|Year : 2014 | Volume
| Issue : 3 | Page : 290-293
Erythromycin-resistant genes in group A β-haemolytic Streptococci in Chengdu, Southwestern China
W Zhou1, YM Jiang1, HJ Wang1, LH Kuang1, ZQ Hu1, H Shi1, M Shu2, CM Wa2
1 Department of Clinical Labortory, West China Second Hospital, Sichuan University, Chengdu, Sichuan, The People's Republic of China
2 Department of Pediatrics, West China Second Hospital, Sichuan University, Chengdu, Sichuan, The People's Republic of China
|Date of Submission||26-May-2013|
|Date of Acceptance||29-Dec-2013|
|Date of Web Publication||10-Jul-2014|
C M Wa
Department of Pediatrics, West China Second Hospital, Sichuan University, Chengdu, Sichuan, The People's Republic of China
Source of Support: None, Conflict of Interest: None
Context: The management of Group A β-haemolytic Streptococci (Streptococcus pyogenes or GAS) infection include the use of penicillins, cephalosporins or macrolides for treatment. A general increase in macrolides resistance in GAS has been observed in recent years. Differences in rates of resistance to these agents have existed according to geographical location and investigators. Aims: To investigate the antibiotic pattern and erythromycin-resistant genes of GAS isolates associated with acute tonsillitis and scarlet fever in Chengdu, southwestern China. Settings and Design: To assess the macrolide resistance, phenotype, and genotypic characterization of GAS isolated from throat swabs of children suffering from different acute tonsillitis or scarlet fever between 2004 and 2011 in the city of Chengdu, located in the southwestern region of China. Materials and Methods: Minimal inhibitory concentration with seven antibiotics was performed on 127 GAS isolates. Resistance phenotypes of erythromycin-resistant GAS isolates were determined by the double-disk test. Their macrolide-resistant genes (mefA, ermB and ermTR) were amplified by PCR. Results: A total of 98.4% (125/127) of the isolates exhibited resistance to erythromycin, clindamycin and tetracycline. All isolates were sensitive to penicillin G and cefotaxime. Moreover, 113 ermB-positive isolates demonstrating the cMLS phenotype of erythromycin resistance were predominant (90.4%) and these isolates showed high-level resistance to both erythromycin and clindamycin (MIC 90 > 256 μg/ml); 12 (9.6%) isolates demonstrating the MLS phenotype of erythromycin resistance carried the mefA gene, which showed low-level resistance to both erythromycin (MIC 90 = 8 μg/ml) and clindamycin (MIC 90 = 0.5 μg/ml); and none of the isolates exhibited the M phenotype. Conclusions: The main phenotype is cMLS, and the ermB gene code is the main resistance mechanism against macrolides in GAS. Penicillin is the most beneficial for treating GAS infection, and is still used as first-line treatment. And macrolide antibiotics are not recommended for treatment of GAS infection in children because of the high rates of antimicrobial resistance in mainland China.
Keywords: Child/children, genotype, group A β-haemolytic streptococcus, phenotype, resistence, streptococcus pyogenes
|How to cite this article:|
Zhou W, Jiang Y M, Wang H J, Kuang L H, Hu Z Q, Shi H, Shu M, Wa C M. Erythromycin-resistant genes in group A β-haemolytic Streptococci in Chengdu, Southwestern China. Indian J Med Microbiol 2014;32:290-3
|How to cite this URL:|
Zhou W, Jiang Y M, Wang H J, Kuang L H, Hu Z Q, Shi H, Shu M, Wa C M. Erythromycin-resistant genes in group A β-haemolytic Streptococci in Chengdu, Southwestern China. Indian J Med Microbiol [serial online] 2014 [cited 2019 Nov 22];32:290-3. Available from: http://www.ijmm.org/text.asp?2014/32/3/290/136568
| ~ Introduction|| |
The Group A β-haemolytic streptococcus (GAS) is a form of β-haemolytic Streptococcus bacteria responsible for many common infections including acute tonsillitis and scarlet fever. The management of GAS infection include the use of penicillins, cephalosporins or macrolides for treatment. A general increase in macrolides resistance in GAS has been observed in recent years. Although differences in rates of resistance to these agents have existed according to geographical location and investigators. ,,, The purpose of the present study was to investigate the erythromycin resistance and its molecular mechanisms of GAS isolates associated with acute tonsillitis and scarlet fever in Chengdu, southwestern China.
| ~ Materials and Methods|| |
Among clinical isolates of GAS collected between 2004 and 2011 at West China Second University Hospital, Chengdu City, southwestern China, a total of 127 isolates were obtained from community-acquired acute pharyngitis (n = 7) and scarlet fever (n = 120) in this study. All of the isolates were recovered from throat swabs of different patients. GAS were identified on the basis of β-haemolysis, group A latex agglutination with specific antibodies (SLIDEX ® Strepo Plus) and available regents from French bioMerieux company. The study population consisted of children 2-14-year-old, with signs or symptoms of acute pharyngitis (fever, sore throat and exudate, tender cervical lymph nodes, or headache) and scarlet fever (sore throat, fever and a characteristic red rash). These patients were treated with penicillin, amoxicillin or azithromycin. All 127 cases recovered from bacterial pharyngitis and scarlet fever.
Determination of erythromycin-resistant phenotypes
Resistance phenotypes of erythromycin-resistant GAS isolates were determined by the double-disk test with erythromycin and clindamycin (French bioMerieux Company) disks as described previously.  Blunting of the clindamycin zone of inhibition proximal to the erythromycin disk indicated an inducible type of MLSB resistance (IR), and resistance to both disks indicated a constitutive type of MLSB resistance (CR). Susceptibility to clindamycin with no blunting indicated the new erythromycin-resistant phenotype (M phenotype).
Determination of MICs
MICs of different antimicrobial agents were determined by the agar dilution method, following CLSI recommendations and criteria. , S. pyogenes ATCC 10389, Enterococcus faecalis ATCC 29212, Escherichia More Details coli ATCC 25922 and Staphylococcus aureus ATCC 29213 were used as controls in MIC determinations.
Detection of erythromycin-resistant genes
For the identification of erythromycin-resistant genes in erythromycin-resistant isolates, PCR was performed with the oligonucleotide primer pairs speciﬁc to mefA, ermB, and ermTR. Total DNA was extracted as described in UltraClean ® Microbial DNA Isolation kit specification (MOBIO, USA). The PCR conditions for primer sets, PCR mixture, PCR conditions, and restriction endonuclease analysis of the PCR product were as described previously. , The expected sizes of PCR products were 640 bp for ermB, 348 bp for mefA, and 530 bp for ermTR. Each two mefA, ermB-positive strains'DNA were selected sent to the Invitrogen China Limited to sequencing analysis, confirmed the amplification products for the purpose.
| ~ Results|| |
Antimicrobial susceptibility patterns of 127 GAS isolates
98.4% of GAS isolates were resistant to erythromycin, clindamycin and tetracycline. The overall resistance rates of the isolates were found to be 15% for trimethoprim-sulfamethoxazole; 7.9% for chloramphenicol; whereas all isolates were susceptible to penicillin G and cefotaxime [Table 1].
Distribution of MLS B -resistant phenotypes and MICs of erythromycin and clindamycin for 125 erythromycin-resistant GAS isolates
Of 125 erythromycin-resistant GAS isolates, 90.4% exhibited the cMLS phenotype; and 9.6% had the MLS phenotype; whereas the M phenotype was not found in our study [Table 2]. MICs of different MLS phenotypes of GAS isolates was shown in [Table 3].
|Table 2: Distribution of MLSB-resistant phenotypes among 125 erythromycin - resistant GAS isolates |
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Genotypes among 125 isolates of erythromycin-resistant GAS
Three erm types were obtained for the 125 erythromycin-resistant isolates [Table 4]. Among 113 isolates of the cMLSB-resistant phenotype, 109 strains carried the ermB gene, 4 strains carried the ermB and mefA gene. All 12 MLSB-resistant isolates carried the mefA gene and none of isolates carried ermTR gene [Table 4].
|Table 4: Distribution of MLSB-resistant phenotypes and genotypes among 125 isolates of erythromycin-resistant GAS|
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| ~ Discussion|| |
GAS is the most common cause of bacterial pharyngitis in children. In USA, the prevalence of GAS infection was from 15% to 30%.  Kim et al., reported that the isolation rate was up to 50.8% in the paediatric outpatients with acute pharyngitis in South Korean in 2002.  Antibiotic treatment has been demonstrated to be effective against GAS infection. As the first-line therapy, penicillin has been shown effective in treating GAS infection. Macrolide antibiotics are the preferred alternatives for patients with allergy to beta-lactam antibiotics of GAS infection.  However, recent studies have shown that changing strains of GAS resistant to these agents have emerged. The resistance to macrolide has increased substantially, especially. The prevalence of erythromycin-resistant GAS is varying among different countries and from year to year. Our results show that the isolates from paediatric patients in Chengdu city, Sichuan, China were resistant to erythromycin and clindamycin with 98.4%. The resistance rate was similar to that reported in two Children's Hospitals in Beijing and Shanghai in 2008 (97.9%), , but much higher than those in Sichuan province in the late 1990s (35.8%), French (22.4%)  Children's Hospital of Pittsburgh (38%)  and Italy (35.8%).  Meanwhile, our data shows that the isolates of GAS were resistant to tetracycline with 98.4%. It was similar to 92% in four Children's Hospitals in mainland China in 2008, but much higher than in Europe (1.8-5.2%), , in USA (4%),  and in India (27.4%). 
It is generally known that resistance rates will increase with increasing antibiotic use. In 1990, erythromycin-resistance of GAS isolates in Korea had increased to 44.7%, corresponding to a marked increase in macrolide use during the time. , Use of macrolides since then has declined, and a marked decrease in rates of erythromycin resistance has followed.  Since 1990, also the usage of antibiotics in mainland China increased markedly. Therefore, we speculate that the high macrolide-resistant rate of GAS isolates in mainland China might be associated with a marked increase in macrolide use in recent years.
The distribution of frequencies of MLSB-resistance phenotypes and genotypes of GAS varied widely between countries and geographical regions. A global, longitudinal study showed that 46.1% of 143 GAS detected were mefA, 30.8% were ermB, 23.1% were ermA subclass ermTR. In Europe, in Germany, Greece, Spain, Finland and Italy, the mefA-positive isolates demonstrating the M phenotype of erythromycin resistance were predominant; ,,,, in France, the eermB and ermTR-positive isolates demonstrating the MLS phenotype of erythromycin resistance were predominant;  in Taiwan, Korea and Hongkong, the ermB-positive isolates demonstrating the MLS phenotype of erythromycin resistance were predominant. ,, However, in our study, 113 ermB-positive isolates demonstrating the cMLS phenotype of erythromycin resistance were predominant (90.4%) and these isolates showed high-level resistance to both erythromycin and clindamycin (MIC 90 > 256 μg/ml); 12 (9.6%) isolates demonstrating the MLS phenotype of erythromycin resistance carried the mefA gene, which showed low-level resistance to both erythromycin (MIC 90 = 8 μg/ml) and clindamycin (MIC 90 = 0.5 μg/ml); and none of the isolates exhibited the M phenotype.
| ~ Conclusion|| |
High macrolide resistance in GAS strains was observed in paediatric patients in the city of Chengdu, located in the southwestern region of China. The cMLS-resistant phenotype was dominant among erythromycin-resistant GAS. Penicillin is the most beneficial for treating GAS infection, and is still used as first-line treatment. And macrolide antibiotics are not recommended for treatment of GAS infection in children because of the high rates of antimicrobial resistance to these agents among GAS strains from paediatric patients in mainland China.
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[Table 1], [Table 2], [Table 3], [Table 4]