Prevalence and Macrolide Resistance Phenotypes

REPRINT Journal of Chemotherapy

Vol. 19 - n. 3 (327-?) - 2007

REVIEW Prevalence and Macrolide Resistance Phenotypes and Genotypes Among Clinical Isolates of Streptococcus pneumoniae Collected in Sofia, Bulgaria from 2001 to 2005 L.P. SETCHANOVA - V. OUZOUNOVA-RAYKOVA - G.Z. ZHELEZOVA - I.G. MITOV Department of Microbiology, Faculty of Medicine, Medical University of Sofia, Sofia, Bulgaria. Corresponding author: Assoc. Prof. Lena Setchanova, M.D., Ph.D., Department of Microbiology, Faculty of Medicine, Medical University of Sofia, 2, Zdrave str., 1431-Sofia, Bulgaria. Tel: + 359 2 91 72 720; Fax: + 359 2 951 53 17. E-mail: [email protected]

Summary A total of 328 clinical isolates of Streptococcus pneumoniae were analyzed to determine the rate of macrolide and penicillin resistance as well as macrolide resistance phenotypes and genotypes. Erythromycin resistance was found in 81 pneumococcal isolates (24.7%) and 10.7% of isolates were clindamycin resistant. The prevalence of penicillin G-intermediate (minimum inhibitory concentrations, MICs, 0.125 to 1 g/ml) and penicillin-resistant (MICs, ≥2 g/ml) S. pneumoniae isolates was 25.6% and 13.7%, respectively. The rate of ceftriaxone-intermediate and ceftriaxone-resistant strains was 2.7% and 1.2%, respectively. Among erythromycin-resistant S. pneumoniae isolates, strains harboring mef(A) genes (n=42; 51.8%) were found to be predominant over strains with erm(B) genes (n=34; 42.0%). One (1.2%) isolate carried both erm(B) and mef(A), while 4 (4.9%) isolates carried L4 protein mutations. By using the erythromycin, clindamycin and rokitamycin triple-disk test, 42 strains were assigned to the M phenotype of macrolide resistance, 31 isolates were assigned to the partially inducible (iMcLS) phenotype, 4 were assigned to the constitutive (cMLS) phenotype. Four strains with L4 gene showed a rare phenotype with the triple-disk test. Serotyping of S. pneumoniae isolates suggested that serotype (or serogroup) 14, 6 and 19 were predominant (81.5%) among erythromycin-resistant strains. Among mef(A) positive isolates serotype 14 was predominant, among erm(B) positive isolates serogroups 6 and 19 were the most prevalent. Key words: Macrolide resistance, erm(B), mef(A), L4, resistance, Bulgaria, Streptococcus pneumoniae.

INTRODUCTION Streptococcus pneumoniae is the major cause of bacterial pneumonia, otitis media and sinusitis and it is an important cause of meningitis and bacteraemia in the community. Treatment of pneumococcal infections is becoming difficult because of dissemination of penicillin-resistant

© E.S.I.F.T. srl - Firenze

strains and the rapid development of resistance to other antibiotics, including macrolides 1. In a 19992000 surveillance study from Central and Eastern European Countries, among tested Bulgarian S. pneumoniae strains, 39.6% were nonsusceptible to penicillin G, and the macrolide resistance rate was documented to be 19.8% 2. Pneumococcal macrolide resistance is mediated

ISSN 1120-009X

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L.P. SETCHANOVA - V. OUZOUNOVA-RAYKOVA - G.Z. ZHELEZOVA - I.G. MITOV

were isolated from cerebrospinal fluid (CSF), 49 were isolates from nasopharynx (all from patients with respiratory tract infections), 47 were isolates from ear, 35 strains were isolated from blood, 23 were isolates from eye, and 17 strains were isolated from sinus pus. Organisms were isolated from all patient age groups.

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mainly by two mechanisms. The first is modification of the ribosomal target site (23S rRNA), mediated by methylases, which are encoded mainly by erm(B) and, rarely erm(A) 3. Phenotypically, this results in the MLSB resistance phenotype, with resistance to all macrolides plus lincosamides and streptogramin B antibiotics. MLSB resistance can be expressed either constitutively (cMLSB phenotype) or inducibly (iMLSB phenotype) 4. The second mechanism is active efflux of macrolides from the cell. For the efflux mechanism, the acquired gene mef(E) is necessary 5 . Strains with mef have the M phenotype and are only resistant to 14- and 15-membered macrolides, but not to 16-membered macrolides, plus lincosamides and streptogramin B antibiotics. Other mechanisms of macrolide resistance among clinical isolates of S. pneumoniae have been described and include mutations in 23S rRNA and in genes that encode the L4 and L22 ribosomal proteins 3,6,7. The prevalence of the macrolide resistance genotypes varies substantially among countries 8 . In France, Spain, Switzerland, Poland, Belgium, the Netherlands and Italy 8,9,10 strains are predominantly erm(B), whereas higher levels of mef(A)- or mef(E)positive strains have been reported from the USA, Greece, Germany and Finland 8,11,12,13. In addition, ribosomal mutations conferring resistance to macrolides in Streptococcus pneumoniae have been shown to account for resistance in pneumococci 2,3,7,9,10,14. The aims of this study were to determine the frequency of resistance to macrolides and penicillin G and to investigate the phenotype and genotype distribution of macrolide resistance in clinical isolates of S. pneumoniae collected in Sofia, Bulgaria between 2001-2005.

MATERIALS AND METHODS

Bacterial isolates Between 2001 and 2005, 328 pneumococcal strains consecutively isolated from various clinical specimens were collected at the central laboratory of the Department of Microbiology, Medical University of Sofia. Strains were obtained from specimens of patients with respiratory tract infections or with invasive pneumococcal diseases (meningitis and bacteremia). Isolates were collected from three Microbiology Laboratories in Sofia (270 strains, 82.3%), and from two laboratories in other University Hospitals (Pleven and Plovdiv) in Bulgaria (58 isolates, 17.7%). Strains were confirmed as S. pneumoniae by both methods - Optochin disk test and the bile solubility method as well. Duplicate organisms from different specimens from the same patient were eliminated. Among the 328 pneumococcal isolates, 83 were isolates from sputum, pleural fluid or tracheobronchial fluid, 74 pneumococci

Susceptibility testing

Susceptibility of S. pneumoniae to erythromycin and penicillin G was first determined by the disk diffusion method. Minimum inhibitory concentration (MIC) testing was performed on Mueller-Hinton II agar (BBL Microbiology Systems) supplemented with 5% sheep blood for all erythromycin resistant strains (≤15mm) and all penicillin non-susceptible strains (≤19mm) 15. Antibiotics used for MIC testing were: erythromycin, clarithromycin, azithromycin, clindamycin, penicillin G and ceftriaxone. The antimicrobial agents tested were obtained from commercial sources (ICN Biomedicals Inc.). Penicillin G and ceftriaxone were included in order to estimate susceptibilities to these agents and because it is known that the rate of penicillin resistance is high in our country 2,16. Strain preparation, MIC testing by agar dilution method and incubation were performed by the methods published elsewhere 15,17. The lowest concentration of antibiotics that resulted in no growth was read as the MIC. Standard quality control strain S. pneumoniae ATCC 49619 was included with each run. Breakpoints were those approved by the CLSI for S. pneumoniae 18.

Determination of macrolide resistance phenotype The macrolide-resistance phenotypes for all erythromycin A resistant strains were determined by disk diffusion using the triple-disk test (erythromycin plus clindamycin and rokitamycin) as described by Montanari et al. 4. Test strains were assigned to the constitutive (cMLS), the partially inducible (iMcLS), the truly inducible (iMLS), or the efflux-mediated (M type) macrolide resistance phenotypes.

Determination of macrolide resistance genes All erythromycin-resistant S. pneumoniae strains were analyzed further by PCR. Each preparation of DNA was assayed for the presence of erm(B) and mef(A) by PCR. Primer sets of known genotype 02J 1095 for erm(B) and 02J 1175 for mef(A) were as described previously 19,20. Isolates that were negative for erm and mef genes were checked for the presence of mutations in ribosomal protein L4, because this mutation exists in Bulgaria, as was shown previously 2. PCR primer pair for L4 yielded a 720-bp product as was described previously 6. Gene amplification was performed using a Techgene - thermal cycler (Techne, England) at 94°C for 5 min, fol-

PREVALENCE AND MACROLIDE RESISTANCE PHENOTYPES AND GENOTYPES AMONG CLINICAL ISOLATES OF STREPTOCOCCUS PNEUMONIAE ...

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lowed by 35 cycles of 94°C for 1 min, 53°C or 55°C for 1 min, 72°C for 1 min, and final elongation at 72°C for 10 min.

MIC testing. The rate of erythromycin resistance in 328 patients was higher in children - 34.8% versus 11.4% in patients more than 14 years old.

Serotyping of S. pneumoniae

MIC distribution for S. pneumoniae isolates and macrolide resistance mechanisms

Serotyping of macrolide-resistant pneumococcal strains was performed by the standard Quellung method (the capsular swelling test) with antisera from Statens Seruminstitut (Copenhagen, Denmark). S. pneumoniae isolates were serotyped/serogrouped by using 12 pooled antisera with chessboard typing system 21, following the procedure given by manufacturer (Pneumotest Kit, Statens Seruminstitut, Copenhagen, Denmark).

RESULTS

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The highest rate of pneumococcal infection was in children ages 15 days to 14 years (187 isolates, 57.0%), while the rate of infection in patients older than 14 years was 43.0% (141 strains). Of the 328 S. pneumoniae strains, 81 were erythromycin-resistant (24.7%), and 35 isolates were clindamycin-resistant (10.7%), both by disk diffusion method and by

The prevalence of macrolide resistance mechanisms among the 81 erythromycin-resistant strains, and MIC distribution for different erythromycin resistance genotypes of erythromycin, clarithromycin, azithromycin and clindamycin against tested pneumococcal isolates are shown in Table 1. Of the 81 erythromycin-resistant S. pneumoniae, 42 (51.8%) had the mef(A) gene only, and 34 (42.0%) had the erm(B) gene only. One isolate had both erm(B) and mef(A) genes (1.2%). Four isolates, obtained from respiratory specimens, that were negative for erm(B) and mef(A) genes in repeated assays were further analyzed for mutations in L4 ribosomal protein. All 4 strains (4.9%) were positive for L4 gene by PCR analyzes. In this study we did not find erythromycinresistant S. pneumoniae isolates negative for the mechanisms tested. The correlations between the MICs and different erythromycin resistance genotypes is given in Table

TABLE 1 - MICs for different erythromycin resistance genotypes and MICs of penicillin, and ceftriaxone against S. pneumoniae isolates. E resistance genotype (%)

N. tested

Antibiotic

mef (A) (51.8)

42

Erythromycin Clarithromycin Azithromycin Clindamycin

erm (B) (42.0)

34

erm (B) + mef (A) (1.2)

L4 (4.9 )

MIC (mg/L) %S

%I

%R

0.5->64 0.03-32 0.06->64 0.01-0.12

2.4 2.4 100

2.4 7.1 4.8 -

97.6 90.5 92.9 -


16->64 2->64 >64 0.5->64

-

2.9

100 100 100 97.1

1


>64 >64 >64 >64

-

-

100 100 100 100

4

Erythromycin Clarithromycin Azithromycin Clindamycin Penicillin G * Ceftriaxone *

64->64 16-64 >64 0.06-0.12 0.03-8 0.01-8

100 60.7 96.0

25.6 2.7

100 100 100 13.7 1.2

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Range

E- Erythromycin; S-susceptible; I-intermediate; R-resistant. * MICs to penicillin G and ceftriaxone were performed only for 134 strains that were penicillin-nonsusceptible by disk diffusion method (≤19 mm).

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cline, and 49.4% to chloramphenicol (results not shown). All erythromycin-resistant pneumococcal strains, except two strains (97.5%), were multidrug resistant (three or more antimicrobials). The MIC distribution of penicillin G and the third generation cephalosporin (ceftriaxone) for the tested 134 pneumococcal isolates is also shown in Table 1. Of the 328 S. pneumoniae isolates, 134 strains were penicillin nonsusceptible by the disk diffusion method. MIC testing of these 134 strains showed that penicillin G nonsusceptibility was confirmed in 129 strains of all pneumococci (39.3%). Penicillin resistance (MIC ≥2 g/ml) was found in 13.7% and intermediate resistance (MICs, 0.1-1 g/ml) in 25.6%. The MIC90 of penicillin G for tested pneumococcal isolates was 2 g/ml. Almost half (47.3%) of the penicillin-resistant or -intermediate isolates were also macrolide resistant. Overall, 4% of the isolates (meningitis and nonmeningitis strains) were ceftriaxone nonsusceptible (2.7% ceftriaxone intermediate and 1.2% ceftriaxone resistant strains). The MIC90 of ceftriaxone was 0.5 g/ml.


1. Of the 81 strains, only one was intermediately susceptible to erythromycin (MIC=0.5 g/ml), this isolate had mef(A) gene. The remaining 80 isolates had MICs for erythromycin ≥1 g/ml. For strains with mef(A) gene, the MICs of erythromycin, clarithromycin and azithromycin were generally 1 to 8 g/ml and MIC of clindamycin was ≤0.12 g/ml. However, erythromycin MICs for two mef(A)-positive S. pneumoniae strains were ≥64 g/ml, and both these strains had clarithromycin MIC of 32 g/ml and azithromycin MICs were 64 and >64, respectively. These two isolates did not carry erm(B) or L4 genes. Erythromycin MICs of isolates harboring the erm(B) gene were >64 g/ml, and as expected, cross-resistance to MLSB antimicrobials were typical for isolates expressing erm(B) gene. However, one isolate harboring the erm(B) gene was not resistant to clindamycin (MIC=0.5 g/ml), and displayed erythromycin MIC of 16 g/ml, clarithromycin MIC of 2 g/ml, but azithromycin MIC was >64 g/ml. One S. pneumoniae isolate that harbored both erm(B) and mef(A) genes had MICs of >64 g/ml for all tested macrolides and lincosamides (erythromycin, clarithromycin, azithromycin and clindamycin). This isolate cannot be distinguished from erm(B) alone using MIC data. The four isolates with L4 gene had MICs of erythromycin ≥64 g/ml, clarithromycin MICs were 16-64 g/ml, and azithromycin MICs were >64 g/ml, but all strains were susceptible to clindamycin (MICs, 0.06 to 0.125). Of the 81 erythromycin-resistant isolates 61 (75.3%) were also nonsusceptible to penicillin G. Six of these strains had penicillin MIC of ≥2 g/ml (3 isolates with L4 gene, 2 with mef(A), and 1 erm(B)+mef(A)genes), while the remaining 55 strains were intermediately resistant (MICs 0.1-1 g/ml). Among the 81 erythromycin-resistant isolates, 43.2% were clindamycin nonsusceptible (all erm(B) alone isolates and one with erm(B)+mef(A) genes). Erythromycin-resistant pneumococci were also resistant to other antimicrobial drugs, apart from MLS antibiotics. Thus, of the 81 erythromycinresistant strains, 82.7% were nonsusceptible to trimethoprim-sulfamethoxazole, 55.6% to tetracy-

Macrolide resistance phenotypes Macrolide resistance phenotypes and correlations with erythromycin resistance genes and serotypes/serogroups in 81 erythromycin-resistant S. pneumoniae are summarized in Table 2. On the basis of the erythromycin-clindamycin-rokitamycin triple disk test (TDT), 42 (51.8%) strains were assigned to the M phenotype. All 42 M phenotype isolates had the mef(A) gene. Methylase-mediated coresistance to MLS antibiotics was found in 35 (43.2%) strains, 31 (38.3%) were assigned to the iMcLS phenotype, 4 (4.9%) were assigned to the cMLS phenotype, and we did not observe true iMLS-type in this collection of erythromycin-resistant S. pneumoniae isolates. All iMcLS and cMLS isolates had the erm(B) gene, including one cMLS isolate that had both erm(B)+mef(A) genes. Four (4.9%) strains with L4 mutation had a completely different phenotype with TDT (unique for our collection). These 4 strains were fully erythromycin resistant with no zone of inhibition around the ery-

T ABLE 2 - Macrolide resistance phenotypes and correlations with erythromycin resistance genes and serotypes/serogroups of 81 erythromycin-resistant S. pneumoniae. Phenotype of macrolide resistance

N. (%) of strains

N. of strains with gene erm (B) mef (A) L4

Serotype/serogroups (n. of strains)

M

42 (51.8)

-

42

-

14(22); 6(5); 23(5); 15(3); 19(3); 7(1); 11(1); 18(1); 33(1);

iMcLS

31 (38.3)

31

-

-

6(18); 19(11); 23(2);

cMLS

4 (4.9)

4

1

-

19(3); 11(1)

L4

4 (4.9)

-

-

4

19(4)


thromycin disk; they all were clindamycin susceptible (displayed a large zone of inhibition), but they had a reduced zone of inhibition around 16-membered macrolide rokitamycin - 15 to 16 mm in diameter, without blunting of this zone. This L4 phenotype with TDT was different from the M phenotype.

Serotyping

DISCUSSION

microbiological treatment failures 25,26. Treatment failure occurred only in those patients infected with a macrolide-resistant pneumococcus 25. The prevalence of penicillin G-intermediate and penicillin-resistant S. pneumoniae isolates were 25.6% and 13.7%, respectively. Therefore, the prevalence of non-susceptible pneumococcal isolates in the present study (39.3%) still appeared to be similar to the one published in 2000 2. Ceftriaxone nonsusceptibility appeared to be low (4%). Erythromycin and penicillin resistance were related strongly, in that almost half of the isolates with reduced penicillin susceptibility were also macrolide resistant, and as many as 75% of the macrolideresistant isolates showed reduced susceptibility to penicillin. Such a relationship was found in other surveys 10,13. For the treatment of pneumococcal infections in Bulgaria, -lactam antimicrobials, such as high doses amoxicillin or a third generation cephalosporin, are still first-line therapy, because our isolates are >90% susceptible to these drugs (results are not shown for amoxicillin). In the present study, 51.8% of erythromycinresistant isolates carried mef(A) efflux gene, target modification mediated by erm(B) was the second most prevalent mechanism found in 42% of the strains, and 1.2% carried both genes erm(B)+mef(A). Therefore, the prevalence of mef(A) gene in Bulgaria is not too high and further investigation of macrolide resistance genotypes is needed. The rate of the mef(A) genotype even seems to go down in comparison with the prevalence reported in 1999-2000 (60% vs. 51.8% here) 2. Alteration in the L4 protein continues to play a role in macrolide resistance of pneumococci (4.9% in this study). It is well known that macrolide resistance mechanisms are inferred from macrolide and lincosamide MICs 2. The genotyping data show that macrolide and clindamycin susceptibilities of isolates with erm(B) and mef(A) genotypes were broadly consistent with the expected phenotype, but exceptions were observed 13. The data presented here showed that two mef(A) positive isolates had erythromycin MICs ≥64 g/ml, and one isolate harboring the erm(B) gene was not resistant to clindamycin (MIC=0.5 g/ml), and displayed an erythromycin MIC of 16 g/ml. Such isolates have been described previously in a surveillance study where discrepancies were also observed 13. Wide geographical differences in the distribution of erm(B) and mef(A) throughout the world have been reported, with erm(B) predominating in Europe, and mef(A) in North America 13,27 . However, in several European countries (Germany, Greece, Finland and Bulgaria) efflux-mediated (M type) macrolide resistance is predominant 2,8,12. Thus, the prevalence of macrolide resistance genotypes also varies greatly between European countries 22. Since clinical laboratories do not test


Correlations between macrolide resistance genes and serotypes/serogroups are shown in Table 2. Twenty-two of the 42 isolates carrying mef(A) belonged to serotype 14 (52.4%), serogroups 6 and 23 contained each five isolates, and the remaining 10 strains were distributed over six other serogroups (Table 2). Of the 35 strains carrying the erm(B) gene, all of the iMcLS and cMLS phenotypes, 18 belonged to serougroup 6 (51.4%), 14 strains belonged to serogroup 19 (40.0%), and 3 isolates belonged to the two serogroups 23 and 11, respectively. All 4 isolates carrying the L4 gene belonged to serogroup 19. Overall, 9 different serotypes/serogroups were represented in the 81 serotyped S. pneumoniae strains, of which three were more prevalent 6, 14 and 19, accounting for 81.5% of all macrolide-resistant S. pneumoniae. Thus, 100% of the examined erythromycin-resistant strains belonged to vaccine related types or groups included in the 23-valent pneumococcal vaccine. The vast majority of erythromycin-resistant isolates (75/81) were in vaccine serotype/groups included in the pneumococcal conjugate vaccines.

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The present study evaluated the in-vitro resistance of 328 S. pneumoniae isolates from Bulgaria (mainly Sofia) to macrolides, penicillin G and ceftriaxone. The study analyzed the mechanisms of macrolide resistance, macrolide resistance phenotypes and their association with erythromycin resistance genes, and serotypes/serogroups distribution. Erythromycin resistance of S. pneumoniae was found to have increased from 19.8% in 2000 2 to 24.7% in the present survey. Macrolides are important alternatives to -lactams for the treatment of pneumococcal infections, but the level of macrolide resistance in S. pneumoniae is increasing in our country. In comparison with other European countries, the level of macrolide resistance presented here is moderate. The highest macrolide resistance rates are reported in Spain, France and Italy, >40% of pneumococcal isolates 22,23,24. Erythromycin resistance rates of >20% were reported from Hungary, Romania, Poland, Greece, Belgium and Finland 2,8,9,12 . Surveillance studies have shown a link between increased use of macrolides and increased rates of pneumococcal resistance 25. The clinical importance of macrolide-resistant S. pneumoniae has only recently been described for clinical and

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6 Tait-Kamradt A, Davies T, Cronan M. et al. Mutation in 23S rRNA and ribosomal proten L4 account for resistance in pneumococcal strains selected in vitro by macrolide passage. Antimicrob Agents Chemother 2000; 44: 2118-2125. 7 Tait-Kamradt A, Davies T., Appelbaum PC et al. Two new mechanisms of macrolide resistance in clinical strains of S. pneumoniae from Eastern Europe and North America. Antimicrob Agents Chemother 2000; 44: 3395-3401. 8 Reinert RR, Ringelstein A, van der Linden M, et al. Molecular epidemiology of macrolide-resistant Streptococcus pneumoniae isolates in Europe. J Clin Microbiol 2005; 43:1294-1300. 9 Van Eldere J, Meekers E, Lagrou K et al. Macrolideresistance mechanisms in Streptococcus pneumoniae isolates from Belgium. Clin Microbiol Infect 2005; 11: 332-334. 10 Neeleman C, De Valk JA, Klaassen CHW et al. In-vitro susceptibility and molecular characterization of macrolide resistance mechanisms among Streptococcus pneumoniae isolates in the Netherlands: the duel 2 study. Clin Microbiol Infect 2005; 11: 312-318. 11 Fotopoulou N, Tassios PT, Beste DV, et al. A common clone of erythromycin-resistant Streptococcus pneumoniae in Greece and the UK. Clin Microbiol Infect 2003; 9: 924-929. 12 Rantala M, Huikko S, Huovinen P, Jalava J. Prevalence and molecular genetics of macrolide resistance among Streptococcus pneumoniae isolates collected in Finland in 2002. Antimicrob Agents Chemother 2005; 49: 4180-4186. 13 Farrell DJ and Jenkins SG. Distribution across the USA of macrolide resistance and macrolide resistance mechanisms among Streptococcus pneumoniae isolates collected from patients with respiratory tract infections: PROTEKT US 20012002. J Antimicrob Chemother 2004; 54 (Suppl S1); i17i22. 14 Reinert RR, Wild A, Appelbaum P et al. Ribosomal mutations conferring resistance to macrolides in Streptococcus pneumoniae clinical strains isolated in Germany. Antimicrob Agents Chemother 2003; 47: 23192322. 15 Davies TA, Kelly LM, Jacobs MR, Appelbaum PC. Antipneumococcal activity of telithromycin by agar dilution, microdilution, E-test and disk diffusion. J Clin Microbiol 2000; 38:1444-1448. 16 Setchanova L. Clinical Isolates and Nasopharyngeal Carriage of Antibiotic-Resistant Streptococcus pneumoniae in Hospital for Infectious Diseases, Sofia, Bulgaria, 1991-1993. Microb Drug Resist 1995; 1: 79-84. 17 Fasola EL, Bajaksouzian S, Appelbaum PC and Jacobs MR. Variation in erythromycin and clindamycin susceptibilities of Streptococcus pneumoniae by four test methods. Antimicrob Agents Chemother 1997; 41: 129-134. 18 Clinical and Laboratory Standards Institute. 2005. Performance standards for antimicrobial susceptibility testing: 15 th informational supplement. CLSI/NCCLS document M100-S15. Clinical and Laboratory Standards Institute, Wayne, Pa. 19 Sutcliffe J, Tait-Kamradt A, Wondrack L. Streptococcus pneumoniae and Streptococcus pyogenes resistant to macrolides but sensitive to clindamycin: a common resistance pattern mediated by an efflux system. Antimicrob Agents Chemother 1996; 40: 1817-1824. 20 Sutcliffe J, Grebe T, Tait-Kamradt A et al. Detection of erythromycin-resistant determinants by PCR. Antimicrob Agents Chemother 1996; 40: 2562-2566. 21 Sorensen UBS. Typing of pneumococci by using 12 pooled antisera. J Clin Microbiol 1993; 31: 2097-2100. 22 Reinert RR, Reinert S, Van der Linden M, et al. Antimicrobial susceptibility of Streptococcus pneumoniae in eight European countries from 2001 to 2003. Antimicrob Agents Chemother 2005; 49: 2903-2913.


streptogramin B 7, use of erythromycin, clindamycin and rokitamycin ????????? is more accurate method to determine the macrolide resistance phenotype than the erythromycin and clindamycin double-disk diffusion test (DDT). (AUTHOR, SOMETHING SEEMS TO BE MISSING IN THIS SENTENCE.) In our study, the use of TDT analysis to determine constitutively or inducible resistance to MLS antibiotics was helpful, not only for predicting the likely mechanism responsible for macrolide resistance, but for finding a rare phenotype that corresponds to the L4 gene. Serotyping of S. pneumoniae isolates indicated that serotype (or serogroup) 14, 6 and 19 were predominant (81.5%) among this collection of erythromycin-resistant strains. This surveillance has confirmed the potential public health benefits that could be achieved by use of pneumococcal conjugate vaccines. Among mef(A) positive isolates serotype 14 was predominant, among erm(B) positive isolates serogroups 6 and 19 were the most prevalent. Resistance patterns of erythromycin-resistant S. pneumoniae isolates to multiple antibacterial drugs (results not shown), the correlation of serotyping data and distribution of macrolide resistance mechanisms suggest that some strains might be clonally related. This suggestion requires further investigation. In conclusion, resistance of S. pneumoniae to macrolides is of growing importance in Bulgaria. Knowledge of the resistance mechanisms is crucial for the empiric antibiotic treatment of pneumococcal community-acquired infections. ACKNOWLEDGEMENTS: We gratefully acknowledge the Bulgarian Pneumo Study Group that provided S. pneumoniae isolates for this study. This work was supported in part by a grant from the Medical University, Sofia, Bulgaria (Grant No.46/23.06.2004).

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Appelbaum PC. Resistance among Streptococcus pneumoniae: implications for drug selection. Clin Infect Dis 2002; 34: 1613-1620. 2 Nagai K, Appelbaum PC, Davies TA et al. Susceptibilities to telithromycin and six other agents and prevalence of macrolide resistance due to L4 ribosomal protein mutation among 992 pneumococci from 10 Central and Eastern European countries. Antimicrob Agents Chemother 2002; 46 (2): 371-377. 3 Leclercq R, Courvalin P. Resistance to macrolides and related antibiotics in Streptococcus pneumoniae. Antimicrob Agents Chemother 2002; 46: 2727-2734. 4 Montanari MP, Mingoia M, Giovanetti E, Varaldo PE. Differentiation of resistance phenotypes among erythromycinresistant pneumococci. J Clin Microbiol 2001; 39:13111315. 5 Tait-Kamradt A, Clancy J, Cronan M et al. MefE is necessary for the erythromycin-resistant M phenotype in S. pneumoniae. Antimicrob Agents Chemother 1997; 41: 22512255.


23 Felmingham D, Reinert RR, Hirakata Y, Rodloff A. Increasing prevalence of antimicrobial resistance among isolates of Streptococcus pneumoniae from the PROTEKT surveillance study, and comparative in vitro activity of the ketolide, telithromycin. J Antimicrob Chemother 2002; 50 (Suppl.S1): 25-37. 24 Marchese A, Gualco L, Cochetti I, et al. Antibiotic susceptibility and serotype distribution in Streptococcus pneumoniae circulating in Italy: results of the SEMPRE surveillance study (2000-2002). Int J Antimicrob Agents 2005; 26: 138145.

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2007 n.3,