Antimicrobial Resistance of Streptococcus pneumoniae Isolates of ...

Microbiology Chemotherapy 2004;50:184–189 DOI: 10.1159/000080692

Received: June 3, 2003 Accepted after revision: January 4, 2004 Published online: September 3, 2004

Antimicrobial Resistance of Streptococcus pneumoniae Isolates of Outpatients in Germany, 1999–2000

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ANY DISTRIBUTION OF THIS ARTICLE WITHOUT WRITTEN CONSENT FROM S. KARGER Ralf René Reinert a Rudolf Lütticken a Susanne Reinert c Adnan Al-Lahham a AG, BASEL IS A VIOLATION OF THE COPYRIGHT. Sebastian Lemmen b a Institute

of Medical Microbiology, National Reference Center for Streptococci and b Department of Infection Control, University Hospital, Aachen, and c SR Medical Communications GmbH, Frechen, Germany

Key Words Streptococcus pneumoniae W Macrolide W Resistance W Germany W Telithromycin W Serotype

Abstract Background: The aim of the study was to determine the prevalence of antimicrobial resistance among clinical isolates of Streptococcus pneumoniae during the winter of 1999–2000 in Germany. Methods: Pneumococcal isolates were prospectively collected by 14 different clinical microbiology laboratories. Minimal inhibitory concentrations of penicillin G, erythromycin A, clarithromycin, roxithromycin, azithromycin, clindamycin, levofloxacin and telithromycin were determined by the broth microdilution method. Results: Among 328 strains 4.6% were nonsusceptible to penicillin G (intermediate and resistant strains) and 9.5% were resistant to erythromycin A. Analysis of erythromycin-resistant strains for the underlying

This study was presented in part at the 40th Interscience Conference on Antimicrobial Agents and Chemotherapy, Toronto, Canada (abstract 693).

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resistance determinants revealed that 12 (38.7%) belonged to the erm(B) and 19 (61.3%) to the mef(E) type of resistance. Among the macrolide-resistant strains, serotypes 19F (n = 9) and 14 (n = 8) were the predominant types. Conclusions: Macrolide resistance in Germany is of growing concern and mainly due to the high prevalence of pneumococci expressing the mef(E) type of resistance. Copyright © 2004 S. Karger AG, Basel

Introduction

Streptococcus pneumoniae continues to be a significant cause of morbidity and mortality in humans [1]. The worldwide increase in antibiotic resistance in these species has become a serious infectious disease problem within the last 20 years [2]. Despite the emergence of this problem in many parts of Europe, e.g. Spain [3], and France [4], penicillin resistance has remained uncommon among clinical isolates of S. pneumoniae in Germany. In contrast, decreased susceptibility to macrolides in S. pneumoniae has been increasingly reported in Germany [5, 6].

R.R. Reinert Institute for Medical Microbiology, National Reference Center for Streptococci University Hospital, Pauwelsstrasse 30 DE–52057 Aachen (Germany) Tel. +49 241 8089787, Fax +49 241 8082483, E-Mail [email protected]

Telithromycin (HMR 3647) is the first of a novel family of antimicrobials, the ketolides, developed specifically for the treatment of community-acquired respiratory tract infections. Ketolides are characterized by a ketone group, which replaces the cladinose sugar at position 3 of the macrolactone ring [7]. The aim of the current multicenter study was to determine the prevalence of antimicrobial resistance among clinical isolates of S. pneumoniae during the winter of 1999–2000. Sampling was performed prospectively to provide large numbers of consecutive isolates from community-acquired infections representative of different geographic areas. In addition, the study was aimed at identification of macrolide resistance mechanisms and of the serotype distribution in Germany. Material and Methods Study Design A total of 328 isolates of S. pneumoniae were collected from 14 different medical microbiology laboratories between November 1999 and April 2000 in Germany. All participating laboratories were asked to send up to 30 consecutive pneumococcal strains. All strains included in the study were isolated from infections of nonhospitalized patients. Only pneumococcal isolates of at least probable clinical significance were included. Strains were sent immediately in a transport medium (Port-A-Cul; Difco, Germany) to the National Reference Center for Streptococci for species identification and susceptibility testing. Confirmation of S. pneumoniae strains was performed by optochin sensitivity and bile solubility testing. Susceptibility Testing Minimal inhibitory concentration (MIC) testing was performed using the broth microdilution method as recommended by the National Committee for Clinical Laboratory Standards (NCCLS) [8]. Commercially manufactured microtiter plates (Micronaut-S; Merlin Diagnostics, Bornheim, Germany) containing penicillin G, erythromycin A, clarithromycin, roxithromycin, azithromycin, clindamycin, levofloxacin and telithromycin with cation-adjusted MuellerHinton broth (Oxoid, Wesel, Germany) plus 5% lysed horse blood (Oxoid) were used. The final inoculum was 5 ! 105 CFU/ml. MICs were determined following 35 ° C incubation for 24 h in ambient air. S. pneumoniae ATCC 49619 was used as a control strain. Current NCCLS interpretive criteria were used to define antimicrobial resistance. Isolates were stored at –70 ° C on porous beads (Microbank; Mast Diagnostics, Rheinfeld, Germany). PCR Experiments PCR was performed as described previously [5]. For the detection of erm(A) and mef(E) the primers described by Trieu-Cuot et al. [9] and by Tait-Kamradt et al. [10] with the sequences [erm(B)] 5) CGA GTG AAA AAG TAC TCA ACC 3) (position: 362–382), 5) GGC GTG TTT CAT TGC TTG ATG 3) (position 978–958) and the sequences [mef(E)] 5) AGT ATC ATT AAT CAC TAG TGC 3) (position: 57–77), 5) GTA ATA GAT GCA ATC ACA GC 3) (position 551–532) were chosen.

Pneumococcal Resistance in Germany

Pneumococcal strains were serotyped by Neufeld’s Quellung reaction using type and factor sera provided by the Statens Serum Institut, Copenhagen, Denmark.

Results

A total of 328 isolates were collected by 14 centers. Strains were isolated from the following sources: nasopharyngeal swabs from patients with respiratory tract infections (n = 121, 36.9%), bronchial secretions or lavages (n = 62, 18.9%), ear swabs (n = 49, 14.9%), sputum (n = 31, 9.5%), eye swabs (n = 23, 7.0%), blood (n = 21, 6.4%), sinus aspirates (n = 6, 1.8%), pleural puncture (n = 3, 0.9%), paracentesis (n = 2, 0.6%), wounds (n = 5, 1.5%), and normally sterile body sites (n = 2, 0.6%). Data on the source were not available in three cases. Data on age were available for 311 patients. Pneumococci were predominantly isolated from infants and young children ^5 years of age (n = 107, 34.4% of cases). Fifty-two (16.7%) and 152 (48.9%) cases were seen in the age groups 15 to ^16 and 116 years of age, respectively (mean age: 30.4 years, range: !1 month to 88 years). The male:female patient ratio was 58.6:41.4%. Data on antibiotic resistance are presented in table 1. 95.4% of isolates were found to be susceptible to penicillin G (MIC ^0.06 mg/l) and 4.3% to be penicillin-intermediate (MICs ^0.1–1 mg/l). Only one strain was resistant to penicillin G (MIC 62 mg/l). 9.5% of isolates were resistant to erythromycin A. MICs of the reference strain S. pneumoniae ATCC 49619 were consistently within the normal range. MIC distributions are presented in figure 1. At centers where 20 or more isolates were included in the study (n = 13) the combined percentages of intermediate and resistant strains varied between 0 and 16.7% and 0 and 20.8% for penicillin G and erythromycin A, respectively (table 2). Centers were categorized as belonging to one of four geographic regions in Germany: northern Germany (centers: Hamburg, Osnabrück and Plön), central and western Germany (centers: Bonn, Dillingen, Leverkusen, Trier, Weiden and Wülfrath), eastern Germany (centers: Bad Hersfeld and Berlin II) and southern Germany (centers: Augsburg and Stuttgart). The average resistance rates (intermediate and resistant strains) observed for penicillin G and erythromycin A in these regions were: northern Germany: 3.9 and 15.4%, central and western Germany: 7.8 and 8.5%, eastern Germany: 0 and 12.0%, and southern Germany: 1.8 and 1.8%.

Chemotherapy 2004;50:184–189

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Table 1. MIC range, MIC90, and resistance rates of 328 pneumococcal strains isolated in Germany in 1999–2000

Antibiotic

Penicillin G Erythromycin A Clarithromycin Roxithromycinb Azithromycin Clindamycin Levofloxacin Telithromycin

Number of strains found to bea

MIC range mg/l

MIC, mg/l 50%

90%

susceptible

intermediate resistant

^0.016–2 ^0.03–632 ^0.03–632 ^0.03–632 ^0.03–632 ^0.03–632 0.5–8 ^0.03–0.5

^0.016 ^0.03 ^0.03 ^0.03 ^0.06 ^0.03 0.5 ^0.03

^0.03 ^0.5 ^0.5 ^0.5 ^0.5 ^0.125 1 ^0.03

313 (95.4) 295 (89.9) 295 (89.9) 297 (90.5) 298 (90.9) 315 (96.6) 326 (99.4) 328 (100)

14 (4.3) 2 (0.6) 2 (0.6) 0 (0) 0 (0) 1 (0.3) 1 (0.3) 0 (0)

1 (0.3) 31 (9.5) 31 (9.5) 31 (9.5) 30 (9.1) 12 (3.7) 1 (0.3) 0 (0)

Figures in parentheses represent percentages. Breakpoints (I, R) according to NCCLS: penicillin G: 0.1–1 mg/l, 62 mg/l; erythromycin A: 0.5 mg/l, 61 mg/l; clarithromycin: 0.5 mg/l, 61 mg/l; azithromycin: 1 mg/l, 62 mg/l; clindamycin: 0.5 mg/l, 61 mg/l; levofloxacin: 4 mg/l, 68 mg/l; telithromycin: ^1 mg/l, 64 mg/l. b Roxithromycin breakpoints are not NCCLS-approved. The breakpoints ^1 mg/l, 62 mg/l were used (Swedish breakpoints). a

Table 2. Penicillin G and erythromycin A

resistance of pneumococcal isolates in 13 laboratories in Germanya

Town

Bonn Dillingen Leverkusen Trier Weiden Wülfrath Bad Hersfeld Berlin Hamburg Osnabrück Plön Augsburg Stuttgart Total

Regionb

C-W C-W C-W C-W C-W C-W E E N N N S S

Total number of strains

Macrolide-resistant strains

Penicillin G-resistant strains

numberc

numberc

22 24 28 24 20 23 26 24 29 24 25 27 28

1 (4.5) 4 (16.7) 2 (7.1) 2 (8.3) 1 (5.0) 2 (8.7) 3 (11.5) 3 (12.5) 2 (6.9) 5 (20.8) 5 (20.0) 0 (0.0) 1 (3.6)

324

31 (9.5)

difference from 1998–1999d

–16.8 –4.2 –6.7 –0.5 +0.1 –2.6 +10 –2.7 –7.4 +5.1 +6.3 +1.4 –1.2 –0.5

3 (13.6) 1 (4.2) 2 (7.1) 4 (16.7) 0 (0.0) 1 (4.3) 0 (0.0) 0 (0.0) 3 (10.3) 0 (0.0) 0 (0.0) 0 (0.0) 1 (3.6) 15 (4.6)

difference from 1998–1999d

–1.9 –9.8 +1.6 +16.7 –14.7 +1.2 –6.1 –8.7 +4.2 –10.9 –9.2 –3.4 –2.4 –2.0

Figures in parentheses represent percentage. Centers with less than 20 isolates (n = 1) were not evaluated. b Centers were categorized into the following geographic regions: N = northern Germany; C-W = central/western Germany; E = eastern Germany; S = southern Germany. Cumulative resistance data see text. c Breakpoints (I, R) according to NCCLS [8]: penicillin G: 0.1–1 mg/l, 62 mg/l; erythromycin A: 0.5 mg/l, 61mg/l. d Difference in percentage of penicillin G and macrolide resistance calculated compared to 1998–1999 data published by Reinert et al. [5]. Difference in macrolide resistance was calculated by differences in combined rates of clarithromycin-intermediate and clarithromycinresistant strains, as these data were given in the previous publication. a

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Reinert/Lütticken/Reinert/Al-Lahham/ Lemmen

Fig. 1. a MIC distributions for erythromycin, roxithromycin, azithromycin, clarithromycin, clindamycin, and telithromycin against S. pneumoniae (n = 328) isolated in Germany in 1999–2000. b MIC distributions for penicillin G and levofloxacin against S. pneumoniae (n = 328) isolated in Germany in 1999–2000.

Analysis of erythromycin-resistant strains (n = 31) for the underlying resistance determinants revealed that 12 (38.7%) belonged to the erm(B) and 19 (61.3%) to the mef(E) type of resistance, respectively. Among the macrolide-resistant strains, serotypes 19F (n = 9, 29% of macrolide-resistant strains), 14 (n = 8, 25.8%), 6B (n = 6, 19.4%), and 23F (n = 6, 19.4%) were the predominant types (table 3).

Pneumococcal Resistance in Germany

Discussion

In the present study, only 4.3% of strains were found to be penicillin-intermediate and only one isolate was highly penicillin-resistant, underscoring the relatively favorable level of beta-lactam resistance of S. pneumoniae in Germany. In addition, the resistance level of S. pneumoniae in Germany at the moment seems to be stable, and a further increase, which had been feared in the late 1990s [5] and has been observed in some European countries [4], cannot be documented for this country. Our findings are in agree-


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Table 3. Serotype distribution of pneumococcal respiratory tract iso-

lates in Germany in 1999–2000 Serotype

Number of erythromycin A-susceptible strains

Number of erythromycin A-resistant strains

3 4 6A 6B 9N 9V 10A 11A 11C 12F 14 15A 15B 15C 17F 18C 19A 19F 23A 23F 24F Rough

8 (8.7) 1 (1.1) 12 (13.0) 12 (13.0) 3 (3.3) 2 (2.2) 3 (3.3) 6 (6.5) 1 (1.1) 1 (1.1) 1 (1.1) 1 (1.1) 1 (1.1) 1 (1.1) 1 (1.1) 3 (3.3) 1 (1.1) 19 (20.7) 1 (1.1) 11 (12.0) 1 (1.1) 2 (2.2)

0 (0) 0 (0) 1 (3.2) 6 (19.4) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 8 (25.8) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 9 (29.0) 0 (0) 6 (19.4) 0 (0) 1 (3.2)

Total

92 (100.0)

31 (100.0)

Figures in parentheses represent percentages.

ment with those of the PROTEKT study, which reported 6.2 and 2.2% of pneumococcal isolates to be penicillinintermediate and penicillin-resistant in the 1999–2000 winter season in Germany, respectively [5]. In contrast, the level of macrolide resistance is high in Germany, with 9.5% of strains being found to be macrolide-resistant in the present study. The present level was somewhat lower than the 15.7% reported by the PROTEKT study but nearly identical to what was found by our group in 1998–1999 [5]. Resistance rates can show pronounced variations between geographic regions and study centers within Germany. In the present study the combined percentages of intermediate and resistant strains ranged from 0 to 16.7 and 4.5 to 16.7% for penicillin G and erythromycin A, respectively. In comparison with a previous study undertaken by the German National Reference Center for Streptococci, the total level of macrolide resistance remained stable between 1998–1999 and the period covered by the pres-

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ent study (1999–2000), but pronounced differences were recorded within single laboratories (table 2). It is noteworthy that a 2% decrease in the level of penicillin resistance (I + R) was recorded. Mechanisms of macrolide resistance were found to vary widely between countries and different geographical regions in general, with mef(A) predominating in North America and erm(B) in Europe [11]. As in the US, macrolide resistance in Germany is now mainly due to the high prevalence of pneumococci expressing an M phenotype (61.3% of erythromycin A-resistant strains). Interestingly, in 1998–1999 it was the MLSB phenotypes that were predominant (74% of macrolide-resistant strains) but, as in other countries, the M phenotypes are now emerging [5]. Furthermore, a similar observation was made in a recent study on invasive pneumococcal disease, which showed that 43.4 and 56.1% of the erythromycin-resistant strains belonged to the erm(B) and mef(A) genotypes, respectively [6]. In the present study, erythromycin A-resistant strains were seen mainly among serotypes 6B, 14, 19F and 23F, confirming previous findings of our working group [5]. The latter may indicate the genetic relatedness of isolates and offers further evidence that the increase of macrolide resistance may be due to oligoclonal spread of pneumococci. Resistance to newer fluoroquinolones is extremely rare in Germany [12, 13] and in the present study only one strain exhibiting a levofloxacin MIC of 8 mg/l was isolated. Nevertheless, the level of pneumococcal fluoroquinolone resistance should be carefully monitored as the resistance level may increase with the more widespread use of these drugs [14]. Telithromycin showed excellent activity against macrolide-resistant S. pneumoniae possessing the mef(E) or erm(B) genotype and may therefore be an attractive alternative for the treatment of respiratory tract infections caused by pneumococci.

Acknowledgment We thank the following persons and institutions for their cooperation and for providing isolates: B. Wille, Institut für Krankenhaushygiene und Infektionskontrolle, Giessen, Germany; G. Schonard, Laborarztpraxis, Bad Hersfeld, Germany; U. Grimmer, Laborarztpraxis, Chemnitz, Germany; M. Seewald, Institut für Medizin Diagnostik, Berlin, Germany; R. Pfüller, Medizinisch-Diagnostisches Institute, Berlin, Germany; J. Ungeheuer, Labor Frohreich und Partner, Hamburg, Germany; J. Enzenhauer, Osnabrück, Germany; Untersuchungsamt, Hannover, Germany; A. Krenz-Weinreich, Plön, Germany; E. Kühnen, Trier, Germany; H.G. Enders, Stuttgart, Germa-

Reinert/Lütticken/Reinert/Al-Lahham/ Lemmen

ny; U. Walter, Wülfrath, Germany; J. Lenzen, Bonn, Germany; M. Jacobs, Mikrobiologisches Labor, Dillingen, Germany; W. Dirr, Augsburg, Germany; H. Hofmeister, Weiden, Germany; J. Matthes, Neuötting, Germany; F. Pranada, Gemeinschaftspraxis für Labormedizin, Dortmund, Germany; N. Schöngen, Gemeinschaftspraxis für Labormedizin, Leverkusen, Germany, and B. Hövener, Aachen, Germany.

The authors thank Nelli Neuberger, Maria Lemperle, Jutta Bungenstab, and Claudia Cremer for excellent technical assistance, and Susan Griesbach, Münster, for copy editing. The study was supported in part by Aventis, Romainville, France and in part by grant RKI-415/1369235 from the German Ministry of Health (Bundesministerium für Gesundheit).

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11 Farrell DJ, Morrissey I, Bakker S, Felmingham D: Molecular characterization of macrolide resistance mechanisms among Streptococcus pneumoniae and Streptococcus pyogenes isolated from the PROTEKT 1999–2000 study. J Antimicrob Chemother 2002;50(suppl S1):39– 47. 12 Lemmen S, Al-Lahham A, Lütticken R, Reinert RR: In vitro activity of gemifloxacin against Streptococcus pneumoniae isolates in Germany. Chemotherapy 2001;47:104–109. 13 Reinert RR, Al-Lahham A, Lütticken R, Boos M, Schmitz FJ: Characterization of clinical Streptococcus pneumoniae strains from Germany with decreased susceptibility to fluoroquinolones. J Antimicrob Chemother 2002;49: 1015–1018. 14 Klugman KP: The successful clone: The vector of dissemination of resistance in Streptococcus pneumoniae. J Antimicrob Chemother 2002; 50(suppl C):1–6.

© Free Author Copy - for personal use only ANY DISTRIBUTION OF THIS ARTICLE WITHOUT WRITTEN CONSENT FROM S. KARGER AG, BASEL IS A VIOLATION OF THE COPYRIGHT. Written permission to distribute the PDF will be granted against payment of a permission fee, which is based on the number of accesses required. Please contact [email protected] Pneumococcal Resistance in Germany


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