Molecular Mechanisms of Tetracycline and Macrolide Resistance and emm Characterization of Streptococcus pyogenes Isolates in Tunisia

Abstract

Streptococcus pyogenes or group A Streptococcus, a major human pathogen, remains susceptible to beta-lactams, but resistance to other antibiotics is becoming more common. The purpose of this study was to characterize both phenotypic and genotypic epidemiological markers of group A Streptococcus and to identify the mechanisms of resistance to macrolides and tetracyclines. A total of 103 strains, isolated at Charles Nicolle University Hospital of Tunis, were investigated. The rate of resistance to erythromycin was low (5%), whereas a high rate of tetracycline resistance was found (70%). All the macrolide-resistant isolates expressed the constitutive macrolide, lincosamide, and streptograminB phenotype and harbored the erm(B) gene. Resistance to tetracycline was mainly due to the tet(M) gene, which is commonly associated with the conjugative transposon Tn916. No significant association was found between erm(B) and tet(M) genes. The tetracycline-resistant strains belonged to 28 distinct emm types. Among them, emm118 was the most prevalent type, followed by emm42, std432, emm76, and emm18. However, emm1, emm4, emm6, emm28, and emm3 were the most frequent types among tetracycline susceptible isolates. Only emm118 and emm42 types (p ≤ 0.05) were significantly associated with resistance to tetracycline.

Introduction

Streptococcus pyogenes or group A Streptococcus (GAS) is one of the most common and important etiological agents of bacterial infections in humans, especially in children. It causes both mild infections, such as pharyngitis and impetigo, and severe diseases, such as toxic shock syndrome and necrotizing fasciitis. GAS infections can also lead to sequelae such as acute rheumatic fever, rheumatic heat disease, and glomerulonephritis.¹⁰

Penicillin has been the therapy of choice for S. pyogenes infections for decades and resistance of S. pyogenes to penicillin has never been observed.²⁸ Macrolide and lincosamide antibiotics, a proven, safe, efficient alternative, are commonly prescribed, particularly for patients allergic to β-lactams.¹⁹ However, emergence of macrolide resistance has been observed among S. pyogenes in many countries.^21,25 Macrolide resistance in S. pyogenes can be caused by an efflux system that is encoded by the mef(A) gene.⁵ This mechanism is responsible for cross resistance to 14- and 15-membered macrolides, but does not affect the activity of 16-membered macrolides and lincosamides (M phenotype).⁴³ A second macrolide resistance mechanism is the target site modification mediated by the presence of the erm genes (ermB or ermTR) encoding an rRNA methylase that modifies an adenine residue in 23 rRNA and reduces the binding of macrolide, lincosamide, and streptograminB (MLS_B) antibiotics to their target site in the 50S ribosomal subunit. The phenotypic expression of MLS_B resistance can be inducible or constitutive.²⁵

Although tetracycline is not used in the treatment of GAS diseases, the resistance of GAS to tetracycline has been reported in many countries.^5,35 Resistance to tetracycline is conferred by ribosome protection genes such as tet(M), tet(O), tet(S), and tet(T). Tetracycline resistance genes can reside on mobile genetic elements that carry macrolide resistance genes; so co-occurrence of resistance to both classes of drugs can be observed. To our knowledge, no study has been previously done in our country to characterize the mechanism of resistance to this species.

The aim of this study was to characterize phenotypic and genotypic markers of GAS isolates and to investigate the molecular mechanisms of tetracycline and macrolide resistance.

Materials and Methods

Bacterial strains

A total of 103 nonduplicated GAS isolates collected between January 2000 to December 2006 at the Laboratory of Microbiology of Charles Nicolle University Hospital of Tunis were investigated. Patients ranged in age from 2 to 93 years; 12 isolates were recovered from children (aged 0–15 years) and 91 isolates from adults aged 16 years and older. The sources of isolates and sites of infection were skin (n = 43), respiratory tract (n = 41), blood (n = 12), fluids (n = 4), endometrium (n = 1), vagina (n = 1), and urine (n = 1).

All GAS strains were grown on blood agar plates incubated in 5% CO₂ at 37°C overnight. They were identified by their colony morphology, beta-hemolysis, absence of catalase, group A agglutination (bioMérieux, Marcy l'Etoile, France), and presence of pyrrolidonyl arylamidase tested by the disk method.

Antimicrobial susceptibility testing

Antimicrobial susceptibility testing was done using disk diffusion method on Mueller-Hinton agar supplemented with 5% of defibrinated horse blood according to the guidelines of the “Comité de l'Antibiogramme de la Société Française de Microbiologie.” Eleven antimicrobial agents were tested: penicillin G (10 IU), amoxicillin (25 μg), vancomycin (30 μg), teicoplanin (30 μg), rifampin (30 μg), tetracycline (30 IU), gentamicin (500 μg), erythromycin (15 IU), clindamycin (2 IU), and pristinamycin (15 μg). Resistance to bacitracin was determined by an absence of inhibition zone around the disk of bacitracin (0.04 IU). Staphylococcus aureus ATCC 29213 and Enterococcus faecalis ATCC 29212 were used for quality control.

The phenotypes of macrolide-lincosamide-streptogramin B resistance [constitutive MLS_B (cMLS_B), inducible MLS_B (iMLS_B), and M phenotype] were determined by the double disk diffusion method using erythromycin and clindamycin disks as described by Seppälä et al.⁴²

Minimum inhibitory concentrations (MICs) of macrolides (erythromycin, clarithromycin, and azithromycin), lincosamides (clindamycin), streptogramins (quinupristin-dalfopristin), oxazolidinone (linezolid), and tetracycline were determined for resistant isolates using E-test (AB Biodisk, Solna, Sweden) according to the manufacturer's instructions.

T-typing

The serotype T was determined by slide agglutination of trypsin-digested suspension of washed bacterial cells in the presence of type-specific antisera (Sevapharma, Prague, Czech Republic).¹⁸

emm gene typing

The emm types were determined by sequencing the variable 5′ end of the emm gene after PCR amplification with the MF (5′-ATA AGG AGC ATA AAA ATG GCT-3′) and MR (5′-AGC TTA GTT TTC TTC TTT GCG-3′) primers.³⁶

The DNA sequences were compared with published sequences in CDC database using the BLAST 2 program available at http://www.cdc.gov/ncidod/biotech/strep/strepblast.htm.

A sequence was considered as belonging to a specific emm gene when, over the first 160 bases of the sequence, it had 95% or greater identity with that of the reference emm gene.

Detection of resistance genes

Macrolide-resistant strains were subjected to a multiplex PCR for detection of the resistance determinants erm(TR), erm(B), and mef(A) with the oligonucleotides previously described.⁴⁴

The tetracycline-resistant isolates were tested for presence of the tet(M), tet(O), tet(S), tet(T), tet(L), and tet(K) tetracycline resistance determinants and the int-Tn gene, encoding the integrase of Tn916, by PCR.³⁸ Control strains used in PCR experiments were (Enterococcus fecalis BM4110:Tn1545P6 for the detection of tet(M) gene,³⁷ Streptococcus anginosus MG23 for tet(O) gene,⁶ Listeria monocytogenes BM4210/pIP811 for tet(S) gene,³⁹ Streptococcus pyogenes A498 for tet(T) gene,⁶ Staphylococcus aureus RN4220/pT181 for tet(K) gene,²³ Listeria monocytogenes BM4212/pIP812 for tet(L) gene,⁴⁰ and Enterococcus fecalis BM4110::Tn1545 for int-Tn gene⁶).

Statistical analysis

Chi square or Fisher's exact test was used for statistical analysis. A p-value of ≤0.05 was considered significant.

Results

All isolates were fully susceptible to penicillin G, amoxicillin, pristinamycin, vancomycin, and teicoplanin.

About 0.9% and 2.9% of isolates were resistant to rifampin and bacitracin, respectively. No high level of resistance to gentamicin was found.

Only five strains (5%) were resistant to erythromycin and clindamycin. They expressed the cMLS_B phenotype and harbored the erm(B) gene. They showed high MICs for all macrolides and clindamycin (MICs >256 mg/L). All strains were susceptible to quinupristin-dalfopristin and linezolid (MIC₅₀/MIC₉₀ values of quinupristin-dalfopristin and linezolid were 0.25/0.25 and 0.38/0.75 mg/L, respectively). They exhibited different T and emm types (Table 1).

Table 1.

Distribution of emm and T Types According to Erythromycin and Tetracycline Susceptibility

	emm types
	emm118	emm42	emm1	st432	emm28	emm76	emm18	emm77	emm81	st3757	emm11	emm12	Others
Tetracycline resistant (n = 72)
tet(M) (n = 63)	10	9	0	5	2	6	4	0	1	2	1	1	22
tet(O) (n = 1)	0	0	0	0	0	0	0	1	0	0	0	0	0
tet(M)+tet(O) (n = 2)	0	0	0	0	1	0	0	1	0	0	0	0	0
tet(M)+Tet(L) (n = 2)	0	0	0	0	0	0	0	0	1	1	0	0	0
Tetracycline resistant gene unknown	0	0	0	2	0	0	0	0	0	0	2	0	0
T types	NT	T3/13/B3264 or NT		T8/14/25, T9/14/B3264 or NT	T3/T13/B3264 or NT	T12 or NT	T3/13/B3264 orNT	NT	T6/23 or NT	NT	T11	T12
Tetracycline susceptible (n = 31)	-	-	8	-	3	-	-	-	-	-	-	2	18
T types	-	-	T1	-	T3/T13/B3264, T28/T11 or NT	-	-	-	-	-	-	T11/T12
Erythromycin resistant (n = 5)	0	0	0	0	2	0	0	0	0	0	2	1	0
erm (B)	0	0	0	0	2	0	0	0	0	0	2	1	0
T types	-	-	-	-	T3/T13/B3264 or NT	-	-	-	-	-	T11	T12
Erythromycin susceptible (n = 98)	10	9	8	7	4	6	4	2	2	3	1	2	40
T types	NT	T3/13/B3264, or NT	T1	T8/14/25, B3264/T14/T9 or NT	T3/T13/B3264 or T28/T11	T12 or NT	T3/13/B3264 or NT	NT	T6/23 or NT	NT	T11	T12

NT, non–T typable.

Resistance to tetracycline was expressed by 70% of the isolates, with MIC range from 12 to >256 mg/L (MIC₅₀: 24 mg/L; MIC₉₀: 32 mg/L).

Ribosome protection mechanism, associated with the presence of tet(M) or tet(O) genes, was identified in 87.5% and 1.3% of tetracycline-resistant GAS isolates, respectively; two strains were detected to have tet(O) in association with tet(M). Efflux mechanism, associated with tet(L) gene, was detected only in two strains, which also harbored the tet(M) gene. The tet(K), tet(S), and tet(T) determinants were not detected in any strain. Four isolates did not have any of the tet genes tested. No significant association was found between erm(B) and tet(M) genes.

Consistently, the int-Tn gene, encoding the integrase of Tn916, was found in 94% of the strains harboring tet(M).

Of the 103 GAS isolates, 61 were typable (59%). Twenty different T patterns were observed. T3/13/B3264, T1, T4, and T11 were the most dominant and accounted for 59% of all T-typable isolates.

Thirty-eight distinct emm types were observed. The most predominant emm types were emm118 (9.7%), emm42 (8.7%), emm1 (7.8%), st432 (6.8%), emm28 (5.8%), and emm76 (5.8%).

Among the 12 isolates collected from children, the most frequent emm types were emm1 (25%) and emm106 (16.6%). Among adult patients, the most prevalent types were emm118 (10.9%), emm42 (9.8%), st432 (7.6%), emm76 (6.5%), emm1 (5.4%), and emm28 (5.4%). Of the 72 tetracycline-resistant strains, 41.6% were T typable and belonged to nine different T patterns, the majority being types T3/13/B3264 (43.3%), T11 (16.6%), and T4 (3%); 58% of the strains were non–T typable (NT). Among these tetracycline-resistant strains, 28 distinct emm types were observed; emm118 was the most prevalent type (13.8%), followed by emm42 (12.5%), std432 (9.7%), emm76 (8.3%), and emm18 (5.5%) (Table 1).

The group of 31 tetracycline-susceptible isolates was also heterogenous. The 25 T-typable strains belonged to nine different T patterns, of which T1 (32%), T3/13/B3264 (20%), and T6 (16%) were the most common; 19% of the strains were non–T typable (NT). Thirteen different emm types were represented within this group; emm1 (25.8%), emm4 (12.9%), emm6 (12.9%), emm28 (9.6%), and emm3 (9.6%) types were the most frequent (Table 1).

Only emm118 (p ≤ 0.03) and emm42 (p ≤ 0.05) types were significantly associated with resistance to tetracycline.

Discussion

S. pyogenes has remained fully susceptible to penicillin, despite the continued use of this agent as first-line antibiotic in the treatment of streptococcal infections.²⁸ The present data confirm the continuing susceptibility of S. pyogenes to penicillin and other β-lactam antibiotics.

Erythromycin and other macrolides have been widely used as alternative treatment for patients allergic to penicillin. In our hospital, macrolide resistance in S. pyogenes has remained low as in many parts of the world including Iran, Argentina, Turkey, and the United Kingdom, which is probably due to a reduced macrolide consumption.^8,12,16,26 Conversely, it is an emerging problem in several countries such as Italy (47%), Korea (51%), and Taiwan (70%).^8,15,24 It was reported that the rapid emergence of erythromycin resistance has been related to the clonal spread of resistant strains belonging to a limited number of T and M genotypes.^20,34

The association quinupristin-dalfopristin and linezolid were proved to be highly active against all the GAS isolates tested. The MICs obtained were comparable to those reported by others.²⁹

In the present study, all erythromycin-resistant isolates expressed a cMLS_B phenotype and harbored ermB. Similar finding was reported in France and Portugal, where the ermB gene was the main resistance mechanism,^1,3 whereas the M phenotype encoded by mefA gene predominates in many other European countries such as Finland, Spain, and Italy as well as in the United States and Canada.^{2,20,22,30,33} Differences among the countries could be due to different patterns in use of antimicrobials, as suggested by the correlation of mef(A) expansion with the use of macrolides in Spain.^13,31

Our results showed a high rate of tetracycline resistance (70%), as it was reported in Denmark, Brazil, Islamic Republic of Iran, and China.^11,16,17,26 Although tetracycline has not been recommended for the therapy of GAS infections, selective pressure from the intensive use of tetracycline to treat a variety of human and veterinary infections may have contributed to the emergence of this resistance among GAS isolates around the world. Another possible key driving force in the high level of tetracycline resistance could be the increase of its use in food animals, with transfer of genes from animals to humans. The fact that GAS strains share common resistance genes with various species of the oral flora suggest that the transfers could occur in the oral cavity.^11,31 More than 40% of nongroupable streptococci were resistant to tetracycline in our hospital.

According to current knowledge, the mechanisms of resistance to tetracycline in gram-positive bacteria are mainly associated with the presence of tet(M) gene and more rarely with tet(O), tet(S), and tet(T), which all encode ribosomal protection proteins.⁴⁰ Tetracycline resistance is often encoded by the efflux genes tet(K) and tet(L), which are much less common.⁹ In our study, 93% of tetracycline-resistant isolates harbor the tet(M) gene and all the tet(M)-positive strains but one harbor the int-Tn gene, encoding the integrase of Tn916. These results demonstrate that the tet(M) is carried by the Tn916-related transposons, which can translocate from chromosome to chromosome. These data might explain the large distribution of the tet(M) gene among the tetracycline-resistant GAS isolates.

The tet(O) gene was less frequently present (only three strains) and two isolates harbored both tet(M) and tet(O) genes. The tet(L) determinant was detected in two strains in association with tet(M). In four tetracycline-resistant isolates, none of the known tetracycline-resistant genes was found. It is possible that these isolates carried another tet determinant.

A linkage between the erm(B) and the tet(M) genes has been well established in many reports.^4,7 In this study, the absence of association between tet(M) and erm(B) genes could be related to the small number of macrolide-resistant strains.

emm118, emm42, emm1, st432, emm28, and emm76 were the most common emm types, which constituted 44.6% of all isolates. This distribution is different from emm type distributions reported in different countries such as America, Asia, and Europe.^17,27,32

However, it was of interest to examine the relationship of tetracycline resistance and emm types. In the present data, an association between emm118, emm42, and tetracycline resistance was found. Conversely, all emm1 isolates were susceptible to tetracycline. Ho et al. from the United States reported that emm1 isolates were significantly less likely to have resistance to tetracycline.¹⁴

In conclusion, these data showed a low rate of macrolide resistance and a high level of tetracycline resistance mainly due to the presence of the tet(M) gene, which was commonly associated with the presence of conjugative transposon (Tn916). These results emphasize the need to monitor the epidemiology and genetic basis of antibiotic resistance in GAS. Indeed, increase of resistance rates has been shown to be able to rapidly change in relation with the emergence of various mechanisms.⁴¹

Footnotes

Acknowledgments

This study was done with the financial support of the Ministry of Scientific Research and Technology of Tunisia. The authors thank Gislène Collobert for excellent technical assistance.

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