In Vitro Activities of Tigecycline against Erythromycin-Resistant Streptococcus pyogenes and Streptococcus agalactiae: Mechanisms of Macrolide and Tetracycline Resistance

Abstract

The activity of tigecycline was tested against erythromycin-resistant streptococci (107 Streptococcus pyogenes and 98 Streptococcus agalactiae strains). The presence of erythromycin and tetracycline resistance genes was determined by PCR. Among S. pyogenes strains the most prevalent gene was mef(A) (91.6%). The erm(B) gene was the most prevalent (65.3%) among S. agalactiae strains. Tigecycline proved to be very active against all the isolates tested (MIC at which 90% of the isolates tested were inhibited, 0.06 μg/ml), including those resistant to tetracycline.

An increased incidence of macrolide resistance among beta-hemolytic streptococci has been reported in several countries during the past 2 decades (1, 5, 6, 11, 18). Every day, fewer therapeutic options for penicillin-allergic patients are available. The present rates of erythromycin resistance in our area of Spain are about 30% for Streptococcus pyogenes (3) and 18% for Streptococcus agalactiae (2). Tigecycline (formerly GAR-936) is a novel glycylcycline antibiotic that was shown to have potent activity against a wide spectrum of gram-positive and gram-negative bacteria, including strains resistant to other antimicrobials (4, 8, 10, 13). The purpose of this study was to evaluate the in vitro activities of tigecycline and comparator agents against macrolide-resistant S. pyogenes and S. agalactiae. The mechanisms of macrolide and tetracycline resistance have also been determined.

A total of 107 S. pyogenes and 98 S. agalactiae isolates resistant to erythromycin were included, regardless of their resistance to tetracycline. Organisms were collected at the Hospital Clínico San Carlos during the period 1994 to 2001. The sources of S. pyogenes isolates were the upper respiratory tract (93 isolates), skin and soft tissues (8 isolates), vagina (4 isolates), blood (1 isolate), and cerebrospinal fluid (1 isolate). The 98 S. agalactiae isolates were recovered from the following sources: skin and soft tissues (51 isolates), urine (24 isolates), vagina (11 isolates), abdomen (5 isolates), blood (4 isolates), and upper respiratory tract (3 isolates). Organisms were identified by standard methods, including agglutination with latex (Slidex Strepto A and Slidex Strepto B; bioMérieux, Marcy l'Etoile, France). Only one isolate per patient was studied to avoid duplication.

Susceptibility was tested by the agar dilution method according to the National Committee for Clinical Laboratory Standards (12) with Mueller-Hinton agar supplemented with 5% sheep blood. The plates were incubated overnight at 35°C in ambient air. The MIC was defined as the lowest antibiotic concentration that completely inhibits the growth of the organism as detected by the unaided eye. The preliminary breakpoints of tigecycline are ≤2 μg/ml for susceptibility, 4 μg/ml for intermediate status, and ≥8 μg/ml for resistance (7). Streptococcus pneumoniae ATCC 49619 and Staphylococcus aureus ATCC 29213 were used as control strains. The following antimicrobial agents were included in the study: tigecycline (Wyeth Pharmaceuticals, Philadelphia, Pa.), erythromycin (Abbott Laboratories S.A., Madrid, Spain), clindamycin (Pharmacia S.A., Barcelona, Spain), quinupristin-dalfopristin (Aventis Pharma S.A., Madrid, Spain), penicillin (Cepa Schwarz Pharma S.L., Madrid, Spain), and minocycline and tetracycline (Sigma-Aldrich Química S.A., Madrid, Spain).

Determination of macrolide resistance phenotypes was performed by the double-disk method (15). The presence of erythromycin resistance genes was determined by PCR (16). The DNAs of the erythromycin-resistant isolates were amplified with primers specific for the erm(A), erm(B), and mef(A) genes. The PCR conditions for the primer sets were as described previously (9, 16). DNA preparation and electrophoresis of PCR products were carried out by established procedures (9, 16). The 14 S. pyogenes and 88 S. agalactiae isolates resistant to tetracycline were tested for the presence of tetracycline resistance genes tet(M), tet(O), tet(K), and tet(L) by PCR as described by Trzcinski et al. (17).

The results of the susceptibility studies are shown in Table 1. The resistance rate for tetracycline among S. agalactiae isolates was 89.8%, while among S. pyogenes this rate was 13.1%. Quinupristin-dalfopristin had excellent activity (MIC at which 90% of the isolates tested were inhibited, 0.5 μg/ml). All isolates tested were exquisitely susceptible to penicillin, and penicillin MIC ranges for S. pyogenes were slightly lower than those for S. agalactiae.

TABLE 1.

In vitro activity of tigecycline compared with those of other agents against 205 erythromycin-resistant streptococci

Organism (no. of isolates) and antimicrobial agent	MIC (μg/ml)
	Range	50%	90%
S. pyogenes (107)
Tigecycline	0.016-0.06	0.03	0.06
Minocycline	≤0.06-8	≤0.06	4
Tetracycline	≤0.06-32	0.12	16
Penicillin	≤0.008-0.03	≤0.008	0.016
Erythromycin	1->64	8	64
Clindamycin	0.016->64	0.06	2
Quinupristin-dalfopristin	0.12-1	0.25	0.5
S. agalactiae (98)
Tigecycline	0.03-0.25	0.06	0.12
Minocycline	≤0.06-16	8	16
Tetracycline	0.12-64	32	64
Penicillin	0.01-0.06	0.03	0.06
Erythromycin	1->64	64	>64
Clindamycin	0.03->64	>64	>64
Quinupristin-dalfopristin	0.25-1	0.5	0.5

Tigecycline demonstrated similar in vitro activities against S. pyogenes and S. agalactiae. The new glycylcycline tested had better activities than did the comparative antibiotics, except penicillin, against most resistant organisms. Tigecycline, based on comparison of MICs at which 90% of the isolates were inhibited, was 12 to 14 times more active than minocycline and 4 to 8 times more active than quinupristin-dalfopristin against both streptococcal species tested. Our results agree with those described previously by several authors (7, 8, 10, 13), despite the fact that they studied a smaller number of streptococcal isolates.

Tigecycline is known to overcome the two major determinants of tetracycline resistance, ribosomal protection and active efflux (13, 19). In our study, no differences in the activities of tigecycline between the tetracycline-susceptible and tetracycline-resistant streptococcal isolates tested were noted. Tetracycline-resistant S. agalactiae and S. pyogenes strains were inhibited by ≤0.25 and ≤0.06 μg/ml of tigecycline, respectively.

The distribution of erythromycin and tetracycline resistance genes is shown in Table 2. The constitutive macrolide-lincosamide-streptogramin B resistance (cMLS_B) phenotype was observed in 9.3% of S. pyogenes and 68.4% of S. agalactiae isolates. Strains with the M phenotype accounted for 82.3 and 6.1% of S. pyogenes and S. agalactiae isolates, respectively.

TABLE 2.

Distribution of erythromycin and tetracycline resistance genes among 205 erythromycin-resistant streptococci

Organism	Erythromycin resistance phenotypea	No. of isolates	No. of isolates with gene(s):
			mef(A)	erm(B)	erm(A)	tet(M)	tet(O)
S. pyogenes	M	88	88		9	2
	iMLSB	9	1	1	9	5
	cMLSB	10		8	1	4
S. agalactiae	M	6	6		3	4
	iMLSB	25	4	16	18	16	6
	cMLSB	67		48	37	46	13

^aM, efflux; iMLS_B, inducible; cMLS_B, constitutive.

PCR of the macrolide resistance determinants in S. pyogenes isolates detected mef(A) in 89 isolates and erm(B) in 9 isolates. Both mef(A) and erm(A) were found in 10 isolates. The 88 M-phenotype isolates harbored the mef(A) gene, and nine of them also had the erm(A) gene. Erythromycin resistance in one constitutive-phenotype isolate was not associated with either the mef or the erm gene.

Among S. agalactiae strains, the erm(B) gene was the most prevalent (in 65.3% of isolates) followed by erm(A) (59.2%). The erm(A) gene was found in 37 cMLS_B phenotype isolates, in 18 isolates with the inducible MLS_B phenotype, and in 3 M-phenotype isolates. Twenty-seven isolates possessed both erm(A) and erm(B) determinants.

We also investigated the molecular basis of tetracycline resistance. The majority (76.5%) of tetracycline-resistant streptococcal isolates included in the study harbored tet(M), which confers resistance to both tetracycline and minocycline. The 12 S. pyogenes isolates resistant to tetracycline have been found to be positive only for tet(M). Of the 88 tetracycline-resistant S. agalactiae isolates, 66 (75%) carried the tet(M) gene and 19 (21.6%) carried the tet(O) gene. In five tetracycline-resistant isolates (two of S. pyogenes and three of S. agalactiae), none of the tetracycline resistance genes tested were found. Isolates carrying both tet(M) and tet(O) genes, as Poyart et al. (14) recently reported, were not detected in our study. Neither the tet(L) nor the tet(K) gene was found in the present study.

The potent activity of tigecycline against all the strains tested, irrespective of their phenotype of resistance to erythromycin or their resistance to tetracycline, indicates that this agent could be considered an alternative to penicillin for the treatment of infections caused by erythromycin-resistant S. pyogenes and S. agalactiae.