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Keywords:

  • Streptococcus pyogenes;
  • macrolide antibiotics;
  • erythromycin resistance

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. References

Objective: To assess the incidence of resistance to erythromycin and to the three other macrolide antibiotics most extensively used in Italy (azithromycin, clarithromycin and roxithromycin) among clinical strains of Streptococcus pyogenes freshly isolated from throat cultures of pediatric patients in an area of Central Italy.

Method: Two sets of isolates were examined. The strains of the first set (n= 100) were collected according to a protocol admitting only throat swabs from untreated patients with symptoms of acute pharyngotonsillitis. The second set (n= 180) consisted of strains isolated from throat cultures during the routine activity of diagnostic laboratories, no particular protocol being applied.

Results: A trimodal distribution of strains was observed in relation to their macrolide susceptibility levels: two clusters were constituted by highly susceptible and highly resistant strains, respectively; a third, middle cluster consisted of strains displaying low-level resistance (or even intermediate susceptibility, in a minority of isolates, to clarithromycin). The distribution of individual isolates in the three modal clusters was the same with all four drugs. Both MIC ranges and MIC50s almost overlapped in the isolates of the two sets, whereas MIC90s were far higher in the strains of the second set (4 μg/mL for clarithromycin, 8 μg/mL for erythromycin and azythromycin, and 16 μg/mL for roxithromycin) than in those of the first (0.125 μg/mL for all four drugs). Resistant strains were 5% among the isolates of the first set and three times as many among those of the second.

Conclusions: The lower incidence of macrolide resistance recorded in the first set is probably more reliable: the threefold incidence observed in the second set may be overestimated due to the lower frequency of strains involved in drug-responsive infections and to the increased occurrence of strains from unsuccessfully treated patients.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. References

A re-emergence of severe Streptococcus pyogenes infections and of their suppurative and non-suppurative sequelae has been reported over the last decade in Europe and North America [1,2]. All S. pyogenes isolates are susceptible to penicillin, the drug of choice for the treatment of group A streptococcal infections. Penicillin failures may, however, occur due to causes which include inadequate dosage, poor patient compliance or tolerance phenomena [3]; a possible role for β-lactamase production by the surrounding oropharyngeal flora [4] is more controversial.

Erythromycin is usually recommended as the alternative treatment for group A streptococcal pharyngotonsillitis only in patients allergic to penicillins or in cases of penicillin failure [5]. In fact, however, macrolide antibiotics are extensively used in these infections, and generally in infections of the upper respiratory tract, indeed even more so since the introduction of new molecules with an expanded spectrum (including in particular Haemophilus influenzae), better pharmacologic properties and fewer gastrointestinal side effects as compared with erythromycin [6]. The resistance of S. pyogenes to erythromycin was first reported in the late 1950s [7]. Since then, this resistance trait has been found, with variable but generally low incidence, in several countries [8,9]. Increasing rates, with peaks exceeding 80%, associated with the diffusion of new types, were reported in Japan in the 1970s [10–13]. In more recent years, while resistance rates are still quite low according to some studies [14–17], an increased incidence of erythromycin resistance has been reported in other surveys from different parts of the world [18–22]. For Italy, only preliminary and rather heterogeneous data are so far available [23–25].

In this study, two different sets of S. pyogenes isolates obtained from throat cultures of patients living in the same area of Central Italy were examined. One set of strains came from untreated patients with symptoms of acute pharingotonsillitis. The second set of strains was isolated during the routine activity of diagnostic laboratories of the same area, no particular protocol being applied. All strains were tested for their susceptibility to erythromycin and to the three other macrolide antibiotics most extensively used in Italy (azithromycin, clarithromycin and roxithromycin). The different resistance rates recorded in the isolates of the two sets, in spite of substantial cross-response to the four macrolide antibiotics, suggest that the criteria for specimen collection are likely to influence the reported incidence of macrolide resistance.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. References

Bacterial strains

Two separate sets of S. pyogenes strains were examined. All strains were isolated over a 6-month period (October 1995 to March 1996) from throat cultures of pediatric patients living in an area of Central Italy (part of Marche and Umbria, with a population of approximately one million). Multiple isolates from the same patient were avoided. The first set, designed to be representative of the variety of S. pyogenes strains causing acute pharingotonsillitis in the community, included 100 isolates collected according to a protocol requiring throat cultures to be obtained from untreated patients (who had not received antibiotics for at least 1 month prior to throat sampling) diagnosed with this infection. The second set included 180 throat strains isolated in different diagnostic laboratories in the area in the course of their routine activity, without the application of any protocol.

β-Hemolytic colonies were identified as S. pyogenes using bacitracin disks (Difco Laboratories, Detroit, MI, USA) and a latex agglutination assay (Streptex; Wellcome, Dartford, UK). The strains were maintained in glycerol at −70°C until all isolates were collected, and subcultured twice on blood agar before susceptibility testing.

Antibiotics

Erythromycin and roxithromycin were purchased from Sigma Chemical Co., St Louis, MO, USA. Azithromycin and clarithromycin were obtained from Pfizer Inc., New York, NY, USA, and Abbott Laboratories, Abbott Park, IL, USA, respectively.

Susceptibility testing

Minimum inhibitory concentrations (MICs) were determined by a standard microdilution procedure [26] using Mueller-Hinton II broth (Becton Dickinson Microbiology Systems, Cockeysville, MD, USA) supplemented with 3% lysed horse blood as the test medium. The inoculum was 5 × 105 CFU/mL. The antibiotics were tested at final concentrations (prepared from twofold dilutions) ranging from 0.03 to 128 μg/mL. The MIC was defined as the lowest concentration which yielded no visible growth. The following MIC breakpoints suggested by the National Committee for Clinical Laboratory Standards [27] were considered for erythromycin, clarithromycin, and roxithromycin (susceptible, ≦0.25 μg/mL; intermediate, 0.5 μg/mL; resistant, ≧1 μg/mL) and for azythromycin (susceptible, ≦0.5 μg/mL; intermediate, 1 μg/ml; resistant, ≧2 μg/mL). Enterococcus faecalis ATCC 29212 and Staphylococcus aureus ATCC 29213 were used as controls.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. References

The activities of the four macrolide antibiotics against the S. pyogenes isolates of the two sets are summarized in Tables 1 and 2.

Table 1.  Activities of erythromycin, azithromycin, clarithromycin and roxithromycin against 100 S. pyogenes isolates of the first set (i.e. collected according to a protocol requiring throat swabs to be obtained from untreated patients with symptoms of acute pharyngotonsillitis)
 No. (cumulative no.) and cumulative % of strains inhibited at MIC (μg/mL)
Antibiotic≦0.030.060.120.250.51248163264128128
Erythromycin39 (39)36 (75)19 (94)1 (95)     2 (97)   3 (100)
 39%75%94%95%    97%    100%
Azithromycin35 (35)40 (75)20 (95)     1 (96)1 (97)   3 (100)
 35%75%95%     96%97%   100%
Clarithromycin45 (45)38 (83)12 (95)      2 (97)   3 (100)
 45%83%95%      97%   100%
Roxithromycin34 (34)38 (72)21 (93)2 (95)     2 (97)   3 (100)
 34%72%93%95%     97%   100%

A comparison of these results shows that both MIC ranges and MIC50s almost overlapped in the two sets, whereas MIC90s were far higher in the strains of the second set (4 μg/mL for clarithromycin, 8 μg/mL for erythromycin and azithromycin, and 16 μg/mL for roxithromycin) than in those of the first (0.12 μg/mL for all four drugs). Among the 100 isolates of the first set, the same five strains were cross-resistant to all four macrolide antibiotics, and no intermediate value was observed. Among the 180 isolates of the second set, the same 27 strains were resistant to erythromycin, azithromycin and roxithromycin, and resistant (25 strains) or intermediate (two strains) to clarithromycin. The difference in the resistance rate between the first set isolates (five of 100, 5%) and the second set isolates (27 of 180, 15%) was statistically significant (p=0.0109).

Table 2.  Activities of erythromycin, azithromycin, clarithromycin and roxithromycin against 180 S. pyogenes strains of the second set (i.e. isolated in the routine activity of diagnostic laboratories, no particular protocol being applied)
 No. (cumulative no.) and cumulative % of strains inhibited at MIC (μg/mL)
Antibiotic≦0.030.060.120.250.51248163264128>128
Erythromycin80 (80)69 (149)4 (153) 2 (155)1 (156)1 (157)6 (163)10 (173)  7 (180)  
 44.4%82.8%85% 86.1%86.7%87.2%90.6%96.1%100%    
Azithromycin72 (72)73 (145)8 (153)  2 (155)4 (159)10 (169)4 (173)    7 (180)
 40%80.6%85%    86.1%88.3%93.9%96.1%  100%
Clarithromycin94 (94)59 (153)    2 (155)1 (156)8 (164)9 (173)   7 (180)
 52.2%85%   87.6%86.7%91.1%96.1%    100%
Roxithromycin70 (70)73 (143)10 (153)   2 (155)3 (158)3 (161)6 (167)6 (173)  7 (180)
 38.9%79.4%85%   86.1%87.8%89.4%92.8%96.1%  100%

A trimodal distribution in relation to macrolide susceptibility was evident in the isolates of both sets. Two clusters consisted of highly susceptible and highly resistant strains, respectively. The modal MIC was ≦0.12 μg/mL for azithromycin and clarithromycin and ≦0.25 μg/mL for erythromycin and roxithromycin within the highly susceptible cluster, and >128 μg/mL for all drugs within the highly resistant cluster. A third, middle cluster was separated from both highly susceptible and highly resistant strains by a variable number of MIC values not recorded for any test strain. Among the first set strains, this middle cluster was represented by two isolates (2%) displaying low-level resistance (8 to 16 μg/mL) to all four macrolides. Among the second set strains, it was represented by 20 isolates (11.1%) displaying low-level resistance (1 to 32 μg/mL) to erythromycin, azithromycin and roxithromycin, and low-level resistance (2 to 8 μg/mL) or, less often, intermediate susceptibility (0.5 μg/mL) to clarithromycin. The three modal clusters were distributed rather homogeneously within the geographic area considered.

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. References

There was substantial cross-response of the S. pyogenes test strains to the four macrolide antibiotics, in the sense that the distribution of individual isolates over the three distinct modal clusters (highly susceptible, middle and highly resistant) was the same with all four macrolide antibiotics. It is worth stressing that the occurrence, among resistant strains, of two distinct degrees of resistance (high or low) separated by a clear solution of continuity had already been noted in early studies of erythromycin resistance in S. pyogenes [8,9].

Interestingly, resistant strains were three times more frequent within the second than the first set, this difference being essentially accounted for by low-level resistant strains. By virtue of the criterion applied for collection, the isolates of the first set — i.e. those obtained from untreated patients with symptoms of acute pharyngotonsillitis — are likely to be representative of the actual variety of S. pyogenes strains currently involved in acute pharyngotonsillitis in the community living in the area covered by our survey. This is less true of the second set strains, isolated from throat swabs routinely processed in diagnostic laboratories of the same area, with no particular protocol applied. In fact, in patients receiving macrolide antibiotics for acute pharyngotonsillitis or other acute infections of the higher respiratory tract, therapy is given in most instances without prior microbiological investigation. This empirical therapy is successful in the majority of patients, and often only in case of failure is the diagnostic laboratory applied to for help. This is why the isolates of the first set probably reflect more closely the incidence of macrolide resistance occurring in the community. The threefold incidence observed in the second set may be overestimated due to the lower frequency of strains involved in drug-responsive infections and to the increased occurrence of strains from unsuccessfully treated patients in the throat swabs routinely processed in diagnostic laboratories.

References

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. References
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