Abstract
The aims of this study were to determine the susceptibilities to macrolide and tetracycline antibiotics and emm type distribution of Streptococcus pyogenes strains isolated in the Kocaeli University Hospital, Turkey. A total of 127 S. pyogenes clinical isolates were tested. Eleven (9%) isolates were resistant to erythromycin, and 23 (18%) isolates were resistant to tetracycline. Ten of the erythromycin-resistant isolates were also resistant to tetracycline. By the triple-disk test, all erythromycin-resistant isolates showed the inducible macrolide-lincosamide-streptogramin-C phenotype and harbored erm(TR) gene. tet(O) was the most common tetracycline resistance gene. Among erythromycin-tetracycline coresistant isolates, seven harbored the tet(O) gene. emm 4, emm 1, emm 2,114, and emm 89 were the most common emm types. These isolates were more susceptible to erythromycin. There was considerable emm type heterogeneity in macrolide or tetracycline resistant isolates. According to our knowledge, this is the first study in which emm type distribution is investigated in Turkey. More comprehensive studies are needed to obtain true information about the epidemiology of macrolide and tetracycline resistance and emm type distribution in Turkey.
Introduction
Streptococcus pyogenes (group A β-haemolytic streptococcus) is a common human pathogen that causes a wide spectrum of diseases, and penicillins remain the drugs of choice for their treatment. Macrolides are also used for patients who are allergic or nontolerant to penicillins. The increasing rates of macrolide resistance in S. pyogenes have been reported from different parts of the world.26
There are two main phenotypes of macrolide resistance in S. pyogenes: the M phenotype (efflux-mediated), mediated by the mef genes, which confers low levels of resistance to 14- and 15-membered macrolides but not to 16-membered macrolides, lincosamides, or streptogramin B; and the macrolide-lincosamide-streptogramin B (MLS) phenotype, mediated by the erm genes, which confers resistance to macrolides, lincosamides, and streptogramin B antibiotics. This latter phenotype can be constitutive (cMLS), which is generally mediated by the erm(B) gene, or inducible (iMLS), which is generally mediated by the erm(A) subclass TR gene.24,36,38 iMLS strains show susceptibility to lincosamides but turn to high-level resistance after induction.18
There are several reports about the association between erythromycin, and tetracycline resistance has been considered to be an important cofactor in the selection of erythromycin resistance.5,28
The M protein is a major virulence factor of S. pyogenes. The emm gene encodes the M protein. emm gene sequencing is presently used for routine typing of S. pyogenes.6
The aims of this study were to determine the susceptibilities to macrolide and tetracycline antibiotics of S. pyogenes isolates and in the case of resistant isolates, to ascertain their patterns and genetic mechanisms of resistance. We also determined the prevalence of emm types among S. pyogenes isolates. According to our knowledge, this is the first study about emm typing of S. pyogenes in Turkey.
Methods
Bacterial isolates
A total of 127 clinical isolates of S. pyogenes isolated between October 2005 and July 2008 in the Clinical Microbiology Laboratory of Kocaeli University Hospital were tested. A total of 103 (81%) isolates were cultured from pharyngeal specimens, and 24 (19%) were cultured from other sources (18 from wound, 3 from sputum, 2 from urine, and 1 from pericardial fluid). Duplicates were excluded.
The isolates were identified as S. pyogenes by colony morphology, β-hemolysis on sheep blood agar, bacitracin disc test (0.04 U), and pyrrolidonyl arylamidase test. The strains were stored at −70°C and then subcultured twice on blood agar before susceptibility testing.
Antimicrobial susceptibility testing and macrolide resistance phenotype
Penicillin, vancomycin, levofloxacin, chloramphenicol, tetracycline, erythromycin, clarithromycin, azithromycin, telithromycin, and clindamycin susceptibilities were investigated by disk diffusion method. Minimum inhibitory concentrations (MICs) were determined using Etest (AB Biodisk) for isolates demonstrating intermediate susceptibility or resistace to erythromycin and/or tetracycline. Interpretation of the results was basically as outlined in the Clinical and Laboratory Standards Institute (CLSI) guidelines.11 Since CLSI telithromycin breakpoints for group A β-haemolytic streptococcus are not available, the results were interpreted according to the breakpoints proposed by the French Society for Microbiology.13 Streptococcus pneumoniae ATCC 49616 was used as the quality control strain.
The erythromycin resistance phenotypes were classified by triple-disk test with erythromycin (15 μg), clindamycin (2 μg), and spiramycin (100 μg) disks, as previously described.18
Detection of erythromycin and tetracycline resistance genes
Erythromycin-resistant and intermediate-susceptible isolates were screened for the erm(B), erm(TR), and mef(A) genes, and tetracycline-resistant and intermediate-susceptible isolates were screened for the tet(K), tet(L), tet(M), and tet(O) genes by real-time polymerase chain reaction (PCR) (iCycler IQ; v3.0a-BioRad Laboratories). Total genomic DNA was isolated from streptococcal strains with QIAamp® DNA Mini Kit (Qiagen). DNA in the reaction mixture (25 μl) was denatured at 93°C for 3 min, and the amplification cycles consisted of elongation at 72°C for 60 sec, denaturation at 93°C for 60 sec, and annealing at 52°C for 60 sec. After 35 amplification cycles, the last elongation step was performed at 72°C for 5 min.36 Primer sets specific for the several genes were used as seen in Table 1.3,16 Two reference Escherichia coli strains carrying erm(A) and erm(B) genes and one S. pyogenes strain positive for mef(A) (kindly provided by Dr. Ziya Cibali Acikgoz) were used as positive PCR controls.
Table 1.
List of Primers Used in Real-Time Polymerase Chain Reaction
| Gene | Primer target | Sequence (5′-3′) | Product size (bp) |
|---|---|---|---|
| erm(A) | TR-up | ATAGAAATTGGGTCAGGAAAAGG | 530 |
| TR-rev | TTGATTTTTAGTAAAAAG | ||
| erm(B) | ERMB-up | GAAAAGGTACTCAACCAAATA | 639 |
| ERMB-rev | AGTAACGGTACTTAAATTGTTTAC | ||
| mef(A) | mefA-up | AGTATCATTAATCACTAGTGC | 348 |
| mefA-rev | TTCTTCTGGTACTAAAAGTGG | ||
| tet(M) | tetM-up | ACAGAAAGCTTATTATATAAC | 171 |
| tetM-rev | TGGCGTGTCTATGATGTTCAC | ||
| tet(O) | tetO-up | ACGGARAGTTTATTGTATACC | 171 |
| tetO-rev | TGGCGTATCTATAATGTTGAC | ||
| tet(K) | tetK-up | TATTTTGGCTTTGTATTCTTTCAT | 1,159 |
| tetK-rev | GCTATACCTGTTCCCTCTGATAA | ||
| tet(L) | tetL-up | ATAAATTGTTTCGGGTCGGTAAT | 1,077 |
| tetL-rev | AACCAGCCAACTAATGACAATGAT |
emm typing
A total of 91 S. pyogenes isolates were emm typed. S. pyogenes emm sequence database (National Centers for Disease Control Biotechnology Core Facility Computing Laboratory, Blast 2.0) was used for Streptococci Group A Subtyping. The emm type-specific sequence databases have been downloaded from ftp://ftp.cdc.gov/pub/infectious_diseases/biotech/tsemm/(for DNA). All PCR products were purified using the High Pure PCR Product Purification Kit® (Roche Diagnostics GmbH) and directly sequenced with the ABI PRISM 310 Genetic Analyzer® equipment using the DYEnamic ET Terminator Cycle Sequencing Kit® (Amersham Pharmacia Biotech, Inc.). The electropherogram-obtained sequences were assembled using Vector NTI® v5.1 (InforMax™ Invitrogen™ life science software).
Statistical analysis was performed by chi-square test using Epi Info (TM) 3.5.1 program.
Results
Among 127 isolates tested, 11 (9%) were resistant to erythromycin, 7 (6%) were resistant to clarithromycin, and 23 (18%) were resistant to tetracycline. All of the erythromycin-resistant isolates were also resistant to azithromycin. Among 11 erythromycin-resistant isolates, 10 were also resistant to tetracycline, and 7 were also resistant to clarithromycin. All of the seven clarithromycin-resistant isolates were also resistant to erythromycin, azithromycin, and tetracycline. All 127 isolates were susceptible to other tested antibiotics including telithromycin.
By the triple-disk test, all of the erythromycin-resistant isolates were assigned to the iMLS-C phenotype. None of the isolates had M, cMLS, iMLS-A, or iMLS-B resistance phenotypes.
MIC ranges were between 0.75 and 1 μg/ml in erythromycin-resistant (and intermediate) isolates and 4–32 μg/ml in tetracycline-resistant (and intermediate) isolates.
Genotype analysis of erythromycin resistance revealed that 8 of the 11 erythromycin-resistant isolates carried the erm(TR) gene. In the other three isolates, macrolide resistance genes were not detected. None of the erythromycin-resistant isolates had the erm(B) and mef(A) genes. erm(TR) was strongly correlated to tet(O).
Seven of 23 tetracycline-resistant isolates had the tet(O) gene, 3 isolates had the tet(L) gene, 2 isolates had the tet(K) gene, 1 isolate had the tet(M) gene, 2 isolates had both tet(K) and tet(L) genes, and 1 isolate had both tet(K) and tet(O) genes. Seven isolates did not have any of these tet genes. Among erythromycin-tetracycline coresistant isolates, seven carried the tet(O) gene, one carried both tet(K) and tet(O) genes, and two did not have any of these tet genes (Table 2).
Table 2.
Distribution of Resistance Genes Among 127 Streptococcus pyogenes Isolates
| |
|
No. of isolates |
|
|
|||||
|---|---|---|---|---|---|---|---|---|---|
| Macrolide susceptibility | Susceptible to tetracycline | tet(K) | tet(L) | tet(M) | tet(O) | tet(K)+tet(L) | tet(K)+tet(O) | Tetracycline resistant, gene unknown | Total |
| Susceptible to erythromycin | 103 | 2 | 2 | 1 | 0 | 2 | 0 | 6 | 116 |
| erm(TR) | 1 | 0 | 1 | 0 | 6 | 0 | 0 | 0 | 8 |
| erm(B) | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| mef(A) | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| Macrolide resistant, gene unknown | 0 | 0 | 0 | 0 | 1 | 0 | 1 | 1 | 3 |
| Total | 104 | 2 | 3 | 1 | 7 | 2 | 1 | 7 | 127 |
A total of 37 different emm types were found. The most prevalent emm types were emm4 (n = 10, 11%), emm1 (n = 9, 10%), emm2,114 (n = 8, 9%), and emm89 (n = 7, 6%). There was no predominant emm type among erythromycin-resistant and tetracycline-resistant isolates. However, emm1, emm4, emm89, and emm2,114 were detected more in erythromycin-susceptible isolates (n = 8, n = 10, n = 6, n = 6, respectively) than in erythromycin-resistant isolates (n = 1, n = 0, n = 0, n = 0, respectively) (p < 0.05) (Table 3). Further, emm2,114 was observed more in pharyngeal isolates (n = 7) than in the other isolates (n = 0) (p < 0.05) (data not shown). There were no differences in distribution of emm types among pharyngeal and nonpharyngeal isolates with the exception of emm2,114, which was found to be more frequent among pharyngeal isolates (n = 7 vs. n = 0) (p < 0.05) (data not shown).
Table 3.
emm Type Distribution of 91 Streptococcus pyogenes Isolates
| emm type | Fully sensitive | Erythromycin-resistant | Tetracycline-resistant | Erythromycin and tetracycline-resistant | Total |
|---|---|---|---|---|---|
| 1 | 8 | — | — | 1 | 9 |
| 4 | 10 | — | — | — | 10 |
| 5 | 3 | — | 1 | — | 4 |
| 6 | 3 | — | — | — | 3 |
| 8 | 1 | — | — | — | 1 |
| 9 | 1 | — | — | — | 1 |
| 11 | — | — | 1 | — | 1 |
| 12 | 4 | — | — | — | 4 |
| 14 | 1 | — | — | — | 1 |
| 17 | — | — | 1 | 1 | 2 |
| 18 | 1 | — | — | — | 1 |
| 19 | 2 | — | — | 1 | 3 |
| 22 | — | — | — | 1 | 1 |
| 29 | 2 | — | — | — | 2 |
| 49 | 1 | — | — | — | 1 |
| 52 | — | — | 2 | — | 2 |
| 73 | — | — | — | 1 | 1 |
| 79 | 1 | — | — | 1 | 2 |
| 81 | 2 | — | — | — | 2 |
| 82 | 1 | — | 1 | — | 2 |
| 87 | 3 | — | — | — | 3 |
| 89 | 6 | — | — | — | 6 |
| 98 | — | — | 1 | — | 1 |
| 99 | — | — | 1 | — | 1 |
| 100 | 1 | — | — | — | 1 |
| 102 | 1 | — | — | 2 | 3 |
| 118 | 1 | — | — | — | 1 |
| 2, 114 | 6 | — | 1 | — | 7 |
| 17, 41 | — | — | 1 | — | 1 |
| 22, 78 | 1 | — | — | — | 1 |
| 33, 41 | — | — | 1 | — | 1 |
| 41, 75 | 3 | — | — | — | 3 |
| 44, 65 | 2 | — | — | — | 2 |
| 65, 69 | 1 | — | — | — | 1 |
| 77, 102 | — | 1 | — | 2 | 3 |
| 79, 87 | 1 | — | — | — | 1 |
| sts 104 | 2 | — | — | — | 2 |
| Total | 69 | 1 | 11 | 10 | 91 |
Discussion
Macrolide resistance and the distribution of the macrolide resistance genes of S. pyogenes vary widely in different regions of the world. In this study, erythromycin resistance rate (9%) was lower compared with that in many countries (21.3%-45%).8,29,33,39 Low erythromycin resistance rates (0.6%–6.8%) were reported in some countries.14,15,21,23,25,28,30,37 Our erythromycin resistance rate was similar to rates in the United States (9.6%),19 Germany (12.7%),4 and Greece (11.9%).34 The low erythromycin resistance rates (%3–7) were reported from different parts of Turkey.1,2,12 Previously, we found low-level erythromycin resistance in the European section of Turkey.2 Moreover, all resistant isolates had low-level resistance to erythromycin (MIC ranges: 0.75–1 μg/ml) in the present study.
In our study, iMLS phenotype was detected in all erythromycin-resistant isolates. iMLS strains are susceptible to lincosamides but turn to high-level resistance after induction.18 In the previous studies performed in Turkey, the M phenotype was the most common phenotype followed by the iMLS phenotype.1,2 The M phenotype is the predominant phenotype in many countries,7,15,19,34,37 whereas iMLS is the most common phenotype in some countries4,14,25,30. Differences among the countries could be due to different antibiotic using patterns. In our study, 3 of 11 erythromycin-resistant isolates did not have erm(A), erm(B), and mef(A) genes. This may be due to the ribosomal mutations.28
Telithromycin resistance has particularly been reported in erm(B)-positive (cMLS) S. pyogenes isolates, although telithromycin retained activity against strains possessing the other macrolide resistance genotypes, such as erm(A) and mef(A).17,20,30 This explains the full susceptibility to telithromycin in all of the isolates we studied, in which erythromycin resistance was due to erm(TR).
In gram-positive bacteria, tetracycline resistance is mediated mainly by two mechanisms: ribosomal protection (tet M and tet O gene) and efflux (tet K and tet L gene).3,10 In some studies, tetracycline resistance in S. pyogenes was found to be associated with the tetM gene and, less frequently, with the tetO gene.16,29,34 Since tetracycline resistance genes can reside in mobile genetic elements that carry macrolide resistance genes, the cooccurrence of resistance to both classes of drugs is possible.5 Particularly, a linkage between erm(B) and tet(M) is well known.9 In many studies, erm(B) or both erm(B) and mef(A) were found to be associated with the tet(M) gene.5,9,25,28 Further, tet(M) can be found in a silent form in tetracycline-susceptible isolates.9 In the present study, we investigated the resistance genes in only resistant isolates. In this study, tet(O) was the most common tetracycline resistance gene. However, the most of the tet(O) positive isolates were also erm(TR) positive. There are several studies that reported the erm(TR)-tet(O) linkage.25,28 Seven tetracycline-resistant isolates did not have tet(M), tet(O), tet(K), and tet(L) genes. These isolates warrant further investigation on the other resistance genes.
This is the first study where in which mm types were investigated in S. pyogenes isolates from Turkey. emm 4, emm 1, emm 2,114, and emm 89 were the most common emm types, which accounted for 37% of all isolates. In a review that investigated the global emm type distribution, emm1, emm12, emm28, emm3, and emm4 were reported to be the most common emm types. However, significant differences in emm type distribution by region and clinical disease state were also reported.35
In the present study, emm1, emm4, emm89, and emm2,114 typed isolates were more susceptible to erythromycin (p < 0.05). However, we did not detect any significant distribution in macrolide or tetracycline-resistant isolates. The heterogeneity of emm types among macrolide-resistant and tetracycline-resistant isolates in the present study is compatible with some reports37 and contrasts with the others14,19,26,27,30,39. Montes et al. reported an association between emm77 and tet(M). They also reported that the emm22 isolates were the most frequent clone among the inducible resistant isolates in Spain.27 Green et al. reported that emm75 and emm12 accounted for more than half of the macrolide-resistant isolates.19 Fluctuations in the serotype distribution over time were also reported.22,31,32
In conclusion, prevalence of macrolide and tetracycline resistance in S. pyogenes isolates is low in our region and mainly due to erm(TR) and tet(O) genes, respectively. Although the number of the isolates is low in this study, a high frequency of macrolide-tetracycline coresistance is found. Therefore, tetracycline resistance can be considered as a cofactor in selection of macrolide-resistant strains. This is the first study in which emm type distribution is investigated in Turkey. Detected considerable emm type heterogeneity mainly in macrolide and tetracycline resistant isolates is probably due to the limited number of the studied isolates. More comprehensive and multicentre surveillance studies are needed to obtain true information about the epidemiology of macrolide and tetracycline resistance and emm type distribution in Turkey.
Acknowledgment
This study was supported by Research Foundation of Kocaeli University.
Disclosure Statement
No competing financial interests exist.
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