JAC
Journal of Antimicrobial Chemotherapy (1999) 44, 461–464
Macrolide resistance mechanisms and expression of phenotypes among Streptococcus pneumoniae circulating in Italy A. Marchese, E. Tonoli, E. A. Debbia and G. C. Schito* Institute of Microbiology, University of Genoa, Largo R. Benzi 10, 16132 Genoa, Italy In Italy, macrolide-resistant pneumococci have been isolated at a rate increasing from 6% in 1993 to 31.7% in 1998. A collection of 161 erythromycin-resistant Streptococcus pneumoniae recovered between 1993 and 1997 has now been phenotypically and genotypically characterized. Approximately 90% of these microorganisms possessed a constitutive MLSB mechanism of resistance. PCR detected ermB and mefE genes in strains showing MLSB and M phenotypes, respectively. Using pulsed-field gel electrophoresis of chromosomal DNA, one dominant restriction profile and its variations were detected in 51 S. pneumoniae isolates collected from different locations, indicating the circulation of a clone characterized by the possession of a great ability to spread.
Introduction Erythromycin resistance in Streptococcus pneumoniae has increased over recent years in several geographical areas, Italy included.1,2 Currently, 31.7% of all pneumococci circulating in Italy are resistant to macrolides,2 compared with 6% in 1993.3 In Canada4 and the USA,5 the M phenotype specified by the mefE gene6 represents the prevailing mechanism, while in Spain7 an MLSB phenotype is observed almost exclusively. Since M phenotype strains display low-level macrolide resistance, their local preponderance might have an impact on antibiotic choice for non-meningeal infections. Their incidence has therefore been evaluated using a large collection of S. pneumoniae isolated in Italy by phenotypic determination and PCR analysis. In addition, to ascertain whether spreading of resistance is due to clonal expansion, the nature of all strains has been analysed by pulsed-field gel electrophoresis (PFGE).
supplied by Abbott S.p.a. (Campoverde, Italy), clavulanic acid by SmithKline Beecham Pharmaceuticals (Milan, Italy), imipenem by the hospital pharmacy, and clindamycin, penicillin G, amoxycillin, cefotaxime, ceftriaxone, co-trimoxazole, tetracycline, chloramphenicol, rifampicin and vancomycin were purchased commercially (SigmaAldrich, Milan, Italy). Resistance phenotypes were classified using a doubledisc test with erythromycin and clindamycin discs. After 18–42 h of incubation at 35°C, a blunting of the clindamycin zone of inhibition proximal to the erythromycin disc was taken to indicate inducible resistance (I), while resistance to clindamycin with no blunting indicated constitutive resistance (C). The M phenotype was characterized by susceptibility to clindamycin with no blunting of the zone of inhibition around the clindamycin disc. ermB and mefE genes were amplified by PCR as described previously,10 and PFGE of chromosomal DNA and pattern analysis were performed as reported elsewhere.10
Materials and methods
Results and discussion
Erythromycin-resistant S. pneumoniae (161) selected from among 1046 respiratory strains collected between 1993 and 1997, and originating from widely dispersed laboratories in Italy (Ancona, Bologna, Genoa, Florence, Milan, Naples, Parma, Turin and Vercelli), were studied. Susceptibility to antimicrobial drugs was assessed by microdilution assay as detailed in NCCLS guidelines (1997).8,9 Erythromycin was
Table I shows the susceptibility patterns of erythromycinresistant S. pneumoniae. Rifampicin and vancomycin emerged as the most potent drugs tested (100% susceptible strains) followed by third-generation cephalosporins ( 90%), amoxycillin, co-amoxiclav and imipenem (84%), penicillin (79%), chloramphenicol (60%), co-trimoxazole (40%), tetracycline (13%) and clindamycin (10%).
*Corresponding auuthor. Tel:
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461 © 1999 The British Society for Antimicrobial Chemotherapy
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A. Marchese et al. Table I. Susceptibilities to selected antibiotics of 161 erythromycin-resistant S. pneumoniae
Microorganism S. pneumoniae Ery-R (161)
Antimicrobial agent
Range
erythromycin clindamycin penicillin amoxycillin co-amoxiclav cefotaxime ceftriaxone imipenem co-trimoxazole tetracycline chloramphenicol rifampicin vancomycin
1–128 0.06– 128 0.06–8 0.06–4 0.06–4 0.06–1 0.06–1 0.06–1 0.12–8 0.06– 64 0.06–16 0.06 0.06–0.5
MIC50
MIC90
Percentage susceptiblea
64 64 0.06 0.06 0.06 0.06 0.06 0.06 4 64 4 0.06 0.25
128 128 1 1 1 0.5 0.5 0.5 4 64 16 0.06 0.25
0 10 79 84 84 92 98 84 40 13 60 100 100
a
Breakpoints suggested by NCCLS (1998)9 for S. pneumoniae, in mg/L: erythromycin, 0.25; clindamycin, 0.25; penicillin, 0.06; amoxycillin, 0.5; co-amoxiclav, 0.5/0.25; cefotaxime, 0.5; ceftriaxone, 0.5; imipenem, 0.12; co-trimoxazole, 0.5/9.5; tetracycline, 2; chloramphenicol, 4; rifampicin, 1; vancomycin, 1. ery-R, erythromycin-resistant.
Table II summarizes the antibiotype, phenotype and genotype distribution in this collection of strains. The majority (65%) of microorganisms (105/161) carried resistance to two or more additional drugs with the pattern: erythromycin, -co-trimoxazole–tetracycline–chloramphenicol being the most represented (46 strains out of 161; 28.6%). In total, 87% of the 161 S. pneumoniae strains tested were resistant to tetracycline, 60% to co-trimoxazole, 40% to chloramphenicol and 21% to penicillin (12.4% and 8.6% lowand high-level resistance, respectively). As expected, penicillin resistance in erythromycin-resistant S. pneumoniae (21%) was higher than that observed in the general S. pneumoniae population circulating in Italy (12.7%).2 The high incidence of associated erythromycin and tetracycline resistance is not surprising since ermB and TetM determinants appear to be carried by transposon Tn1545. Erythromycin resistance alone was rare (14 strains, 8.6%), the majority (11/14) of these showing the M phenotype (Table II). Most S. pneumoniae (145 out of 161; 90%) expressed a constitutive phenotype in keeping with MIC results, while 15 displayed the M phenotype. Only one isolate possessed an inducible mechanism of resistance (Table II). All strains belonging to the MLSB phenotype were PCR-positive for the ermB gene and 15 strains categorized as M carried the mefE gene (Table II). M phenotype S. pneumoniae showed erythromycin MIC90 not exceeding 8 mg/L, as expected,6 while erythromycin and clindamycin MIC90 values for those with a constitutive mechanism were 128 mg/L (Table III). Overall, 38 different clones could be distinguished by
Table II. Distribution of antibiotic resistance patterns, macrolide phenotypes and genotypes among the 161 S. pneumoniae studied Phenotype/genotype Antibiotype (number of strains) E (14) EP (1) E Sxt (4) E Tc (37) E Tc P (6) E Sxt Tc (22) E Sxt Ch (1) E Tc Ch (3) E Tc Ch P (4) E Sxt Tc Ch (46) E Sxt Tc Ch P (9) E Sxt Tc P (13) E Sxt Ch P (1) Total strains
C/ermB 2 1 4 37 5 22 1 3 4 46 7 13 145 (90%)
I/ermB
M/mefE
1
11
1
2
1 (0.7%)
1 15 (9.3%)
E, erythromycin; Sxt, co-trimoxazole; Tc, tetracycline; Ch, chloramphenicol; P, penicillin; C, constitutive; I, inducible; M, M-type.
PFGE, indicating a substantial heterogeneity among the erythromycin-resistant isolates. The most prevalent are illustrated in the Figure. The most represented PFGE profile, designated L, was found in 17 strains, and its variations in 34 other microorganisms. S. pneumoniae belonging to
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Macrolide resistance in S. pneumoniae in Italy Table III. In-vitro activities of erythromycin and clindamycin against macrolide constitutive (C), inducible (I) and M-type resistant (M) S. pneumoniae Phenotype (number of strains) C (145) I (1) M (15)
Antibiotic
Range
erythromycin clindamycin erythromycin clindamycin erythromycin clindamycin
1–128 4–128 1 0.06 1–16 0.06–0.12
Figure. Most prevalent PFGE patterns abserved among erythromycin resistant pneumococcal isolates. Lanes 1 and 13: molecular weight marker ladder; lanes 2–12: profiles A, A , B, G, L, L , L , LV, LVI, O, P.
this clone circulate in all Italian centres. The majority (32/51) of strains characterized by profile L or by its variations were also resistant to co-trimoxazole, tetracycline and chloramphenicol. The second most represented profile (11 strains) corresponded to the Italian autochthonous clone described previously.10 These microorganisms were isolated in northern and central Italy exclusively, and were resistant to erythromycin, penicillin, co-trimoxazole and tetracycline. The third profile, found in four isolates and in eight other variants, is identical to that displayed by the Spanish/USA clone whose presence in Italy has been reported before.10 Among the 15 M phenotypes
MIC50 64 64
8 0.06
MIC90 128 128
8 0.06
of S. pneumoniae, six different profiles emerged. Only one strain was characterized by restriction pattern L, dominating among constitutive macrolide-resistant microorganisms. In 1993, percentages of penicillin and erythromycin resistance were very similar (5.5 and 6%, respectively) while erythromycin resistance (31.7%) currently exceeds the rate of penicillin resistance (12.7%):2 it therefore seems clear that, in Italy, the evolution of erythromycin resistance is driven by independent forces, possibly including the spread of clone L (penicillin-susceptible). The molecular mechanisms underlying macrolide resistance in S. pneu moniae appear to be similar in Spain7 and Italy, where ermB genes confer the MLSB phenotype (mostly in the constitutive variant) to the vast majority of strains. The situation is reversed in Canada4 and the USA,5 where the mefE gene predominates. Our observation that the ermB gene is carried by S. pneumoniae clones that are only rarely colonized by mefE genes may implicate a different host range of the two genetic determinants and/or a geographic segregation of S. pneumoniae subtypes. Widely divergent prescription habits, rates of overall resistance, amounts of antibiotic consumption and gene pool sizes may also be responsible for the differences observed. Resistance to antibiotics is a complex phenomenon and actual levels of expression markedly influence the clinical significance of this trait. There is now unanimous agreement on the contention that -lactam resistance in S. pneu moniae does not necessarily equate with treatment failures in non-meningeal infections.1 Moreno et al.11 observed surprising cure rates among a small number of patients with pneumonia caused by erythromycin-resistant S. pneu moniae treated with erythromycin. In addition, response to the drug was found to be independent of both MIC values for the isolated strains and presence of bacteraemia. These findings, while limited and requiring confirmation, are extremely interesting and point to the possibility that the clinical response described for -lactam-resistant S. pneu moniae may also hold true for other classes of drug. Given these possible therapeutic implications and in analogy with what is currently done with -lactams, assess-
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A. Marchese et al. ment of macrolide resistance phenotypes should routinely be performed in clinical microbiology laboratories.
1997. Abstract C-77b, p. 59, American Society for Microbiology, Washington, DC.
Acknowledgement
6. Sutcliffe, J., Grebe, T., Tait-Kamradt, A. & Wondrack, L. (1996). Detection of erythromycin-resistant determinants by PCR. Antimicrobial Agents and Chemotherapy 40, 2562–6.
This study was supported in part by Abbott S.p.a., Campoverde, Italy.
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7. Lantero, M., Portillo, A., Gastanares, M. J., Ruiz-Larrea, F., Zarazaga, M., Olarte, I. et al. (1998). MLS resistance phenotypes and mechanisms in S. pneumoniae. In Program and Abstracts of the Fourth International Conference on the Macrolides, Azalides, Streptogramins and Ketolides. Barcelona, Spain, 1998. Abstract 3.10, p. 34. 8. National Committee for Clinical Laboratory Standards. (1997). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically—Fourth Edition: Approved Standard M7-A4. NCCLS, Wayne, PA. 9. National Committee for Clinical Laboratory Standards. (1998). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically—Fourth Edition: Approved Standard M100S8. NCCLS, Wayne, PA.
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4. Johnston, N. J., De Azavedo, J., Kellner, J. D. & Low, D. E. (1998). Prevalence and characterization of the mechanisms of macrolide, lincosamide and streptogramin resistance in Streptococcus pneumoniae. Antimicrobial Agents and Chemotherapy 42, 2425–6.
11. Moreno, S., Garcià-Leoni, M. E., Cercenado, E., Diaz, M. D., Bernaldo de Quiros, J. C. L. & Bouza, E. (1995). Infections caused by erythromycin-resistant Streptococcus pneumoniae: incidence, risk factors and response to therapy in a prospective study. Clinical Infectious Diseases 20, 1195–200.
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Received 11 December 1998; returned 11 March 1998; revised 19 April 1999; accepted 12 May 1999
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