Prevalence of macrolide-resistance genes in Staphylococcus aureus and Enterococcus faecium isolates from 24 European university hospitals

Franz-Josef Schmitza,b,*, Ralf Sadurskia, Angela Kraya, Mechthild Boosa, Roland Geisela, Karl Köhrera, Jan Verhoefb and Ad C. Fluitb

a Institute for Medical Microbiology and Virology, Heinrich-Heine Universität Düsseldorf, Universitätsstraße 1, Geb. 22.21, 40225 Düsseldorf, Germany; b Eijkman-Winkler Institute for Clinical Microbiology, University Medical Center Utrecht, The Netherlands


    Abstract
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 Abstract
 Introduction
 Material and methods
 Results and discussion
 References
 
The polymerase chain reaction (PCR) was used to study the prevalence of the macrolide resistance genes ermA, ermB, ermC, msrA/msrB, ereA and ereB, in 851 clinical isolates of Staphylococcus aureus and 75 clinical isolates of Enterococcus faecium that were erythromycin resistant. The isolates were from 24 European university hospitals. In S. aureus, the ermA gene was more common in methicillin-resistant S. aureus (MRSA) isolates (88%) than in methicillin-susceptible S. aureus (MSSA) isolates (38%), and occurred mainly in strains with constitutive MLSB expression. In contrast, ermC was more common in MSSA (47%) than in MRSA (5%), occurring mainly in strains with inducible expression. The ereB gene was only found in MRSA isolates expressing a constitutive MLSB phenotype (1%). The ereA gene was not detected. Macrolide resistance by efflux due to the msrA/msrB gene was only detected in MSSA isolates (13%). In contrast to S. aureus, erythromycin resistance in E. faecium was almost exclusively due to the presence of the ermB gene (93%).


    Introduction
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 Abstract
 Introduction
 Material and methods
 Results and discussion
 References
 
Macrolide resistance can be caused by several mechanisms,1 the predominant form being target modification mediated by one or more erm genes encoding a 23S rRNA methylase. The addition of two methyl residues to a highly conserved adenine residue in domain V, the peptidyl transferase centre of 23S rRNA, leads to a conformational change in the ribosome, rendering the strain resistant to most macrolides, lincosamides and streptogramin B compounds. Phenotypically, this resistance pattern is known as MLSB resistance.1 Resistance to MLS antibiotics caused by the presence of macrolide efflux pumps in staphylococci (encoded by msrA or msrB) has also been documented.2 Furthermore, inactivation has been described in several organisms.3 For example, enzymes (EreA and EreB) that hydrolyse the lactone ring of the macrocyclic nucleus and phosphotransferases that inactivate macrolides have been reported in Staphylococcus aureus.4

In an attempt to update our knowledge of the status of MLS resistance in Europe, we recently investigated the prevalence of resistance to macrolides, clindamycin and quinupristin/dalfopristin.5 The present investigation was undertaken to study the prevalence of the macrolide resistance genes, ermA, ermB, ermC, msrA/msrB, ereA and ereB, using the polymerase chain reaction (PCR), in the first 851 unrelated clinical isolates of erythromycin-resistant S. aureus and the first 75 unrelated clinical isolates of erythromycin-resistant Enterococcus faecium. These isolates were sent from 24 different European university hospitals as part of the SENTRY Antimicrobial Surveillance Programme.


    Material and methods
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 Abstract
 Introduction
 Material and methods
 Results and discussion
 References
 
Bacterial isolates

The study included clinical isolates collected since the initiation of the European SENTRY programme in April 1997 through to December 1998. The protocol for this study has been described previously.5

PCR for the detection of macrolide resistance genes

Every isolate of S. aureus and E. faecium demonstrating resistance to erythromycin was screened for the presence of macrolide resistance genes. In total, 851 erythromycin-resistant S. aureus (358 methicillin-susceptible S. aureus (MSSA) and 493 methicillin-resistant S. aureus (MRSA)) and 75 erythromycin-resistant E. faecium isolates were analysed. In addition, 50 random isolates that were fully susceptible to macrolides (40 S. aureus and 10 E. faecium) were screened for the presence of macrolide resistance genes as described below.

Oligonucleotide primers for ermA, ermB, ermC, msrA/ msrB, ereA and ereB for use in the PCRs were selected from the DNA sequences published by Sutcliffe et al.3 The specificity of each set of primers was tested using DNA extracts of reference strains containing ermA, ermB, ermC, msrA/msrB, ereA and ereB (ermA: S. aureus RN 1389; ermB: Streptococcus pyogenes AC1/pAC1; ermC: S. aureus RN4220/pE194 and msrA: S. aureus RN4220/pAT10) all kindly supplied by Joyce Sutcliffe (Central Research Division, Pfizer, Groton, CT, USA). In addition, strains containing ereA (Escherichia coli/pIP1100) and ereB (E. coli/pAT72) kindly supplied by Patrice Courvalin (Institute Pasteur, Paris, France) were used. A random sample of PCR products with each set of primers was sequenced. Primers specific for conserved regions of the 16S rRNA gene were used as additional internal controls.6

Genomic DNA was isolated and two multiplex PCRs (primer set for ermA, ermB and ermC, together with msrA/msrB, as well as a primer set for ereA and ereB in a second separate PCR) were performed as described by Sutcliffe et al.3 The expected PCR products for ermA, ermB and ermC were between 639 and 645 bp. Therefore, after confirmation of the presence of an erm gene, single PCRs were performed in order to verify the class of the erm gene, either ermA, ermB or ermC.


    Results and discussion
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 Abstract
 Introduction
 Material and methods
 Results and discussion
 References
 
We reported recently that the percentage of MSSA isolates from European university hospitals that showed susceptibility to erythromycin was about 20 times higher than that of MRSA isolates (66.9% versus 3.4%). In 93% of the erythromycin-resistant MRSA and 44% of the erythromycin-resistant MSSA, expression of MLSB resistance was constitutive.5 Only 6.6% of the E. faecium isolates tested were susceptible to erythromycin, with all erythromycin-resistant isolates displaying the constitutive MLSB resistance phenotype.5

This present survey constitutes the largest collection of clinical isolates of S. aureus and E. faecium studied for the prevalence of macrolide resistance genes. The multiplex PCRs described by Sutcliffe et al.3 for research purposes can also be routinely applied to survey macrolide resistance mechanisms present in large collections of clinical isolates. Using their method we failed to detect non-specific PCR products. Furthermore, macrolide resistance genes were not detected in any of the 50 erythromycin-sensitive S. aureus and E. faecium isolates tested. As shown in Table IGo, the most prevalent resistance gene in S. aureus was ermA (571/851; 67%), followed by ermC (192/851; 23%) and msrA/msrB (48/851; 6%). Less common were ermB and ereB, each occurring in 0.6% of the erythromycin-resistant S. aureus isolates tested. The ereA gene was not detected in any of the isolates.


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Table I. Prevalence of macrolide resistance genes in erythromycin-resistant Staphylococcus aureus and Enterococcus faecium isolates
 
The ermA gene was more common in MRSA isolates (88% in MRSA versus 38% in MSSA), while ermC was more common in MSSA (5% in MRSA versus 47% in MSSA). Within the S. aureus collection, ermA was predominant in strains expressing a constitutive MLSB phenotype, while ermC was predominant in MSSA isolates with an inducible MLSB phenotype. One erm gene was detected in 716 of 851 S. aureus isolates (84%), whereas the combination ermA and ermC was found in only 26 of 851 isolates (3%). In general, our observations are in line with the findings of Lina et al.,7 who studied 144 MLSB-resistant S. aureus strains originating from French hospitals in 1995. They found that the ermA gene was more common in MRSA isolates (57.6%), mainly in strains with constitutive MLSB expression, than in MSSA isolates (5.6%), whereas ermC was more common in MSSA isolates (20.1%), mainly in strains with inducible expression, than in MRSA isolates (4.9%).7 Similar findings to ours were also reported from Denmark, where ermA and ermC genes were responsible for erythromycin resistance in 98% of the 428 S. aureus isolates studied.8 The ermA gene was solely responsible for erythromycin resistance until 1971, while ermC became dominant between 1984 and 1988.8 In accordance with the observations from Denmark, Nicola and colleagues detected the ermA gene in 15 of 16 erythromycin-resistant S. aureus isolates originating from the USA and isolated between 1958 and 1969.9 Thus, ermC has only recently become prevalent in the S. aureus population. Our results on the low prevalence of ermB are also in line with earlier studies.7,9 Although ermB was present in only a minority of strains, it was formerly found only in animal strains.10 In contrast to Lina et al.7 and Nicola et al.,9 we found an association between different erm genes, namely ermA in combination with ermC, in S. aureus isolates.

The ereB gene, coding for a macrolide-inactivating enzyme, was only found in MRSA isolates expressing the constitutive MLSB phenotype (5/458; 1%) (Table IGo). Neither ereA nor ereB in combination with other macrolide resistance determinants was found. We are not aware of any other surveillance study describing the prevalence of ereA and ereB in erythromycin-resistant S. aureus isolates.

Macrolide resistance by efflux due to the msrA/msrB gene was only found in MSSA isolates (14/358; 13%). This is in contrast to the results of Lina et al.,7 who detected the msrA/msrB gene in both MSSA and MRSA isolates. Moreover, they found the gene in only 2.1% of the 144 S. aureus strains tested. In line with their observations, however, we found no combination of msrA/msrB with other macrolide resistance determinants. To date, three S. aureus isolates have been found to harbour esterase activity-hydrolysing macrolides and a macrolide efflux system.4

The ermB gene was the most prevalent resistance determinant found in erythromycin-resistant E. faecium isolates, followed by ermA (93% versus 4%). The combination of ermA and ermB was detected in two of 75 isolates (3%). Jensen and colleagues recently analysed 113 erythromycin-resistant enterococcal isolates of human and animal origin and found the ermB gene to be present in 88%.11

The frequency of isolates that displayed erythromycin resistance in the absence of one of the six resistance genes tested for ranged between 4.5% and 8.6% in the five groups of isolates analysed. This implies that other mechanisms contribute to macrolide resistance in S. aureus and E. faecium.

In summary, resistance to erythromycin in S. aureus isolates from French hospitals was due mainly to the presence of ermA and ermC genes. The ermA gene was more common in MRSA isolates, mainly in strains with a constitutive MLSB expression, than in MSSA isolates, whereas ermC was more common in MSSA isolates, mainly in strains with inducible expression. Only a few strains had the ereB or ermB gene, while macrolide resistance by efflux due to the msrA gene was more common, but only detectable in MSSA. In contrast to S. aureus, erythromycin resistance in E. faecium was almost exclusively due to the presence of the ermB gene.


    Acknowledgments
 
We thank Alice Florijn, Miriam Klootwijk, Carlijn Kusters and Stefan de Vaal for their expert technical assistance. We also thank the following members of the SENTRY Participants Group for referring isolates and epidemiological data for use in this study: Professor Helmut Mittermayer, Professor Marc Struelens, Professor Jacques Acar, Professor Vincent Jarlier, Professor Jerome Etienne, Professor Rene Courcol, Professor Franz Daschner, Professor Ulrich Hadding, Professor Nikos Legakis, Professor Gian-Carlo Schito, Professor Carlo Mancini, Professor Piotr Heczko, Professor Waleria Hyrniewicz, Professor Dario Costa, Professor Evilio Perea, Professor Fernando Baquero, Dr Rogelio Martin Alvarez, Professor Jacques Bille, Professor Gary French, Dr Nathan Keller, Dr Volkan Korten, Dr Deniz Gür and Dr Serhat Unal. This work was funded in part by Bristol-Myers Squibb Pharmaceuticals via the SENTRY Antimicrobial Surveillance Programme.


    Notes
 
* Corresponding author. Tel/Fax: +49-2132-72040; E-mail: schmitfj{at}uni-duesseldorf.de Back


    References
 Top
 Abstract
 Introduction
 Material and methods
 Results and discussion
 References
 
1 . Leclercq, R. & Courvalin, P. (1991). Bacterial resistance to macrolide, lincosamide, and streptogramin antibiotics by target modification. Antimicrobial Agents and Chemotherapy 35, 1267–72.[ISI][Medline]

2 . Ross, J. I., Eady, E. A., Cove, J. H., Cunliffe, W. J., Baumberg, S. & Wootton, J. C. (1990). Inducible erythromycin-resistance in staphylococci is encoded by a member of the ATP-binding transport super-gene family. Molecular Microbiology 4, 1207–14.[ISI][Medline]

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

4 . Wondrack, L., Massa, M., Yang, B. V. & Sutcliffe, J. (1996). Clinical strain of Staphylococcus aureus inactivates and causes efflux of macrolides. Antimicrobial Agents and Chemotherapy 40, 992–8.[Abstract]

5 . Schmitz, F.-J., Verhoef, J. & Fluit, A. C. (1999). Prevalence of resistance to MLS antibiotics in 20 European university hospitals participating in the European SENTRY surveillance programme. SENTRY Participants Group. Journal of Antimicrobial Chemotherapy 43, 783–92.[Abstract/Free Full Text]

6 . Greisen, K., Loeffelholz, M., Purohit, A. & Leong, D. (1994). PCR primers and probes for the 16S rRNA gene of most species of pathogenic bacteria, including bacteria found in cerebrospinal fluid. Journal of Clinical Microbiology 32, 335–51.[Abstract]

7 . Lina, G., Quaglia, A., Reverdy, M.-E., Leclercq, R., Vandenesch, F. & Etienne, J. (1999). Distribution of genes encoding resistance to macrolides, lincosamides, and streptogramins among staphylococci. Antimicrobial Agents and Chemotherapy 43, 1062–6.[Abstract/Free Full Text]

8 . Westh, H., Hougaard, D. M., Vuust, J. & Rosdahl, V. T. (1995). Prevalence of erm gene classes in erythromycin-resistant Staphylococcus aureus strains isolated between 1959 and 1988. Antimicrobial Agents and Chemotherapy 39, 369–73.[Abstract]

9 . Nicola, F. G., McDougal, L. K., Biddle, J. W. & Tenover, F. C. (1998). Characterization of erythromycin-resistant isolates of Staphylococcus aureus recovered in the United States from 1958 through 1969. Antimicrobial Agents and Chemotherapy 42, 3024–7.[Abstract/Free Full Text]

10 . Eady, E. A., Ross, J. I., Tipper, J. L., Walters, C. E., Cove J. H. & Noble, W. C. (1993). Distribution of genes encoding erythromycin ribosomal methylases and an erythromycin efflux pump in epidemiologically distinct groups of staphylococci. Antimicrobial Agents and Chemotherapy 31, 211–17.

11 . Jensen, L. B., Frimodt-Moller, N. & Aarestrup, F. M. (1999). Presence of erm gene classes in gram-positive bacteria of animal and human origin in Denmark. FEMS Microbiology Letters 170, 151–8.[ISI][Medline]

Received 13 October 1999; returned 2 December 1999; revised 4 January 2000; accepted 24 January 2000