The objective of this study is to characterize Streptococcus pyogenes isolates with a mucoid phenotype and to compare them with non-mucoid isolates obtained between April and August 2016.
Material and methodsIdentification and antimicrobial susceptibility were performed in all isolates. The emm type and exotoxin genes speA, speB, speC, speF, speG, speH, speJ, speZ and ssa were analyzed. Clinical and demographic data were collected.
ResultsFrom 96 isolates analyzed, 47% had a mucoid phenotype and 95.5% of them presented speA-speB-speF-speG-ssa genes and emm3 genotype. The main clinical manifestation was pharyngotonsillitis (77.1%) evolving to scarlet fever in 67.5% of the cases.
ConclusionThis study describes the circulation of a mucoid phenotype strain with a speA-speB-speF-speG-ssa toxin profile and emm3.1 genotype considered one of the most frequent and virulent of SGA.
El objetivo de este estudio es la caracterización de cepas de Streptococcus pyogenes con fenotipo mucoide y su comparación con las cepas no mucoides aisladas entre abril y agosto de 2016.
Material y métodosSe llevó a cabo la caracterización y el estudio de sensibilidad antimicrobiana de todos los aislados. Se determinó el tipo emm y se analizaron los genes de exotoxinas speA, speB, speC, speF, speG, speH, speJ, speZ y ssa. Se recogieron datos clínicos y demográficos.
ResultadosDe 96 aislados analizados, el 47% presentaron un fenotipo mucoide, y de estos últimos, el 95,5% presentaron los genes speA-speB-speF-speG-ssa y genotipo emm3. La principal manifestación clínica entre todos los pacientes fue faringoamigdalitis (77,1%) que evolucionó a escarlatina en el 67,5% de los casos.
ConclusiónSe describe la circulación de una cepa de aspecto mucoide con perfil de toxinas speA-speB-speF-speG-ssa y genotipo emm3.1 considerado de los más frecuentes y más virulentos de SGA.
Streptococcus pyogenes or group A beta-haemolytic streptococcus (GAS) causes numerous infectious processes that range from pharyngitis or skin infections to invasive diseases such as bacteraemia, septic arthritis, pneumonia, toxic shock syndrome or puerperal sepsis1.
The M protein, encoded by the emm gene, has been described as the main virulence factor of GAS, and is used to classify strains into various genotypes2. Other determinants of virulence are streptococcal pyrogenic exotoxins, encoded by the spe genes, and the expression of hyaluronic acid capsular polysaccharide, which confers a mucoid appearance to the colonies3.
In April 2016, a sudden increase in S. pyogenes isolates with a mucoid phenotype was observed in the samples received at the Microbiology Service of the Hospital Universitario Reina Sofía [Reina Sofía University Hospital] (HURS) in Cordoba. The objective of this study was the epidemiological and molecular characterisation of these strains with mucoid phenotype and their comparison with non-mucoid strains isolated in the same period of time.
Material and methodsGAS isolates obtained from clinical samples received at the HURS Microbiology Service between April and August 2016 were studied. The samples were seeded on blood agar plates with a bacitracin disk and incubated for 48 h in an atmosphere enriched with 5% CO2 at 35 ± 2 °C. Identification and antimicrobial susceptibility was carried out by microdilution with panels of the semi-automated Wider system (Panel 94B MIC/ID, Francisco Soria Melguizo SA, Madrid, Spain). The results were interpreted according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Isolates resistant to erythromycin were classified into three phenotypes based on the sensitivity pattern shown in the double-disk test with erythromycin (15 μg) and clindamycin (2 μg): M phenotype (erythromycin resistant and clindamycin sensitive), constitutive MLSB phenotype (erythromycin and clindamycin resistant) and inducible MLSB phenotype (erythromycin resistant and inducible clindamycin resistance detected by flattening of the clindamycin inhibition halo near the erythromycin disk).
Molecular characterisation of the strains was carried out at the Carlos III Health Institute. The emm genotype of all isolates was determined by PCR amplification and subsequent sequencing of the emm gene following the Centers for Disease Control and Prevention (CDC) protocol (https://www.cdc.gov/streplab/protocol-emm-type.html). The exotoxin genes speA, speB, speC, speF, speG, speH, speJ, speZ and ssa were analysed by PCR4. Clinical and demographic data of the patients in whom the studied strains were isolated were collected. The data were analysed in the SPSS® programme, version 19.0.
ResultsA total of 96 GAS isolates obtained from pharyngeal exudate (74; 77.1%), sputum (4; 4.2%), vaginal discharge (8; 8.3%), wound exudate (3; 3.1%), blood (2; 2.1%), ear discharge (1; 1%), bronchial aspirate (1; 1%), lymph node biopsy (1; 1%), pleural fluid (1; 1%) and lochia (1; 1%). The samples came from 14 primary healthcare centres (PHCs) (70; 72.9%), paediatric emergency departments (9; 9.4%), specialised clinics (10; 10.4%), internal medicine (4; 4.2%), the intensive care unit (2; 2.1%) and thoracic surgery (1; 1%). The mean age of the study patients was 16 years (range: <1–76 years); 61.5% were women and 38.5% were men.
Colonies from 45 (46.9%) of the isolates had a mucoid appearance and 51 (53.1%) had the classic appearance of shiny flat colonies.
In the set of 96 isolates, 13 genotypes were identified, the most frequent type being emm3 (49.4%), followed by emm4 (10.4%), emm6 (6.3%) and emm12 (5.2%). A new emm3 subtype, classified by the CDC as emm3.166, was identified in 11 patients. Among the isolates with a mucoid phenotype, 44 (97.8%) belonged to the emm3 type, with the majority being the emm3.1 (75.6%) and emm3.166 (15.6%) subtypes. The other mucoid isolate corresponded to the emm77 type. In all, 31.4% of the non-mucoid isolates presented the emm3 genotype, including emm3.1 (23.5%) and emm3.166 (7.8%), as well as 10 other genotypes, the most frequent being emm4 (19.6%), emm6 (11.8%) and emm12 (9.8%).
19 different toxin profiles were identified. The most common spe genes were speB (92.7%), speF (91.6%) and speG (86.5%). A total of 95.5% of the strains with a mucoid phenotype presented the speA-speB-speF-speG-ssa genes. In strains with a non-mucoid phenotype, the most common toxin profiles were speA-speB-speF-speG-ssa and genotype emm3 (25.5%), followed by speB-speC-speF-speG-speH and genotype emm6 (21.6 %).
All isolates were sensitive to penicillin, ampicillin, clindamycin and vancomycin. Three isolates showed resistance to erythromycin with the M phenotype of macrolide resistance, presenting the genotypes emm77 with a mucoid phenotype, emm3 with a mucoid phenotype, and emm94 with a non-mucoid phenotype.
The main clinical manifestation was pharyngotonsillitis in 77.1% of the cases, which progressed to scarlet fever in 67.5% of them. Some 65% of these cases were in children under 5 years of age. No significant differences (p = 0.220) were observed between the mucoid and non-mucoid phenotypes regarding the development of scarlet fever. Of the scarlet fever-producing isolates, 67.3% presented the emm3 type, although this trend did not reach a statistically significant association (p = 0.17).
Six invasive diseases were diagnosed, four of them (one bacteraemia, one mediastinitis, one knee arthritis and one cervical adenopathy) caused by isolates with a mucoid phenotype and emm3.1 genotype, with no statistically significant association (p = 0.316). Two of these patients died. The other two invasive diseases (a bacteraemia that ended in death and a puerperal sepsis) were caused by isolates with a non-mucoid phenotype with genotypes emm87 and emm89. Another death occurred in a patient with lung cancer in whom GAS (non-mucoid phenotype with genotype emm1) was isolated in a sputum sample. The rest of the clinical manifestations were vaginitis (eight cases), pneumonia (three cases), wound infection (two cases) and ear infection (one case).
Table 1 shows the distribution of isolated cases by phenotype, sample type, syndrome, emm genotype and exotoxin gene profile.
Relationship between phenotype, sample type, syndrome, emm genotype and exotoxin gene profile in 96 group A streptococcus isolates.
| Phenotype (n) | Sample type (n) | Syndrome (n) | emm genotype (n) | Exotoxin profile (n) |
|---|---|---|---|---|
| Mucoid (45) | Pharyngeal exudate (34) | Pharyngotonsillitis (9) | 3.1 (6) | A,B,F,G,J,ssa (2) |
| A,B,F,G,ssa (4) | ||||
| 3.93 (1) | A,B,F,G,ssa (1) | |||
| 3.166 (1) | A,B,F,G,ssa (1) | |||
| 77 (1) | B,C,F (1) | |||
| Scarlet fever (25) | 3.1 (18) | A,B,F,G,J,ssa (4) | ||
| A,B,F,G,ssa (13) | ||||
| A,G,ssa (1) | ||||
| 3.166 (6) | A,B,F,G,ssa (6) | |||
| 3.93 (1) | A,B,F,G,ssa (1) | |||
| Sputum (2) | Pneumonia (1) | 3.1 (1) | A,B,F,G,J,ssa (1) | |
| Bronchiectasis (1) | 3.1 (1) | A,B,F,G,ssa (1) | ||
| Pleural fluid (1) | Mediastinitis (1)a | 3.1 (1) | A,B,F,G,ssa (1) | |
| Vaginal discharge (4) | Vulvovaginitis (4) | 3.1 (3) | A,B,F,G,J,ssa (1) | |
| A,B,F,G,ssa (1) | ||||
| G,ssa (1) | ||||
| 3.24 (1) | A,B,F,G,ssa (1) | |||
| Ear discharge (1) | Middle ear infection (1) | 3.1 (1) | A,B,F,G,J,ssa (1) | |
| Blood (1) | Bacteraemia (1)a | 3.1 (1) | A,B,F,G,ssa (1) | |
| Purulent discharge (1) | Knee arthritis (1)a | 3.1 (1) | A,B,F,G,ssa (1) | |
| Lymph node biopsy (1) | Cervical adenopathy (1)a | 3.1 (1) | A,B,F,G,ssa (1) | |
| Non-mucoid (51) | Pharyngeal exudate (40) | Pharyngotonsillitis (16) | 1.0 (1) | A,B,F,G,J,Z (1) |
| 3.1 (5) | A,B,F,G,J,ssa (1) | |||
| A,B,F,G,ssa (3) | ||||
| B,F,G,ssa (1) | ||||
| 3.166 (1) | A,B,F,G,ssa (1) | |||
| 4.0 (4) | B,C,F,J,Z,ssa (1) | |||
| B,C,F,Z,ssa (2) | ||||
| B,C,F,G (1) | ||||
| 6.0 (1) | B,C,F,G,H (1) | |||
| 12.0 (2) | Negative (1) | |||
| B,C,F,G (1) | ||||
| 82.0 (1) | A,B,C,F,G,H (1) | |||
| 89.0 (1) | B,F,G (1) | |||
| Scarlet fever (24) | 1.0 (1) | A,B,F,G,J,Z (1) | ||
| 3.1 (6) | A,B,F,G,ssa (6) | |||
| 3.166 (3) | Negative (1) | |||
| A,B,F,G,ssa (2) | ||||
| 4.0 (6) | C,Z,ssa (2) | |||
| B,C,F,Z,ssa (4) | ||||
| 6.0 (3) | B,C,F,G,H (3) | |||
| 12.0 (2) | B,C,F,G,H (2) | |||
| 21.0 (1) | B,C,F,G (1) | |||
| 87.0 (1) | B,C,F,G,J,Z,ssa (1) | |||
| 94.1 (1) | B,C,F,G,H (1) | |||
| Sputum (2) | Pneumonia (1) | 1.0 (1) | Negative (1) | |
| Bronchiectasis (1) | 6.0 (1) | B,C,F,G,H (1) | ||
| Bronchial aspirate (1) | Pneumonia (1) | 12.0 (1) | B,C,F,G,H (1) | |
| Blood (1) | Bacteraemia (1)a | 87.0 (1) | B,F,G,J,Z,ssa (1) | |
| Vaginal discharge (4) | Vulvovaginitis (4) | 2.0 (1) | B,C,F,G,J (1) | |
| 3.1 (1) | A,B,F,G,ssa (1) | |||
| 6.0 (1) | B,C,F,G,H,J (1) | |||
| 12.36 (1) | B,C,F,G,H (1) | |||
| Purulent discharge (2) | Groin wound (1) | 75.0 (1) | B,C,F,G (1) | |
| Pressure ulcer (1) | 6.4 (1) | B,C,F,G,H (1) | ||
| Lochia (1) | Puerperal sepsis (1)a | 89.0 (1) | B,C,G (1) |
The most prevalent emm genotypes of GAS globally are emm1, emm28, emm3, emm89 and emm4. This distribution varies significantly according to geographic region and clinical manifestation, with the emm1, emm3, emm28 and emm18 types being the ones most associated with invasive disease5,6. In Spain, the most common emm genotypes are emm1, emm3, emm4, emm12, emm28 and emm89, with emm1 and emm3 being the ones mainly associated with invasive disease7,8.
Of the six invasive diseases observed in this study, four of them were caused by the emm3 type with a mucoid phenotype, showing a non-statistically significant trend probably due to the low number of isolates producing invasive diseases obtained in this series.
One case of puerperal sepsis was detected due to the emm89 genotype, previously associated with this disease3.
In other parts of the world, such as in the US, mucoid strains are widely distributed, but there are few studies on this phenotype in Spain. Several works associate this virulence factor with the emm3 genotype9,10. A total of 97.8% of the isolates with a mucoid phenotype in this study presented the emm3 genotype (emm3.1, emm3.24, emm3.93 and the newly described emm3.166), with the majority being emm3.1 with a speA-speB-speF-speG-ssa toxin profile. The non-mucoid population presented up to 11 different emm genotypes, with emm3.1 being one of the most frequent, as well as different toxin profiles.
Some 51% of the patients developed scarlet fever, 65% being under five years of age. This could make it difficult to differentiate this condition from viral infections more typical of this age. These data coincide with those obtained by studies in other countries that have observed an increase in the rate of scarlet fever in recent years11. In Spain, as scarlet fever is not a notifiable disease, it is difficult to determine its real incidence.
Some authors associate the production of speA pyrogenic exotoxin with the appearance of scarlet fever12. In this study, 69% of the scarlet fever-producing isolates carried the speA gene compared to 60% of the pharyngitis-producing isolates, with no significant differences observed (p = 0.419) between both groups. These results coincide with those obtained by other authors where no differences were observed13.
The three isolates resistant to erythromycin (emm77, emm3 and emm94) presented the M phenotype, this being the most frequent in GAS isolated in Spain. Antibiotic resistance in GAS is concentrated in some genotypes, with emm77 being one of the most resistant emm types. The emm94 genotype is less frequent in Spain, but also shows high resistance to erythromycin.
The circulation of a mucoid strain with a speA-speB-speF-speG-ssa toxin profile and emm3.1 genotype, considered to be one of the most frequent and most virulent GAS strains, has been described3,5.
Conflicts of interestThe authors declare that they have no conflicts of interest.
Please cite this article as: Guzmán-Puche J, Tejero-Garcia R, Villalón P, Pino-Rosa S, Martínez-Martínez L. Caracterización de estreptococos beta-hemolíticos del grupo A con fenotipo mucoide aislados en un hospital de tercer nivel. Enferm Infecc Microbiol Clin. 2022;40:381–384.
