1 Laboratorio di Batteriologia e Micologia Medica, Istituto Superiore di Sanità, viale Regina Elena 299, 00161 Rome, Italy; 2 Respiratory and Systemic Infection Laboratory, PHLS Central Public Health Laboratory, London, UK
Received 20 December 2001; returned 13 March 2002; revised 22 April 2002; accepted 2 May 2002
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Abstract |
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Introduction |
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Current guidelines state that non-toxigenic C. diphtheriae should be regarded as a potential pathogen and be treated with antibiotics if the patient is symptomatic.4 Although C. diphtheriae has been shown to be susceptible to a wide range of antimicrobial agents,5 benzylpenicillin and erythromycin remain the recommended drugs for the antimicrobial treatment and control of diphtheria.4 Failure to eliminate C. diphtheriae from cases of diphtheria and carriers treated with penicillin has been reported in the past, but no resistance to penicillin has been documented.6 In contrast, inducible resistance to erythromycin has been reported in the USA and Vietnam.7
This study was carried out to determine the susceptibility of clinical isolates of non-toxigenic C. diphtheriae biotype gravis to benzylpenicillin and erythromycin. In several of the cases it was documented, on a clinical basis, that penicillin failed to cure the pharyngitis and therefore a second course of antibiotic treatment with erythromycin had to be administered. This second course of treatment was usually successful, and the patients did not return to their general practitioner.
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Materials and methods |
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The MIC and MBC of penicillin (benzylpenicillin-G, 1670 U/mg; Sigma-Aldrich) were determined by the broth macrodilution method according to the NCCLS guidelines.8 Exponentially growing cells were suspended at c. 5 x 105 cfu/mL in MH-II broth supplemented with two-fold serial dilutions of penicillin (320.03 mg/L). Following incubation under agitation at 37°C for 24 h, viable counts were carried out by plating in triplicate a 100 µL sample on Columbia blood agar (supplemented with 5% sheep blood). Plates were incubated for 2448 h at 37°C before enumeration. The lower limit of reproducibly quantifiable cfu was 50 cfu/mL. Staphylococcus aureus (ATCC 29213) was used as a control for both methods. MIC50s and MIC90s were calculated using cumulation and interpolation.9 A timekill assay was carried out by incubating c. 5 x 104 to 5 x 105 cfu/mL in MH-II broth supplemented with 0 x, 2 x, 4 x, 8 x and 16 x MIC of penicillin or erythromycin, at 37°C with agitation.10 At pre-determined time points, 100 µL samples were taken for determination of viable colony counts, as described above. All experiments were conducted in duplicate. Bactericidal activity was defined as the minimum concentration of antibiotic required to achieve a reduction of the inoculum by at least 99.9% within 24 h.
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Results and discussion |
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The number of strains examined in our study is not large. Nonetheless, the data clearly show that a high percentage of isolates of non-toxigenic C. diphtheriae gravis are tolerant to penicillin and that there are no differences in the MIC between susceptible and tolerant strains. MBCs are high, very often above the penicillin serum level of c. 24 mg/L.11 A significant correlation between penicillin MBC or penicillin tolerance and therapy outcome was not found. However, a therapeutic failure was often observed when a tolerant C. diphtheriae strain was isolated in the absence of group A streptococci (9/17). Thus, it is important to consider that in cases of pharyngitis/tonsillitis where C. diphtheriae is isolated as the sole pathogen, it could be the aetiological agent of infection and that penicillin is not the most appropriate antibiotic. Therefore, treatment of these infections with erythromycin is recommended as it was always shown to eliminate the symptoms. Tolerance of C. diphtheriae to ß-lactams should also be considered in systemic infections, as C. diphtheriae tolerant to amoxicillin have been isolated from a case of endocarditis.12
In addition to erythromycin, a number of other antimicrobial agents were examined by Etest and were active against the isolates tested in this study. These included clindamycin (MIC50 0.094 mg/L, MIC90 0.125 mg/L), ciprofloxacin (MIC50 0.064 mg/L, MIC90 0.094 mg/L), rifampicin (MIC50 < 0.002 mg/L, MIC90 0.03 mg/L) and tetracycline (MIC50 0.25 mg/L, MIC90 0.38 mg/L). Because antibiotic breakpoints for C. diphtheriae are not currently available, the percentage of isolates susceptible to each antibiotic has not been determined. Nonetheless, the present study indicates that antibiotics other than penicillin may have to be considered for the treatment of non-toxigenic C. diphtheriae pharyngitis/tonsillitis.
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Acknowledgements |
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Footnotes |
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References |
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