbapenems in our study may be a result of the rare use of these substances. There is no difference in resistance induction by either elective or symptomatic parenteral antibiotic therapy, as found by Elborn et al. [19]. A correlation between the number of courses of oral ciprofloxacin and the development of resistance could not be shown in the study presented here, possibly owing to the small sample size. Intermittent inhalation of aminoglycoside antibiotics improves the pulmonary function and decreases the density of P. aeruginosa in sputum [20]. Our results suggest that long-term inhalation of aminoglycosides may lead to an accumulation
ß 2003 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, CMI, 9, 370–379
GEN, Gentamicin; AMK, Amikacin; TOB, Tobramycin; CIP, Ciprofloxacin; LEV, Levofloxacin; MOX, Moxifloxacin; TROVA, Trovafloxacin; IPM, Imipenem; MEM, Meropenem; CAZ, Ceftazidim; CPM, Cefepim; PIP, Piperacillin; GM, geometric mean value
0% 0% 7.7% 12.5% 11.1% 0% 0% 20% 7.7% 18.8% 22.2% 42.3% 100% 80% 84.6% 68.8% 66.7% 57.7% 0% 10% 15.4% 18.8% 27.8% 30.8% 41% 80% 76.9% 56.3% 38.9% 50% 0% 20% 15.4% 31.3% 27.8% 38.5% 5g (n¼22) 10g (n¼10) 15g (n¼13) 20g (n¼16) 25g (n¼18) >30g (n¼26)
41% 10% 0% 25% 22.2% 19.2%
59% 70% 84.6% 43.8% 50% 42.3%
59% 10% 7.7% 25% 33.3% 19.2
resistant intermediate sensitive resistant Amount of inhaled gentamicin (g)
sensitive
intermediate
sensitive
intermediate
resistant
TOB AMK GEN
Table 5 MICs (mg/l) for the P. aeruginosa clones with 38 different PFGE patterns (¼38 isolates) and geometric mean values (GM) at the beginning of the observation; light grey, sensitive; middle grey, intermediate; dark grey, resistant.
378 Clinical Microbiology and Infection, Volume 9 Number 5, May 2003
of resistant strains. However, it has to be taken into account that the children also received aminoglycosides parenterally. Burns et al. [21] also noted an increase of MICs during chronic intermittent inhalation. The dominant mechanism of aminoglycoside resistance of P. aeruginosa isolated from cystic fibrosis patients is reduced permeability [22]. The concentration of inhaled drugs can be many times higher than systemic concentrations [19]. Inhaled antibiotic can lead to clinical improvement when the P. aeruginosa isolates are above the threshold of susceptibility for systemic application [20]. As parenteral antibiotic therapy, consisting as a rule of a betalactam and an aminoglycoside, still constitutes an important component in the management of P. aeruginosa infections in CF patients, consideration of how to avoid the accumulation of resistant strains is needed. Further studies are necessary in order to investigate the influence of inhalation therapy on the development of resistance. In summary, our results indicate that a moderate increase of MICs for P. aeruginosa of several therapeutically valuable compounds is detectable over time. This increase affected least of all ceftazidime, imipenem and meropenem. Long-term inhalation of aminoglycosides may lead to an increase of resistant isolates of P. aeruginosa, especially with regard to amikacin and gentamicin, but less distinctly against tobramycin. It may be advisable to overcome these trends by using suitable antibiotic combinations and by introducing new and innovative substances in the treatment of patients with CF. ACKNOWLEDGEMENTS This paper was partially presented as a poster at the 9th ECCMID, Berlin, March 21–24, 1999 (abstract number P588).
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23.
fibrosis patients. Antimicrob Agents Chemother 2000; 44: 710–2. Spencker F-B. Antibakterielle Aktivita¨ t von Meropenem in Vergleich mit sieben anderen Antibiotika gegenu¨ ber Sta¨ mmen von Pseudomonas aeruginosa, Stenotrophomonas maltophilia und Burkholderia cepacia isoliert von Kindern mit Mukoviszidose. Z Antimikr Antineoplast Chemother 1997; 15: 111–4. Ballestro S, Fernandez-Rodriguez A, Villaverde R, Escobar H, Perez-Diaz JC, Baquero F. Carbapenem resistance in Pseudomonas aeruginosa from cystic fibrosis patients. J Antimicrob Chemother 1996; 38: 39–45. Chen HY, Livermore DM. In vitro activity of biapenem, compared with imipenem and meropenem, against Pseudomonas aeruginosa strains and mutants with known resistance mechanisms. J Antimicrob Chemother 1994; 33: 949–58. Bonfiglio G, Marchetti F. In vitro activity of ceftazidime, cefepime and imipenem on 1,005 Pseudomonas aeruginosa clinical isolates either susceptible or resistant to beta-lactams. Chemotherapy 2000; 46: 229–34. Carmeli Y, Troillet N, Eliopoulos GM, Samore MH. Emergence of antibiotic-resistant Pseudomonas aeruginosa: Comparison of risks associated with different antipseudomonal agents. Antimicrob Agents Chemother 1999; 43: 1379–82. Elborn JS, Prescott RJ, Stack BHR, et al. Elective versus symptomatic treatment in cystic fibrosis patients with chronic Pseudomonas infection of the lungs. Thorax 2000; 55: 355–8. Ramsey BW, Pepe MS, Quan JM, et al. Intermittent administration of inhaled Tobramycin in patients with cystic fibrosis. N Engl J Med 1999; 340: 23–30. Burns JL, van Dalfsen JM, Shawar RM, et al. Effect of chronic intermittent administration of inhaled tobramycin on respiratory microbial flora in patients with cystic fibrosis. J Infect Dis 1999; 179: 1190–6. MacLeod DL, Nelson LE, Shawar RM, et al. Aminoglycoside-resistance mechanisms for cystic fibrosis Pseudomonas aeruginosa isolates are unchanged by long-term, intermittent, inhaled Tobramycin treatment. J Infect Dis 2000; 181: 1180–4.
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Objective Determination of the extent of changes in quantitative resistance in Pseudo-
monas aeruginosa isolates from patients with cystic fibrosis over a period of approximately 2 years. Methods Three hundred and ninety nine isolates of P. aeruginosa collected from 34 pediatric patients in the period between April 1994 and April 1996 were investigated. During the 2 years the children were treated with a combination of a betalactam and an aminoglycoside, approximately every 3 months. In between they received ciprofloxacin orally, when required. The minimal inhibitory concentrations (MICs) of 38 clones of P. aeruginosa defined by different patterns in macrorestriction analysis (pulse field gel electrophoresis, PFGE) were established for 12 antibiotics: gentamicin, amikacin, tobramycin, ciprofloxacin, levofloxacin, moxifloxacin, trovafloxacin, imipenem, meropenem, ceftazidime, cefepime, and piperacillin by means of broth microdilution tests according to DIN 58940. Results Twenty-four of the 38 clones developed increased MIC values during the time of observation especially for aminoglycosides and quinolones. Comparatively less affected were ceftazidime, imipenem and meropenem. An association between the number of the intravenous treatment courses and the increase of the MIC values could not be verified. Conclusions A trend towards an increase of the MICs against antipseudomonal agents was observed over a limited period of time. It is necessary to prevent this development possibly by employing suitable combinations of antibiotics and the introduction of new substances. Keywords Pseudomonas aeruginosa, antibiotic resistance, minimal inhibitory concentrations (MICs), cystic fibrosis Accepted 30 April 2002
Clin Microbiol Infect 2003; 9: 370–379
INTRODUCTION Cystic fibrosis (CF) is an autosomal recessive, multisystem disease with Pseudomonas aeruginosa as the major prognostic factor in chronic respiratory infection [1]. Once colonization is Corresponding author and reprints request: Prof Dr med F.B. Spencker, Institute of Medical Microbiology and Epidemiology of Infectious Diseases, University of Leipzig, 04103 Leipzig, Liebigstr. 24, Germany Tel: þ49 341 9715204 Fax: þ49 341 9715209 E-mail: [email protected]
established the eradication of this pathogen is almost impossible owing to the nature of cystic fibrosis itself and the appearance of mucoid phenotypes of P. aeruginosa forming microcolonies on the mucosal surfaces which are surrounded by an abundant mass of the microbial exopolysaccharide alginate [2,3]. This unique bacterial product may present a polyanionic barrier to antimicrobial agents so that their action is prevented. As a result of the repeated use of antimicrobial agents in the management of cystic fibrosis, there may be a subsequent emergence of resistant
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>500
SUBJECTS AND METHODS
2.7732 (0.59; L3.03) 6.6659 (0.93; 17.46) 0.6942 (0.28; 1.75) 0.2355 (0.09; 0.64) 0.5781 (0.2; 1.67) 1.7281 (0.52; 5.78) 0.2731 (0.08; 0.91) 1.0565 (0.47; 2.38) 0.2152 (0.07; 0.67) 1.667 (0.6; 4.66) 1.9997 (0.76; 5.26) 3.7062 (0.8; 17.18)
P. aeruginosa [8]. The objective of this study was to investigate the gradual development of in vitro antibiotic resistance over a period of 2 years in P. aeruginosa isolates from CF patients and to determine changes in relation to the way the antimicrobial agents used were applied.
1
GM (standard deviation) (mg/L)
Spencker et al Development of resistance of pseudomonas aeruginosa in CF 371
1
64 2
>32
2 9
1
5 9
1
3 2 3
6
1 1 1
2
32 16 >8
1 6
2 2 8
8 13 3 1 3 9 2 2 8 5 2 2 2 8 1 11 11
6
2
13
8 2
1 3
GEN AMK TOB CIP LEV MOX TROVA IPM MEM CAZ CPM PIP
1
4 1
1 9 3
4 15 8 2 4 4 3
21 5 12 5 12 7 13 7
13 1 6 1 8 7 3 13 3 11 10 3 2
14 19 3
8 1 0.063 0.031
Table 1 Patient demographics
0.125
0.25
0.5
2
4
>4
Bacteriologic methods Sputum samples or deep-throat swab specimens were collected before and after the antibiotic treatment. Isolated strains were assessed by morphologic features, including pigment production, and identified as P. aeruginosa by a positive oxidase reaction and appropriate results with the API-20 NE system (Bio-Me´ rieux, Nu¨ rtingen, Germany). The strains were stored in special vials (Microbank, Mast, Liverpool, UK) at 208. The P. aeruginosa strains were analyzed for different pyocin types by the spotting method as described by Fyfe et al. [4] and pulse field gel electrophoresis (PFGE) patterns by contourclamped homogeneous electric field electrophoresis as described earlier [5]. Genomic DNA was subjected to the rare-cutting restriction enzyme SpeI. Twenty-two patients were chronically colonized with strains with the same PFGE pattern, seven were transiently colonized with two or more different types and five were permanently colonized with two different types in the observed period (details have been published elsewhere) [5]. As a result of the variable number of colonizing strains we investigated 38 P. aeruginosa clones with
2
>64
500
Isolates of P. aeruginosa, 399 in number, collected from 34 children, who regularly attended the CF center at the Department of Pediatrics of the University of Leipzig between April 1994 and April 1996, were investigated. Most of the patients were hospitalized either for regular 2-week courses or for the treatment of acute exacerbations as a rule with an intravenous combination of ceftazidime and gentamicin. Altogether there were 83 intravenous treatment courses in the period of investigation, for details see Figure 1. When deemed necessary the children received oral ciprofloxacin in between and nebulized aminoglycosides for inhalation. The patients were followed up in the outpatient clinic of the above mentioned department.
1
Patients
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GEN
AMK
n-muc (n¼122) 5.6746 11.5075 (0.97; 33.25) (4.1; 32.3) Muc (n¼277) 3.1677 7.4038 (0.78; 12.79) (2.99; 18.36) P-value 0.0001 0.0001
TOB
CIP
LEV
MOX
TROVA
IPM
MEM
CAZ
CPM
PIP
0.9493 (0.32; 2.78) 0.6811 (1.5; 0.31) 0.006
0.2997 (0.09; 0.97) 0.3237 (0.1; 1.07) 0.697
0.6859 (0.19; 2.5) 0.7945 (0.22; 2.83) 0.278
2.4181 (0.76; 7.67) 2.4473 (6.84; 0.88) 0.803
0.3116 (0.07; 1.43) 0.386 (0.08; 1.81) 0.185
0.7364 (0.27; 1.99) 1.1275 (2.89; 0.44) 0.0001
0.1977 (0.06; 0.65) 0.2157 (0.72; 0.06) 0.268
1.3979 (0.34; 5.73) 1.4681 (0.55; 3.93) 0.090
2.3655 (0.73; 7.68) 2.4968 (0.97; 6.44) 0.842
3.3368 (0.49; 8.32) 3.7062 (0.86; 18.8) 0.119
n-muc, non-mucoid phenotype; muc, mucoid phenotype; P-value, Mann–Whitney U-test; GEN, Gentamicin; AMK, Amikacin; TOB, Tobramycin; CIP, Ciprofloxacin; LEV, Levofloxacin; MOX, Moxifloxacin; TROVA, Trovafloxacin; IPM, Imipenem; MEM, Meropenem; CAZ, Ceftazidim; CPM, Cefepim; PIP, Piperacillin.
Table 3 MICs (mg/L), geometric mean, standard deviation in brackets, of 38 P. aeruginosa clones at the end of observation in relation to the duration of colonization in April 96.
G1 G2 G3 G4 P-value
GEN
AMK
TOB
CIP
LEV
MOX
TROVA
IPM
1.68 (4.8; 0.6) 1.68 (7.3; 0.4) 1.99 (4.4; 0.9) 27.97 (328.9; 2.4) 0.0056
5.66 (11.3; 2.8) 5.19 (25.9; 1.0) 5.79 (14.8; 2.3) 19.34 (51.8; 7.2) 0.033
0.71 (1.4; 0.4) 0.71 (2.3; 0.2) 0.57 (1.2; 0.3) 1.76 (6.3; 0.5) 0.071
0.18 (0.3; 0.1) 0.23 (0.8; 0.1) 0.16 (0.8; 0.0) 0.53 (2.9; 0.1) 0.262
0.42 (1.0; 0.2) 0.65 (2.5; 0.2) 0.38 (1.7; 0.1) 1.29 (5. 6; 0.3) 0.256
1.41 (2.8; 0.7) 1.41 (8.1; 0.2) 1.45 (5.8; 0.4) 3.99 (9.6; 1.7) 0.178
0.21 (0.5; 0.1) 0.29 (1.3; 0.1) 0.21 (1.2; 0.0) 0.78 (4.2; 0.2) 0.218
1.42 (2.1; 0.71 (2.6; 0.69 (1.9; 1.07 (3.5; 0.3
0.9) 0.2) 0.2) 0.3)
MEM
CAZ
CPM
PIP
0.18 (0.6; 0.1) 0.18 (0.4; 0.1) 0.17 (0.6; 0.1) 0.21 (1.1; 0.0) 0.835
1.68 (3.27; 0.86) 1.54 (2.58; 0.92) 1.32 (4.99; 0.35) 1.99 (10.61; 0.38) 0.65
1.41 (2.1; 0.9) 1.99 (4.6; 0.9) 1.59 (5.8; 0.4) 5.14 (15.2; 1.7) 0.056
2.38 (14.7; 4.76 (11.8; 2.76 (16.9; 3.75 (26.3; 0.623
0.4) 1.9) 0.5) 0.5)
GEN, Gentamicin; AMK, Amikacin; TOB, Tobramycin; CIP, Ciprofloxacin; LEV, Levofloxacin; MOX, Moxifloxacin; TROVA, Trovafloxacin; IPM, Imipenem; MEM, Meropenem; CAZ, Ceftazidim; CPM, Cefepim; PIP, Piperacillin. Group 1 (G1) 0.5 years, n¼4; group 2 (G2): 0.5 – 2 years, n¼8; group 3 (G3): >2– 4 years, n¼14; group 4 (G4): >4 years, n¼12)
372 Clinical Microbiology and Infection, Volume 9 Number 5, May 2003
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Table 2 Comparison of geometric mean (GM) values of the MICs (mg/L), standard deviation in brackets, between phenotypic non mucoid and mucoid P. aeruginosa isolate
Spencker et al Development of resistance of pseudomonas aeruginosa in CF 373
Figure 1 Antibiotics and combinations of antibiotics used in 83 episodes of i.v. therapy.
Figure 2 MICs of ceftazidime and moxifloxacin against Pseudomonas aeruginosa clones with 38 PFGE-patterns at the beginning of the investigation (t ¼ 0; 38 isolates), after 1 year (t ¼ 1 year; 31 isolates) and after 2 years (t ¼ 2 years; 19 isolates).
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374 Clinical Microbiology and Infection, Volume 9 Number 5, May 2003
Figure 3 MICs of ciprofloxacin and levofloxacin against Pseudomonas aeruginosa clones with 38 PFGE patterns at the beginning of the investigation (t ¼ 0; 38 isolates), after 1 year (t ¼ 1 year; 31 isolates) and after 2 years (t ¼ 2 years; 19 isolates).
different PFGE patterns. Two to 31 isolates per PFGE-pattern were isolated. Minimal inhibitory concentration (MIC) The MIC is defined as the lowest concentration of an antimicrobial agent that prevents visible growth of bacteria [6]. The MICs of 38 strains were established for 12 antibiotics: gentamicin (Serva, Heidelberg, Germany), amikacin (Bristol-Myers Squibb, Munich, Germany), tobramycin (Sigma, Munich, Germany), ciprofloxacin (Bayer, Leuerkusen, Germany), levofloxacin (Daiichi, Aventis, Bad Soden, Germany), moxifloxacin (Bayer), trovafloxacin (Pfizer, Karlsruhe, Germany), imipenem (Merck Sharp & Dohme, Haar, Germany), meropenem (AstraZeneca, Wedel, Germany), ceftazidime (GlaxoSmithKline, Munich, Germany), cefepime (Bristol) and piperacillin
(Lederle, Mu¨ nster, Germany) by means of broth microdilution tests according to DIN 58940 [6]. The interpretative categories with regard to the breakpoints were fixed [6] as follows: gentamicin, tobramycin 1 mg/mL (s), 2–4 mg/ mL (i), 8 mg/mL (r); amikacin 4 mg/mL (s), 8–16 mg/mL (i), 32 mg/mL (r); ciprofloxacin, levofloxacin, moxifloxacin, trovafloxacin 1 mg/mL (s), 2 mg/mL (i), 4 mg/ mL (r); imipenem ¼ 2 mg/mL (s), 4 mg/mL (i), ¼ 8 mg/ mL (r); meropenem ¼ 2 mg/mL (s), 4–8 mg/mL (i), ¼ 16 mg/mL (r); ceftazidime, cefepime ¼ 4 mg/mL (s), 8–16 mg/ mL (i), ¼ 32 mg/mL (r); piperacillin ¼ 4 mg/mL (s), 8–32 mg/mL(i), ¼ 64 mg/mL (r).
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Spencker et al Development of resistance of pseudomonas aeruginosa in CF 375
Figure 4 MICs of gentamicin and tobramycin against Pseudomonas aeruginosa clones with 38 PFGE patterns at the beginning of the investigation (t ¼ 0; 38 isolates), after 1 year (t ¼ 1 year; 31 isolates) and after 2 years (t ¼ 2 years; 19 isolates).
RESULTS Table 1 shows the susceptibility of the 38 P. aeruginosa clones at the beginning of the observation. When the geometric means were compared, tobramycin showed about four times lower MICs than gentamicin and about nine times lower MICs than amikacin. Among the quinolones, ciprofloxacin and trovafloxacin possessed the lowest MIC values. They were two times lower than levofloxacin and seven times lower than moxifloxacin. Cefepime was somewhat more active than ceftazidime. The geometric mean MIC of meropenem showed the lowest value, being about five times lower than that of imipenem. The number of samples with the typical mucoid colony morphology (n ¼ 277) was greater than those without alginate production (n ¼ 122).
The mucoid isolates were, except for the aminoglycosides, less susceptible to the anti-pseudomonal agents, but significance was only achieved by imipenem (Table 2). The mucoid isolates were significantly more susceptible to gentamicin, tobramycin, and amikacin (the Mann– Whitney U-test). The mean time the patients were colonized with P. aeruginosa was 3.8 years in April 1996. Four patients were colonized for less than 0.5 years, eight patients for 0.5–2 years, 14 patients for 2– 4 years and eight patients for more than 4 years. The length of colonization correlated with increasing MIC values, especially for aminoglycosides and quinolones, a statistical significance was reached for gentamicin and amikacin (Kruskal– Wallis one-way test). Isolates from patients colonized for more than 4 years accounted for this
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376 Clinical Microbiology and Infection, Volume 9 Number 5, May 2003
Figure 5 MICs of meropenem and imipenem against Pseudomonas aeruginosa clones with 38 PFGE patterns at the beginning of the investigation (t ¼ 0; 38 isolates), after 1 year (t ¼ 1 year; 31 isolates) and after 2 years (t ¼ 2 years; 19 isolates).
statistical significance (Mann–Whitney-Test, a ¼ 0.05, P <0.0083) (Table 3). During the study period 24 clones showed an increase of MIC values that resulted in alterations of the qualitative susceptibility (from susceptible to intermediate, from susceptible to resistant and from intermediate to resistant), at least against one of the investigated antibiotics. The extent of the development of resistance differed for the investigated antibiotics. Ceftazidime, imipenem and meropenem were less affected than the other substances. Figures 2–5 show, in eight representative examples, the gradual development of resistance during the observed time. The MICs (geometric mean) did not change the qualitative interpretative categories except for levofloxacin (sensitive to intermediate) and moxifloxacin (intermediate to resistant). There were no correlations between the
number of parenteral antibiotic treatment courses or the number of ciprofloxacin applications, respectively, and the development of resistance in 14 clones from 12 patients with at least four parenteral antibiotic courses, and ten clones from nine patients with at least four ciprofloxacin applications (Chi2 Test), respectively (Table 4). The proportion of strains resistant to gentamicin and amikacin increased in proportion to the amount of inhaled gentamicin (Chi2 Test, gentamicin P ¼ 0.0229; amikacin P ¼ 0.01; tobramycin P >0.05, Table 5). For parenteral treatment the patients received an intravenous combination, as a rule consisting of ceftazidime (150 mg/kg/day) and gentamicin (9 mg/kg/day). In between the children were treated with long-term nebulized gentamicin (80 mg/day) as a rule and, when necessary, oral ciprofloxacin for 2 weeks (40 mg/kg/ day).
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Spencker et al Development of resistance of pseudomonas aeruginosa in CF 377
Table 4 Proportion (%) of sensitive, intermediate and resistant P. aeruginosa strains with 14 different PFGE patterns (¼ 14 isolates pro cycle) in relation to the number of parenteral antibiotic courses Number of i.v. therapys 1 sensitive intermediate resistant 2 sensitive intermediate resistant 3 sensitive intermediate resistant 4 sensitive intermediate resistant
GEN
AMK
TOB
CIP
LEV
MOX
TROVA
IPM
MEM
CAZ
CPM
PIP
14.3% 71.4% 14.3%
28.6% 57.1% 14.3%
85.7% 7.1% 7.1%
85.7% 7.1% 7.1%
71.4% 14.3% 14.3%
35.7% 14.3% 50%
85.7% 0% 14.3%
92.9% 0% 7.1%
100% 0% 0%
78.6% 14.3% 7.1%
78.6% 14.3% 7.1%
42.9% 50% 7.1%
14.3% 50% 35.7%
21.4% 57.2% 21.4%
57.1% 35.8% 7.1%
71.4% 21.5% 7.1%
64.3% 14.3% 21.4%
21.4% 28.6% 50%
71.4% 7.2% 21.4%
64.3% 28.6% 7.1%
92.9% 7.1% 0%
71.4% 28.6% 0%
64.3% 21.4% 14.3%
50% 35.7% 14.3%
14.3% 57.1% 28.6%
21.4% 57.2% 21.4%
71.4% 21.5% 7.1%
71.4 21.5% 7.1%
64.3% 14.3% 21.4%
28.6% 35.7% 35.7%
71.4% 7.2% 21.4%
57.1% 28.6% 14.3%
92.9% 7.1% 0%
71.4% 21.5% 7.1%
57.1% 35.8% 7.1%
50% 35.7% 14.3%
7.1% 57.2% 35.7%
7.1% 78.6% 14.3%
71.4% 28.6% 0%
92.9% 0% 7.1%
57.1% 28.6% 14.3%
28.6% 7.1% 64.3%
85.7% 0% 14.3%
85.7% 0% 14.3%
92.9% 7.1% 0%
78.6% 14.3% 7.1%
57.1% 35.8% 0%
50% 42.9% 7.1%
GEN, Gentamicin; AMK, Amikacin; TOB, Tobramycin; CIP, Ciprofloxacin; LEV, Levofloxacin; MOX, Moxifloxacin; TROVA, Trovafloxacin; IPM, Imipenem; MEM, Meropenem; CAZ, Ceftazidim; CPM, Cefepim; PIP, Piperacillin.
DISCUSSION P. aeruginosa strains isolated from patients with cystic fibrosis over time are often multiply resistant. Emergence of resistance in strains isolated from CF patients has been reported earlier [8]. Current epidemiological data on the prevalence of antibiotic resistant P. aeruginosa infecting CF patients in European countries other than Italy [9] and Denmark [7] are rare. Of the aminoglycosides, the P. aeruginosa isolates investigated here were most susceptible to tobramycin, followed by gentamicin and amikacin. Shawar et al. [10] reported that tobramycin has good activity, even against multiply resistant P. aeruginosa strains. Among the quinolones ciprofloxacin showed the highest activity, followed by trovafloxacin, levofloxacin and moxifloxacin. Other authors reported similar results [11,12]. In P. aeruginosa strains isolated from CF the effluxsystem MexC-MexD-OprJ and MexE-MexF-OprN are the dominant mechanisms of resistance [13] to fluoroquinolones. According to previous studies [14], the carbapenems have excellent activities, and meropenem is about five times more active than imipenem. The higher activity of meropenem could be related to the lower induction of chromosomal beta-lactamase, its ability to cross the outer membrane through the OprD-Porin and an additional channel, and the additional target protein PBP3 [15–17].
With increasing time of colonization with P. aeruginosa, the MICs increased, especially for aminoglycosides and the quinolones. It seemed that the susceptibility of the clones remained relatively constant during the first years of colonization. In our study resistant P. aeruginosa isolates were predominantly found in patients who were colonized for more than 2 years. A correlation between the number of courses of parenteral antibiotic therapy and the development of resistance could not be verified. Carmeli et al. [18] suggested that treatment with ceftazidime is associated with a relatively low risk of subsequent emergence of resistance in P. aeruginosa, as compared to other substances such as imipenem. This is in accordance with our results. The low extent of development of resistance to the carbapenems in our study may be a result of the rare use of these substances. There is no difference in resistance induction by either elective or symptomatic parenteral antibiotic therapy, as found by Elborn et al. [19]. A correlation between the number of courses of oral ciprofloxacin and the development of resistance could not be shown in the study presented here, possibly owing to the small sample size. Intermittent inhalation of aminoglycoside antibiotics improves the pulmonary function and decreases the density of P. aeruginosa in sputum [20]. Our results suggest that long-term inhalation of aminoglycosides may lead to an accumulation
ß 2003 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, CMI, 9, 370–379
GEN, Gentamicin; AMK, Amikacin; TOB, Tobramycin; CIP, Ciprofloxacin; LEV, Levofloxacin; MOX, Moxifloxacin; TROVA, Trovafloxacin; IPM, Imipenem; MEM, Meropenem; CAZ, Ceftazidim; CPM, Cefepim; PIP, Piperacillin; GM, geometric mean value
0% 0% 7.7% 12.5% 11.1% 0% 0% 20% 7.7% 18.8% 22.2% 42.3% 100% 80% 84.6% 68.8% 66.7% 57.7% 0% 10% 15.4% 18.8% 27.8% 30.8% 41% 80% 76.9% 56.3% 38.9% 50% 0% 20% 15.4% 31.3% 27.8% 38.5% 5g (n¼22) 10g (n¼10) 15g (n¼13) 20g (n¼16) 25g (n¼18) >30g (n¼26)
41% 10% 0% 25% 22.2% 19.2%
59% 70% 84.6% 43.8% 50% 42.3%
59% 10% 7.7% 25% 33.3% 19.2
resistant intermediate sensitive resistant Amount of inhaled gentamicin (g)
sensitive
intermediate
sensitive
intermediate
resistant
TOB AMK GEN
Table 5 MICs (mg/l) for the P. aeruginosa clones with 38 different PFGE patterns (¼38 isolates) and geometric mean values (GM) at the beginning of the observation; light grey, sensitive; middle grey, intermediate; dark grey, resistant.
378 Clinical Microbiology and Infection, Volume 9 Number 5, May 2003
of resistant strains. However, it has to be taken into account that the children also received aminoglycosides parenterally. Burns et al. [21] also noted an increase of MICs during chronic intermittent inhalation. The dominant mechanism of aminoglycoside resistance of P. aeruginosa isolated from cystic fibrosis patients is reduced permeability [22]. The concentration of inhaled drugs can be many times higher than systemic concentrations [19]. Inhaled antibiotic can lead to clinical improvement when the P. aeruginosa isolates are above the threshold of susceptibility for systemic application [20]. As parenteral antibiotic therapy, consisting as a rule of a betalactam and an aminoglycoside, still constitutes an important component in the management of P. aeruginosa infections in CF patients, consideration of how to avoid the accumulation of resistant strains is needed. Further studies are necessary in order to investigate the influence of inhalation therapy on the development of resistance. In summary, our results indicate that a moderate increase of MICs for P. aeruginosa of several therapeutically valuable compounds is detectable over time. This increase affected least of all ceftazidime, imipenem and meropenem. Long-term inhalation of aminoglycosides may lead to an increase of resistant isolates of P. aeruginosa, especially with regard to amikacin and gentamicin, but less distinctly against tobramycin. It may be advisable to overcome these trends by using suitable antibiotic combinations and by introducing new and innovative substances in the treatment of patients with CF. ACKNOWLEDGEMENTS This paper was partially presented as a poster at the 9th ECCMID, Berlin, March 21–24, 1999 (abstract number P588).
REFERENCES 1. Kerem BS, Rommens JM, Buchanan JA, et al. Identification of the cystic fibrosis gene: genetic analysis. Science 1989; 245: 1073–80. 2. Govan JRW, Deretic V. Microbial pathogenesis in cystic fibrosis: mucoid. Pseudomonas Aeruginosa Burkholderia Cepacia Microbiol Rev 1996; 60: 539–74. 3. Pedersen SS, Høiby N, Espersen F, Kock C. Role of alginate in infection with mucoid Pseudomonas aeruginosa in cystic fibrosis. Thorax 1992; 47: 6–13.
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