Telithromycin and Quinupristin-Dalfopristin Resistance in Clinical Isolates of Streptococcus pyogenes: SMART Program 2001 Data

Abstract

This study evaluated the current status of antimicrobial resistance in clinical isolates of Streptococcus pyogenes in Taiwan as part of the SMART (Surveillance from Multicenter Antimicrobial Resistance in Taiwan) program. In 2001, 419 different isolates of S. pyogenes, including 275 from respiratory secretions, 87 from wound pus, and 31 from blood, were collected from nine hospitals in different parts of Taiwan. MICs of 23 antimicrobial agents were determined at a central location by the agar dilution method. All of the isolates were susceptible to penicillin (MIC at which 90% of the isolates were inhibited [MIC₉₀], ≤0.03 μg/ml), cefotaxime (MIC₉₀, ≤0.03 μg/ml), cefepime (MIC₉₀, 0.06 μg/ml), meropenem (MIC₉₀, ≤0.03 μg/ml), moxifloxacin (MIC₉₀, 0.25 μg/ml), vancomycin (MIC₉₀, 0.5 μg/ml), and linezolid (MIC₉₀, 1 μg/ml). Overall, 78% of isolates were not susceptible to erythromycin (54% were intermediate, and 24% were resistant), and 5% were not susceptible to clindamycin. Of the 101 erythromycin-resistant isolates, 80.2% exhibited the M phenotype (mefA gene positive), 18.9% exhibited the cMLS (constitutive resistance to macrolides-lincosamides-streptogramin B [MLS]) phenotype (ermB gene positive), and 1% exhibited the iMLS (inducible resistance to MLS) phenotype (ermB gene positive). Fluoroquinolones (sitafloxacin > moxifloxacin > ciprofloxacin = levofloxacin = gatifloxacin > gemifloxacin) demonstrated potent activity against nearly all of the isolates of S. pyogenes tested. Thirty-two isolates (8%) were not susceptible to quinupristin-dalfopristin. Seventeen percent of isolates had telithromycin MICs of ≥1 μg/ml, and all of these isolates exhibited erythromycin MICs of ≥32 μg/ml. The high prevalence of resistance to telithromycin (which is not available in Taiwan) limits its potential use in the treatment of S. pyogenes infections, particularly in areas with high rates of macrolide resistance.

Streptococcus pyogenes is the most common bacterial etiology of acute tonsillopharyngitis and skin and soft tissue infections that may result in life-threatening invasive diseases in both children and adults (7, 15, 16). S. pyogenes is still susceptible to penicillin but has shown increasing resistance to macrolides recently (3, 6, 20, 29, 30, 38). Resistance to macrolides in S. pyogenes is mediated by ermA, ermB, ermTR, and mefA genes, and the presence of resistance mechanisms varies in different countries (2, 6, 11, 13, 22, 31, 32, 34, 36).

Telithromycin has been reported to be active against S. pyogenes isolates with inducible ermAM(B)- and mefA-mediated resistance. However, telithromycin has lower activity against isolates with the constitutive ermAM(B) resistance mechanism (2, 10, 19, 21, 25, 26, 33). Other newly developed antimicrobial agents, such as fluoroquinolones, linezolid, quinupristin-dalfopristin, and tigecycline, have also been demonstrated to have excellent in vitro activity against S. pyogenes isolates regardless of their macrolide resistance mechanisms (1, 4, 5, 8, 9, 13, 24).

In previous studies of isolates from Taiwan, the prevalence of macrolide resistance in S. pyogenes isolates was high (about 50%), and isolates possessing mefA-mediated resistance tended to predominate (>90%) (14, 17, 18, 37). To better understand the current and nationwide resistance status of S. pyogenes, we examined the in vitro activities of telithromycin, newer fluoroquinolones, tigecycline, and other antimicrobial agents against isolates that were recently collected from different parts of Taiwan. This study is part of the SMART (Surveillance from Multicenter Antimicrobial Resistance in Taiwan) programs conducted in 2001.

MATERIALS AND METHODS

Bacterial isolates.

From January 2000 to December 2001, a total of 419 isolates of S. pyogenes were collected for this study. The isolates were recovered from clinical specimens from patients treated at nine hospitals (with 800 to 3,000 beds) located in different parts of Taiwan. The nine hospitals in Taiwan were as follows: the National Taiwan University Hospital (NTUH), Taipei (187 isolates); Chang-Gung Memorial Hospital, LinKou (57 isolates); Mackay Memorial Hospital, Taipei (74 isolates); Tri-service General Hospital, Taipei (18 isolates); Taichung Veterans General Hospital, Taichung (11 isolates); China Medical College Hospital, Taichung (27 isolates); National Cheng-Kung University Hospital, Tainan (6 isolates); Chi-Mei Medical Center, Tainan (11 isolates); and Kaohsiung Veterans General Hospital, Kaohsiung (28 isolates). Of the 419 isolates tested, 64% were recovered from respiratory specimens, 21% were from skin and soft tissue specimens (wound pus or abscess), and 14% were from blood or other sterile body fluids. The age of the patients ranged from 1 to 75 years old, with the age group of 3 to 15 years predominant (48% of the 370 patients whose ages were known) (Table 1). All of the isolates were further identified by conventional methods at the NTUH.

TABLE 1.

Sources of 419 isolates of S. pyogenes recovered from 419 patients from nine major hospitals from different parts of Taiwan in 2001

Source of isolates and age group of patients	No. (%) of isolates
Sources of isolates
Throat swab	225 (54)
Other respiratory secretions (sputum, sinus, ear discharge)	41 (10)
Pus (wound, abscess drainage)	87 (21)
Blood	40 (10)
Urine	10 (2)
Other body fluids (synovial fluid, ascitic fluid, pleural effusion)	16 (4)
Total no. of isolates	419
Age groups (yr)
≤3	3 (1)
3-15	176 (48)
16-64	141 (38)
≥65	50 (13)
Total no. of patients	370a

^aThe age of the patient was available for 370 of the 419 patients.

Trend in resistance.

To determine any trends in the erythromycin resistance patterns, data on the disk diffusion susceptibility to erythromycin for S. pyogenes isolates recovered from patients treated at the NTUH from 1993 to 2002 were also analyzed (17, 18).

Antimicrobial susceptibility testing.

The following antimicrobial agents were provided by their manufacturers for use in this study: penicillin, erythromycin, clindamycin, tetracycline, and chloramphenicol (Sigma Chemical Co., St. Louis, Mo.); amoxicillin and gemifloxacin (GlaxoSmithKline, Philadelphia, Pa.); vancomycin (Eli Lilly & Co., Indianapolis, Ind.); cefotaxime, cefpirome, teicoplanin, quinupristin-dalfopristin, and telithromycin (Aventis Pharma, Romainville, France); cefepime and gatifloxacin (Bristol-Myers Squibb, Princeton, N.J.); imipenem and ertapenem (Merck Sharp & Dohme, Rahway, N.J.); meropenem (Sumitomo Pharmaceuticals, Tokyo, Japan); azithromycin (Pfizer Inc., New York, N.Y.); tigecycline (Wyeth-Ayerst, Pearl River, N.Y.); linezolid (Pharmacia, Kalamazoo, Mich.); levofloxacin and sitafloxacin (Daiichi Pharmaceuticals, Tokyo, Japan); and ciprofloxacin and moxifloxacin (Bayer Co., Leverkusen, Germany).

MICs of these antimicrobial agents for all 419 isolates of S. pyogenes were determined by the agar dilution method and interpreted according to the guidelines established by the National Committee for Clinical Laboratory Standards (NCCLS) (27, 28). The isolates were grown overnight on Trypticase soy agar plates supplemented with 5% sheep blood (BBL Microbiology Systems, Cockeysville, Md.) at 37°C in ambient air (2, 27). Bacterial inocula were prepared by suspending the freshly grown bacteria in sterile normal saline and adjusting the solution to a McFarland standard of 0.5. For susceptibility testing of the isolates, we used Mueller-Hinton agar supplemented with 5% sheep blood (BBL Microbiology Systems). Using a Steers replicator, an organism density of 10⁴ CFU/spot was inoculated onto the appropriate plate with various concentrations of antimicrobial agents. The following organisms were included as control strains: Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, and Streptococcus pneumoniae ATCC 49619.

Erythromycin-clindamycin double-disk test.

The resistance phenotypes of erythromycin-resistant S. pyogenes were determined by the double-disk test with an erythromycin disk (15 μg) and a clindamycin disk (2 μg) (BBL Microbiology Systems) as previously described (18, 31). A blunting of the clindamycin zone of inhibition proximal to the erythromycin disk indicated an inducible resistance to macrolides-lincosamides-streptogramin B (MLS) (iMLS phenotype). The absence of a significant zone of inhibition around the two disks was interpreted as constitutive resistance to MLS (cMLS phenotype). An M phenotype was characterized by a susceptibility to clindamycin with no blunting of the zone of inhibition around the clindamycin disk.

Detection of erythromycin resistance genes.

All of the erythromycin-resistant isolates were tested by PCR for the presence of ermAM(B), ermTR, and mefA genes as described previously (11, 18, 34-36).

RESULTS

Erythromycin resistance trend.

Figure 1 shows the yearly percentages of erythromycin-resistant isolates of the S. pyogenes isolates obtained from 1993 to 2002 at the NTUH. A stepwise decrease in the rate of this resistance has been seen since 1995, though a transient peak of resistance in 1998 was noted. In 2002, only 17% of S. pyogenes isolates were resistant to erythromycin at the NTUH.

FIG. 1.

Trend in erythromycin resistance determined by the routine disk diffusion method for S. pyogenes isolates recovered from patients treated at the NTUH from 1993 to 2002.

Antimicrobial susceptibilities.

All of the S. pyogenes isolates were susceptible to penicillin (MIC at which 90% of the isolates were inhibited [MIC₉₀], ≤0.03 μg/ml), cefotaxime (MIC₉₀, ≤0.03 μg/ml), cefepime (MIC₉₀, 0.06 μg/ml), meropenem (MIC₉₀, ≤0.03 μg/ml), vancomycin (MIC₉₀, 0.5 μg/ml), and linezolid (MIC₉₀, 1 μg/ml) (Table 2). Amoxicillin , cefepime, cefpirome, imipenem, ertapenem, and teicoplanin also had good in vitro activities against these isolates. All of the isolates were inhibited by 0.12 μg of tigecycline per ml. Fluoroquinolones (sitafloxacin > moxifloxacin > ciprofloxacin = levofloxacin = gatifloxacin > gemifloxacin) demonstrated potent activity against nearly all isolates of S. pyogenes tested. Thirty-two isolates (8%) were not susceptible to quinupristin-dalfopristin, and 7 (2%) of these isolates were fully resistant (MICs of ≥4 μg/ml). Isolates not susceptible to quinupristin-dalfopristin were more predominant in southern Taiwan (16%) than in central or northern Taiwan (Fig. 2). All of the four gatifloxacin-intermediate isolates (MICs of 2 μg/ml) were found in patients from northern Taiwan.

TABLE 2.

In vitro susceptibilities for 419 clinical isolates of S. pyogenes^a

Antibiotic	MIC (μg/ml)b			% of isolatesc
	Range	50%	90%	S	I	R
Penicillin	<0.03-0.12	<0.03	<0.03	100	0	0
Amoxicillin	<0.03-0.25	<0.03	0.06	100	0	0
Cefotaxime	<0.03-0.5	<0.03	<0.03	100	0	0
Cefpirome	<0.03-0.25	<0.03	<0.03	—d	—	—
Cefepime	<0.03-1	<0.03	0.06	100	—	—
Imipenem	<0.03	<0.03	<0.03	—	—	—
Meropenem	<0.03-0.12	<0.03	<0.03	100	0	0
Ertapenem	<0.03-0.25	<0.03	<0.03	—	—	—
Tigecycline	<0.03-0.12	0.06	0.12	—	—	—
Erythromycin	0.06->128	0.5	>128	22	54	24
Azithromycin	0.12->128	1	>128	22	54	24
Clindamycin	0.06->128	0.12	0.25	95	0	5
Chloramphenicol	1-64	2	4	93	2	5
Trimethoprim-sulfa- methoxazole	0.5->128	>128	>128	—	—	—
Vancomycin	0.12-0.5	0.25	0.5	100	0	0
Teicoplanin	<0.03-0.5	0.12	0.25	—	—	—
Quinupristin-dalfo- pristin	0.12-4	0.5	1	92	6	2
Linezolid	0.12-2	1	1	100	0	0
Ciprofloxacin	0.12-4	0.5	1	—	—	—
Levofloxacin	0.12-4	0.5	1	99	1	0
Moxifloxacin	<0.03-0.5	0.12	0.25	100	0	0
Gatifloxacin	<0.03-2	0.25	1	99	1	0
Gemifloxacin	<0.03-2	<0.03	2	—	—	—
Sitafloxacin	<0.03-0.12	<0.03	0.06	—	—	—
Telithromycin	0.03->32	0.06	1	83e	14e	3e

^aIsolates were collected from different parts of Taiwan in 2001. Susceptibility was determined by the agar dilution method.

^b50% and 90%, MICs at which 50 and 90% of isolates are inhibited, respectively.

^cSusceptibility results (S, susceptible; I, intermediate; R, resistant) were interpreted according to the MIC interpretive standards established by the National Committee for Clinical Laboratory Standards (NCCLS) for Streptococcus spp. other than S. pneumoniae (28).

^d—, interpretive criteria for defining susceptibility for Streptococcus spp. other than S. pneumoniae to cefpirome, imipenem, ertapenem, tigecycline, trimethoprim-sulfamethoxazole, teicoplanin, ciprofloxacin, gemifloxacin, and sitafloxacin, were not provided by the NCCLS (28).

^eSusceptibility data of telithromycin were interpreted using the following breakpoints: susceptible, ≤0.5 μg/ml; intermediate, 1 to 2 μg/ml; and resistant, ≥4 μg/ml (25).

FIG. 2.

Percentages of S. pyogenes isolates from different parts of Taiwan that were resistant to erythromycin or clindamycin and not susceptible to quinupristin-dalfopristin (Q/D) or telithromycin (MICs of ≥1 μg/ml). The number of isolates exhibiting resistance or nonsusceptibility to the indicated antimicrobial agent is shown in each bar, and the percentage of isolates exhibiting resistance or nonsusceptibility to the antimicrobial agent is shown above the bar.

Overall, 78% of the S. pyogenes isolates were not susceptible to both erythromycin and azithromycin (54% were intermediate, and 24% were resistant), and 5% were not susceptible to clindamycin. Isolates resistant to erythromycin were more common in patients aged 3 to 15 years (28%), followed by patients aged 16 to 64 years (21%), ≥65 years (20%), and ≤3 years (0%) (only three patients in this age group). Thirty percent of isolates from throat swab or respiratory specimens were resistant to erythromycin, followed by 20% from blood samples, and 16% from pus (wound and abscess) specimens.

The distribution of telithromycin and erythromycin MICs for S. pyogenes isolates tested is illustrated in Fig. 3. Erythromycin MICs displayed a bimodal distribution (MICs concentrated in the ranges of 0.25 to 0.5 μg/ml and ≥16 μg/ml). A trimodal distribution of the telithromycin MICs was also noted. Telithromycin susceptibility data according to the European MIC breakpoints (i.e., susceptible, ≤0.5 μg/ml; intermediate, 1 to 2 μg/ml; and resistant, ≥4 μg/ml) showed that 58 (14%) isolates were intermediate, and 14 (3%) were resistant (MICs of ≥32 μg/ml for all 14 isolates) (25). Of the 101 erythromycin-resistant isolates (MICs, ≥1 μg/ml), 72% (72 isolates) exhibited telithromycin MICs of ≥1 μg/ml (58% with MICs of 1 to 2 μg/ml and 14% with MICs of ≥32 μg/ml).

FIG. 3.

Distribution of erythromycin and telithromycin MICs for 419 isolates of S. pyogenes.

Erythromycin-resistant phenotypes.

Of the erythromycin-resistant isolates, 81 (80.2%) exhibited the M phenotype, 19 (18.9%) exhibited the cMLS phenotype, and one (1%) exhibited the iMLS phenotype (one isolate in northern Taiwan) (Table 3). M-phenotype isolates predominated in the northern (80.5%) and central (90.9%) parts of Taiwan compared to southern Taiwan (37.5%).

TABLE 3.

MIC distribution by mechanism of resistance in 101 isolates of erythromycin-resistant S. pyogenes^a

Drug	MIC (μg/ml) of isolates with erythromycin-resistant phenotypeb:
	cMLS (n = 19)			M (n = 81)
	Range	50%	90%	Range	50%	90%
Erythromycin	128->128	128	>128	1-64	32	64
Azithromycin	128->128	128	>128	2-64	64	128
Clindamycin	1->128	64	>128	0.06-0.5	0.12	0.25
Telithromycin	2-64	32	64	0.5-1	0.5	1

^aOne isolate exhibiting the iMLS phenotype had erythromycin, azithromycin, and telithromycin MICs of 64, 64, and 0.06 μg/ml, respectively.

^bIsolates with erythromycin MIC of ≥1 μg/ml. 50% and 90%, MICs at which 50 and 90% of isolates are inhibited, respectively.

All isolates with the cMLS phenotype had erythromycin MICs of ≥128 μg/ml and telithromycin MICs of 2 to 64 μg/ml (MIC₅₀ and MIC₉₀, 32 and 64 μg/ml, respectively). Isolates with the M phenotype had erythromycin MICs of 1 to 64 μg/ml and telithromycin MICs of 0.5 to 1 μg/ml (MIC₅₀ and MIC₉₀, 0.5 and 1 μg/ml, respectively). Isolates with the iMLS phenotype had erythromycin, azithromycin, clindamycin, and telithromycin MICs of 64, 64, 0.12, and 0.06 μg/ml, respectively, and had no zone of inhibition around the erythromycin disk (iMLS-B).

Erythromycin resistance genes.

All of the isolates with the M phenotype had the mefA gene, while all of the isolates with the cMLS phenotype and the iMLS phenotype had the ermAM(B) gene. The isolate with the iMLS phenotype harbored the ermTR gene. The mefA gene was not found in isolates with the iMLS and cMLS phenotypes.

DISCUSSION

Five aspects of this study on recent S. pyogenes isolates from Taiwan are of particular importance. First, a large number of S. pyogenes isolates from Taiwan, particularly those exhibiting resistance to macrolides, were not susceptible to telithromycin. Second, the high rate of telithromycin nonsusceptibility and the emergence of resistance to quinupristin-dalfopristin in S. pyogenes isolates are of great concern, because these two agents are not yet available in Taiwan (23). Third, given the persistently high consumption of macrolides in clinical settings, the declining prevalence of erythromycin resistance in S. pyogenes from the 10 years of data from the NTUH and the present resistance data from the nationwide surveillance was impressive (14, 17, 18). Interestingly, the majority (71%) of the non-erythromycin-susceptible isolates found in this study were actually intermediate to erythromycin. Fourth, the predominance of isolates with the cMLS phenotype in southern Taiwan and the predominance of isolates with the M phenotype in northern and central Taiwan indicated the presence of different erythromycin resistance mechanisms in S. pyogenes, varying not only in different countries but also in different areas of one country. Finally, the newly developed agents, linezolid, sitafloxacin, and tigecycline, exhibited excellent in vitro activity against S. pyogenes isolates in Taiwan.

Previous studies showed that telithromycin is very active against macrolide-susceptible and -resistant S. pneumoniae isolates irrespective of macrolide resistance mechanisms: ermB-positive MLS-B phenotype or mefA-positive M phenotype (26, 33). The excellent in vitro activity of telithromycin, together with a lower capacity to select resistant mutants and the lack of resistance induction, make it a promising alternative for treatment of infections caused by S. pneumoniae when macrolides are indicated (26, 33). Unfortunately, our previous study also showed that similar rates (15%) of recently collected isolates of S. pneumoniae were not susceptible to telithromycin. For S. pyogenes isolates, telithromycin is active against susceptible and moderately erythromycin-resistant efflux-mediated isolates. However, it does not overcome the erm(B)-mediated macrolide-resistant isolates (telithromycin MICs, 8 to 64 μg/ml) (11, 19). Our results support previous findings. In the present study, the MICs of all antimicrobial agents were determined in ambient air. A previous study has clearly demonstrated that the presence of 5 to 6% of CO₂ during incubation results in increasing telithromycin MICs for S. pyogenes isolates, remarkably against isolates possessing the ermB gene (increasing from 1 to 6 twofold dilutions) (1).

The results of the present study and our previous study both demonstrated that the M phenotype was predominant in erythromycin-resistant isolates (18). There were few S. pyogenes or S. pneumoniae isolates with the iMLS phenotype in Taiwan (18). Using the triple-disk assay (erythromycin, clindamycin, and josamycin) and induction of MLS resistance, three different subtypes (iMLS-A, -B, and -C) in the isolates with the iMLS phenotype have been previously described (11, 12). The erythromycin and telithromycin MIC data for the iMLS phenotype isolate reported in this study were in accordance with those for the iMLS-B phenotype reported previously (11). Giovanetti et al. clearly demonstrated that, in addition to the presence of erm-encoded methylase activity, the iMLS-B isolates had a novel, transferable efflux system, not associated with mefA or with other known macrolide efflux genes (12). Further studies, including conjugation experiment, should be performed to elucidate the resistance mechanism.

In the present study, the MICs of the newer fluoroquinolones were lower than those of the older fluoroquinolones (ciprofloxacin and levofloxacin) against different macrolide-resistant phenotypes of S. pyogenes isolates. Of the newer fluoroquinolones (moxifloxacin, gemifloxacin, and gatifloxacin), sitafloxacin had the lowest MIC₉₀. Few of our isolates were intermediate to gatifloxacin (four isolates) and levofloxacin (one isolates) according to the NCCLS breakpoints. Our results were in agreement with previous findings (7, 9, 24).

Linezolid and quinupristin-dalfopristin both have excellent in vitro activities against beta-hemolytic streptococci with MIC₅₀s (MIC₉₀s shown in parentheses) of 1 μg/ml (2 μg/ml) and 0.25 μg/ml (0.25 μg/ml), respectively (1, 13). In contrast, quinupristin-dalfopristin had two- to fourfold-less activity against our S. pyogenes isolates than those reported previously (18). The emergence of quinupristin-dalfopristin resistance in S. pyogenes isolates in Taiwan in 2001 is alarming, because no such isolates were found before, although resistance to quinupristin-dalfopristin in other gram-positive bacteria has existed for many years (23). Further studies should be conducted to elucidate the mechanism(s) of resistance to quinupristin-dalfopristin and the clonality of these resistant S. pyogenes isolates.

Tigecycline has shown promising activity against a wide spectrum of aerobic and anaerobic bacteria, including bacteria resistant to older tetracyclines (4). Tigecycline MIC₅₀ and MIC₉₀ for S. pyogenes isolates were reported to be 0.12 and 0.12 μg/ml, respectively. The present study confirmed these findings (5, 8). Although susceptibility to tetracycline was not examined in this study, our previous study showed that 72.2% of S. pyogenes isolates collected in Taiwan from 1979 to 1998 were resistant to tetracycline (18). If the proposed tigecycline susceptibility criteria of ≤2 μg/ml for S. pyogenes were adopted (8), all of the isolates tested in this study and other studies would be considered susceptible.

Given the persistently high selective pressure for macrolides in the community in Taiwan (16, 23), the reasons for the substantial upsurge in the prevalence of erythromycin resistance in S. pneumoniae (16, 23) but a remarkable decrease in the prevalence of erythromycin resistance in S. pyogenes (or a increasing number of isolates with MICs shifting from the resistance category to the intermediate category) with time are not understood. Although it is likely that the disappearance of some resistant clones may contribute to the decline in resistance, a previous study had demonstrated that erythromycin-resistant S. pyogenes isolates with the M phenotype in Taiwan originated in multiple clones (37). Studies regarding the clonality of our erythromycin-resistant S. pyogenes isolates should be performed.

The high prevalence of resistance to telithromycin limits its potential use in the treatment of S. pyogenes infections, particularly those caused by isolates with the constitutive erm resistance gene and in areas with a high burden of macrolide resistance.