Journal of Antimicrobial Chemotherapy (2001) 47, 239-240
© 2001 The British Society for Antimicrobial Chemotherapy
Correspondence |
Resistance to tetracycline and distribution of tetracycline resistance genes in European Staphylococcus aureus isolates
a Institute for Medical Microbiology and Virology, Heinrich-Heine University Düsseldorf, Düsseldorf, Universitätsstrasse 1. Geb. 22.21, 40225 Düsseldorf, Germany; b Eijkman-Winkler Institute, University Medical Center Utrecht, Utrecht, The Netherlands.
Sir,
We read with great interest the recent article by Trzcinski et al.,1 in which the analysis of the expression of tetracycline resistance in 66 methicillin-resistant Staphylococcus aureus (MRSA) isolates was reported. In that study, they used the polymerase chain reaction (PCR) to screen for the presence of four tetracycline-resistance determinants, tetK, tetL, tetM and tetO. They also determined the MICs of tetracycline, doxycycline and minocycline, with or without preincubation with subinhibitory concentrations of tetracycline or minocycline. Despite the results of their susceptibility tests, Trzcinski et al.1 suggested that all tetracycline-resistant S. aureus isolates also be considered doxycycline resistant, and that all tetM-positive isolates be treated as resistant to all tetracyclines.
In our study, we investigated the tetracycline susceptibility of 3052 S. aureus isolates collected from April 1997 to February 1999 at 25 university hospitals participating in the European SENTRY programme.2 We also determined the distribution of the four tetracycline resistance genes in 600 tetracycline-resistant S. aureus isolates, 400 MRSA and 200 methicillin-susceptible S. aureus (MSSA) isolates.
We found that 764 of the 3052 isolates (25%) were methicillin resistant and that there was an obvious relationship between oxacillin resistance and resistance to tetracyclines. Susceptibility to tetracycline was 89.7% in the MSSA isolates and 42.9% in the MRSA isolates.
Two mechanisms of tetracycline resistance have been identified in Staphylococcus species: (i) active efflux resulting from the acquisition of the tetK and tetL genes located on a plasmid; and (ii) ribosomal protection mediated by tetM or tetO determinants located on either a transposon or the chromosome.3,4 S. aureus strains carrying tetK only have been described as being tetracycline resistant, but minocycline susceptible. The tetM gene, in contrast, is believed to confer resistance to all drugs of the tetracycline group, including tetracycline and minocycline.1,3,4
In our study, we screened 400 MRSA and 200 MSSA isolates, all tetracycline resistant and randomly selected, for the presence of the four tetracycline resistance genes, tetK and tetM according to Warsa et al.3 and tetL and tetO according to Trzcinski et al.1 We also screened 50 randomly selected isolates (30 MRSA and 20 MSSA) that were fully susceptible to tetracycline for the presence of the tetracycline resistance genes. Primers specific for the conserved regions of the 16S rRNA gene were used as additional internal controls5 and the PCR products of the tetracycline resistance genes were randomly sequenced to ensure specificity. Failure of an amplification to produce a product corresponding to the 16S rRNA gene target sequence indicated an error in the experimental conditions and the necessity of repeating the procedure.
Although the primers were designed to be specific by the authors,1,3 it is sometimes possible to detect closely related components. However, we were not able to detect any non-specific PCR products in any of our tetracycline-resistant isolates. Moreover, none of the tested genes encoding tetracycline resistance and no non-specific PCR products were detectable in any of the 50 fully tetracycline-susceptible S. aureus isolates tested.
Of the 66 MRSA isolates, mainly from Eastern Europe, in the study by Trzcinski et al.1, 24 displayed tetM only, 21 had tetK only and 21 had both tetK and tetM. In the present study, the tetM gene was detected in 304/400 (76%) and the tetK gene in 292/400 (73%) tetracycline-resistant MRSA isolates. Approximately half of the MRSA isolates (202/ 400, 50.5%) carried both the tetM and tetK genes. The tetL gene was detected in 6/400 (1.5%) MRSA isolates. The tetM gene was detected in 20/200 (10%) and the tetK gene in 192/200 (96%) tetracycline-resistant MSSA isolates. Twelve (6%) of the MSSA isolates carried both the tetM and tetK genes. The tetL gene was not detected in any of the MSSA isolates. The tetO gene was not found in any of the isolates tested. Our results show that the tetM and tetK genes were the most prevalent single tetracycline resistance determinants in MRSA and MSSA, respectively, and that the combination tetM/tetK was approximately 10 times more prevalent in tetracycline-resistant MRSA isolates than in tetracycline-resistant MSSA isolates. All of the isolates carrying the tetM gene showed in vitro resistance to both tetracycline and minocycline, confirming the results reported by others.1,3,4 Furthermore, isolates harbouring both the tetK and tetM genes displayed higher MIC values than the isolates containing just one of the genes. The respective MIC50/MIC90 values (in mg/L) and geometric means (in mg/L) for the tetK and/or tetM gene-containing isolates were determined according to NCCLS guidelines:6 tetK: tetracyclines 64/128 and 71, minocycline 0.25/0.25 and 0.20; tetM: tetracyclines 64/128 and 68, minocycline 4/4 and 3.95; tetK and tetM: tetracyclines 256 and 512, minocycline 8/8 and 6.5. In general, these MIC results are in line with those published by Trzcinski et al.1
In summary, the tetK gene was the most prevalent tetracycline resistance determinant in MSSA isolates, while the tetM gene was most frequent in MRSA isolates. The combination tetM/tetK was approximately 10 times more prevalent in tetracycline-resistant MRSA isolates than in tetracycline-resistant MSSA isolates. All isolates carrying the tetM gene showed in vitro resistance to tetracycline and minocycline, and the strains harbouring both the tetK and tetM genes displayed higher MIC values than those carrying just one of the genes. This survey on the prevalence of tetracycline resistance genes in tetracycline-resistant MRSA and MSSA strains constitutes the largest collection of clinical S. aureus isolates studied so far.
Acknowledgements
This work was funded in part by the SENTRY Antimicrobial Surveillance Programme, which is funded by an educational grant from Bristol-Myers Squibb Pharmaceutical Company, and by the European Network for Antimicrobial Resistance and Epidemiology (ENARE) with a grant (ERBCHRCT940554) from the European Union. European SENTRY participants: H. Mittermayer (Austria), M. Struelens (Belgium), F. Goldstein (France), V. Jarlier (France), J. Etienne (France), P. R. Courcol (France), F. Daschner (Germany), U. Hadding (Germany), N. Legakis (Greece), G.-C. Schito (Italy), G. Raponi (Italy), P. Heczko (Poland), W. Hyrniewicz (Poland), D. Costa (Portugal), E. Perea (Spain), F. Baquero (Spain), R. Martin Alvarez (Spain), J. Bille (Switzerland), G. French (UK), V. Korten (Turkey), S. Unal (Turkey), D. Gür (Turkey) and N. Keller (Israel).
Notes
J Antimicrob Chemother 2001; 47: 239–240
* Corresponding author. Tel/Fax: +49-2132-72040; E-mail: schmitfj{at}uni-duesseldorf.de 
Other SENTRY participants are listed in the Acknowledgements.
References
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Trzcinski, K., Cooper, B. S., Hryniewicz, W. & Dowson, C. G. (2000). Expression of resistance to tetracyclines in strains of methicillin-resistant Staphylococcus aureus. Journal of Antimicrobial Chemotherapy 45, 763–70.
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Schmitz, F.-J., Verhoef, J., Fluit, A. C. & the SENTRY Participants Group. (1999). Prevalence of resistance to MLS antibiotics in 20 European university hospitals participating in the European SENTRY surveillance programme. Journal of Antimicrobial Chemotherapy 43, 783–92.
3 . Warsa, U. C., Nonoyama, M., Ida, T., Okamoto, R., Okubo, T., Shimauchi, C. et al. (1996). Detection of tet(K) and tet(M) in Staphylococcus aureus of Asian countries by the polymerase chain reaction. Journal of Antibiotics 49, 1127–32.[Medline]
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