Predominance of a methicillin-resistant Staphylococcus aureus clone susceptible to erythromycin and several other non-ß-lactam antibiotics in a Greek hospital

A. Polyzoua, A. Slavakisa, S. Pournarasb, A. N. Maniatisb, D. Sofianoua and A. Tsakrisc,*

a Department of Microbiology, Hippokration General Hospital, Thessaloniki; b Department of Medical Microbiology, University of Thessalia, Larissa; c Department of Microbiology, Medical School, Aristotle University of Thessaloniki, 54 006 Thessaloniki, Greece


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A clone of heterogeneously methicillin-resistant Staphylococcus aureus (MRSA) isolates susceptible to erythromycin, ciprofloxacin, clindamycin, co-trimoxazole, nitrofurantoin, rifampicin, tetracycline and vancomycin, predominated in a Greek hospital with a high incidence of MRSA, representing 69.1% of the total MRSA isolates. All isolates of this clone lacked the conserved genes ermA and aadD. Two subtypes of this clone were detected, the more common being resistant to aminoglycosides and carrying the bi-functional gene aacA–aphD, while an aminoglycoside-susceptible variant, lacking this gene, lost a larger SmaI macrorestriction DNA fragment and gained a smaller fragment.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Since their first recognition in the early 1960s, methicillin-resistant Staphylococcus aureus (MRSA) strains have become a major cause of nosocomial infections worldwide. The overall incidence of MRSA isolation has gradually increased to reach levels of around 30% or more in some countries.1 MRSA strains are usually resistant to a wide range of non-ß-lactam antibiotics, such as macrolides, lincosamides, quinolones, tetracyclines and aminoglycosides. In particular, the majority of MRSA strains are not susceptible to macrolides and aminoglycosides, because the genes ermA and aadD encoding resistance to these drugs are usually conserved within mec DNA, and located upstream and downstream, respectively, of the mecA gene.2 Nevertheless, MRSA isolates susceptible to several, or virtually all, non-ß-lactam antibiotics have appeared in some European regions.35 This study reports the predominance of clonally related MRSA strains susceptible to multiple antistaphylococcal non-ß-lactam antibiotics in a Greek hospital that has a high incidence of MRSA.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A total of 110 non-duplicate MRSA isolates were recovered consecutively from patients in Hippokration General Hospital, Thessaloniki, Greece, between November 1999 and August 2000, representing 50.7% of the total S. aureus isolated during this period. In Hippokration Hospital, which has 1012 beds (51 with acute care facilities), more than 55 000 patients are admitted each year. All MRSA isolates were recovered from clinical samples of patients hospitalized for at least 3 days before the isolation of the organism. Identification and initial antimicrobial susceptibility testing was performed with the Vitek automated system (bioMérieux Vitek, Hazelwood, MO, USA). The antimicrobial agents tested were ciprofloxacin, clindamycin, co-trimoxazole, erythromycin, gentamicin, nitrofurantoin, oxacillin, penicillin, rifampicin, tetracycline, tobramycin and vancomycin. Isolates that were susceptible to erythromycin were selected for further study. The MICs for these isolates of clindamycin, erythromycin, gentamicin, oxacillin and tobramycin were also determined by an agar dilution method in accordance with NCCLS recommendations.6 Testing of heterogeneity or homogeneity of the methicillin resistance phenotype was performed initially by a 1 µg oxacillin disc diffusion test using plates containing Mueller–Hinton agar (Difco, Detroit, MI, USA) supplemented with 4% NaCl, which were incubated at 37°C for 24 h. The preliminary classification was confirmed by population analysis in order to determine oxacillin resistance classes as described previously.3 The erythromycin-susceptible MRSA isolates were also tested for high-level resistance to spectinomycin (500 mg/L), which has been associated with carriage of the transposon Tn554.

PCR was performed on MRSA isolates in order to amplify a 449 bp product from within the mecA gene.7 PCR was also performed on the multi-susceptible MRSA isolates, for amplification of genes ermA (139 bp product), aadD (165 bp product) and aacA–aphD (220 bp product) using published primers.8,9

The erythromycin-susceptible isolates were typed by pulsed-field gel electrophoresis (PFGE) after genomic DNA macrorestriction. Chromosomal DNA was prepared and digested with SmaI nuclease as described previously.5 Digested chromosomal DNA was separated with a CHEF DRIII apparatus (Bio-Rad, Birmingham, UK) for 20 h with ramp pulse times ranging from 5 to 34.9 s. Banding patterns of the strains were compared visually, on the basis of up to three band differences for related isolates.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Seventy-six of the 110 MRSA (69.1%) isolated during the study period were susceptible to erythromycin and concomitantly to several other antistaphylococcal non-ß-lactam antibiotics (ciprofloxacin, clindamycin, co-trimoxazole, nitrofurantoin, rifampicin, tetracycline and vancomycin). The multi-susceptible MRSA isolates were recovered from various clinical materials of patients hospitalized in 12 different departments (four special ICUs, two medical, three paediatric and three surgery units). They were recovered from the following specimens (number of specimens in parentheses): blood (24), pus (19), bronchial tube (15), intravascular catheter (9), umbilical cord (4), urine (3), knee fluid (1) and conjunctiva (1). All 76 isolates were characterized as heterogeneously resistant to oxacillin, belonging to class II or II/III, and were susceptible to spectinomycin at 500 mg/L. The MICs for isolates of oxacillin ranged from 16 to 64 mg/L, and of erythromycin and clindamycin from <=0.12 to 0.5 mg/L. The majority of the erythromycin-susceptible isolates (47, 61.8%) exhibited resistance to tobramycin and gentamicin, while the remaining 29 (38.2%) were susceptible to both aminoglycosides. The MICs for the aminoglycoside-resistant isolates were 32–128 mg/L of gentamicin and 32–64 mg/L of tobramycin, and the respective MICs for the susceptible isolates of these antibiotics were <=0.5–1 mg/L.

A 449 bp product of the mecA gene was detected in all 76 isolates. PCR failed to amplify any product for genes ermA and aadD in all erythromycin-susceptible MRSA isolates, while a 220 bp product for gene aacA–aphD was amplified in the 47 aminoglycoside-resistant isolates. The latter gene was not detected in any of the 29 aminoglycoside-susceptible isolates (Figure 1Go). The MRSA isolates belonged to the same major PFGE type, which included two subtypes (Ia and Ib) differing from each other by two bands (Figure 2Go). The isolates within each subtype were indistinguishable. Subtype Ia corresponded to the predominant aminoglycoside-resistant strain, while subtype Ib included the aminoglycoside-susceptible isolates. The PFGE pattern of subtype Ia included a band of approximately 340 kb, while with the pattern of subtype Ib this band was substituted by a smaller band of approximately 295 kb.



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Figure 1. PCR analysis with primers specific for the detection of gene aacA–aphD in representative isolates of the study. Lanes 1, 2, 5: MRSA isolates susceptible to all non-ß-lactam antibiotics. Lanes 3, 4, 6–10: MRSA isolates susceptible to all non-ß-lactam antibiotics except aminoglycosides.

 


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Figure 2. SmaI macrorestriction patterns of representative MRSA isolates belonging to subtypes Ia (lanes 2–5) and Ib (lane 1). Lanes M, molecular mass markers.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Multi-resistant MRSA has become one of the commonest causes of nosocomial infections worldwide.1 However, new MRSA strains more susceptible to antibiotics have been reported sporadically in recent years. They have become common only in French hospitals, being susceptible to gentamicin but generally resistant to tobramycin and erythromycin, carrying the conserved genes aadD and ermA.4 In other regions, multi-susceptible MRSA was confined to paediatric or community settings.3,10 In contrast to these reports, the multi-susceptible MRSA of the present study, isolated in a large general hospital, did not contain aadD and ermA, and resistance to aminoglycosides was attributed to the presence of aacA–aphD. The latter gene confers resistance to most clinically available aminoglycosides. Thus, although aminoglycosides cannot be used for the treatment of infections caused by these MRSA, a variety of alternative antibiotics, such as macrolides, quinolones, tetracyclines, clindamycin and co-trimoxazole, may be used against such strains. Also, the existence of MRSA susceptible to all non-ß-lactam antibiotics, including aminoglycosides, may provide an opportunity for the re-introduction of these drugs against MRSA, further reducing reliance on vancomycin.

The isolates that exhibited susceptibility to all non-ß- lactam antibiotics differed from the aminoglycosideresistant isolates by two bands, and belonged to the same clone. Multi-susceptible MRSA isolates have spread in our hospital during 1999.5 However, aminoglycoside-resistant multi-susceptible MRSA isolates were scarcely detected previously but gradually became predominant, and currently represent more than 60% of the multi-susceptible isolates. This observation could suggest that the aminoglycoside-resistant strain possibly resulted from the susceptible strain after a genetic event, obtaining an element that contained the gene aacA–aphD. This shift is possibly caused by the substitution of a smaller by a larger PFGE band. It should be noted that strains with a PFGE pattern identical to that of the subtype Ib have been identified previously in another hospital, suggesting the circulation of this strain in our region.5 In contrast, strains with the antibiotic resistance phenotype of subtype Ia have not been identified in other northern Greek hospitals (J. Douboyas, personal communication). Thus, it could be postulated that the aminoglycoside-susceptible strain might have been the ancestor of the aminoglycoside-resistant strain and the genetic event possibly occurred intra-hospitally. The gradual predominance of isolates belonging to subtype Ia might have been facilitated by the high consumption of aminoglycoside antibiotics in our setting. It is also of interest that the PFGE patterns of Greek strains seem to differ substantially from those of other multi-susceptible MRSA clones,3,4 although these strains were not run under the same conditions.

The clone of the present report, predominated in a hospital with a high prevalence of MRSA, representing 69.1% of the total MRSA. The remaining MRSA isolates exhibited various multi-resistant phenotypes. It seems that in our hospital, the susceptible clone has replaced the classical multi-resistant MRSA clones, as the overall frequency of MRSA among S. aureus has not changed considerably during the last decade (data not shown). Changes in use of antibiotics cannot in themselves account for the emergence of this clone and the contribution of factors influencing the colonization or resistance of this particular strain to environmental stress cannot be excluded.


    Acknowledgments
 
We thank Dr A. P. Johnson for helpful suggestions.


    Notes
 
* Corresponding author. Tel: +30-31-999-091; Fax: +30-31-999-149; E-mail: atsakris{at}med.auth.gr Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Ayliffe, G. A. J. (1997). The progressive intercontinental spread of methicillin-resistant Staphylococcus aureus. Clinical Infectious Diseases 24, Suppl. 1, S74–9.[ISI][Medline]

2 . Chambers, H. F. (1997). Methicillin resistance in staphylococci: molecular and biochemical basis and clinical implications. Clinical Microbiology Reviews 10, 781–91.[Abstract]

3 . Sa-Leao, R., Santos Sanches, I., Dias, D., Peres, I., Barros, R. M. & de Lencastre, H. (1999). Detection of an archaic clone of Staphylococcus aureus with low-level resistance to methicillin in a pediatric hospital in Portugal and in international samples: relics of a formerly widely disseminated strain? Journal of Clinical Microbiology 37, 1913–20.[Abstract/Free Full Text]

4 . Lelievre, H., Lina, G., Jones, M. E., Olive, C., Forey, F., Roussel-Delvallez, M. et al. (1999). Emergence and spread in French hospitals of methicillin-resistant Staphylococcus aureus with increasing susceptibility to gentamicin and other antibiotics. Journal of Clinical Microbiology 37, 3452–7.[Abstract/Free Full Text]

5 . Pournaras, S., Slavakis, A., Polyzou, A., Sofianou, D., Maniatis, A. N. & Tsakris, A. (2001). Nosocomial spread of an unusual methicillin-resistant Staphylococcus aureus clone that is sensitive to all non-ß-lactam antibiotics, including tobramycin. Journal of Clinical Microbiology 39, 779–81.[Abstract/Free Full Text]

6 . National Committee for Clinical Laboratory Standards. (1997). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically—Fourth Edition: Approved Standard M7-A4. NCCLS, Wayne, PA.

7 . Bignardi, G. E., Woodford, N., Chapman, A., Johnson, A. P. & Speller, D. C. E. (1996). Detection of the mec-A gene and phenotypic detection of resistance in Staphylococcus aureus isolates with borderline or low-level methicillin resistance. Journal of Antimicrobial Chemotherapy 37, 53–63.[Abstract]

8 . van Asselt, G. J., Vliegenthart, J. S., Petit, P. L., van de Klundert, J. A. & Mouton, R. P. (1992). High-level aminoglycoside resistance among enterococci and group A streptococci. Journal of Antimicrobial Chemotherapy 30, 651–9.[Abstract]

9 . Martineau, F., Picard, F. J., Lansac, N., Menard, C., Roy, P. H., Ouellette, M. et al. (2000). Correlation between the resistance genotype determined by multiplex PCR assays and the antibiotic susceptibility patterns of Staphylococcus aureus and Staphylococcus epidermidis. Antimicrobial Agents and Chemotherapy 44, 231–8.[Abstract/Free Full Text]

10 . Nimmo, G. R., Schoonveldt, J., O'Kane, G., McCall, B. & Vickery, A. (2000). Community acquisition of gentamicin-sensitive methicillin-resistant Staphylococcus aureus in Southeast Queensland, Australia. Journal of Clinical Microbiology 38, 3926–31.[Abstract/Free Full Text]

Received 9 February 2001; returned 10 April 2001; revised 4 May 2001; accepted 10 May 2001