a College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul 151-742; b School of Biological Sciences, Seoul National University, Seoul 151-742; c Department of Clinical Pathology, Yonsei University College of Medicine, CPO Box 8044, Seoul, Korea
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Abstract |
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Introduction |
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Resistance to MLS in Staphylococcus spp. is mediated by a methylase encoded by erythromycin resistance methylase (erm) genes.5 Methylases cause a conformational change in the prokaryotic ribosome, leading to reduced binding of MLS antibiotics to the target site in the 50S ribosomal subunit. The phenotypic expression of MLS resistance can be either inducible or constitutive.68
A new mechanism of resistance to macrolides, based on an efflux system, has recently emerged among Streptococcus pyogenes and Streptococcus pneumoniae. It is due to the presence of macrolide efflux (mef) genes conferring the M phenotype, which is characterized by resistance to 14- and 15-membered macrolides and sensitivity to lincosamide and streptogramin antibiotics. Genes of the mef class have also been found in other Gram-positive genera, including Corynebacterium, Enterococcus, Micrococcus and Streptococcus.9,10
In order to investigate the prevalence of MLS resistance in a Korean hospital, we compared the in vitro activities of several MLS antibiotics against 1097 clinical isolates of Staphylococcus aureus, coagulase-negative staphylococci (CNS) and enterococci. In addition, the resistance phenotype was determined by the double-disc test, using erythromycin and clindamycin. The resistance genotype was determined by PCR analysis of the erythromycin-resistant isolates for the presence of four representative MLS resistance genes, erm(A), erm(B), erm(C) and mef.
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Materials and methods |
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A total of 1097 clinical isolates of Gram-positive cocci, comprising 467 methicillin-resistant S. aureus (MRSA), 169 methicillin-susceptible S. aureus (MSSA), 100 methicillin-resistant CNS, 86 methicillin-susceptible CNS, 180 Enterococcus faecalis and 95 Enterococcus faecium, were collected from the Severance Hospital in Seoul, Korea between May 1999 and January 2000. Multiple isolates from the same patient were avoided. The strains were stored in brain heart infusion (BHI) broth plus 20% glycerol at 70°C until studied.
Antibiotics
Erythromycin and clindamycin were purchased from Sigma Chemical Co. (St Louis, MO, USA). The other antibiotics were obtained as follows: clarithromycin, Abbott Laboratories (Abbott Park, IL, USA); azithromycin, Pfizer Inc. (New York, NY, USA); josamycin, ICN Biomedicals (Costa Mesa, CA, USA) and pristinamycin, Rhône-Poulenc Rorer (Paris, France).
Determination of MICs
MICs were determined by a standardized agar dilution method11 with MuellerHinton (MH) agar for staphylococci and BHI agar for enterococci. The MIC resistance breakpoints used, based on the guidelines from the NCCLS12 and the French Society for Microbiology,13 were as follows: erythromycin, clarithromycin, azithromycin and josamycin 8 mg/L, clindamycin
4 mg/L and pristinamycin
2 mg/L. S. aureus ATCC25923 and E. faecalis ATCC29212 were used as controls in the MIC determinations. The bacterial suspensions (104 cfu/spot) were inoculated using a microinoculator (Sakuma Co. Ltd, Tokyo, Japan).
Determination of resistance phenotypes
The resistance phenotypes of erythromycin-resistant isolates were determined by the double-disc test with erythromycin (15 µg) and clindamycin (2 µg) discs, as described previously.14 After 18 h incubation at 37°C, blunting of the clindamycin zone of inhibition proximal to the erythromycin disc indicated an inducible type of MLS resistance (IR), and resistance to both erythromycin and clindamycin indicated a constitutive type of MLS resistance (CR).
Detection of resistance genotypes
The presence of genes encoding MLS resistance due to alteration of the ribosome target site was determined by multiplex PCR amplification of erm genes using primers specific for erm(A), erm(B) and erm(C). In addition, the presence of the gene involved in the macrolide efflux system was determined by PCR with primers for mef. Genomic DNA was extracted as described previously,15 and was used as the template for amplification. Primers were designed from published GenBank sequences to provide specific PCR products (Table 1). We selected an Enterococcus isolate from this study as a control strain harbouring mef, and confirmed its presence by Southern blot hybridization using the mef PCR product as a probe. PCR was carried out on the 552 isolates (174 MRSA, 20 MSSA, 75 methicillin-resistant CNS, 50 methicillinsusceptible CNS and 233 enterococci) displaying resistance to erythromycin, as well as on the control strains for each genetic determinant (Table 1
). All PCR amplifications consisted of an initial cycle of 5 min of denaturation at 95°C, followed by 35 cycles of 30 s of denaturation at 95°C, 30 s of annealing at 54°C, 1 min of elongation at 72°C and one cycle of 5 min of extensive elongation at 72°C on a DNA thermal cycler (PTC-200; MJ Research, Inc., Watertown, MA, USA). After amplification, electrophoresis and visualization of the PCR products were carried out by established procedures.16
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Results and discussion |
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The MIC range and the MIC50 and MIC90 values for the three groups of test organisms are displayed in Table 2. The S. aureus and CNS strains were mostly resistant to the macrolide and lincosamide antibiotics. The MIC90 values of pristinamycin for S. aureus and CNS strains were relatively low. However, the high rates of resistance to pristinamycin in S. aureus and CNS do not support pristinamycin being regarded as a first-line agent for staphylococcal infections. The enterococcal isolates were also resistant to MLS antibiotics. Pristinamycin was ineffective against the E. faecalis isolates because of natural resistance to streptogramins, but the MIC50 of pristinamycin for E. faecium was lower (1 mg/L) than those of the other macrolide and lincosamide antibiotics (
64 mg/L). MLS antibiotics cannot be considered as efficient therapeutic agents for enterococcal infections in Korea. Antibiotic resistance, including MLS, is usually closely related to the extent to which these agents are used, and some reports have shown that a decrease in the use of these antibiotics has led to a decrease in the prevalence of resistance.1719 The relatively low incidence of resistance to pristinamycin may be related to the low usage of this antibiotic in Korea.
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The resistance phenotypes of 851 erythromycin-resistant isolates (comprising 493 S. aureus, 125 CNS and 233 enterococci) were determined according to the results of double-disc tests. The IR phenotype was demonstrated in 72 S. aureus isolates (14.6%) and 12 CNS (9.6%), with the remainder exhibiting the CR phenotype. The IR phenotype in enterococci was rare, being found in only two of 233 isolates. In conclusion, the resistance rate to MLS antibiotics of the Gram-positive cocci isolated from the Severance Hospital was very high, and the most prevalent MLS resistance phenotype of the three Gram-positive groups was CR.
MLS resistance genotypes
The distribution of resistance genes among 552 isolates resistant to erythromycin (194 S. aureus, 125 CNS and 233 enterococci) as determined by PCR amplification of the genes erm(A), erm(B), erm(C) and mef, is displayed in Table 3. The most prevalent resistance determinant in S. aureus was erm(A), which was detected in 82.5% of the isolates. The erm(C) determinant was found as a single MLS resistance gene in five (2.6%) isolates while 16 isolates contained both erm(A) and erm(C). No S. aureus harbouring different mechanisms of resistance (i.e. a methylase and a macrolide efflux protein) was found. Neither erm(B) nor mef was identified in S. aureus. The distribution of resistance determinants in S. aureus was less complex than those in the CNS and enterococcal strains. This point could be explained by the selection of a high proportion of clonal MRSA in this study. One hundred and seventy four of 194 erythromycin-resistant S. aureus isolates selected from 260 clinical isolates were methicillin resistant (89.7%).
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erm(B) was the most prevalent gene in enterococci, whereas it was rarely identified in Staphylococcus spp.:19,23 it was detected alone in 55.4% of the isolates and with erm(A) (5.2%) or mef less frequently (3.5%). The erm(B) gene has previously been demonstrated to be involved in macrolide resistance in various Gram-positive bacteria, such as Enterococcus, S. pneumoniae, S. pyogenes and S. aureus.19,2326 Our results support the fact that the erm(B) gene is most frequently found among the enterococci. In contrast, the erm(C) gene was not detected in enterococci, whereas it was the most prevalent resistance genotype in CNS. This result is consistent with other reports of enterococci lacking erm(C).27,28 In general, erythromycinresistant enterococci have a more complex distribution of resistance genotypes than staphylococci. For example, three enterococcal isolates were detected that each harboured the three genetic determinants, erm(A), erm(C) and mef.
The mef determinant was found, either alone or with the erm gene, in CNS and enterococci, but was not detected in the S. aureus isolates. The mef gene has also been detected in a variety of Gram-positive genera, including S. pyogenes, S. pneumoniae and Streptococcus agalactiae, as well as in Micrococcus luteus, Corynebacterium jeikeium, Corynebacterium spp. and viridans streptococci, indicating widespread distribution.2931
In 13 S. aureus, 22 CNS and 51 enterococci that were macrolide resistant, erm and mef genes were not detected. It is likely that these isolates harbour other resistance genes, such as msrA/B, linA/linA', vga, vgb or vat. The msrA/B (macrolide and streptogramin resistance) efflux pump gene differs from the mef gene, in that msrA/B confers resistance to both macrolide and streptogramin antibiotics. Lincomycin nucleotidyltransferase (linA/linA'), virginiamycin factor A (vga), virginiamycin factor B hydrolase (vgb) and virginiamycin factor A acetyltransferase (vat) have been identified previously in several Grampositive bacteria.3235
In conclusion, the three groups of Gram-positive cocci used in this study showed a relatively high rate of resistant to MLS antibiotics. The MLS resistance genes were species specific with erm(A) dominant in S. aureus, erm(B) in enterococci and erm(C) in CNS.
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Acknowledgements |
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Notes |
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References |
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Received 4 June 2001; returned 3 September 2001; revised 22 October 2001; accepted 28 November 2001