PCR amplification of virulence factors (emm, sagA, speA, speB, scpA, slo, hylA and ska genes) of Streptococcus pyogenes isolated from tonsillitis and pharyngitis patients in a local population of Lahore, Pakistan

Paper Details

Research Paper 01/12/2020
Views (314) Download (13)

PCR amplification of virulence factors (emm, sagA, speA, speB, scpA, slo, hylA and ska genes) of Streptococcus pyogenes isolated from tonsillitis and pharyngitis patients in a local population of Lahore, Pakistan

Sehr Syed, Javeria Zaheer, Asif Ali, Fiaz Ahmed, Sahrish Tabassum, Naila Ashraf, Maryam Khan, Saba Shamim
Int. J. Biosci.17( 6), 317-327, December 2020.
Certificate: IJB 2020 [Generate Certificate]


Tonsillitis and pharyngitis are infections often caused by Streptococcus pyogenes, a member of the group A Streptococcus. The aim of this study was to isolate S. pyogenes and identify the virulence genes associated with its pathogenesis in a local population of Lahore, Pakistan. The samples (n=110) were collected from patients suffering from tonsillitis and pharyngitis. They were proceeded on 5 % sheep blood agar and crystal violet agar for the subsequent isolation and purification of S. pyogenes. The biochemical characterization, determination of antibiogram and serum opacity factor (SOF) was performed by using standard procedures. The amplification of the selected virulent genes was carried out by PCR. Results demonstrated β- hemolytic colonies of S. pyogenes in 18.8 % (n=20) of the samples, which was also confirmed by coagulation with specific antisera. The 62.5 % of the strains were notably tested positive for serum opacity factor (SOF), whereas 37.5 % of the strains were tested negative. The antibiogram of S. pyogenes revealed sensitivity to cefotaxime (62.5 %), gentamicin (37.5 %), penicillin G (87. 5 %), clindamycin (87.5 %) and erythromycin (100 %). All strains were found to be resistant to tetracycline. PCR amplification yielded product sizes of 700 bp, 290 bp, 780 bp, 820 bp, 510 bp, 409 bp, 550 bp and 580 bp for emm, sagA, speA, speB, scpA, slo, hylA and ska genes, respectively. The presence of these virulent genes confirmed their incidence in the local population, which can be studied further to gain deeper insights into the molecular mechanisms of S. pyogenes pathogenicity.


Al-Kareem FEA, Abbas AK, Hussein MA. 2014. Comparative study of the antibody responses to Streptococcus pyogenes between school children carriers and patients with tonsilitis. Iraqi Journal of Science 55, 403-410.

Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA. 2003. Current protocols in molecular biology. In: Biochemistry and Molecular Biology Education (Eds. Struhl K). Volume 24, Issue 1. Page: 68. Publisher John Wiley & Sons, Inc. http://dx.doi.org/10.1002/bmb.1996.5690240143

Biswas I, Germon P, McDade K, Scott JR. 2001. Generation and surface localization of intact M protein in Streptococcus pyogenes are dependent on sagA. Infection and Immunity 69, 7029-7038. http://dx.doi.org/10.1128/IAI.69.11.7029-7038.2001

Borek AL, Obszańska K, Hryniewicz W, Sitkiewicz I. 2012. Detection of Streptococcus pyogenes virulence factors by multiplex PCR. Virulence 3(6), 529-533. http://dx.doi.org/10.4161/viru.21540

Buckley SJ, Timms P, Davies MR, McMillan DJ. 2018. In silico characterization of the two-component system regulators of Streptococcus pyogenes. PLoS One 13, 1-23. http://dx.doi.org/10.1371/journal.pone.0199163

Cantón R, Loza E, Morosini MI, Baquero F. 2002. Antimicrobial resistance amongest isolates of Streptococcus pyogenes and Staphylococcus aureus in the PROTEKT antimicrobial surveillance programme during 1999-2000. Journal of Antimicrobial Chemotherapy 50(Suppl_2), 9-24. http://dx.doi.org/10.1093/jac/dkf811

Carapetis JR, Steer AC, Mulholland EK, Weber M. 2005. The global burden of Group A Streptococcal diseases. The Lancet Infectious Diseases 5(11), 685-694. http://dx.doi.org/10.1016/S1473-3099(05)70267-X

Carroll RK, Musser JM. 2011. From transcription to activation: How group A Streptococcus, the flesh-eating pathogen, regulates SpeB cysteine protease production. Molecular Microbiology 81(3), 588-601. http://dx.doi.org/10.1111/j.1365-2958.2011.07709.x

Cheesbrough M. 2002. Biochemical tests to identify bacteria. In: Laboratory Practice in Tropical Countries, Cambridge, UK. p 63-70.

Chiang-Ni C, Wu JJ. 2008. Effects of streptococcal pyrogenic exotoxin B on pathogenesis of Streptococcus pyogenes. Journal of the Formosan Medical Association 107(9), 677-685. http://dx.doi.org/10.1016/S0929-6646(08)60112-6

Cleary PP, Prahbu U, Dale JB, Wexler DE, Handley J. 1992. Streptococcal C5a peptidase is a highly specific endopeptidase. Infection and Immunity 60(12), 5219-5223.

CLSI. 2011. Performance standards for antimicrobial susceptibility testing: 20th Informational Supplement M100-S20. Wayne, USA p 1-15.

Courtney HS, Hasty DL, Dale JB. 2003. Serum opacity factor (SOF) of Streptococcus pyogenes evokes antibodies that opsonize homologous and heterologous SOF-positive serotypes of group A streptococci. Infection and Immunity 71(9), 5097-5103. http://dx.doi.org/10.1128/iai.71.9.5097-5103.2003

Courtney HS, Pownell HJ. 2010. The structure and function of serum opacity factor: a unique streptococcal virulence determinant that targets high-density lipoproteins. BioMed Research International 2010, 1-16. http://dx.doi.org/10.1155/2010/956071

Dao VA, Overhagen S, Bilstein A, Kolot C, Sonnemann U, Mösges R. 2019. Ectoine lozenges in the treatment of acute viral pharyngitis: A prospective, active-controlled clinical study. European Archives of Otorhinolaryngology 276(3), 775-783. http://dx.doi.org/10.1007/s00405-019-05324-9

Dmitriev A, Suvorov A, Shen AD, Yang YH. 2004. Clinical diagnosis of group B streptococci by scpB gene based PCR. Indian Journal of Medical Research 119, 233-236.

Dunne EM, Marshall JL, Baker CA, Manning J, Gonis G, Danchin MH, Smeesters PR, Satzke C, Steer AC. 2013. Detection of group a streptococcal pharyngitis by quantitative PCR. BMC Infectious Diseases 13, 312-318. http://dx.doi.org/10.1186/1471-2334-13-312

Fontaine MC, Lee JJ, Kehoe MA. 2003. Combined contributions of streptolysin O and streptolysin S to virulence of serotype M5 Streptococcus pyogenes strain Manfredo. Infection and Immunity 71(7), 3857-3865. http://dx.doi.org/10.1128/IAI.71.7.3857-3865.2003

Gera K, McIver KS. 2013. Laboratory growth and maintenance of Streptococcus pyogenes (the Group A Streptococcus, GAS). In: Current Protocols in Microbiology 30, 9D.2.1-9D.2-13. http://dx.doi.org/10.1002/9780471729259.mc09d02s30

Hoffmann S. 1985. The throat carrier rate of group A and other beta hemolytic streptococci among patients in general practice. Acta Pathologica Microbiologica Scandinavica Series B: Microbiology 93, 347-351. http://dx.doi.org/10.1111/j.16990463.1985.tb02899.x

Hynes WL, Dixon AR, Walton SL, Aridgides LJ. 2000. The extracellular hyaluronidase gene (hylA) of Streptococcus pyogenes. FEMS Microbiology Letters 184(1), 109-112. http://dx.doi.org/10.1111/j.15746968.2000.tb08999.x

Hynes WL, Lynn H, Ferretti JJ. 1995. Analysis of a second bacteriophage hyaluronidase gene from Streptococcus pyogenes: Evidence for a third hyaluronidase involved in extracellular enzymatic activity. Infection and Immunity 63(8), 3015-3020.

Jing H-b, Ning BB, Hao HJ, Zheng YL, Chang D, Jiang W, Jiang YQ. 2006. Epidemiological analysis of group A streptococci recovered from patients in China. Journal of Medical Microbiology 55(8), 1101-1107. http://dx.doi.org/10.1099/jmm.0.46243-0

Kasper KJ, Zeppa JJ, Wakabayashi AT, Xu SX, Mazzuca DM, Welch I, Baroja ML, Kotb M, Cairns E, Cleary PP, Haeryfar SMM, McCormick JK. 1992. Bacterial superantigens promote acute nasopharyngeal infection by Streptococcus pyogenes in a human MHC Class II-dependent manner. PLoS Pathogens 10(5), 1-11. http://dx.doi.org/10.1371/journal.ppat.1004155

Khosravi AD, Ebrahimifard N, Shamsizadeh A, Shoja S. 2016. Isolation of Streptococcus pyogenes from children with pharyngitis and emm type analysis. Journal of the Chinese Medical Association 79(5), 276-280. http://dx.doi.org/10.1016/j.jcma.2016.01.002

Liang Y, Shen X, Huang G, Wang C, Shen Y, Yang Y. 2008. Characteristics of Streptococcus pyogenes strains isolated from Chinese children with scarlet fever. Acta Paediatrica 97(12), 1681-1685. http://dx.doi.org/10.1111/j.1651-2227.2008.00983.x

Mayfield JA, Liang Z, Agrahari G, Lee SW, Donahue DL, Ploplis VA, Castellino FJ. 2014. Mutations in the control of virulence sensor gene from Streptococcus pyogenes after infection in mice lead to clonal bacterial variants with altered gene regulatory activity and virulence. PLoS One 9, 1-12. http://dx.doi.org/10.1371/journal.pone.0100698

McMillan DJ, Vu T, Bramhachari PV, Kaul SY, Bouvet A, Shaila MS, Karmarkar MG, Sriprakash KS. 2010. Molecular markers for discriminating Streptococcus pyogenes and S. dysgalactiae subspecies equisimilis. European Journal of Clinical Microbiology and Infectious Diseases 29(5), 585-589. http://dx.doi.org/10.1007/s10096-010-0899-x

Mirjamali NA, Soufian S, Molaee N, Abbasian SS, Abtahi H. 2014. Cloning and expression of the enzymatic region of Streptococcal hyaluronidase. Iranian Journal of Basic Medical Sciences 17(9), 667-672.

Molaee N, Abtahi H, Mosayebi G. 2013. Expression of recombinant streptokinase from Streptococcus pyogenes and its reaction with infected human and murine sera. Iranian Journal of Basic Medical Sciences 16(9), 985-989.

Molloy EM, Cotter PD, Hill C, Mitchell DA, Ross RP. 2011. Streptolysin S-like virulence factors: the continuing sagA. Nature Reviews Microbiology 9, 670-681.

Musser JM, Kapur V, Szeto J, Pan X, Swanson DS, Martin DR. 1995. Genetic diversity and relationships among Streptococcus pyogenes strains expressing serotype M1 protein: Recent intercontinental spread of a subclone causing episodes of invasive disease. Infection and Immunity 63(3), 994-1003. http://dx.doi.org/10.1128/IAI.63.3.994-1003.1995.

Nizet V, Beall B, Bast DJ, Datta V, Kilburn L, Low DE, de Azavedo JC. 2000. Genetic locus for streptolysin S production by group A Streptococcus. Infection and Immunity 68(7), 4245-4254. http://dx.doi.org/10.1128/iai.68.7.4245-4254.2000

Patterson MJ. 1996. Streptococcus. In: Medical Microbiology (Baron S, Ed.). Chapter 13. 4th edition. University of Texas Medical Branch at Galveston.

Pérez-Trallero E, Montes M, Orden B, Tamayo E, García-Arenzana JM, Marimón JM. 2007. Phenotypic and genotypic characterization of Streptococcus pyogenes isolates displaying the MLSB phenotype of macrolide resistance in Spain, 1999 to 2005. Antimicrobial Agents and Chemotherapy 51(4), 1228-1233. http://dx.doi.org/10.1128/AAC.01054-06

Pichichero ME, Marsocci SM, Murphy ML, Hoeger W, Green JL, Sorrento A. 1999. Incidence of streptococcal carriers in private pediatric practice. Archives of Pediatrics and Adolescent Medicine 153(6), 624-628. http://dx.doi.org/10.1001/archpedi.153.6.624

Ray D, Saha S, Sinha S, Pal NK, Bhattacharya B. 2016. Molecular characterization and evaluation of the emerging antibiotic-resistant Streptococcus pyogenes from eastern India. BMC Infectious Diseases 16, 753-764. http://dx.doi.org/10.1186/s12879-016-2079-9

Sambrook J, Russell D. 2001. Molecular Cloning: A laboratory manual. 3rd Ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.

Savic DJ, Ferretti JJ. 2003. Novel genomic rearrangement that affects expression of the Streptococcus pyogenes streptolysin O (slo) gene. Journal of Bacteriology 185(6), 1857-1869. http://dx.doi.org/10.1128/JB.185.6.1857-1869.2003

Shea PR, Ewbank AL, Gonzalez-Lugo JH, Martagon-Rosado AJ, Martinez-Gutierrez JC, Rehman HA, Serrano-Gonzalez M, Fittipaldi N, Beres SB, Flores AR, Low DE, Willey BM, Musser JM. 2011. Group A Streptococcus emm gene types in pharyngeal isolates, Ontario, Canada, 2002–2010. Emerging Infectious Diseases 17(11), 2010-2017. http://dx.doi.org/10.3201/eid1711.110159

Shumba P, Mairpady-Shambat S, Siemens N. 2019. The role of streptococcal and staphylococcal exotoxins and proteases in human necrotizing soft tissue infections. Toxins 11(6), 332-361. http://dx.doi.org/10.3390/toxins11060332

Slotved H-C, Hoffmann S. 2017. Evaluation of procedures for typing of group B Streptococcus: a retrospective study. Peer J 5, e3105. http://dx.doi.org/10.7717/peerj.3105.

Sriskandan S, Unnikrishnan M, Krausz T, Cohen J. 1999. Molecular analysis of the role of streptococcal pyrogenic exotoxin A (SPEA) in invasive soft-tissue infection resulting from Streptococcus pyogenes. Molecular Microbiology 33(4), 778-790. http://dx.doi.org/10.1046/j.1365-2958.1999.01525.x

Starr CR, Engleberg NC. 2006. Role of hyaluronidase in subcutaneous spread and growth of group A streptococcus. Infection and Immunity 74(1), 40-48. http://dx.doi.org/10.1128/IAI.74.1.40-48.2006

Sykes EA, Wu V, Beyea MM, Simpson MTW, Beyea JA. 2020. Pharyngitis: Approach to diagnosis and treatment. Canadian Family Physician Medecin de Famille Canadien 66(4), 251–257.

Tamayo J, Pérez-Trallero E, Gómez-Gracés JL, Alós JI. 2005. Resistance to macrolides, clindamycin and telithromycin in Streptococcus pyogenes isolated in Spain during 2004. Journal of Antimicrobial Chemotherapy 56(4), 780-782. http://dx.doi.org/10.1093/jac/dki286

Terao Y. 2012. The virulence factors and pathogenic mechanisms of Streptococcus pyogenes. Journal of Oral Biosciences 54(2), 96-100. http://dx.doi.org/10.1016/j.job.2012.02.004

Timmer AM, Kristian SA, Datta V, Jeng A, Gillen CM, Walker MJ, Beall B, Nizet V. 2006. Serum opacity factor promotes group A streptococcal epithelial cell invasion and virulence. Molecular Microbiology 62(1), 15-25. http://dx.doi.org/10.1111/j.1365-2958.2006.05337.x

Vlaminckx BJM, Mascini ME, Schellekens J, Schouls LM, Paauw A, Fluit AC, Novak R, Verhoef J, Schmitz FJ. 2003. Site-specific manifestations of invasive Group A Streptococcal disease: type distribution and corresponding patterns of virulence determinants. Journal of Clinical Microbiology 41(11), 4941-4949. http://dx.doi.org/10.1128/JCM.41.11.49414949.2003

Walker MJ, Barnett TC, McArthur JD, Cole JN, Gillen CM, Henningham A, Sriprakash KS, Sanderson-Smith ML, Nizet V. 2014. Disease manifestations and pathogenic mechanisms of Group A Streptococcus. Clinical Microbiology Reviews 27, 264-301. http://dx.doi.org/10.1128/CMR.00101-13

Wexler DE, Cleary PP. 1985. Purification and characteristics of the streptococcal chemotactic factor inactivator. Infection and Immunity 50(3), 757-764.

Wilson K. 2001. Preparation of genomic DNA from bacteria. In: Current Protocols in Molecular Biology 56(1), 2.4.1-2.4.5. http://dx.doi.org/10.1002/0471142727.mb0204s56

Yu CE, Ferretti JJ. 1991. Frequency of the erythrogenic toxin B and C genes (speB and speC) among clinical isolates of group A streptococci. Infection and Immunity 59(1), 211-215.

Zhu L, Olsen RJ, Lee JD, Porter AR, DeLeo FR, Musser JM. 2017. Contribution of secreted NADase and streptolysin O to the pathogenesis of epidemic serotype M1 Streptococcus pyogenes infections. The American Journal of Pathology 187(3), 605-613. http://dx.doi.org/10.1016/j.ajpath.2016.11.003.