In Vitro Assessment of Biofilm Formation by Streptococcus pyogenes Isolates From Invasive and Non-invasive Samples With Diverse emm Type Profiles
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Abstract
Introduction: Biofilm is one of the important virulence factors that is responsible for the severity and progression of the Streptococcus pyogenes diseases. M-protein is involved in the irreversible attachment of S. pyogenes to surfaces during biofilm development. This study aims to determine the propensity of S. pyogenes to form biofilms and the molecular epidemiology of S. pyogenes isolates by emm typing. Methods: We screened 45 S. pyogenes isolates for the biofilm formation by Congo red agar (CRA) and quantified the biofilms by crystal violet microtiter-plate methods (CVMtP). The emm typing of all isolates was performed by conventional PCR with established primers according to the CDC protocol. Results: Majorities of S. pyogenes were isolated from non-invasive, 27 (60.0%) than invasive sources, 18 (40.0%). Regardless of invasiveness, 40 (88.9%) S. pyogenes isolates formed black colonies on CRA, while 43 (95.6%) of the isolates demonstrated various degrees of biofilm formation by CVMtP method. A total of 30 different emm types and subtypes were identified. No new emm types/subtypes were detected. The predominant emm types/subtypes were emm1, emm63, emm18.21, emm91, and emm97.4 which each gene accounted for 7.0%. All emm types/subtypes of S. pyogenes produced biofilms by CVMtP method except emm17.2 and emm57 which were isolated from non-invasive sources. Conclusions: Biofilm-producing S. pyogenes strains of various sources are genetically diverse and biofilm phenotypes are inherent to individual characteristic rather than specific emm type. Nonetheless, higher propensity of GAS to form biofilms warrants better management strategies to avoid treatment failures in the future.
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Bisno AL, Brito MO, Collins CM. Molecular basis of group A streptococcal virulence. The Lancet Infectious Diseases. 2003;3(4):191–200. PubMed PMID: 12679262.
Cunningham MW. Pathogenesis of group A streptococcal infections. Clinical Microbiology Reviews. 2000;13(3):470–511. PubMed PMID: 10885988.
Centres for Disease Control and Prevention. Active Bacterial Core Surveillance Report, Emerging Infections Program Network, Group A Streptococcus. Atlanta, US. 2020. Available from: https://www2.cdc.gov/vaccines/biotech/strepblast.asp;
Public Health England. Group A streptococcal infections: third report on seasonal activity in England, 2017/18. Health Protect Rep. 2018;12(13):5–7.
Tyrrell GJ, Fathima S, Kakulphimp J, Bell C. Increasing rates of invasive group a streptococcal disease in Alberta, Canada; 2003-2017. Open Forum Infect Dis. 2018;5(8):177.
Public Health England. Group A streptococcal infections: fourth update on seasonal activity, 2015/2016. May 6, 2016. https://assets.publishing.service.gov.uk/government/uploads/system/uploads/attachment_data/file/521550/hpr1616_SF-GAS2.pdf (accessed April 17, 2019).
Siemens N, Chakrakodi B, Shambat SM, et al. Biofilm in group A streptococcal necrotizing soft tissue infections. JCI Insight. 2016; 1(10) e87882. http://dx.doi.org/10.1172/jci.insight.87882.
Costerton JW. Cystic fibrosis pathogenesis and the role of biofilms in persistent infection. Trends Microbiol. 2001;9: 50–52.
Baldassarri L, Creti R, Recchia S, Imperi M, Facinelli B, Giovanetti E, et al. Therapeutic failures of antibiotics used to treat macrolide-susceptible Streptococcus pyogenes infections may be due to biofilm formation. J Clin Microbiol. 2006;44: 2721– 2727.
Manetti AG, Zingaretti C, Falugi F, Capo S, Bombaci M, Bagnoli F, et al. Streptococcus pyogenes pili promote pharyngeal cell adhesion and biofilm formation. Mol Microbiol. 2007;64: 968–983.
Riani C, Standar K, Srimuang S, Lembke C, Kreikemeyer B, Podbielski A. Transcriptome analyses extend understanding of Streptococcus pyogenes regulatory mechanisms and behavior toward immunomodulatory substances. Int J Med Microbiol. 2007;297: 513–523.
Conley J, Olson ME, Cook LS, Ceri H, Phan V, Davies HD. Biofilm formation by group A streptococci: is there a relationship with treatment failure? J Clin Microbiol. 2003; 41(9): 4043-8. http://dx.doi.org/10.1128/JCM.41.9.4043-4048.2003.
Lembke C, Podbielski A, Hidalgo-Grass C, Jonas L, Hanski E, Kreikemeyer B. Characterization of biofilm formation by clinically relevant serotypes of group A streptococci. Appl Environ Microbiol. 2006; 72(4): 2864-75. http://dx.doi.org/10.1128/AEM.72.4. 2864-2875.2006. PMID: 16597993.
Ogawa T, Terao Y, Okuni H, et al. Biofilm formation or internalization into epithelial cells enable Streptococcus pyogenes to evade antibiotic eradication in patients with pharyngitis. Microb Pathog. 2011; 51(1-2): 58-68. http://dx.doi.org/10.1016/j.micpath.2011.03.009.
Thenmozhi R, Balaji K, Kumar R, Rao TS, Pandian SK. Characterization of biofilms in different clinical M serotypes of Streptococcus pyogenes. J Basic Microbiol. 2011;51:196–204.
Kalgo HM, Jasni AS, Hadi SRA, Umar NH, Hamzah SNA, Hamat RA. Extremely low prevalence of erythromycin-resistant Streptococcus pyogenes isolates and their molecular characteristics by M protein gene and multilocus sequence typing methods. Jundishapur J Microbiol. 2018;115.
Creti R, Gherardi G, Imperi M, Huolstein CV, Baldassarri L, Pataracchia M, et al. Association of group A streptococcal emm types with virulence traits and macrolide- resistance genes is independent of the source of isolation. J Med Microbiol. 2005;54:10:913-917.
Mathur T, Singhal S, Khan S, Upadhyay DJ, Fatma T, Rattan A. Detection of biofilm formation among isolates of staphylococci: an evaluation of three different screening methods. Indian J Med Microbiol. 2006;24(1):25–9.
Cotter JJ, O’gara JP, Mack D, Casey E. Oxygen- mediated regulation of biofilm development is controlled by the alternative sigma factor σβ in Staphylococcus epidermidis. Appl Environ Microbiol, 2009;75(1):261–4.
Freeman DJ, Falkiner FR, Keane CT. New method for detecting slime production by coagulase negative staphylococci. Journal of clinical pathology. 1989;42(8): 872-4.
Stepanovic S, Vukovic D, Daki I, Savic B, Vlahovic- Svabic M. A modified microtiter- plate test for quantification of staphylococcal biofilm formation. J Microbiol Methods. 2000;40:175-179.
Centres for Disease Control and Prevention. Active Bacterial Core Surveillance Report, Emerging Infections Program Network, Group A Streptococcus. Atlanta, US. 2020. Available from: http://www.cdc.gov/ncidod/biotech/strep/protocols.htm.
Facklam R, Beall B, Efstratiou A, Fischetti V, Johnson D, Kaplan E, et al. emm typing and validation of provisional M types for group A streptococci. Emerging Infectious Diseases. 1999;5(2):247–253.
Maddocks SE, Wright CJ, Nobbs AH, Brittan JL, Franklin L, Strömberg N, Kadioglu A, Jepson MA, Jenkinson HF. Streptococcus pyogenes antigen I/II- family polypeptide AspA shows differential ligand- binding properties and mediates biofilm formation. Mol Microbiol. 2011;81(4):1034-49. doi: 10.1111/j.1365-2958.2011.07749.x.
Melo PC, Ferreira LM, Nader Filho A, Zafalon LF, Vicente HIS, Souza V. Comparison of methods for the detection of biofilm formation by Staphylococcus aureus isolated from bovine subclinical mastitis. Brazilian Journal of Microbiology. 2013;44: 119-
doi:10.1590/S1517-83822013005000031.
Jain A, Agarwal A. Biofilm production, a marker of pathogenic potential of colonizing and commensal staphylococci. J Microbiol Methods. 2009;76:88- 92.
Hassan A, Usman J, Kaleem F, Omair M, Khalid A, Iqbal M. Evaluation of different detection methods of biofilm formation in the clinical isolates. Braz J Infect Dis. 2011;15(4):305-311.
Ruchi T, Sujata B, Anuradha D. Comparison of Phenotypic Methods for the Detection of Biofilm Production in Uro-Pathogens in a Tertiary Care Hospital in India. Int J Curr Microbiol App Sci. 2015;4: 840-849.
Aleksandra , Ina G, LJiljana B, Lazar R. Adherence and Biofilm Production of Streptococcus pyogenes [Internet]. IntechOpen Limited. 5 Princes Gate Court, London; 2016. Available from: https://www.intechopen.com/books/microbial-biofilms-importance-and-applications/adherence-and-biofilm-production-of-streptococcus-pyogenes.
Ismael NF. “Vinegar” as anti-bacterial biofilm formed by Streptococcus pyogenes isolated from recurrent tonsillitis patients, in vitro. Jordan J Biol Sci. 2013;6(3):191– 197.
Al-kafaween MA, Mohd Hilmi AB, Jaffar N, et al. Determination of optimum incubation time for formation of Pseudomonas aeruginosa and Streptococcus pyogenes biofilms in microtiter plate. Bull Natl Res Cent. 2019;43: 100. doi:10.1186/s42269-019-0131-9.
Kostakioti M, Hadjifrangiskou M, Hultgren SJ. Bacterial biofilms: development, dispersal, and therapeutic strategies in the dawn of the postantibiotic era. Cold Spring Harb Perspect Med. 2013; 3(4)a010306. doi: 10.1101/cshperspect.a010306.
Donlan RM. Biofilms: microbial life on surfaces. Emerg Infect Dis. 2002; 8(9): 881- 90. http://dx.doi.org/10.3201/eid0809.020063.
Lemos M, Borges A, Teodósio J, Araújo P, Mergulhão F, Melo L, et al. The effects of ferulic and salicylic acids on Bacillus cereus and Pseudomonas fluorescens single-and dual-species biofilms. Int Biodeterior Biodegradation. 2014;86:42–51.
Marks LR, Reddinger RM, Hakansson AP. Biofilm formation enhances fomite survival of Streptococcus pneumoniae and Streptococcus pyogenes. Infect Immun. 2014; 82(3): 1141-6. doi:10.1128/IAI.01310-13.
Beall B, Facklam R, Thompson T. Sequencing emm- specific PCR products for routine and accurate typing of group A streptococci. J Clin Microbiol. 1996;34(4):953-8. [PubMed: 8815115]. [PubMed Central: PMC228924].