Assessing the Role of Pharyngeal Cell Surface Glycans in Group A Streptococcus Biofilm Formation

Group A Streptococcus (GAS) causes 700 million infections and accounts for half a million deaths per year. Antibiotic treatment failure rates of 20–40% have been observed. The role host cell glycans play in GAS biofilm formation in the context of GAS pharyngitis and subsequent antibiotic treatment failure has not been previously investigated. GAS serotype M12 GAS biofilms were assessed for biofilm formation on Detroit 562 pharyngeal cell monolayers following enzymatic removal of all N-linked glycans from pharyngeal cells with PNGase F. Removal of N-linked glycans resulted in an increase in biofilm biomass compared to untreated controls. Further investigation into the removal of terminal mannose and sialic acid residues with α1-6 mannosidase and the broad specificity sialidase (Sialidase A) also found that biofilm biomass increased significantly when compared to untreated controls. Increases in biofilm biomass were associated with increased production of extracellular polymeric substances (EPS). Furthermore, it was found that M12 GAS biofilms grown on untreated pharyngeal monolayers exhibited a 2500-fold increase in penicillin tolerance compared to planktonic GAS. Pre-treatment of monolayers with exoglycosidases resulted in a further doubling of penicillin tolerance in resultant biofilms. Lastly, an additional eight GAS emm-types were assessed for biofilm formation in response to terminal mannose and sialic acid residue removal. As seen for M12, biofilm biomass on monolayers increased following removal of terminal mannose and sialic acid residues. Collectively, these data demonstrate that pharyngeal cell surface glycan structures directly impact GAS biofilm formation in a strain and glycan specific fashion.

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Paediatric empyema: worsening disease severity and challenges identifying patients at increased risk of repeat intervention

Archives of Disease in Childhood ◽

10.1136/archdischild-2019-318219 ◽

2020 ◽

Vol 105 (9) ◽

pp. 886-890 ◽

Cited By ~ 1

Author(s):

Stuart Haggie ◽

Hasantha Gunasekera ◽

Chetan Pandit ◽

Hiran Selvadurai ◽

Paul Robinson ◽

...

Keyword(s):

Treatment Failure ◽

Initial Treatment ◽

Group A Streptococcus ◽

Thoracoscopic Surgery ◽

Tertiary Hospital ◽

Fibrinolytic Therapy ◽

Younger Age ◽

Increased Risk ◽

Group A ◽

Single Intervention

ObjectiveEmpyema is the most common complication of pneumonia. Primary interventions include chest drainage and fibrinolytic therapy (CDF) or video-assisted thoracoscopic surgery (VATS). We describe disease trends, clinical outcomes and factors associated with reintervention.Design/setting/patientsRetrospective cohort of paediatric empyema cases requiring drainage or surgical intervention, 2011–2018, admitted to a large Australian tertiary children’s hospital.ResultsDuring the study, the incidence of empyema increased from 1.7/1000 to 7.1/1000 admissions (p<0.001). We describe 192 cases (174 CDF and 18 VATS), median age 3.0 years (IQR 1–5), mean fever duration prior to intervention 6.2 days (SD ±3.3 days) and 50 (26%) cases admitted to PICU. PICU admission increased during the study from 18% to 34% (p<0.001). Bacteraemia occurred in 23/192 (12%) cases. A pathogen was detected in 131/192 (68%); Streptococcus pneumoniae 75/192 (39%), S. aureus 25/192 (13%) and group A streptococcus 13/192 (7%). Reintervention occurred in 49/174 (28%) and 1/18 (6%) following primary CDF and VATS. Comparing repeat intervention with single intervention cases, a continued fever postintervention increased the likelihood for a repeat intervention (OR 1.3 per day febrile; 95% CI 1.2 to 1.4, p<0.0001). Younger age, prolonged fever preintervention and previous antibiotic treatment were not associated with initial treatment failure (all p>0.05).ConclusionWe report increasing incidence and severity of empyema in a large tertiary hospital. One in four patients required a repeat intervention after CDF. Neither clinical variables at presentation nor early investigations were able to predict initial treatment failure.

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Biofilm formation by group A Streptococcus: a role for the streptococcal regulator of virulence (Srv) and streptococcal cysteine protease (SpeB)

Microbiology ◽

10.1099/mic.0.021048-0 ◽

2009 ◽

Vol 155 (1) ◽

pp. 46-52 ◽

Cited By ~ 41

Author(s):

Christopher D. Doern ◽

Amity L. Roberts ◽

Wenzhou Hong ◽

Jessica Nelson ◽

Slawomir Lukomski ◽

...

Keyword(s):

Cysteine Protease ◽

Biofilm Formation ◽

Group A Streptococcus ◽

Immunoblot Analysis ◽

Wild Type ◽

Flow Conditions ◽

Group A ◽

Streptococcus A ◽

Insight Into ◽

Western Immunoblot Analysis

Recently, biofilms have become a topic of interest in the study of the human pathogen group A Streptococcus (GAS). In this study, we sought to learn more about the make-up of these structures and gain insight into biofilm regulation. Enzymic studies indicated that biofilm formation by GAS strain MGAS5005 required an extracellular protein and DNA component(s). Previous results indicated that inactivation of the transcriptional regulator Srv in MGAS5005 resulted in a significant decrease in virulence. Here, inactivation of Srv also resulted in a significant decrease in biofilm formation under both static and flow conditions. Given that production of the extracellular cysteine protease SpeB is increased in the srv mutant, we tested the hypothesis that increased levels of active SpeB may be responsible for the reduction in biofilm formation. Western immunoblot analysis indicated that SpeB was absent from MGAS5005 biofilms. Complementation of MGAS5005Δsrv restored the biofilm phenotype and eliminated the overproduction of active SpeB. Inhibition of SpeB with E64 also restored the MGAS5005Δsrv biofilm to wild-type levels.

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Characterization of Biofilm Formation by Clinically Relevant Serotypes of Group A Streptococci

Applied and Environmental Microbiology ◽

10.1128/aem.72.4.2864-2875.2006 ◽

2006 ◽

Vol 72 (4) ◽

pp. 2864-2875 ◽

Cited By ~ 92

Author(s):

Cordula Lembke ◽

Andreas Podbielski ◽

Carlos Hidalgo-Grass ◽

Ludwig Jonas ◽

Emanuel Hanski ◽

...

Keyword(s):

Biofilm Formation ◽

Laser Scanning ◽

Group A Streptococcus ◽

Three Dimensional ◽

Positive Control ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Subsequent Formation ◽

Group A

ABSTRACT Streptococcus pyogenes (group A streptococcus [GAS]) is a frequent cause of purulent infections in humans. As potentially important aspects of its pathogenicity, GAS was recently shown to aggregate, form intratissue microcolonies, and potentially participate in multispecies biofilms. In this study, we show that GAS in fact forms monospecies biofilms in vitro, and we analyze the basic parameters of S. pyogenes in vitro biofilm formation, using Streptococcus epidermidis as a biofilm-positive control. Of nine clinically important serotype strains, M2, M6, M14, and M18 were found to significantly adhere to coated and uncoated polystyrene surfaces. Fibronectin and collagen types I and IV best supported primary adherence of serotype M2 and M18 strains, respectively, whereas serotype M6 and M14 strains strongly bound to uncoated polystyrene surfaces. Absorption measurements of safranin staining, as well as electron scanning and confocal laser scanning microscopy, documented that primary adherence led to subsequent formation of three-dimensional biofilm structures consisting of up to 46 bacterial layers. Of note, GAS isolates belonging to the same serotype were found to be very heterogeneous in their biofilm-forming behavior. Biofilm formation was equally efficient under static and continuous flow conditions and consisted of the classical three steps, including partial disintegration after long-term incubation. Activity of the SilC signaling peptide as a component of a putative quorum-sensing system was found to influence the biofilm structure and density of serotype M14 and M18 strains. Based on the presented methods and results, standardized analyses of GAS biofilms and their impact on GAS pathogenicity are now feasible.

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Biofilm Formation by Group A Streptococci: Is There a Relationship with Treatment Failure?

Journal of Clinical Microbiology ◽

10.1128/jcm.41.9.4043-4048.2003 ◽

2003 ◽

Vol 41 (9) ◽

pp. 4043-4048 ◽

Cited By ~ 70

Author(s):

J. Conley ◽

M. E. Olson ◽

L. S. Cook ◽

H. Ceri ◽

V. Phan ◽

...

Keyword(s):

Treatment Failure ◽

Biofilm Formation ◽

Group A Streptococci ◽

Group A

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Identification and Characterization of an Antigen I/II Family Protein Produced by Group A Streptococcus

Infection and Immunity ◽

10.1128/iai.00493-06 ◽

2006 ◽

Vol 74 (7) ◽

pp. 4200-4213 ◽

Cited By ~ 44

Author(s):

Shizhen Zhang ◽

Nicole M. Green ◽

Izabela Sitkiewicz ◽

Rance B. LeFebvre ◽

James M. Musser

Keyword(s):

Cell Surface ◽

Group A Streptococcus ◽

Bacterial Pathogen ◽

Severe Infection ◽

Genomic Islands ◽

Oral Streptococci ◽

Group B Streptococci ◽

Gram Positive ◽

Group A

ABSTRACT Group A Streptococcus (GAS) is a gram-positive human bacterial pathogen that causes infections ranging in severity from pharyngitis to life-threatening invasive disease, such as necrotizing fasciitis. Serotype M28 strains are consistently isolated from invasive infections, particularly puerperal sepsis, a severe infection that occurs during or after childbirth. We recently sequenced the genome of a serotype M28 GAS strain and discovered a novel 37.4-kb foreign genetic element designated region of difference 2 (RD2). RD2 is similar in gene content and organization to genomic islands found in group B streptococci (GBS), the major cause of neonatal infections. RD2 encodes seven proteins with conventional gram-positive secretion signal sequences, six of which have not been characterized. Herein, we report that one of these six proteins (M28_Spy1325; Spy1325) is a member of the antigen I/II family of cell surface-anchored molecules produced by oral streptococci. PCR and DNA sequence analysis found that Spy1325 is very well conserved in GAS strains of distinct M protein serotypes. As assessed by real-time TaqMan quantitative PCR, the Spy1325 gene was expressed in vitro, and Spy1325 protein was present in culture supernatants and on the GAS cell surface. Western immunoblotting and enzyme-linked immunosorbent assays indicated that Spy1325 was produced by GAS in infected mice and humans. Importantly, the immunization of mice with recombinant Spy1325 fragments conferred protection against GAS-mediated mortality. Similar to other antigen I/II proteins, recombinant Spy1325 bound purified human salivary agglutinin glycoprotein. Spy1325 may represent a shared virulence factor among GAS, GBS, and oral streptococci.

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