Plasma activated water as a pre-treatment strategy in the context of biofilm-infected chronic wounds.

Healing and treatment of chronic wounds are often complicated due to biofilm formation by pathogens. Here, the efficacy of plasma activated water (PAW) as a pre-treatment strategy has been investigated prior to the application of topical antiseptics polyhexamethylene biguanide, povidone iodine, and MediHoney, which are routinely used to treat chronic wounds. The efficacy of this treatment strategy was determined against biofilms of Escherichia coli formed on a plastic substratum and on a human keratinocyte monolayer substratum used as an in vitro biofilm-skin epithelial cell model. PAW pre-treatment greatly increased the killing efficacy of all the three antiseptics to eradicate the E. coli biofilms formed on the plastic and keratinocyte substrates. However, the efficacy of the combined PAW-antiseptic treatment and single treatments using PAW or antiseptic alone was lower for biofilms formed in the in vitro biofilm-skin epithelial cell model compared to the plastic substratum. Scavenging assays demonstrated that reactive species present within the PAW were largely responsible for its anti-biofilm activity. PAW treatment resulted in significant intracellular reactive oxygen and nitrogen species accumulation within the E. coli biofilms, while also rapidly acting on the microbial membrane leading to outer membrane permeabilisation and depolarisation. Together, these factors contribute to significant cell death, potentiating the antibacterial effect of the assessed antiseptics.

An optimised GAS-pharyngeal cell biofilm model.

Group A Streptococcus (GAS) causes 700 million infections and accounts for half a million deaths per year. Biofilm formation has been implicated in both pharyngeal and dermal GAS infections. In vitro, plate-based assays have shown that several GAS M-types form biofilms, and multiple GAS virulence factors have been linked to biofilm formation. Although the contributions of these plate-based studies have been valuable, most have failed to mimic the host environment, with many studies utilising abiotic surfaces. GAS is a human specific pathogen, and colonisation and subsequent biofilm formation is likely facilitated by distinct interactions with host tissue surfaces. As such, a host cell-GAS model has been optimised to support and grow GAS biofilms of a variety of GAS M-types. Improvements and adjustments to the crystal violet biofilm biomass assay have also been tailored to reproducibly detect delicate GAS biofilms. We propose 72 h as an optimal growth period for yielding detectable biofilm biomass. GAS biofilms formed are robust and durable, and can be reproducibly assessed via staining/washing intensive assays such as crystal violet with the aid of methanol fixation prior to staining. Lastly, SEM imaging of GAS biofilms formed by this model revealed GAS cocci chains arranged into three-dimensional aggregated structures with EPS matrix material. Taken together, we outline an efficacious GAS biofilm pharyngeal cell model that can support long-term GAS biofilm formation, with biofilms formed closely resembling those seen in vivo.

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.

Streptococcus pyogenes M1T1 Variants Induce an Inflammatory Neutrophil Phenotype Including Activation of Inflammatory Caspases.

Invasive infections due to group A Streptococcus (GAS) advance rapidly causing tissue degradation and unregulated inflammation. Neutrophils are the primary immune cells that respond to GAS. The neutrophil response to GAS was characterised in response to two M1T1 isolates; 5448 and animal passaged variant 5448AP. Co-incubation of neutrophils with 5448AP resulted in proliferation of GAS and lowered the production of reactive oxygen species when compared with 5448. Infection with both strains invoked neutrophil death, however apoptosis was reduced in response to 5448AP. Both strains induced neutrophil caspase-1 and caspase-4 expression in vitro, with inflammatory caspase activation detected in vitro and in vivo. GAS infections involving strains such as 5448AP that promote an inflammatory neutrophil phenotype may contribute to increased inflammation yet ineffective bacterial eradication, contributing to the severity of invasive GAS infections.

Current Understanding of Group A Streptococcal Biofilms.

BACKGROUND: It has been proposed that GAS may form biofilms. Biofilms are microbial communities that aggregate on a surface, and exist within a self-produced matrix of extracellular polymeric substances. Biofilms offer bacteria an increased survival advantage, in which bacteria persist, and resist host immunity and antimicrobial treatment. The biofilm phenotype has long been recognized as a virulence mechanism for many Gram-positive and Gram-negative bacteria, however very little is known about the role of biofilms in GAS pathogenesis. OBJECTIVE: This review provides an overview of the current knowledge of biofilms in GAS pathogenesis. This review assesses the evidence of GAS biofilm formation, the role of GAS virulence factors in GAS biofilm formation, modelling GAS biofilms, and discusses the polymicrobial nature of biofilms in the oropharynx in relation to GAS. CONCLUSION: Further study is needed to improve the current understanding of GAS as both a monospecies biofilm, and as a member of a polymicrobial biofilm. Improved modelling of GAS biofilm formation in settings closely mimicking in vivo conditions will ensure that biofilms generated in the lab closely reflect those occurring during clinical infection.