Characterization of the translational attenuator of 20 methicillin-resistant, quinupristin/dalfopristin-resistant Staphylococcus aureus isolates with reduced susceptibility to glycopeptides

F.-J. Schmitza,b,*, W. Wittec, G. Wernerc, J. Petridoua, A. C. Fluitb and S. Schwarzd

a Institute for Medical Microbiology and Virology, University Hospital Düsseldorf, Germany; b Eijkman Winkler Institute for Medical Microbiology, University Hospital Utrecht, The Netherlands; c Robert Koch Institute, Wernigerode Branch, Wernigerode; d Institut für Tierzucht und Tierverhalten der Bundesforschungsanstalt für Landwirtschaft (FAL), Celle, Germany

Sir,

Macrolide, lincosamide and streptogramin antibiotics are chemically distinct inhibitors of bacterial protein synthesis. Quinupristin/dalfopristin is a combination of streptogramin B and A compounds (SB/SA) with a synergic activity against most Gram-positive bacteria. Expression of MLS resistance in staphylococci may be constitutive or inducible. When expression is constitutive, the strains are resistant to all macrolides, lincosamides and streptogramin B-type antibiotics. Streptogramin A-type antibiotics escape resistance, and synergy with streptogramin B-type antibiotics is retained. Resistance to the B component is mediated by methylation of 23S rRNA, which confers macrolide-lincosamide-streptogramin B (MLSB) resistance when the erm genes are expressed constitutively. Up until now, two kinds of mechanism mediating resistance to the A compound are known: (i) inactivation of the drug by acetylation and (ii) efflux. In staphylococci, three acetyltransferase genes, vat, vatB and vatC and two genes for efflux pumps (ABC porters), vga and vgaB have been described previously.1

Of 3051 Staphylococcus aureus strains collected by the European SENTRY surveillance study between 1997 and 1999, 35 were non-susceptible to quinupristin/dalfopristin (MIC >= 2 mg/L).2 All but one of these non-susceptible isolates were collected in France. This is most probably owing to selective pressure by the use of pristinamycins in France. Twenty-two isolates from a hospital in Lille were of the vatB/vgaB genotype encoding resistance to streptogramin A and possessed in parallel constitutively expressed erm(A) or erm(C) genes encoding MLSB resistance.2 Of these isolates, 20 strains were resistant to quinupristin/dalfopristin (MIC 8 mg/L) and exhibited an elevated MIC (2–4 mg/L) of vancomycin and teicoplanin. These isolates grew on screening plates for assessing the glycopeptide-intermediate S. aureus (GISA) phenotype. When performing population analysis they were revealed to be hetero-GISA. In addition, these 20 isolates belonged to five different SmaI-restriction patterns when a difference in four or more fragments was used as criterion. For another 12 antimicrobials checked, only MICs of fosfomycin and linezolid were in the susceptible range.2

The purpose of the present study was to analyse alterations within the translational attenuator of the erm genes harboured by these 20 quinupristin/dalfopristin-resistant S. aureus strains displaying the hetero-GISA phenotype. Twelve of these isolates possessed a constitutively expressed erm(A) gene, the remaining eight strains a constitutively expressed erm(C) gene. To our knowledge this is the first description of alterations within the translational attenuator of MRSA isolates with such a unique resistance phenotype.

The type of erm(A) or erm(C) gene expression depends on the structure of the regulatory region, which in both cases is located immediately upstream of the respective erm gene. Based on the close structural relatedness of the regulatory regions of erm(A) and erm(C), similar regulatory processes can be assumed to occur during inducible gene expression via translational attenuation.

The presence of erm(A) or erm(C) genes was confirmed by PCR,3,4 and combined resistance to 14-, 15- and 16-membered macrolides as well as lincosamides was determined by the broth microdilution method.5 To detect structural alterations in the erm(A) or erm(C) regulatory region, we used a previously described PCR assay.3,4

Three slightly different types of structural alteration, i.e. tandem duplications of 25 bp each (Figure, aGo) were seen among all 12 erm(A)-containing S. aureus isolates (type 1 in 10 isolates, types 2 and 3 in one isolate each). The tandem duplications were located almost at the same position in the regulatory region and comprised the erm(A)-associated ribosome-binding site (AGAAGG) as well as the inverted repeat (IR) 6 sequence (GGTTATAATGAAC). Thus, two IR6 sequences, IR6a and IR6b, were present in the respective regulatory regions. In the absence of an inducer, mRNA secondary structures will be formed by pairing of IR3:IR4 and IR5:IR6a, rendering IR6b, which in all cases precedes the complete erm(A) gene, accessible to ribosomes. The pairing of IR4:IR5, as expected to occur in the presence of inducers, will leave both IR6a and IR6b accessible to ribosomes. In the case of this tandem duplication, ribosomes that start translation within the IR6a sequence will stop after three codons.




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Figure. Schematic representation of the alterations observed in the translational attenuators of (a) constitutively expressed erm(A) genes and (b) erm(C) genes with respect to the translational attenuator of the inducibly expressed erm(A) gene of Tn554 (2) and the inducibly expressed erm(C) gene of pT48. SD1, SD2 and SD3 represent the Shine–Dalgarno sequences. The arrows indicate the inverted repeated sequences IR1–IR6 in (a) and IR1–IR4 in (b). The truncated erm(A) gene present in isolates that exhibit the 25 bp tandem duplications is displayed as a stippled box. Numbering refers to the positions in the sequences of Tn554 (EMBL database accession no. K02987) and pT48 (accession no. M19652).

 
The same sequence deletion of 107 bp was found in the translational attenuator of the eight isolates carrying the erm(C) gene. The location and the extent of this deletion with respect to the erm(C) gene area of plasmid pT48 is shown in the Figure (b)Go. Since inducible expression of erm(C) also occurs via translational attenuation, different mRNA secondary structures are formed by the IR sequences IR1–IR4 (Figure, bGo) in the presence and absence of inducers. The 107 bp deletion comprised almost the entire translational attenuator and thus abolished the formation of any mRNA secondary structures. This sequence deletion of 107 bp was identical to those found in naturally occurring staphylococci from human and animal origin.6

In summary, this report is the first description of alterations within the translational attenuator of 20 MRSA isolates carrying constitutively expressed erm(A) or erm(C) genes, being resistant to quinupristin/dalfopristin and displaying the hetero-GISA phenotype. In constitutively expressed erm(A) genes, 25 bp duplications were found, and a 107 bp deletion was found in constitutively expressed erm(C) genes. Although these 20 isolates display a unique resistance phenotype, similar or identical alterations in erm genes have been described in naturally occurring staphylococci. This underlines the importance of continuous surveillance for the detection of multi-resistant MRSA.

Notes

* Correspondence address. Institute for Medical Microbiology and Virology, University Hospital Düsseldorf, Universitätsstraße 1, Geb. 22.21, D-40225 Düsseldorf, Germany. Tel/Fax: +49-2132-72040; E-mail: schmitfj{at}uni-duesseldorf.de Back

References

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2 . Witte, W., Werner, G., Cuny, C. & Schmitz, F.-J. (2001). GISA phenotype in quinupristin/dalfopristin resistant MRSA. European Congress of Clinical Microbiology and Infectious Diseases, Istanbul. Clinical Microbiology and Infection 7, Suppl. 1, Poster 556, p. 95.

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