Induction of telithromycin resistance in Streptococcus pneumoniae

Satoru Kaieda1,2, Naohiro Okitsu1,3, Hisakazu Yano3, Yoshio Hosaka1, Ryuichi Nakano1, Ryouichi Okamoto1, Haruo Takahashi2 and Matsuhisa Inoue1,*

1 Department of Microbiology, Kitasato University School of Medicine, Sagamihara, Kanagawa 228-8555; 2 Department of Otolaryngology, Nagasaki University School of Medicine, Nagasaki 852-8501; 3 Department of Otolaryngology, Tohoku University School of Medicine, Sendai 980-8574, Japan

Keywords: ketolides, inducible resistance, erythromycin-resistant Streptococcus pneuomoniae

Sir,

Ketolides are a new class of semisynthetic macrolide derivatives that show excellent activity against Streptococcus pneumoniae, even against erythromycin-resistant isolates. Previous investigators have not found any telithromycin-resistant isolates among S. pneumoniae with the constitutive macrolide–lincosamide–streptogramin B (cMLSB) phenotype, even though staphylococci or Streptococcus pyogenes with the cMLSB phenotype can develop resistance to this drug.13 The lack of induction of methylase production by this drug is one reason for such a difference.4

Recently, Hamilton-Miller & Shah5 reported that resistance to the ketolide cethromycin (formerly ABT-773) could be induced in S. pneumoniae with the cMLSB phenotype by erythromycin or other related antibiotics. In this study, we examined whether 55 isolates of erythromycin-resistant S. pneumoniae (MIC >= 1 mg/L) carrying the mef(A) and/or erm(B) genes could develop resistance to telithromycin, a ketolide with different substitutions from cethromycin (ABT-773), when exposed to erythromycin.

Fifty-five clinical isolates of erythromycin-resistant S. pneumoniae (obtained from the sputum of patients with lower respiratory tract infections between 1998 and 2000) were used. These isolates were identified by their sensitivity to optochin and the bile solubility test, and by PCR amplification of the lytA gene. S. pneumoniae ATCC 6305 was used as the quality control strain.

Reference samples of the following antimicrobial agents of known potency were kindly supplied in powder form by the indicated manufacturers: erythromycin (Shionogi Pharmaceutical Co., Osaka, Japan), clarithromycin (Taisho Pharmaceutical Co., Tokyo, Japan), azithromycin (Pfizer Laboratories, Groton, CT, USA), rokitamycin (Asahi Kasei, Tokyo, Japan), telithromycin (Nippon Hoechst-Marion-Roussel, Tokyo, Japan) and clindamycin (Upjohn, Tokyo, Japan).

All isolates were tested for susceptibility to the six antibiotics listed above at concentrations between 0.015 and 128 mg/L. MICs were determined by the two-fold agar dilution method using susceptibility test agar (Mueller–Hinton agar medium; Eiken Chemicals, Tokyo, Japan) with 8% Strepto Haemo supplement (SHS; Eiken Chemicals).

DNA was obtained as previously reported, and the presence of macrolide resistance genes was investigated by PCR using primers and amplification conditions that have been described previously.6

Induction of telithromycin resistance was examined by the following two methods.

(i) Agar dilution method: induction of telithromycin resistance was evaluated by comparing the MICs for telithromycin in the presence and absence of a subinhibitory concentration of erythromycin (0.1 mg/L). Two susceptibility agar plates were prepared for each isolate: one contained only telithromycin and the other contained telithromycin plus erythromycin. The MIC was determined in accordance with the susceptibility test.

(ii) Disc diffusion method: A bacterial suspension (2 mL of ~108 cfu/mL) was inoculated onto 10 mL of susceptibility test agar containing 8% SHS and spread over the surface. After excess suspension was removed, paper discs (8 mm diameter high discs; Tokyo Roshi Kaisha, Tokyo, Japan) containing erythromycin or rokitamycin at 20 µg/disc or telithromycin at 5 µg/disc were placed on the surface of the agar plate. Then the plates were incubated overnight at 35°C and induction of telithromycin resistance was assessed from the shape of the zone of inhibition around the telithromycin disc nearest either the erythromycin or the rokitamycin disc.

Fifteen isolates carrying only the mef(A) gene had the M phenotype. Among the other 40 isolates, 25 carried the erm(B) gene, and 15 had both the mef(A) and erm(B) genes. All 40 isolates showed a high level of resistance to clindamycin (MIC >= 128 mg/L) and were also resistant to macrolides, lincosamides and streptogramin B (MLSB phenotype). As shown in Figure 1, 26 isolates with this phenotype showed decreased susceptibility to telithromycin after exposure to erythromycin, although the MICs were still <1 mg/L. The remaining 14 isolates showed a minimal decrease in susceptibility to telithromycin (two-fold dilution). However, upon adding a telithromycin disc to an erythromycin–rokitamycin two-disc diffusion test plate, the zone of inhibition around the telithromycin disc was blunted near the erythromycin disc for all isolates with the MLSB phenotype, including the 25 isolates considered to show a true cMLSB phenotype based on constitutive resistance to rokitamycin (MIC >= 4 mg/L). Furthermore, when this was done for the 16 isolates highly resistant to rokitamycin (MIC >= 64 mg/L), the zone of inhibition around the telithromycin disc was blunted near both the erythromycin and rokitamycin discs.



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Figure 1. MICs of telithromycin in the presence and absence of erythromycin (0.1 mg/L). There was no increase in the MICs for the 15 isolates with the M phenotype (ellipses). There was a slight increase (two-fold dilution) for 14 isolates, including seven with the iMcLSB phenotype (triangles) and seven with the cMLSB phenotype (squares). A marked increase (two-fold dilution or greater) was seen for 26 isolates, comprising eight with the iMcLSB phenotype (triangles) and 18 with the cMLSB phenotype (squares).

 
These results indicate that telithromycin resistance can be induced by erythromycin, even in S. pneumoniae isolates with the MLSB phenotype as Hamilton-Miller & Shah reported.5 However, telithromycin still inhibited all 40 isolates at concentrations <1 mg/L, a result that may reflect the strong affinity of the drug for methylated 23S rRNA.7

Acknowledgements

This work was supported in part by a Grant-in-Aid (09670296) from the Ministry of Science, Education and Culture of Japan.

Footnotes

* Corresponding author. Tel: +81-42-778-9349; Fax: +81-42-778-9350; E-mail: matsu{at}kitasato-u.ac.jp Back

References

1 . Reinert, R. R., Bryskier, A. & Lutticken, R. (1998). In vitro activities of the new ketolide antibiotics HMR 3004 and HMR 3647 against Streptococcus pneumoniae in Germany. Antimicrobial Agents and Chemotherapy 42, 1509–11.[Abstract/Free Full Text]

2 . Jalava, J., Kataja, J., Seppala, H. et al. (2001). In vitro activities of the novel ketolide telithromycin (HMR 3647) against erythromycin-resistant Streptococcus species. Antimicrobial Agents and Chemotherapy 45, 789–93.[Abstract/Free Full Text]

3 . Shortridge, V. D., Zhong, P., Cao, Z. et al. (2002). Comparison of in vitro activities of ABT-773 and telithromycin against macrolide-susceptible and -resistant streptococci and staphylococci. Antimicrobial Agents and Chemotherapy 46, 783–6.[Abstract/Free Full Text]

4 . Bonnefoy, A., Girard, A. M., Agouridas, C. et al. (1997). Ketolides lack inducibility properties of MLSB resistance phenotype. Journal of Antimicrobial Chemotherapy 40, 85–90.[Abstract]

5 . Hamilton-Miller, J. M. T. & Shah, S. (2002). Activity of ketolide ABT-773 (cethromycin) against erythromycin-resistant Streptococcus pneumoniae: correlation with extended MLSK phenotype. Journal of Antimicrobial Chemotherapy 50, 907–13.[Abstract/Free Full Text]

6 . Sutcliffe, J., Grebe, T., Tait-Kamradt, A. et al. (1996). Detection of erythromycin-resistant determinants by PCR. Antimicrobial Agents and Chemotherapy 40, 2562–6.[Abstract]

7 . Zhong, P., Cao, Z., Hammond, R. et al. (1999). Induction of ribosome methylation in MLS-resistant Streptococcus pneumoniae by macrolides and ketolides. Microbial Drug Resistance 5, 183–8.[ISI][Medline]