Penicillin tolerance amongst non-toxigenic Corynebacterium diphtheriae isolated from cases of pharyngitis

C. von Hunolstein1,*, F. Scopetti1, A. Efstratiou2 and K. Engler2

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Twenty-four strains of non-toxigenic Corynebacterium diphtheriae biotype gravis from the throats of patients with pharyngitis/tonsillitis were assayed for susceptibility to penicillin and erythromycin using determination of MIC, MBC and time–kill curves. There were no differences between the MICs of penicillin for susceptible and tolerant strains. All but one strain had penicillin MBCs >= 2 mg/L. Seventy-one per cent (17/24) of the strains were tolerant to penicillin. In contrast, all strains were susceptible to erythromycin (MIC <= 0.016 mg/L). These aspects should be considered when choosing the therapy for treating non-toxigenic C. diphtheriae pharyngitis/tonsillitis.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Although immunization is the most important long-term factor for diphtheria prevention, antibiotics play a significant role in the treatment and control of the disease. Antibiotics are used to prevent dissemination and toxin production in infected patients, to eradicate the organism in asymptomatic carriers and to prevent infection of contacts.1 In countries where anti-diphtheria vaccination is regularly implemented, the spectrum of diseases caused by Corynebacterium diphtheriae has changed. Non-toxigenic isolates of C. diphtheriae have become prevalent. Severe and often fatal systemic diseases by non-toxigenic C. diphtheriae, which were previously quite rare, have been documented in various countries during the past several years.2 Furthermore, during the 1990s cases of acute, often recurrent, pharyngitis and tonsillitis associated with the isolation of non-toxigenic C. diphtheriae, in particular biotype gravis, were described in both the UK and Italy.3

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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Twenty-four clinical strains of non-toxigenic C. diphtheriae biotype gravis, isolated from the throat of patients who presented to their general practitioners with a sore throat, were collected during an enhanced surveillance study conducted in the UK between 1995 and 1996.3 Susceptibility to penicillin and erythromycin was determined using Etest strips (AB Biodisk) on Mueller–Hinton II (MH-II cation-adjusted; BBL Microbiology Systems) blood agar (supplemented with 5% sheep blood) following the manufacturer’s recommendations.

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 (32–0.03 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 24–48 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 time–kill 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.


    Results and discussion
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The MIC50 and MIC90 of penicillin were 0.060 and 0.125 mg/L, respectively, and the MIC range was 0.03–0.25 mg/L (Table 1). These MICs are slightly lower than those reported in previous studies and this may reflect variations in methodologies and/or source of strains.2,5 In this study, the MIC of penicillin was also determined by Etest: the MIC50 was 0.115 mg/L, MIC90 0.125 mg/L and the range was 0.064–0.250 mg/L. The two methods (Etest and broth dilution) showed 96% concordance within one two-fold dilution.


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Table 1.  MIC, MBC and relative MBC/MIC ratio of penicillin for 24 strains of non-toxigenic C. diphtheriae biotype gravis from cases of acute, often recurrent, pharyngitis and tonsillitis
 
The MBC50 and MBC90 of penicillin were 2 and 8 mg/L, respectively, and 17/24 (71%) of the strains were found to have an MBC/MIC ratio >= 32 (Table 1), which has been suggested as indicating tolerance. The time-dependent effect of penicillin on the growth of a representative susceptible and tolerant strain of C. diphtheriae with a MIC of 0.125 mg/L is shown in Figure 1. For the susceptible strain, a 3 log10 decrease in cfu/mL (>99.9% killing) was detected after 24 h incubation at a concentration of 16 x MIC. In comparison, the tolerant strain elicited only a 1.6 log10 decrease after 24 h incubation at 16 x MIC.



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Figure 1. Time–kill plots with benzylpenicillin-G against non-toxigenic C. diphtheriae biotype gravis susceptible strain no. 95-325 (a) and tolerant strain no. 96-133 (b). Data are representative of two independent experiments. Standard deviations (always <10%) have been omitted. Diamonds, control; squares, 2 x MIC; white triangles, 4 x MIC; black triangles, 8 x MIC; asterisks, 16 x MIC.

 
Erythromycin was highly active against all isolates of C. diphtheriae. The MIC was <=0.016 mg/L by Etest and time–kill curves showed a 99.9% killing at 24 h, at 4 x MIC (data for four strains; not shown).

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. 2–4 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.


    Acknowledgements
 
We would like to thank Mrs J. White (PHLS, London, UK) for provision of epidemiological data. We thank Dr A. P. Johnson (PHLS, London, UK), Dr G. Orefici and Professor A. Cassone (Istituto Superiore di Sanità, Rome, Italy) for critical review of the manuscript. This work was funded in part by the European Commission DG RTD programme: BioMed 2 BMH4.CT 98-3793 ‘Microbiological surveillance of diphtheria in Europe’.


    Footnotes
 
* Corresponding author. Tel: +39-06-4990-2036; Fax: +39-06-4938-7112; E-mail: cris.v.h{at}iss.it Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Farizo, K. M., Strebel, P. M., Chen, R. T., Kimbler, A., Cleary T. J. & Cochi, A. L. (1993). Fatal respiratory disease due to Corynebacterium diphtheriae: case report and review guidelines for management, investigation and control. Clinical Infectious Diseases 16, 59–68.[ISI][Medline]

2 . Patey, O., Bimet, F., Riegel, P., Halioua, B., Emond, J. P., Estrangin, E. et al. (1997). Clinical and molecular study of Corynebacterium diphtheriae systemic infections in France. Journal of Clinical Microbiology 35, 441–5.[Abstract]

3 . Reacher, M., Ramsay, M., White, J., De Zoysa, A., Efstratiou, A., Mann, G. et al. (2000). Non-toxigenic Corynebacterium diphtheriae: an emerging pathogen in England and Wales? Emerging Infectious Diseases 6, 640–5.[ISI][Medline]

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8 . National Committee for Clinical Laboratory Standards. (2000). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically—Fifth Edition: Approved Standard M7-A5. NCCLS, Wayne, PA.

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10 . Hindler, J. (1992). Antimicrobial susceptibility testing. In Clinical Microbiology Procedure Handbook (Isenberg H. D., Ed.), Vol. 1, pp. 5.16.1–5.16.33. American Society for Microbiology, Washington, DC, USA.

11 . Chamber, H. F. & Neu, H. C. (1995). Penicillins. In Principles and Practice of Infectious Diseases (Mandell, G., Bennet, J. & Dolin R., Eds), pp. 233–46. Churchill Livingstone, New York, NY, USA.

12 . Dupont, C., Turner, L., Rouveix, E., Nicolas, M. H. & Dorra, M. (1995). Endocardite à Corynebacterium diphtheriae tolerant à l’amoxicillin. Presse Medicale 24, 1135.