Molecular epidemiology of resistance to macrolides–lincosamides–streptogramins in methicillin-resistant Staphylococcus aureus (MRSA) causing bloodstream infections in patients admitted to Belgian hospitals

Olivier Denis1,*, Juana Magdalena1,§, Ariane Deplano1, Claire Nonhoff1, Erik Hendrickx2 and Marc J. Struelens1

1 Staphylococcus Reference Laboratory, Service de Microbiologie, Université Libre de Bruxelles-Erasme Hospital, Brussels; 2 Scientific Institute of Public Health, Brussels, Belgium

Keywords: MRSA, PFGE, macrolide–lincosamide–streptogramin resistance

Sir,

In Staphylococcus aureus, three mechanisms of resistance to macrolides, lincosamides and streptogramins (MLS) are known: target modification, active efflux and drug modification.1 Adenine-specific N-methyltransferases mediated by erm genes modify the A2058 residue of the 23S subunit of rRNA, which confers cross-resistance to macrolides, lincosamides and streptogramin B. This MLSB resistance phenotype can be either inducible or constitutive. MLS resistance is less commonly mediated by efflux systems or inactivating enzymes.1 In this study we characterized genes encoding these MLS resistance mechanisms in methicillin-resistant S. aureus (MRSA) collected in Belgium and determined the resistance gene distribution by PFGE typing profile.

As a part of the European Antimicrobial Resistance Surveillance System (http://www.earss.rivm.nl/), 145 non-duplicate MRSA strains were collected in 1999 from blood cultures in patients admitted to 31 Belgian hospitals. In the reference laboratory, isolates were tested for production of free coagulase, growth on oxacillin agar screen, and by PCR for detection of nuc and mecA.2 Susceptibility to erythromycin, clindamycin and pristinamycin was tested by the disc diffusion method (NeoSensitabs, Rosco, Denmark). Interaction between inhibition zone with erythromycin and clindamycin was characterized to differentiate the inducible from constitutive resistance phenotype. The MIC of erythromycin and clindamycin was determined by the agar dilution method.3 The ribosomal methylases encoded by ermA and ermC, the macrolide efflux pumps encoded by msrA and msrB, and the acetyltransferase inactivating streptogramin A encoded by vatB were characterized by PCR as described previously.1 Strains were typed by PFGE analysis after SmaI macrorestriction.2 PFGE patterns were classified as: (i) group (designated by a capital letter) including PFGE profiles showing six or less DNA fragment differences; (ii) type (designated by a roman numeral suffix) including PFGE profiles showing three or less DNA fragment differences; (iii) subtype (designated by a lower case letter suffix) including any variant PFGE profile within a type (O. Denis, A. Deplano, R. De Ryck, C. Nonhoff & M. J. Struelens, unpublished results).

Seventy-six (53%) MRSA isolates were resistant to both erythromycin and clindamycin and 35 (24%) isolates were resistant to erythromycin only (Table 1). The high proportion of resistance to MLS (77%) is in agreement with findings of recent European surveys.4 All strains were susceptible to pristinamycin. Isolates susceptible to MLS did not reveal MLS resistance genes, whereas all resistant strains harboured either ermA or ermC (Table 1), a finding that is also entirely consistent with previous surveys. All strains expressing the constitutive MLSB resistance phenotype harboured ermA, whereas ermC was associated with the inducible resistance phenotype. msrB was detected in a single strain in addition to ermA. Likewise, msrB is reported in only 2–6% of MRSA strains from French and German hospitals.1,5 No msrA or vatB genes were detected. vatB was described in 8% of S. aureus from French hospitals.1 In contrast, we did not find isolates harbouring this gene, or strains resistant to streptogramins, in the present study, in agreement with previous Belgian surveys that indicated full susceptibility of MRSA strains to the new antimicrobial drug quinupristin–dalfopristin in 1995 and 1997 (O. Denis, A. Deplano, R. De Ryck, C. Nonhoff & M. J. Struelens, unpublished results).


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Table 1. Distribution of MLS resistance genes in MRSA blood isolates (n = 145) by PFGE clonal group (Belgium, 1999)
 
By PFGE analysis, MRSA strains clustered into four major groups: A (n = 55), B (n = 51), C (n = 5) and D (n = 23), and the remainder (n = 11) were distributed in six minor groups. Group A strains were subdivided into eight types and 28 subtypes. Types A1 (n = 21) and A20 (n = 16) together represented 67% of group A strains. Group B included a single type B2, which was divided into 11 subtypes. Group C included two types and five subtypes. Group D included three types and six subtypes. Group A and B strains were disseminated in 19 and 21 hospitals, respectively, which are located across the three regions of the country. Group C strains were isolated from five hospitals in Brussels and Flanders, whereas group D strains were recovered from a single hospital in Brussels. Group A strains (which include the previously designated Belgian epidemic MRSA type 1 strains) have been widespread in Belgium and its neighbouring countries since the 1980s.2 Recent data have confirmed that these strains belong to the ‘Iberian clone’ characterized by multi-locus sequence type (ST) 247/staphylococcal chromosomal cassette mec (SCCmec) type Ia.6 Group B strains, which belong to ST45-SCCmec type IV clone, emerged in Flanders in 1992, and have since disseminated across the whole country, gradually replacing group A strains (O. Denis, A. Deplano, R. De Ryck, C. Nonhoff & M. J. Struelens, unpublished results).6

Analysis of the distribution of MLS resistance genes by PFGE genotype showed that MLS resistance in group A strains was mainly due to ermA or ermC (Table 1). All resistant isolates in types A1 and A20 harboured ermA. In contrast, group A strains carrying ermC were restricted to type A19. The majority of group B strains contained ermC. The increased prevalence of group B strains is paradoxical, as they express less co-resistance than group A strains (inducible versus constitutive MLSB; gentamicin susceptibility versus resistance; O. Denis, A. Deplano, R. De Ryck, C. Nonhoff & M. J. Struelens, unpublished results). There was no correlation between the distribution of MLS resistance determinant and PFGE subtype within group B strains. Group C strains were either susceptible to MLS or contained ermC, whereas all group D strains possessed ermA.

In summary, MLS resistance was seen in the majority of MRSA strains collected from Belgian acute care hospitals in 1999 and was generally encoded by ermA or ermC methylase genes, and more rarely by the msrB efflux system gene. Furthermore, the strong association reported here between PFGE groups and MLS resistance genotype suggests that dissemination of successful clones accounts for a large part of the high prevalence of MLS resistance in MRSA.

Acknowledgements

We thank our clinical microbiologist colleagues for their dedicated participation in the European Antimicrobial Resistance Surveillance System and for referring the isolates for this study. We thank Jérôme Etienne for providing reference control strains S. aureus HM1055, HM290-1, RN4220 and BM 12-235. This study was supported in part by subcontract to Agreement S12.123794 (99CVF4-018) with the European Commission DG Sanco with regard to the EARSS, and by a grant-in-aid from Pharmacia, Belgium.

Footnotes

* Correspondence address. Service de Microbiologie, Hôpital Erasme, 808 route de Lennik, 1070 Brussels, Belgium. Tel: +32-2-555-45-18; Fax: +32-2-555-31-10; E-mail: odenis{at}ulb.ac.be Back

§ Present address. School of Biosciences, Division of Molecular Cell Biology, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK. Back

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