ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Apr. 2004, p. 1124–1127 0066-4804/04/$08.00⫹0 DOI: 10.1128/AAC.48.4.1124–1127.2004 Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Vol. 48, No. 4
In Vitro Activities of 28 Antimicrobial Agents against Staphylococcus aureus Isolates from Tertiary-Care Hospitals in Korea: a Nationwide Survey Hong Bin Kim,1 Hee-Chang Jang,1 Hee Jung Nam,2 Yeong Seon Lee,2 Bong Su Kim,2 Wan Beom Park,1 Ki Deok Lee,1 Young Joo Choi,1 Sang Won Park,1 Myoung-don Oh,1,3* Eui-Chong Kim,3,4 and Kang Won Choe1,3 Departments of Internal Medicine1 and Laboratory Medicine,4 College of Medicine, Seoul National University, and Clinical Research Institute, Seoul National University Hospital,3 Seoul 110-744, and Department of Microbiology, National Institute of Health, Seoul 122-701,2 Republic of Korea Received 6 June 2003/Returned for modification 2 October 2003/Accepted 5 December 2003
Staphylococcus aureus, one of the most frequently isolated pathogens in both hospitals and the community, has been particularly efficient at developing resistance to antimicrobial agents. As methicillin-resistant S. aureus (MRSA) has prevailed and, furthermore, as S. aureus with reduced susceptibility to vancomycin has emerged, the therapeutic options for the treatment of S. aureus infections have become limited. To update the current status of antibiotic resistance, clinical S. aureus isolates were collected from eight university-affiliated hospitals from June 1999 to January 2001. Susceptibility tests with 28 antibiotics were performed by the disk diffusion method. Among a total of 682 isolates, the methicillin resistance rate was 64% (439 of 682), and most of the MRSA isolates were resistant to multiple classes of antibiotics. Although a constitutive macrolidelincosamide-streptogramin B resistance phenotype was common, no isolates were resistant to quinupristindalfopristin or linezolid. Rifampin, fusidic acid, trimethoprim-sulfamethoxazole, and arbekacin showed superior in vitro activity compared with the other antibiotics against the MRSA isolates. No isolates showed reduced susceptibility to vancomycin. tions for the treatment of S. aureus infections have become limited. The aims of the present study were to analyze nationwide data on the susceptibilities of S. aureus isolates from tertiarycare hospitals and to improve the empirical approaches to the therapy of serious infections. (This work was presented in part at the 41st Annual Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, Ill., 16 to 19 December 2001.)
Staphylococcus aureus is an important cause of serious infections in both hospitals and the community (16). S. aureus has been found to be the most frequently isolated pathogen causing bloodstream infections, skin and soft tissue infections, and pneumonia (6, 19, 20). Unfortunately this pathogen has been particularly efficient at developing resistance to antimicrobial agents. Since the first isolation of methicillin-resistant S. aureus (MRSA) in the United Kingdom in 1961 (12), increasing rates of methicillin resistance among S. aureus strains have been a cause for concern. In addition, MRSA has become resistant to multiple other antimicrobial agents. Until recently, vancomycin was believed to have retained activity against all strains of S. aureus; therefore, the spread of MRSA has led to increased vancomycin usage and hence increased selective pressure for the development of resistance. In 1997, the first documented infection caused by S. aureus with reduced susceptibility to vancomycin (vancomycin-intermediate S. aureus) was reported in Japan (9). Subsequently, similar strains with reduced susceptibility to vancomycin were identified in several countries, including Korea (25). Furthermore, two strains of fully vancomycin-resistant S. aureus, carrying the vanA gene cluster, were documented in the United States during 2002 (2, 3). Because of the multidrug resistance of MRSA and the emergence of vancomycin-intermediate S. aureus and vancomycin-resistant S. aureus, the therapeutic op-
MATERIALS AND METHODS Bacterial isolates. Clinical S. aureus isolates were collected from eight university-affiliated hospitals, each hospital representing one of eight provinces in Korea. From June 1999 to January 2001, each participating hospital sent 100 consecutive isolates identified as S. aureus. Only one isolate per patient was included in the study. Upon receipt, isolates were subcultured on blood agar to ensure purity. Initial identification was based on colony morphology and agglutination tests with PS LATEX (Eiken Chemical, Tokyo, Japan). When necessary, further confirmatory tests were done with a Vitek system (bioMerieux, Durham, N.C.). All isolates received were immediately stored at ⫺70°C until required. Antibiotic susceptibility testing. Susceptibility testing was conducted by disk diffusion according to the guidelines of the National Committee for Clinical Laboratory Standards (NCCLS) (18). The following 28 antibiotics were tested: oxacillin, penicillin, amoxicillin-clavulanic acid, cefazolin, cefuroxime, cefotaxime, imipenem, erythromycin, azithromycin, clindamycin, quinupristin-dalfopristin, linezolid, vancomycin, teicoplanin, ofloxacin, sparfloxacin, tetracycline, trimethoprim-sulfamethoxazole, rifampin, fusidic acid, gentamicin, arbekacin, amikacin, tobramycin, streptomycin, neomycin, netilmicin, and kanamycin. The breakpoints for resistance were those recommended by the NCCLS. For fusidic acid, arbekacin, streptomycin, and neomycin disks (Becton Dickinson, Tokyo, Japan), breakpoints of ⱖ21, ⱖ18, ⱖ15, and ⱖ17 mm, respectively, were used to define susceptibility according to the manufacturer’s recommendations included in package inserts. The MIC was determined by the broth microdilution method
* Corresponding author. Mailing address: Department of Internal Medicine, College of Medicine, Seoul National University, 28 Yeongun-dong, Chongro-gu, Seoul 110-744, Republic of Korea. Phone: 82-2-760-2945. Fax: 82-2-762-9662. E-mail:
[email protected]. 1124
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TABLE 1. Antibiotic resistance profiles of 243 MSSA and 439 MRSA isolates from tertiary-care hospitals, as determined by disk diffusiona MSSA Antibiotic
No. of isolates that were:
Penicillin Amoxicillin-clavulanic acid Cefazolin Cefuroxime Cefotaxime Imipenem Erythromycin Azithromycin Clindamycin Quinupristin-dalfopristin Linezolid Vancomycin Teicoplanin Ofloxacin Sparfloxacin Tetracycline Trimethoprim-sulfamethoxazole Rifampin Fusidic acid Gentamicin Arbekacin Amikacin Tobramycin Streptomycin Neomycin Netilmicin Kanamycin a
MRSA No. of isolates that were:
S
I
R
Resistance rate (%)
S
I
R
Resistance rate (%)
24 239 243 238 236 243 154 147 215 243 243 243 243 237 240 190 240 241 166 175 240 215 173 134 221 240 163
— — 0 5 5 0 32 39 16 0 — — 0 3 0 9 2 2 1 0 2 23 3 104 6 1 2
219 4 0 0 2 0 57 57 12 0 — — 0 3 3 44 1 0 76 68 1 5 67 5 16 2 78
90.1 1.6 0 2.1 2.9 0 36.7 39.5 11.5 0 0 0 0 2.4 1.2 21.8 1.2 0.8 31.7 28.0 1.2 11.6 28.8 44.9 9.1 1.2 32.9
0 7 10 2 2 38 10 12 69 439 439 439 439 27 27 46 400 360 377 22 384 46 9 302 163 362 7
— — 2 13 15 2 1 2 7 0 — — 0 1 4 11 10 23 8 1 49 69 0 99 3 19 0
439 432 427 424 422 399 428 425 363 0 — — 0 411 408 382 29 56 54 416 6 324 430 38 273 58 432
100 98.4 97.8 99.6 99.5 91.4 97.7 97.3 84.3 0 0 0 0 93.8 93.8 89.5 8.9 18.0 14.1 95.0 12.6 89.5 97.9 31.3 62.9 17.5 98.4
S, susceptible; I, intermediate; R, resistant. —, not applicable.
according to the guidelines of the NCCLS (17). Quality control was performed by testing with S. aureus ATCC 25923 and ATCC 29213. The resistance rate was calculated as the number of intermediate and resistant isolates divided by the total number of isolates. Multidrug resistance was defined as resistance to penicillin and oxacillin plus three or more of the following agents: erythromycin, clindamycin, rifampin, ofloxacin, gentamicin, tetracycline, and trimethoprim-sulfamethoxazole.
RESULTS A total of 682 isolates, excluding consecutive samples from the same patient, were collected from eight tertiary-care hospitals (range, 62 to 99 per hospital); 199 (29%) were from pus, 152 (22%) were from the respiratory tract, 137 (20%) were from blood, and 38 (6%) were from urine. Overall, the prevalence of MRSA was 64% (439 of 682 isolates) and varied from 55 to 74% by hospital and from 48 to 92% by specimen type. Rates of resistance of methicillin-sensitive S. aureus (MSSA) and MRSA to the other antibiotics tested are shown in Table 1. The majority of MRSA isolates (429 of 439) were multidrug resistant. While oxacillin MICs for the 439 MRSA isolates ranged from 4 to ⱖ256 g/ml, only 4% of the isolates (18 of 439) were inhibited at an MIC of ⱕ64 g/ml. Of the 89 erythromycin-resistant MSSA isolates, 21 (24%) had the constitutive macrolide-lincosamide-streptogramin B (MLSB) resistance phenotype, whereas 367 (86%) of the 429 erythromycin-resistant MRSA isolates had the constitutive MLSB resistance phenotype (Table 2). The other erythromycin-resistant S. aureus isolates had an inducible MLSB resis-
tance phenotype. No isolates were resistant to quinupristindalfopristin or linezolid. Of the 682 isolates, 588 (87%) were resistant to at least one of the eight aminoglycosides tested. More than 90% of the MRSA isolates were resistant to kanamycin (98%), tobramycin (98%), gentamicin (95%), and amikacin (90%). Moreover, most of the MRSA isolates were resistant to multiple aminoglycosides, and 88% were resistant to kanamycin, tobramycin, gentamicin, and amikacin (Table 3). Rates of resistance to arbekacin and netilmicin were less than 20%. Arbekacin was the most active of the aminoglycosides tested.
TABLE 2. Phenotypic resistance patterns of 682 S. aureus isolates for erythromycin, clindamycin, and quinupristin-dalfopristin Resistance patterna
Erythromycin resistant, clindamycin susceptible Erythromycin resistant, clindamycin resistant Erythromycin susceptible, clindamycin resistant Erythromycin susceptible, clindamycin susceptible Total a
No. (%) of: MSSA
MRSA
68 (28)
62 (14)
21 (9)
367 (83)
7 (3)
3 (1)
147 (60)
7 (2)
243
All isolates were susceptible to quinupristin-dalfopristin.
439
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ANTIMICROB. AGENTS CHEMOTHER. TABLE 3. Phenotypic resistance patterns of 682 S. aureus isolates for eight aminoglycosides
Type
1 2 3 4 5 6 7 8 9 10 11b
Resistance patterna
No. of:
KAN
TOB
GEN
AMK
NEO
NET
ABK
STR
MSSA
MRSA
Total
R R S S R R R R R R
R R S S R R R R R R
R R S S R R R R R R
R R S S S R R R R R
R S S S S/R S R R S R
S S S S S R R R R S
S S S S S R R S S R
S/R S/R S R S/R S/R R S/R S/R R
5 17 93 68 43 2 1 0 0 0 14
210 99 1 5 28 33 21 13 10 1 18
215 116 94 73 71 35 22 13 10 1 32
243
439
682
Total a
KAN, kanamycin; TOB, tobramycin; GEN, gentamicin; AMK, amikacin; NEO, neomycin; NET, netilmicin; ABK, arbekacin; STR, streptomycin. R, resistant; S, susceptible; S/R, susceptible or resistant. b Type 11 represents other patterns.
DISCUSSION The prevalence of MRSA has increased worldwide, as is evident from many surveillance studies (5, 8, 19–21, 27). However, there are considerable differences between individual countries. The very highest rates of methicillin resistance among S. aureus isolates have been noted in Western Pacific regions, including Korea (5, 21, 27). To update the current status of antibiotic resistance among S. aureus isolates and to search for other therapeutic alternatives, we conducted a nationwide surveillance study. As expected, the overall prevalence of MRSA was very high, similar to other results (4, 15). For most of the MRSA isolates (96%), the oxacillin MIC was ⱖ128 g/ml, in accordance with the report of Lee and Chong (15). Besides the high level of oxacillin resistance attained through acquisition of the mecA gene, another resistance mechanism, such as overexpression of beta-lactamase (borderline oxacillin-resistant S. aureus), could account for oxacillin resistance in S. aureus (13). However, in all of the MRSA blood isolates, the mecA gene was detected by PCR, irrespective of the MICs (data not shown). Furthermore, a majority of the MRSA isolates were resistant to multiple other antimicrobial agents, as previously noted in the literature (8, 21, 22). In general, elevated rates of multidrug resistance may emerge from diverse isolates of S. aureus under antimicrobial pressure or as a result of widespread person-to-person transmission of multidrug-resistant isolates (1). While the genetic relatedness of MRSA isolates was not extensively studied here, molecular epidemiologic studies by both pulsed-field gel electrophoresis and analysis of mecA regulator genes were done. For 384 mecA-positive MRSA isolates analyzed by multiplex PCR of mec regulator genes, eight genotypes were observed. Of the eight types, one containing the entire mec regulator region (mecA mecR1 mecI pre-mecA) accounted for 81% of isolates and was predominant in all of the hospitals. However, analysis of the PFGE DNA banding patterns for 137 blood isolates revealed 14 distinguishable profiles. Twenty isolates belonged to four subtypes with fewer than five fragment differences, and 37 isolates belonged to three other subtypes differing by five fragments or fewer. Even within
a single mec regulator region genotype, nine different PFGE subtypes were present. These results suggested some clonal relationships. Because infection control policies depend on which mechanism is dominant in the spread of multidrugresistant MRSA, more epidemiologic studies are warranted. Despite the high prevalence of MRSA and the increased use of vancomycin, there were no isolates with reduced susceptibility to glycopeptides. Because of the lower sensitivity of the disk diffusion method, we also screened these isolates by spreading 106 CFU on brain heart infusion agar with 4 g of vancomycin/ml. Although 27 isolates (4%) grew on this medium after 48 h of incubation, none possessed the heteroresistance phenotype on population analysis and revealed a vancomycin MIC ⱖ4 g/ml (14). The rates of resistance of MRSA to rifampin, fusidic acid, and trimethoprim-sulfamethoxazole were much lower (⬍20%) than those to other antibiotics. While parenteral glycopeptide agents are the mainstay of therapy for systemic MRSA infections, not all infections are life-threatening; oral antibiotics provide an alternative mode of therapy, particularly when long-term therapy is required, for example, in the presence of prosthetic material. Rifampin, fusidic acid, and trimethoprimsulfamethoxazole are the only widely available oral agents that demonstrate better tissue penetration than the glycopeptides. However, in view of the high rates of resistance of MRSA to quinolones, quinolone-based combination treatment may be unreliable. MRSA isolates showed much higher rates of resistance to the aminoglycoside agents than did MSSA isolates and were resistant to multiple agents. Based on the phenotypic resistance patterns, most MRSA isolates seemed likely to produce AAC(6⬘)/APH(2⬙), with or without ANT(4⬘)-I, among the five kinds of aminoglycoside-modifying enzymes (10, 23). Arbekacin showed superior in vitro activity compared with the other aminoglycosides against the MRSA isolates, probably because it is modified very little by these enzymes (11). In general, a beta-lactam or vancomycin often is combined with an aminoglycoside due to their synergistic effect and increased rates of killing in serious staphylococcal infections (16). However, con-
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sidering the high rates of resistance to the aminoglycosides, the addition of an aminoglycoside for the treatment of MRSA infections may be unpredictable. Quinupristin-dalfopristin showed excellent activity, equal to that of vancomycin, against all S. aureus isolates. While the bactericidal activity of quinupristin-dalfopristin could be compromised due to the high prevalence of erythromycin-resistant MRSA (7), it might provide a valuable option for the treatment of MRSA infections. In addition, all of the isolates were susceptible to the new agent linezolid, even though a few isolates of linezolid-resistant S. aureus were reported elsewhere (24, 26). In summary, the rate of methicillin resistance among S. aureus isolates from tertiary-care hospitals was very high, and multidrug-resistant MRSA was prevalent. Data generated from this nationwide study could be valuable for the selection of therapeutic alternatives. ACKNOWLEDGMENTS This study was supported by the 2001 National R&D Program (Ministry of Science and Technology, Seoul, Korea). We express our gratitude to S. H. Koo (Chungnam National University), S. J. Kim (Gyeongsang National University), S. Kiem (Sungkyunkwan University), D. H. Shin (Chonnam National University), H. S. Shin (Chungbuk National University), S. H. Lee and C. Chang (Pusan National University), and H. S. Lee (Chonbuk National University) for collecting S. aureus isolates. REFERENCES 1. Ayliffe, G. A. J. 1997. The progressive intercontinental spread of methicillinresistant Staphylococcus aureus. Clin. Infect. Dis. 24(Suppl. 1):S74–S79. 2. Centers for Disease Control and Prevention. 2002. Vancomycin-resistant Staphylococcus aureus—United States, 2002. Morb. Mortal. Wkly. Rep. 51: 565–567. 3. Centers for Disease Control and Prevention. 2002. Vancomycin-resistant Staphylococcus aureus—Pennsylvania, 2002. Morb. Mortal. Wkly. Rep. 51: 902. 4. Chong, Y., and K. Lee. 2000. Present situation of antimicrobial resistance in Korea. J. Infect. Chemother. 6:189–195. 5. Diekema, D. J., M. A. Pfaller, F. J. Schmitz, J. Smayevsky, J. Bell, R. N. Jones, M. Beach, et al. 2001. Survey of infections due to Staphylococcus species: frequency of occurrence and antimicrobial susceptibility of isolates collected in the United States, Canada, Latin America, Europe, and the Western Pacific region for the SENTRY Antimicrobial Surveillance Program, 1997–1999. Clin. Infect. Dis. 32(Suppl. 2):S114–S132. 6. Doern, G. V., R. N. Jones, M. A. Pfaller, K. C. Kugler, M. L. Beach, et al. 1999. Bacterial pathogens isolated from patients with skin and soft tissue infections: frequency of occurrence and antimicrobial susceptibility patterns from the SENTRY Antimicrobial Surveillance Program (United States and Canada, 1997). Diagn. Microbiol. Infect. Dis. 34:65–72. 7. Entenza, J. M., H. Drugeon, M. P. Glauser, and P. Moreillon. 1995. Treatment of experimental endocarditis due to erythromycin-susceptible or -resistant methicillin-resistant Staphylococcus aureus with RP 59500. Antimicrob. Agents Chemother. 39:1419–1424. 8. Fluit, A. C., C. L. C. Wielders, J. Verhoef, and F. J. Schmitz. 2001. Epidemiology and susceptibility of 3,051 Staphylococcus aureus isolates from 25 university hospitals participating in the European SENTRY study. J. Clin. Microbiol. 39:3727–3732.
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9. Hiramatsu, K., H. Hanaki, T. Ino, K. Yabuta, T. Oguri, and F. C. Tenover. 1997. Methicillin-resistant Staphylococcus aureus clinical strain with reduced vancomycin susceptibility. J. Antimicrob. Chemother. 40:135–136. 10. Ida, T., R. Okamoto, C. Shimauchi, T. Okubo, A. Kuga, and M. Inoue. 2001. Identification of aminoglycoside-modifying enzymes by susceptibility testing: epidemiology of methicillin-resistant Staphylococcus aureus in Japan. J. Clin. Microbiol. 39:3115–3121. 11. Inoue, M., M. Nonoyama, R. Okamoto, and T. Ida. 1994. Antimicrobial activity of arbekacin, a new aminoglycoside antibiotic, against methicillinresistant Staphylococcus aureus. Drugs Exp. Clin. Res. 22:233–240. 12. Jevons, M. P. 1961. “Celbenin”-resistant staphylococci. Br. Med. J. 1:124– 126. 13. Jorgensen, J. H. 1991. Mechanisms of methicillin resistance in Staphylococcus aureus and methods for laboratory detection. Infect. Control Hosp. Epidemiol. 12:14–19. 14. Kim, H. B., W. B. Park, K. D. Lee, Y. J. Choi, S. W. Park, M. Oh, E. C. Kim, and K. W. Choe. 2003. Nationwide surveillance for Staphylococcus aureus with reduced susceptibility to vancomycin in Korea. J. Clin. Microbiol. 41: 2279–2281. 15. Lee, M. S., and Y. Chong. 1996. Characteristics of methicillin-resistant Staphylococcus aureus isolated from wounds in Korean patients. J. Infect. Chemother. 2:130–135. 16. Lowy, F. D. 1998. Staphylococcus aureus infections. N. Engl. J. Med. 339: 520–532. 17. National Committee for Clinical Laboratory Standards. 2000. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 5th ed. Approved standard M7-A5. National Committee for Clinical Laboratory Standards, Wayne, Pa. 18. National Committee for Clinical Laboratory Standards. 2000. Performance standards for antimicrobial disk susceptibility tests, 7th ed. Approved standard M2-A7. National Committee for Clinical Laboratory Standards, Wayne, Pa. 19. Pfaller, M. A., R. N. Jones, G. V. Doern, H. S. Sader, K. C. Kugler, M. L. Beach, et al. 1999. Survey of blood stream infections attributable to grampositive cocci: frequency of occurrence and antimicrobial susceptibility of isolates collected in 1997 in the United States, Canada, and Latin America from the SENTRY Antimicrobial Surveillance Program. Diagn. Microbiol. Infect. Dis. 33:283–297. 20. Sader, H. S., R. N. Jones, A. C. Gales, P. Winokur, K. C. Kugler, M. A. Pfaller, G. V. Doern, et al. 1998. Antimicrobial susceptibility patterns for pathogens isolated from patients in Latin American medical centers with a diagnosis of pneumonia: analysis of results from the SENTRY Antimicrobial Surveillance Program (1997). Diagn. Microbiol. Infect. Dis. 32:289–301. 21. Santos Sanches, I. S., R. Mato, H. de Lancastre, A. Tomasz, et al. 2000. Patterns of multidrug resistance among methicillin-resistant hospital isolates of coagulase-positive and coagulase-negative staphylococci collected in the international multicenter study RESIST in 1997 and 1998. Microb. Drug Resist. 6:199–211. 22. Schmitz, F. J., A. Krey, R. Giesel, J. Verhoef, H. P. Heinz, A. C. Fluit, et al. 1999. Susceptibility of 302 methicillin-resistant Staphylococcus aureus isolates from 20 European university hospitals to vancomycin and alternative antistaphylococcal compounds. Eur. J. Clin. Microbiol. Infect. Dis. 18:528– 530. 23. Shaw, K. J., P. N. Rather, R. S. Hare, and G. H. Miller. 1993. Molecular genetics of aminoglycoside resistance genes and familial relationships of the aminoglycoside-modifying enzymes. Microbiol. Rev. 57:138–163. 24. Tsiodras, S., H. S. Gold, G. Sakoulas, G. M. Eliopoulos, C. Wennersten, L. Venkataraman, R. C. Mollering, and M. J. Ferraro. 2001. Linezolid resistance in a clinical isolates of Staphylococcus aureus. Lancet 358:207–208. 25. Walsh, T. R., and R. A. Howe. 2002. The prevalence and mechanisms of vancomycin resistance in Staphylococcus aureus. Annu. Rev. Microbiol. 56: 657–675. 26. Wilson, P., J. A. Andrews, R. Charlesworth, R. Walesby, M. Singer, D. J. Farrell, and M. Robbins. 2003. Linezolid resistance in clinical isolates of Staphylococcus aureus. J. Antimicrob. Chemother. 51:186–188. 27. Witte, W. 1999. Antibiotic resistance in gram-positive bacteria: epidemiological aspects. J. Antimicrob. Chemother. 44:1–9.
