Molecular Characteristics of Erythromycin-Resistant Streptococcus pyogenes Strains Isolated from Children Patients in Tunis,Tunisia

Abstract

Aim:

The aims of our study were to characterize phenotypically and genotypically erythromycin-resistant Streptococcus pyogenes or group A streptococci (ERGAS) isolates, to evaluate macrolide resistance and to analyze the association between emm types and virulence factors. Included in this study were all ERGAS strains isolated from 2000 to 2013 at the Children's hospital of Tunis. Antimicrobial susceptibility was performed according to the CA-SFM guidelines. Macrolide resistance genes were revealed by polymerase chain reaction (PCR) method. Virulence factor genes (pyrogenic exotoxin genes and superantigen gene) were detected by PCR, and the emm types were defined by the sequencing of the variable 5′ end of the emm gene.

Results:

Among the 289 GAS isolates collected, 15 (5.2%) were resistant to erythromycin; 7 of the strains were assigned to the cMLS_B phenotype (46.6%); 5 harbored ermB gene alone (33.3%); and 2 strains coharbored ermB and mefA (13.3%). The remaining (53.4%) were assigned to the M phenotype and harbored the mefA gene. The frequency of detection of each toxin gene among ERGAS was 13.4% for speA (2 strains), 53.4% for speC (8 strains), and 13.4% for ssa (2 strains). Emm types 1, 58, 11, and 78 were the most frequent among ERGAS strains. The distribution of the cMLS_B and M phenotypes changed over the period of investigation with a decrement of cMLS_B phenotype and ermB gene that predominated between 2000 and 2006 and an increase of M phenotype and mefA gene between 2007 and 2013, but this difference was nonstatistically significant because of the low number of resistant strains. Emm types 1, 58, and 4 were only present among strains assigned to the M phenotype. However strains assigned to the cMLS_B phenotype were associated to emm11, emm22, emm28, emm78, or emm76. There was diversity in emm distribution in ERGAS between the two study periods. There was diversity in emm distribution among ERGAS particularly in 2000–2006. Indeed, from 2000 to 2006, the 6 ERGAS belonged to 5 different emm types (22, 28, 76, 11, and 4), while between 2007 and 2013, seven among the nine ERGAS belonged to only 2 emm types 58 and 1. The speA gene was present only among emm1 isolates, and the ssa gene was associated with emm4 and emm78 types. All emm78, emm28, and emm11 strains harbored speC gene.

Conclusions:

Our study revealed a low frequency of ERGAS and few emm types were associated with these strains.

Introduction

S treptococcus pyogenes or group A streptococci (GAS) are the most common bacterial cause of pharyngitis. It is a major cause of morbidity and mortality worldwide.¹

Until today, S. pyogenes remains universally sensitive to penicillin, despite the fact that penicillin has been the drug of choice for streptococcal infections for more than 50 years.² However, resistance to other antimicrobial agents was reported worldwide.³ Macrolides and lincosamides are the primary treatment for GAS infections in patients with beta-lactam hypersensitivity. The prevalence of erythromycin-resistant S. pyogenes strains has been reported from an increasing number of countries in recent years.^4–7 Macrolides–lincosamides–streptogramines B (MLS_B) inducible (iMLS_B) and constitutive (cMLS_B) resistant phenotypes are encoded by erm genes (ermA or ermB). Erythromycin-resistant but clindamycin-susceptible strains have the M phenotype encoded by mefA gene, which codes for a macrolide efflux mechanism.⁸ The prevalence of macrolide resistance varied in many countries and it may be influenced by two factors.⁴ The first one is macrolide consumption, and the second is the presence of certain emm types that are strongly associated with erythromycin resistance genotypes and phenotypes and who differ around the world.^4,9,10 In contrast, a variety of studies reveal that the distribution of emm genotypes is related to pyrogenic exotoxin genes and streptococcal superantigen.^11–13

In this study, we evaluated macrolide resistance of S. pyogenes strains isolated in Tunisian children patients by analyzing antimicrobial susceptibility pattern, genetic determinants of resistance to macrolide, and epidemiological markers of erythromycin-resistant strains.

Materials and Methods

Bacterial strains

The study was conducted at the Children's Hospital of Tunis, which is the only pediatric university teaching hospital in Tunisia. This is a public facility, providing tertiary care for Tunis and surrounding areas. The hospital has 347 beds, admitting patients of all ages (1 day–16 years). In 2013, the number of hospitalized patients was 26,463. We included in our study all erythromycin-resistant GAS (ERGAS) strains collected at the Children's Hospital of Tunis between 2000 and 2013. These strains were provided from two periods: 2000 to 2006 and 2007 to 2013 (Table 1).

Table 1.

Distribution of Erythromycin-Resistant Streptococcus pyogenes According to Specimen Origin (2000–2013)

		2000–2006 no resistant isolates/total (%)	2007–2013 no resistant isolates/total (%)	Total (%) 2000–2013
	Specimen origin	6/148 (4)	9/141 (6.4)	15 (5.2)
No invasive isolates/total No = 60	Blood	0/2 (0)	2/9 (22.2)	2/11 (18.2)
No invasive isolates/total No = 60	Osteoarticular	0/15 (0)	2/27 (7.4)	2/42 (4.8)
	Pleural	0/6 (0)	0/1 (0)	0/7 (0)
No noninvasive isolates/total No = 229	Pus^a	4/59 (6.8%)	3/95 (3.2)	7/154 (4.5)
No noninvasive isolates/total No = 229	Throat	1/63 (1.6%)	2/6 (33.3)	3/69 (4.4)
	Nose	1/3 (33.3%)	0/3 (0)	1/6 (16.6)

Pus: abscess, surgical wound infection, and skin burn infection.

Identification

Identification of GAS strains was performed by conventional methods.⁷ The strains were stored in brain hearth with 10% of glycerol at −80°C for further use.

Antimicrobial susceptibility testing

Antimicrobial susceptibility testing of GAS isolates was performed by disk diffusion assays. GAS strains were tested on Mueller–Hinton agar (Bio-Rad, France) supplemented with 5% of defibrinated horse blood according to the guidelines of the Antibiogram Committee of the French Society for Microbiology (www.sfm.asso.fr).¹⁴ Antibiotics tested were penicillin G (6 μg), amoxicillin (25 μg), erythromycin (15 μg), clindamycin (15 μg), pristinamycin (15 μg), streptomycin (500 μg), kanamycin (1000 μg), gentamicin (500 μg), and tetracycline (30UI) (Bio-Rad). Macrolide–lincosamide–streptogramine phenotypes were determined by the double disk test with erythromycin and clindamycin disks as described previously.¹⁵ Susceptibility to bacitracin was carried out by disk diffusion using 0.04 UI bacitracin-containing disk (Bio-Rad). Isolates with noninhibition zone around the disk was interpreted as resistant to bacitracin.¹⁶ The minimum inhibitory concentrations (MICs) of erythromycin and clindamycin were determined using E-test method (BioMérieux) on Mueller–Hinton agar supplemented with 5% of defibrinated horse blood agar according to the manufacturer's instructions. Control strains used for antimicrobial susceptibility testing were Pseudomonas aeruginosa ATCC 27553, Escherichia coli ATCC 25922, and Staphylococcus aureus ATCC 25923.

Detection of erythromycin resistance genes

All strains were subjected to a multiplex polymerase chain reaction (PCR) for detection of erm(A), erm(B), and mef(A) as previously described.¹⁷ Control strains used were S. pneumoniae (P6 and P8) for the detection of erm(B) and mef(A) genes, respectively, and S. aureus (S7) for the detection of erm(A) gene.¹⁸

Detection of speA, speB, speC, and ssa

A multiplex PCR was performed to detect the presence of speA, speB, speC, and ssa genes using specific primers as previously described.^7,19 The control strain used was a clinical strain producing speA, speB, speC, and ssa genes provided from a previous published collection.⁷

emm sequence typing

The emm type was determined by sequencing the variable 5′ end of the emm gene after amplification by PCR with the MF and MR primers.^7,20 Sequences were analyzed according to the Centers for Disease Control and Prevention (CDC) database: (www.cdc.gov/ncidod/biotech/strep/doc.htm).²¹

Statistical analysis

Statistical analysis was performed by chi-square test and Fisher's exact test. A p-value ≤0.05 was considered to be significant.

Results

Of the 289 unduplicated GAS strains collected during the study, 15 were ERGAS (5.2%). These strains were obtained from pus samples (7/15, 46.7%), blood-culture (2/15, 13.4%), osteoarticular punctures (2/15, 13.4%), throat samples (3/15, 20%), and nose samples (1/15, 6.7%). They were susceptible to penicillin G and amoxicillin. Two ERGAS strains (13.4%) were also resistant to tetracycline. A high level of resistance to kanamycin and streptomycin was observed in two strains, respectively (13.4%). One of these strains was also resistant to bacitracin (6.7%). Seven ERGAS isolates were resistant to clindamycin (46.6%) and assigned to the cMLS_B; five of them harbored ermB gene alone (33.3%) and two strains coharbored ermB and mefA (13.3%). The remaining eight strains were resistant only to erythromycin. They assigned to the macrolide phenotype (M) and harbored mefA gene (53.4%) (Table 2). Strains with cMLS_B phenotype had higher MICs for erythromycin and clindamycin (≥ 256 mg/L, respectively) than those with M phenotype (0.19 and 12 mg/L, respectively). The prevalence of ERGAS varied slightly between 2000–2006 and 2007–2013 (4% vs. 6.4%). The distribution of MLS resistance phenotypes also showed variations. We noted a decrease of cMLS_B phenotype and ermB gene that predominated in 2000–2006 and an increase of M phenotype and mefA gene in 2007–2013, but differences were nonsignificant. Thus cMLS_B phenotype decreased from 83.4% in 2000–2006 to 22.3% in 2007–2013 (p = 0.095), while M phenotype increased from16.6% in 2000–2006 to 77.7% in 2007–2013 (p = 0.467). The iMLS_B phenotype remained absent during the two periods (Table 2).

Table 2.

Correlation Between Phenotypes and Genotypes of Erythromycin-Resistant GAS Strains Collected Between 2000 and 2013

	Phenotype no/total ERGAS (%)		Genotype no/total ERGAS (%)
	cMLS_B	M	ermB	ermB+mefA	mefA
2000–2006	5/6 (83.4)	1/6 (16.6)	3/6 (50)	2/6 (33.4)	1/6 (16.6)
2007–2013	2/9 (22.3)	7/9 (77.7)	2/9 (22.3)	0	7/9 (77.7)
2000–2013	7/15 (46.6)	8/15 (53.4)	5/15 (33.3)	2/15 (13.3)	8/15 (53.4)

ERGAS, erythromycin-resistant group A streptococcus.

The speB gene was detected in all strains tested and was alone in six strains (40%). The frequency of detection of each toxin gene among all strains tested was 13.4% for speA (two strains), 53.4% for speC (eight strains), and 13.4% for ssa (two strains) (Table 3).

Table 3.

Characteristics of the 15 Erythromycin-Resistant GAS Strains in Tunisia (2000–2013)

				MLS genotype			Virulence genes
Period no isolates (% ERGAS)	Specimen	emm type	MLS phenotype	ermA	ermB	mefA	speA	speB	speC	ssa	Antimicrobial resistance profile (no strains)
2000 to 2006 6 (4)	Pus^a	11	cMLS_B	−	+	−	−	+	+	−	Ery, Cli
	Throat	11	cMLS_B	−	+	−	−	+	+	−	Ery, Cli
	Nose	22	cMLS_B	−	+	−	−	+	−	−	Ery, Cli
	Pus^a	28	cMLS_B	−	+	+	−	+	+	−	Ery, Cli, Tet, Str, Kan, Bac
	Pus^a	76	cMLS_B	−	+	+	−	+	−	−	Ery, Cli
	Pus^a	4	M	−	−	+	−	+	−	+	Ery
2007 to 2013 9 (6.4)	Osteoarticular	58	M	−	−	+	−	+	−	−	Ery
	Blood	58	M	−	−	+	−	+	−	−	Ery
	Blood	58	M	−	−	+	−	+	+	−	Ery
	Pus^a	78	cMLS_B	−	+	−	−	+	+	−	Ery, Cli
	Pus^a	78	cMLS_B	−	+	−	−	+	+	+	Ery, Cli, Tet, Str, Kan
	Pus^a	1	M	−	−	+	−	+	−	−	Ery
	Osteoarticular	1	M	−	−	+		+	−	−	Ery
	Throat	1	M	−	−	+	+	+	+	−	Ery
	Throat	1	M	−	−	+	+	+	+	−	Ery

pus: abscess, surgical wound infection, and skin burn infection.

Bac, bacitracin; Cli, clindamycin; Ery, erythromycin; Kan, kanamycin; MLS, macrolide–lincosamide–streptogramine; Str, streptomycin; Tet, tetracycline; +, presence of the gene; −, absence of the gene.

Eight different emm types were found. Emm1 accounted for four strains followed by emm58 (three strains). Each of emm types 11 and 78 is represented by two strains, while emm types 4, 28, 22, and 76 are each represented by a single strain. Emm types 1, 58, and 4 were only present among strains assigned to the M phenotype, while strains assigned to the cMLS_B phenotype were associated with emm types 11, 22, 28, 78, and 76, but there was a nonsignificant association between emm types and MLS phenotypes (p > 0.05). Characteristics of all ERGAS isolates regarding their emm types are listed in Table 3.

Only emm28 ERGAS isolate showed high levels of resistance to streptomycin and kanamycin and was also resistant to tetracycline and bacitracin. One of the emm78 strains was resistant to tetracycline and showed high levels of resistance to streptomycin and kanamycin.

The speA gene was significantly associated with emm1 type (two strains) (p = 0.01) and the ssa gene demonstrated significant association with emm78 type (p = 0.01), but was not significantly associated with emm4 (p = 0.13). Strains harbored speC gene demonstrated nonsignificant association with emm78 (p = 0.467), emm28 (p = 1.000), and emm11 (p = 0.4).

Discussion

The present investigation is the first epidemiological study characterizing erythromycin-resistant S. pyogenes through emm typing in Tunisia. In our study, we investigated macrolide-resistant GAS strains collected during the period 2000–2013 using macrolide resistance genes, emm typing, and virulence factors.

Susceptibility testing showed that ERGAS isolates tested were susceptible to all beta-lactams. In other Tunisian studies, all GAS strains were susceptible to all beta-lactams.^6,18,22 Resistance to penicillin G had not been reported in S. pyogenes in any country worldwide.^4,9,23–26 Therefore, penicillin G is the treatment of choice for GAS infections.^23,27

Among our isolates, 15 (5.2%) were resistant to erythromycin. This result is similar to other previous Tunisian studies.^6,18,22 The low number of ERGAS strains in this study is most likely due to a limited number of strains isolated from throat swabs. Indeed, bacterial suspected pharyngitis is usually treated with probabilistic antibiotics without recourse to an etiologic diagnosis based on the rapid test and the culture of throat swab.²⁸ Many studies showed a low rate of resistance to erythromycin as reported in Norway 2.7%,²⁹ in United States 5.2%,²⁵ in France 6.5%,³⁰ in Italy 7.4%,⁴ in Spain 2.8%,³¹ and in Taiwan 10.7%.⁹ In contrast to these results, other studies showed a high rate of resistance to erythromycin such as Egypt 21.3%,³² Lebanon 23%,²⁴ Greece 22.8%,³³ and Iran 33.9%.³⁴ The high rate of resistance to erythromycin has been correlated with significant increase of erythromycin consumption over time using macrolides for the treatment of pharyngitis caused by GAS or other pathogens.^25,35 Introduction of newer macrolides and their extensive use have also contributed to increased resistance.³⁶ Other studies have shown that decrease in the rate of resistance to erythromycin was due to reduction in macrolide consumption.⁴ In fact, decrease in the prevalence of erythromycin resistance in S. pyogenes has been reported in some countries.^4,10 In the United States, the rate of erythromycin resistance has decreased from 9.6% to 5.2%,^25,37 in France from 14.5% to 6.5%,^30,38 in Spain from 19% to 2.8%,^31,39 and in Italy from 35.8% to 7.4%.^4,40

Among ERGAS strains, 46.6% assigned to the cMLS_B phenotype; 33.3% of them harbored ermB alone; and 13.3% coharbored ermB and mefA genes. However, 53.4% of ERGAS assigned to the M phenotype harbored mefA. In previous Tunisian studies, cMLS_B phenotype harboring the ermB gene was predominant.^6,7,22 The cMLS_B strains harboring the ermB gene were predominant in Italy,⁴ in Korea,³⁵ in India,⁴¹ in Japan,² in Portugal,¹⁰ and in China.⁴² M phenotype strains harboring mefA gene were predominant in Europe such as Spain,^31,39,43 Norway,²⁹ Germany,^26,44 and Greece.³⁶ This phenotype was also predominant in Mexico,^23,25 Serbia,⁴⁵ Taiwan,⁹ and Egypt.³² We showed in the present study an inversion in the dominant phenotype and genotype of resistance to macrolides. There is a decrease in the proportion of the cMLS_B phenotype isolates harboring ermB gene over the period 2000 to 2006 and a concomitant increase of the M phenotype isolates harboring the mefA gene over the period 2007 to 2013. In fact, many studies from multiple countries report significant temporal changes in the prevalence of macrolide resistance phenotypes and genotypes.^4,10,46 These suggest instability of the population of GAS strains, which impact on the prevalence of the macrolide resistance phenotypes and genotypes.²⁷

The iMLS_B phenotype was not detected in our study, suggesting its absence in our country such as in Mexico²³ and Korea.³⁵ This phenotype predominates in India⁴¹ and Hawaii.⁴⁷

Detection of pyrogenic exotoxin genes and streptococcal superantigen genes indicated that all ERGAS isolates expressed the chromosomal speB gene. The same result was reported in other studies.^6,7,48 The frequency of detection of each toxin gene varied around the world. Among all our ERGAS strains tested, speA, speC, and ssa were detected in 13.4%, 53.4%, and 13.4%, respectively. In Portugal, distribution of virulence genes among ERGAS strains was 13% for speA, 23% for speC, and 38% for ssa.⁴⁹

In our study, eight different emm types were identified among ERGAS strains isolated between 2000 and 2013. Among erythromycin susceptible GAS strains collected between 2000 and 2006, emm types 22, 76, 9, 1, and 118 predominated, accounting for 53.4%. There was diversity in emm distribution in ERGAS particularly in 2000–2006. Indeed, from 2000 to 2006, the six ERGAS belonged to five different emm types (22, 28, 76, 11, and 4), while between 2007 and 2013, seven among the nine ERGAS belonged to only two emm types (58, and 1), but this result was nonstatistically significant. In addition to this, all but one of the ERGAS invasive strains belonged to the same emm type 58.

Changes of emm genotypes were found also in other studies.^4,9,10 A variety of studies revealed that the distribution of resistance to macrolides is related to certain emm genotypes.^24,48,50 Few number of emm types are associated to ERGAS strains suggesting that few clones are associated with macrolide resistance.^24,30 In fact, in Spain, emm types 11, 12, and 28 were associated with ERGAS,³¹ emm types 12, 28, 75, and 18 in Japan,² emm types 12, 4, and 11 in Italy,⁴ and emm types 75, 12, and 77 in Serbia.⁴⁵ The emm types 4, 11, 12, and 28 were prevalent genotypes found in Norway;⁴⁸ emm types 110, 104, and stD432 in India;⁴¹ and emm44 in France.⁵¹ The only emm28 ERGAS strain isolated in our work showed high levels of resistance to streptomycin and kanamycin and was also resistant to tetracycline and bacitracin suggesting their close relationship to the multiresistant European clone.⁵²

Correlation between phenotypes and genotypes of resistance to macrolides and emm types was reported in many studies.^4,42 In our study, emm types 1, 58, and 4 were only present among strains assigned to the M phenotype, while strains assigned to the cMLS_B phenotype were of emm types 11, 22, 28, 78, and 76. In Italy,⁴⁶ emm75, emm2, and emm4 were associated with M phenotype. In China, all emm1 strains and the majority of emm12 strains were associated with cMLS_B phenotype.⁴² In Taiwan, cMLS_B phenotype predominated in emm12 strains.⁹ In Japan, emm1, emm12, emm4, and emm75 possessed mefA gene.⁵³ The fact that some emm types are rarely found among resistant isolates is suggestive of the limited transfer of macrolide resistance determinants.⁴ So, emergence of macrolide resistance among GAS around the world is due to the dissemination of a limited number of emm types.^4,10,35

Many studies have shown that exotoxin gene acquisition was associated with emm types and varied in a time-dependent manner among different countries and regions.^7,24,54 In our study, the speA gene was correlated with emm1 (p = 0.01). Many studies reported the same result.^12,48,54 In contrast, in New Caledonia, speA gene was associated with emm15.⁵⁵ In our collection, ssa gene was associated with emm4 and emm78 types (p = 0.01). In Norway, all emm4 and emm3 strains possess ssa gene.⁴⁸ All emm78, emm28, and emm11 strains harbored speC gene in our study. In Germany, speC was found in emm1 strains.¹³ In France, speC gene was detected in the majority of the emm2, emm4, and emm6 types, and ssa gene was detected in the majority of emm3, emm4, emm22, and emm87 strains.³⁰

Despite the low number of ERGAS isolates and the limited number of toxins studied in this work, we provided in this report some useful conclusions. The low frequency of ERGAS may be considered in the treatment of noninvasive GAS infections in cases of allergy to beta-lactams. The overall prevalence of the erythrogenic toxin genes and macrolide resistance genes observed depended on the different distribution and spread of emm types across the ERGAS populations during 2000–2013 suggesting dissemination of a limited number of macrolide-resistant clones. Furthermore, complete microbiological characterization of isolates using multilocus sequence typing and evaluation of a larger bacterial population is required to confirm this hypothesis.

Footnotes

Disclosure Statement

No competing financial interests exist.

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