In Vitro Activities of 28 Antimicrobial Agents against Staphylococcus aureus Isolates from Tertiary-Care Hospitals in Korea: a Nationwide Survey

Abstract

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 macrolide-lincosamide-streptogramin B resistance phenotype was common, no isolates were resistant to quinupristin-dalfopristin 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.

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 options 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 tertiary-care 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.)

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 ≥21, ≥18, ≥15, and ≥17 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 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 ≥256 μg/ml, only 4% of the isolates (18 of 439) were inhibited at an MIC of ≤64 μg/ml.

TABLE 1.

Antibiotic resistance profiles of 243 MSSA and 439 MRSA isolates from tertiary-care hospitals, as determined by disk diffusion^a

Antibiotic	MSSA				MRSA
	No. of isolates that were:			Resistance rate (%)	No. of isolates that were:			Resistance rate (%)
	S	I	R		S	I	R
Penicillin	24	—	219	90.1	0	—	439	100
Amoxicillin-clavulanic acid	239	—	4	1.6	7	—	432	98.4
Cefazolin	243	0	0	0	10	2	427	97.8
Cefuroxime	238	5	0	2.1	2	13	424	99.6
Cefotaxime	236	5	2	2.9	2	15	422	99.5
Imipenem	243	0	0	0	38	2	399	91.4
Erythromycin	154	32	57	36.7	10	1	428	97.7
Azithromycin	147	39	57	39.5	12	2	425	97.3
Clindamycin	215	16	12	11.5	69	7	363	84.3
Quinupristin-dalfopristin	243	0	0	0	439	0	0	0
Linezolid	243	—	—	0	439	—	—	0
Vancomycin	243	—	—	0	439	—	—	0
Teicoplanin	243	0	0	0	439	0	0	0
Ofloxacin	237	3	3	2.4	27	1	411	93.8
Sparfloxacin	240	0	3	1.2	27	4	408	93.8
Tetracycline	190	9	44	21.8	46	11	382	89.5
Trimethoprim-sulfamethoxazole	240	2	1	1.2	400	10	29	8.9
Rifampin	241	2	0	0.8	360	23	56	18.0
Fusidic acid	166	1	76	31.7	377	8	54	14.1
Gentamicin	175	0	68	28.0	22	1	416	95.0
Arbekacin	240	2	1	1.2	384	49	6	12.6
Amikacin	215	23	5	11.6	46	69	324	89.5
Tobramycin	173	3	67	28.8	9	0	430	97.9
Streptomycin	134	104	5	44.9	302	99	38	31.3
Neomycin	221	6	16	9.1	163	3	273	62.9
Netilmicin	240	1	2	1.2	362	19	58	17.5
Kanamycin	163	2	78	32.9	7	0	432	98.4

^aS, susceptible; I, intermediate; R, resistant. —, not applicable.

Of the 89 erythromycin-resistant MSSA isolates, 21 (24%) had the constitutive macrolide-lincosamide-streptogramin B (MLS_B) resistance phenotype, whereas 367 (86%) of the 429 erythromycin-resistant MRSA isolates had the constitutive MLS_B resistance phenotype (Table 2). The other erythromycin-resistant S. aureus isolates had an inducible MLS_B resistance phenotype. No isolates were resistant to quinupristin-dalfopristin or linezolid.

TABLE 2.

Phenotypic resistance patterns of 682 S. aureus isolates for erythromycin, clindamycin, and quinupristin-dalfopristin

Resistance patterna	No. (%) of:
	MSSA	MRSA
Erythromycin resistant, clindamycin susceptible	68 (28)	62 (14)
Erythromycin resistant, clindamycin resistant	21 (9)	367 (83)
Erythromycin susceptible, clindamycin resistant	7 (3)	3 (1)
Erythromycin susceptible, clindamycin susceptible	147 (60)	7 (2)
Total	243	439

^aAll isolates were susceptible to quinupristin-dalfopristin.

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 3.

Phenotypic resistance patterns of 682 S. aureus isolates for eight aminoglycosides

Type	Resistance patterna								No. of:
	KAN	TOB	GEN	AMK	NEO	NET	ABK	STR	MSSA	MRSA	Total
1	R	R	R	R	R	S	S	S/R	5	210	215
2	R	R	R	R	S	S	S	S/R	17	99	116
3	S	S	S	S	S	S	S	S	93	1	94
4	S	S	S	S	S	S	S	R	68	5	73
5	R	R	R	S	S/R	S	S	S/R	43	28	71
6	R	R	R	R	S	R	R	S/R	2	33	35
7	R	R	R	R	R	R	R	R	1	21	22
8	R	R	R	R	R	R	S	S/R	0	13	13
9	R	R	R	R	S	R	S	S/R	0	10	10
10	R	R	R	R	R	S	R	R	0	1	1
11b									14	18	32
Total									243	439	682

^aKAN, kanamycin; TOB, tobramycin; GEN, gentamicin; AMK, amikacin; NEO, neomycin; NET, netilmicin; ABK, arbekacin; STR, streptomycin. R, resistant; S, susceptible; S/R, susceptible or resistant.

^bType 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 ≥128 μg/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 multidrug-resistant 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 μg 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 ≥4 μg/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 trimethoprim-sulfamethoxazole 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, considering 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.