Emergence of macrolide and penicillin resistance among invasive pneumococcal isolates in Germany

Ralf René Reinerta,*, Adnan Al-Lahhama, Maria Lemperlea, Christoph Tenholtea, Claudia Briefsa, Stefan Hauptsa, Hans Hubert Gerardsb and Rudolf Lüttickena

a Institute of Medical Microbiology, National Reference Center for Streptococci, University of Aachen, Pauwelsstrasse 30, D-52074 Aachen b GlaxoSmithKline, Munich, Germany


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Continuous nationwide surveillance of antibiotic resistance in invasive pneumococcal disease was performed in Germany between 1992 and 2000, with a total of 2586 strains being isolated. The average resistance rates to erythromycin and clindamycin were 7.7% and 3.5%, respectively, throughout the study period; 3.3% of strains were found to have intermediate resistance to penicillin. Over the study period an increase in both macrolide and penicillin resistance was observed. The percentage of strains exhibiting reduced susceptibility to penicillin increased from 1.8% in 1992 to 5.8% in 2000. A dramatic increase in resistance was observed with erythromycin, where the resistance rate rose from 3.0% in 1992 to 15.3% in 2000. Of the erythromycin-resistant strains, 86 (43.4%) and 111 (56.1%) belonged to the erm(B) and mef types of resistance, respectively. An analysis of macrolide consumption data during the study period showed that erythromycin resistance was highly correlated to the consumption of newer bd and od macrolides (r = 0.89, P < 0.01).


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
In the past decade macrolide resistance among clinical pneumococcal isolates has become a serious problem and resistance rates to these antibiotics have exceeded resistance rates to ß-lactams in some parts of the world.1 Resistance to erythromycin in pneumococci was very low (<1%) in Germany in the 1980s.2 A significant level of resistance (3.8%) was first observed among 131 pneumococcal strains isolated between 1988 and 1991.3 Continuous surveillance of antibiotic resistance among pneumococcal strains isolated from invasive disease was launched in 1992, and the level of macrolide-resistant strains found among pneumococci (n = 844) isolated between 1992 and 1994 was 3.8%.4 In addition, a recent study on antibiotic resistance of pneumococci isolated mainly from respiratory tract specimens demonstrated resistance to clarithromycin in 10.6% of strains.5

Two main mechanisms are known to account for pneumococcal macrolide resistance. The so-called MLSB phenotype of resistance confers co-resistance to most macrolides, lincosamides and streptogramin B antibiotics. Resistance is associated with the erm gene6 and is due to methylation of a specific adenine residue resulting in reduced affinity between the MLSB antibiotics and the ribosome. The other type of resistance, the so-called M phenotype, results in resistance to the 14- and 15-membered macrolides and is caused by the presence of the mef(E) gene.7 In addition, mutations in 23S rRNA and ribosomal protein L4 have been shown to account for resistance in pneumococci.8

In this report we describe the emergence of macrolide and penicillin resistance among invasive pneumococcal strains isolated in Germany over the period 1992–2000.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The German National Reference Center for Streptococci regularly receives all consecutive invasive pneumococcal isolates from 60 clinical microbiological laboratories throughout Germany. Each isolate is confirmed as Streptococcus pneumoniae by optochin susceptibility and bile solubility testing. MICs of penicillin, cefotaxime, tetracycline, erythromycin, vancomycin, teicoplanin, quinupristin–dalfopristin, ciprofloxacin and clindamycin are determined by the broth microdilution method using Mueller–Hinton broth (Difco Laboratories, Detroit, MI, USA) supplemented with 5% lysed horse blood (Oxoid, Wesel, Germany) as recommended by the NCCLS. Susceptibility testing is performed in ambient air. S. pneumoniae ATCC 49619 is used as a reference strain.9

For the detection of erm(B) and mef(E) the primers described by Trieu-Cuot et al.6 and by Tait-Kamradt et al.10 were chosen.

Preparation of DNA and the PCR were performed as described previously.5 Pneumococcal strains were serotyped by Neufeld's Quellung reaction using type and factor sera provided by the Statens Serum Institut, Copenhagen, Denmark.

Antibiotic consumption data were kindly provided by the Institute for Medical Statistics (IMS), Frankfurt, Germany. These data are based on prescriptions issued by German hospitals and general practitioners. Data were provided as annual consumption units of the total German population for all macrolides (parenteral and oral drugs) for clindamycin and for the long-acting ‘newer macrolides’ azithromycin and clarithromycin. Data were analysed by calculating correlation coefficients (Pearson) between prescription data and resistance data according to Baquero.11

The significance of the increase in resistance was determined by the Cochran–Armitage trend test. The influence of previous antibiotic treatment on resistance was analysed by Fisher's exact test. Subgroups of strains for serotyping were selected randomly.

Clinical data were collected by sending questionnaires covering patients' sex, age, clinical diagnosis, underlying disease and recent previous antibiotic therapy.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
A total of 2586 strains was isolated; of these, 2134 (82.5%) were recovered from blood, 314 (12.1%) from cerebrospinal fluid, 81 (3.1%) from pleural fluid and 57 (2.2%) from other normally sterile body sites.

The mean age of the study population was 57.9 years (range <1 month to 96 years). In adults aged >50, the rate of pneumococcal infection was approximately three-fold that of persons aged between 20 and 50 years; more than two-thirds (69.7%) of all reported infections occurred in the former age group. The male:female patient ratio was 1.2:1. Information on antibiotic therapy before the start of microbiological diagnosis was available for 1708 patients, showing that 23% of patients had been treated. Differences in antibiotic resistance among strains cultured from patients with or without previous antibiotic treatment were observed: resistance rates in the treated (n = 1315; not previously treated, n = 393) patients were as follows: penicillin, 3.3% (2.9%, not significant); erythromycin, 10.4% (6.6%, significant, P = 0.02); tetracycline, 15.8% (11.9%, borderline significant, P = 0.056).

Detailed data on underlying diseases were available in 717 cases. Cancer (11.3%), alcohol abuse (9.9%), diabetes mellitus (7.9%), plasmacytoma (7.8%) and leukaemia (7%) were the leading risk factors for pneumococcal disease. It is interesting to note that 5.4% of patients were HIV infected. In addition, 3.4% of patients had asplenia, and 6.5% underlying immunosuppression other than HIV or leukaemia.

The average rate of resistance to erythromycin and clindamycin was 7.7% and 3.5%, respectively, over the study period; 3.3% of strains were found to have penicillin-intermediate resistance, but only two strains were fully resistant (MIC >= 2 mg/L) (Table 1Go).


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Table 1. Antibiotic susceptibility of 2586 strains of S. pneumoniae from invasive infections in Germany, 1992–2000
 
Over the study period a significant increase in both macrolide and penicillin resistance was observed. The rate of intermediate penicillin resistance rose significantly from 1.8% in 1992 to 5.8% in 2000. A dramatic increase in resistance was observed with erythromycin, where the resistance rate rose from 3.0% in 1992 to 15.3% in 2000 (Figure 1Go).



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Figure 1. Development of antibiotic resistance in invasive pneumococcal strains in Germany 1992–2000. Symbols: {blacksquare}, penicillin; {triangleup}, erythromycin; x, clindamycin; *, tetracycline.

 
A total of 198 erythromycin-resistant strains were analysed for their underlying resistance determinants by PCR. Of these strains, 86 (43.4%) and 111 (56.1%) belonged to the erm(B) and mef(E) type of resistance, respectively. One strain showed no PCR product in repeated assays and needs further investigation.

Four hundred and fifty-eight pneumococcal isolates were selected randomly for serotyping. The predominant types were serotypes 14 (13.5%), 9V (7.6%) and 4 (6.8%) (Table 2Go). Overall, 88.2% of these invasive pneumococcal strains were represented in the 23-valent polysaccharide vaccine types.


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Table 2. Serotype frequency in pneumococcal isolates from invasive disease in Germany
 
Antibiotic resistance was associated with certain serotypes. Of the 198 erythromycin-resistant strains, 84 were selected randomly for serotyping. Of these, serotypes 14 (26.2%), 6B (21.4%), 23F (8.3%) and 9V (6.0%) were found to be predominant (Table 2Go). Among the erm- positive strains, serotype 6B (34.1%) was predominant, whereas serotype 14 was predominant among the mef genotype, indicating the likelihood of clonal relatedness of those strains. Of 64 strains exhibiting reduced susceptibility to penicillin, 49 were serotyped, and resistance to this drug was found predominantly with serotypes 23F (22.4%), 9V (16.3%), 14 (10.2%), 6B (6.1%) and 4 (6.1%).

Table 3Go shows the patterns of co-resistance between different antibiotics. Of the erythromycin-resistant strains, 12.1% were also penicillin intermediate or resistant. However, 19.5% of the erm-positive strains exhibited reduced sensitivity to penicillin (Table 3Go). Eighteen strains were resistant to penicillin (intermediate and resistant), erythromycin and tetracycline. Slight differences in macrolide and penicillin resistance between different geographical regions of Germany were observed (Table 4Go).


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Table 3. Cross-resistance in invasive pneumococcal strains
 

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Table 4. Penicillin G and erythromycin resistance of invasive pneumococcal isolates from different regions in Germany, 1992–2000
 
In the present study 136 strains from children <=16 years of age were randomly selected for serotyping (Table 2Go). Serotypes 14 (13.2%), 6A (11.0%), 18C (8.8%), 9V (8.8%) and 19F (8.1%) were predominant.

Analysis of antibiotic consumption data showed that erythromycin resistance was highly correlated with the consumption of total macrolides (r = 0.94, P < 0.01). When macrolides were divided into subgroups, erythromycin resistance appeared to be related mainly to newer macrolides (r = 0.89, P < 0.01). Among those, the best correlation was seen with azithromycin (r = 0.94, P < 0.01) and clindamycin (r = 0.80, P < 0.01), whereas only a moderate correlation was seen with roxithromycin (r = 0.49, P < 0.01). The consumption of erythromycin was not correlated with erythromycin resistance (r = –0.86). Clindamycin consumption is also correlated with erythromycin resistance in S. pneumoniae in Germany (r = 0.97, P < 0.01) (Figure 2Go).



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Figure 2. Macrolide consumption and erythromycin resistance in Germany. Symbols: {diamondsuit}, all macrolides; {blacksquare}, clindamycin; {blacktriangleup}, erythromycin; x, newer macrolides; •, resistance.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
In the present study of 2586 pneumococcal strains isolated from patients with invasive pneumococcal disease, only 3.3% of strains were found to have intermediate resistance to penicillin. Highly penicillin-resistant strains were only rarely observed (n = 2), underscoring the relatively favourable level of ß-lactam resistance of S. pneumoniae in Germany. The resistance rate to penicillin G found in invasive disease is slightly lower and thus comparable to that reported in pneumococcal respiratory tract infection in both children and adults.5,12

The relatively favourable level of ß-lactam resistance in Germany has also been documented by other investigators. Marre & Trautmann13 reported that >95% of 105 pneumococcal strains isolated in Germany between 1994 and 1995 were susceptible to penicillin. In a study performed on pneumococcal isolates from eastern Germany, 100% of 105 pneumococci were susceptible to penicillin.14 In addition, a worldwide study of respiratory tract infections (the Alexander Project) demonstrated a penicillin resistance rate of about 7.2% (intermediate resistance) among strains isolated in one single institution in southern Germany in 1998.1 A multicentre, collaborative study performed in Asia and Europe during the winter of 1997–1998 on the in vitro activity of selected antimicrobial agents against common respiratory pathogens reported 92% of pneumococcal isolates in Germany to be susceptible to penicillin, whereas only 34% were still susceptible to penicillin G in France and Spain.15

The dramatic increase in macrolide resistance among invasive pneumococcal strains is one of the most important findings of the present study. As in many other countries, macrolide resistance has surpassed the level of ß-lactam resistance in Germany.16

In contrast to invasive disease, the level of macrolide resistance is lower in respiratory tract infections in Germany.5 It is interesting to note that a high rate of macrolide-resistant strains (13.8%), including strains isolated in 1997–1998, was also documented in a population-based study of children.17 In the study on respiratory tract infections by Reinert et al.,5 one notable finding was that macrolide resistance rates (range 1.5–21.3%) can show pronounced variations between geographical regions within Germany, a finding that could not be documented by the present study on invasive disease.

The relevance of antibiotic resistance to the outcome of disease has been widely discussed for ß-lactams.18,19 At present, data on the influence of low-level macrolide resistance on outcome are controversial. A retrospective study from the USA of 41 patients admitted with bacteraemia caused by low-level macrolide-resistant pneumococci demonstrated that four patients had previously taken either azithromycin or clarithromycin for 3–5 days.20 These authors state that even low-level resistance to macrolides can lead to clinical failure, and resistance to macrolides should be considered during the selection of empirical therapy for patients with presumed pneumococcal infections.

In Germany macrolide resistance in invasive disease is caused by the high prevalence of both resistance phenotypes (MLSB phenotypes, 43.4% of macrolide-resistant strains; M phenotype, 56.1% of macrolide-resistant strains), whereas in pneumococcal isolates from respiratory tract infections only 20.5% of macrolide-resistant strains show a macrolide efflux mechanism.5

Cross-resistance between ß-lactams, tetracycline and macrolides, especially in MLSB strains, is widespread in Germany; 12.1% of all macrolide-resistant isolates were also found to be penicillin non-susceptible. Penicillin-intermediate strains showed co-resistance to macrolides in 28.6% of isolates, limiting the use of this class of antibiotics in the treatment of infections caused by penicillin nonsusceptible strains.

The prevalence of these resistance genotypes is subject to major variations between countries. In Italy, macrolide-resistant pneumococci were isolated at a rate increasing from 6% in 1993 to 31.7% in 1998. In a collection of 161 erythromycin-resistant S. pneumoniae, c. 90% of isolates possessed a constitutive MLSB mechanism of resistance.21

In a recent Belgian study of 100 S. pneumoniae isolates, 33 were erythromycin resistant. Of the 33 erythromycin-resistant S. pneumoniae isolates, 9.1% showed the M resistance phenotype.22

In one strain from the current study the resistance determinant could not be identified. This strain needs further investigation and may possess a hitherto unknown resistance determinant or may have mutations in 23S rRNA and ribosomal protein L4, which have recently been shown to account for resistance in pneumococcal strains selected in vitro by macrolide passage.8 In addition, resistance may also be due to the presence of the erm(A) gene, as recently reported from Greece.23

Antibiotic consumption has been identified as contributing to the emergence of resistance in Streptococcus pyogenes and S. pneumoniae.24,25 As in other countries, increased usage of macrolides to be taken once or twice daily is recorded in Germany, whereas the erythromycin consumption level seems to be stable (Figure 2Go). As in Spain, a progressive increase in the erythromycin resistance curve was seen after the consecutive introduction of bd and od macrolides, which contributed to the increase in total macrolide consumption, superseding tds macrolide prescription.24 Although this analysis cannot establish an unequivocal causal relationship between antibiotic consumption and pneumococcal resistance, the data are highly consistent with the hypothesis that widespread use of macrolides, mainly of bd and od macrolides, is a primary contributory factor in the emergence of resistance.11,24,25

As antibiotic resistance is a major problem worldwide, infection prophylaxis by vaccination has been advocated in recent years. The 23-valent pneumococcal polysaccharide vaccine was licensed in Germany in 1983. Recently, vaccination of all persons >60 years has been advocated by the German National Advisory Board for Vaccination (STIKO).26 The 88.2% coverage rate for the 23-valent vaccine in the present study of respiratory tract infections is in the same range as the rates reported from studies of invasive disease by Kaufhold et al.2 (83.1%) and by Reinert et al. (92.2%).4

In summary, this study documents an emergence of macrolide resistance among invasive pneumococci in Germany, possibly driven by uncritical usage of macrolides. Nevertheless, the level of resistance in Germany is lower than those reported from southern and south-eastern Europe. Macrolide resistance in particular may now represent a serious problem for treatment of pneumococcal infection in Germany. Therefore, the resistance profile of pneumococci should be carefully monitored in the future, and broad usage of macrolides should be reconsidered.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We thank the following persons and institutions for their cooperation and for providing isolates: W. Ehret and E. R. Niculescu, Institut für Laboratoriumsmedizin, Mikrobiologie und Umwelthygiene des Zentralklinikums Augsburg; H. Hahn and J. Wagner, Institut für Infektionsmedizin, Mikrobiologie und Infektionsimmunologie der Freien Universität Berlin; T. Runz and S. Bessey, Zentralinstitut für Labormedizin der Universität Tübingen am Kreiskrankenhaus Böblingen; S. Gatermann and A. Müller-Chorus, Institut für Med. Mikrobiologie des Medizinaluntersuchungsamtes der Stadt Bochum; P. D. Marklein, Institut für Med. Mikrobiologie und Immunologie am Universitätsklinikum Bonn; Dr Panofsky-Ham, Mikrobiologisches Labor des Klinikums Chemnitz; J. M. Huber, Institut für Laboratoriumsdiagnostik und Transfusionsmedizin am Klinikum Deggendorf; M. Jacobs, Mikrobiologisches Laboratorium, Dillingen/Saar; U. Hadding and G. Zysk, Institut für Med. Mikrobiologie und Virologie der Universität Düsseldorf; V. Brade and V. Schäfer, Institut für Med. Mikrobiologie am Klinikum der Johann-Wolfgang-von-Goethe-Universität, Frankfurt; W. Bredt and A. Serr, Institut für Med. Mikrobiologie und Hygiene der Universitätsklinik Freiburg; T. Chakraborty and R. Füssle, Institut für Med. Mikrobiologie der Klinik an der Justus-Liebig-Universität, Giessen; U. Groß and O. Zimmermann, Hygiene-Institut der Universität Göttingen; C. Pilz, Zentrallabor/Mikrobiologie am Kreiskranken-haus Grossenhain; W. Sietzen, Abt. Mikrobiologie und Krankenhaushygiene am Allgemeinen Krankenhaus Hamburg-Altona; R. Laufs and I. Sobottka, Institut für Med. Mikrobiologie und Immunologie des Universitätskrankenhauses Hamburg-Eppendorf; A. Windorfer and K. Schwegmann, Niedersächsisches Landesgesundheitsamt, Hannover; Professor Bitter-Suermann, Institut für Med. Mikrobiologie an der Medizinischen Hochschule Hannover; J.-R. Krone, H.-J. Hagedorn, B. Dufaux, M. Zimmer and A. Vogel, Labor Krone und Partner, Herford; C. Retzlaff, Jäger, Löbel, Laborarztpraxis, Jena; U. Ullmann and H. Erichsen, Institut für Med. Mikrobiologie und Virologie des Universitätsklinikums Kiel; U. Massenkeil, Institut für Hygiene und Infektionsschutz des Landesuntersuchungsamtes Koblenz; M. Krönke and H. Schütt-Gerowitt, Institut für Med. Mikrobiologie, Immunologie und Hygiene der Universität zu Köln; Laboratoriumsmedizin Lempfried, Lembke, Laser, Eickhoff, Hütter, Hornei, Haschen, Esser und Gerards, Cologne; G. Rudat and E. Schott, Ärzte für Laboratoriumsmedizin, Leer/Ostfriesland; A. Rodloff, Institut für Med. Mikrobiologie und Epidemiologie der Universität Leipzig; R. N. Schöngen and U. Kürlis, Gemeinschaftspraxis für Laboratoriumsmedizin, Leverkusen; H. Hof and T. Nichterlein, Institut für Med. Mikrobiologie und Hygiene am Klinikum Mannheim; K. Heeg and S. Zimmermann, Institut für Med. Mikrobiologie des Klinikums der Philipps-Universität, Marburg/Lahn; L. Neef, Hygiene-Institut des Ruhrgebiets, Zweiginstitut Menden; K. Schikor, Institut für Laboratoriumsund Transfusionsmedizin, Klinikum Minden; H. Erichsen, Biosciencia GmbH, Regionallabor Moers; J. Heesemann and B. Grabein, Max-von-Pettenkofer-Institut für Hygiene und Med. Mikrobiologie der Universität München, Aussenstelle München-Grosshadern; A. Hartinger and K. Riedel, Institut für Med. Mikrobiologie, Immunologie und Krankenhaushygiene, Städt. Krankenhaus München-Harlaching; W. Junge and F. Strahlendorf-Elsner, Zentrallabor, Friedrich-Ebert-Krankenhaus, Neu-münster; H. Klein and K. Fabricius, Zentrallabor, Städt. Kliniken Offenbach/Main; H. Wolf and N. Lehn, Institut für Med. Mikrobiologie und Hygiene der Universität Regensburg; H. Rodt, U. Mildner and B. Rossmann, Gemeinschaftspraxis, Rosenheim; A. Podbielski, Institut für Med. Mikrobiologie der Universität Rostock; G. Enders and T. Regnath, Medizinisch-Diagnostisches Gemeinschaftslabor, Stuttgart; G. Volmer, Zentrallabor der Dr-Horst-Schmidt-Kliniken, Wiesbaden; P.-G. Höhn and V. Knop-Hammad, Institut für Med. Mikrobiologie und Immunologie des Klinikums Wuppertal-Barmen; S. Bastian, Klinisch-Chemisches Zentrallabor des Kreiskrankenhauses Zittau. The authors thank M. Kresken, Bonn, and N. Barnik, Munich, for their support, and S. Griesbach, Münster for copy editing. The study was supported by Rhone Poulenc Rorer between 1992 and 1999 and in part by grant RKI-415/1369235 from the German Ministry of Health (Bundesminister für Gesundheit). Presented in part at the Second International Symposium on Pneumococci and Pneumococcal Diseases, Sun City, South Africa, 2000 (abstract P81).


    Notes
 
* Corresponding author. Tel: +49-241-8089-787; Fax: +49-241-8888-483; E-mail: reinert{at}rwth-aachen.de Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Received 12 February 2001; returned 29 June 2001; revised 13 August 2001; accepted 20 August 2001