Orals

Introduction: The ability of bacteria to colonize implant surfaces and tissues as a biofilm plays a relevant role in medical-device-associated infections. Staphylococcus aureus strains can produce a biofilm matrix made of the poly-N-acetylglucosamine (PIA/PNAG) exopolysaccharide and/or proteins. PIA/PNAG is synthesised by enzymes encoded by the icaADBC operon whose expression is repressed by the transcriptional regulator IcaR, while the protein-dependent biofilm is commonly associated to fibronectin-biding proteins, FnBPA and FnBPB, encoded by fnbA and fnbB genes. The aim of this work was to identify common genetic features in the regulatory regions of biofilm-related genes among clinical S. aureus strains derived from periprosthetic joint infections (PJI).  

Material and Methods: Genomes of 45 S. aureus strains from PJI were sequenced. Firstly, the sequence comprising the entire icaADBC regulatory region (5’UTR of icaADBC and icaR, the icaR coding sequence and its 3’UTR region) and secondly, the sequence of the promoter region of fnbAB were compared to those of S. aureus MW2 strain. Regulatory regions containing distinctive features were identified, fused to a reporter gene and introduced in a reference strain to analyze differences in gene expression.

Results: In the case of the icaADBC operon, single nucleotide polymorphisms (SNPs) in the icaADBC regulatory region allowed clustering of the strains in five groups from which a representative strain was chosen for further studies: S. aureus MIC 6924 (20% of isolates), MIC 6934 (13%), MIC 6936 (7%), MIC 6948 (2%) and MIC 7018 (4%). Of note, MICs 6948 and 7018 contained mutations in the icaR coding sequence. In this respect, a single nucleotide mutation in icaR (Val176Glu) caused a significant increase in icaADBC transcription and thus, in PIA/PNAG production and biofilm formation. In contrast, none of the rest of the SNPs found in the icaADBC regulatory region modified the transcription levels of the reporter gene. With respect to fnBPA and fnBPB genes and in agreement with previous studies, 100% of the strains contained the fnbA gene whereas only 69% contained the fnbB gene. The promoter region of fnbA was found to be highly conserved. SNPs in the promoter region of fnbB allowed clustering the strains in five groups. From these, the most frequently identified pattern was represented by S. aureus MIC 6948 (53%) and correlated with a lower level of reporter expression, whereas the group containing SNPs in the LexA binding sites was represented by MIC 7014 (4%) and correlated with higher expression levels.

Conclusion:  Our results suggest that S. aureus isolates from periprosthetic joint infections do not share specific features in cis regulatory regions of icaADBC and fnbB genes that may help to predict a higher expression levels of biofilm matrix compounds.

Acknowledgement: This project has received funding from the European Union's H2020 research and innovation programme under Marie Sklodowska-Curie grant agreement No 801586.

How to cite: Morales, L., Echeverz, M., Trobos, M., Solano, C., and Lasa, I.: Diversity in regulatory regions of icaADBCR and fnbAB genes among Staphylococcus aureus strains isolated from periprosthetic joint infections, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-71, https://doi.org/10.5194/biofilms9-71, 2020.

Genus Staphylococcus comprises many greatly pathogenic species like S. aureus, S. epidermidis or S. saprophyticus. The great pathogenicity of stated species is often facilitated by their capability to form thick complex biofilms on various biotic or abiotic surfaces. Biofilm formation together with extracellular hydrolases or toxins represents important virulence factor, which increases persistence of staphylococci in host via enhancing their ability to evade host immune system and further promote the infection development. With an increased emergence of antibiotic resistance among pathogenic bacteria including staphylococci the search for novel antibiotic compounds with antivirulence effect is sought. Such substances might be stilbenes, phenolic compounds isolated from various plants (Vitis spp., Vaccinium spp., Pterocarpus spp., Pinus spp.). They possess strong antioxidant activity and a wide spectrum of beneficial pharmacological effects (antitumor, hypolipidemic, hypoglycemic). Apart from that, they also have great antimicrobial activity with a potent ability to enhance antibiotics action in combination.

Presented work focused on resveratrol, pterostilbene (PTE) and pinosylvine and their effect on S. aureus and S. epidermidis biofilm formation. The effect of stilbene representatives on production of other virulence factors (proteases, phospholipases, haemolysins), cell surface hydrophobicity and morphology was also observed.

PTE was found to be the most effective among studied stilbenes against S. aureus and S. epidermidis biofilm with minimum biofilm inhibitory concentrations (MBIC₈₀) ranging from 40 to 130 mg/l. Its effect on mature staphylococcal biofilm eradication was even greater with 80% eradication rate achieved by 40-75 mg/l. PTE (49 mg/l) was found to have a potent combinatory antibiofilm activity with erythromycin or tetracycline (5 mg/l both) causing more than 80% inhibition in metabolic activity of biofilm cells. It was able to permeabilize cytoplasmic membrane, thus probably enabling antibiotic uptake by the cell. PTE also altered cell surface hydrophobicity and production of haemolysin.

PTE might be the solution to increasing biofilm-related resistance problem and a promising candidate with antibiofilm and antivirulence potential for future antibiotic treatment of staphylococcal infections.

This work was supported by the grant of Specific university research – grant No. A2_FPBT_2020_004.

How to cite: Kašparová, P. and Maťátková, O.: Antibiofilm and antivirulence effect of stilbenes on clinically relevant staphylococci, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-32, https://doi.org/10.5194/biofilms9-32, 2020.

Bacteria colonize most surfaces, forming multicellular, antibiotic-resistant, communities known as biofilms. Biofilms cause chronic infections and persistent biofouling of medical implants, marine vessels, and environmental sensors. Biofilm dispersal by nanomolar nitric oxide (NO) appears to be a general phenomenon, but fundamental questions remain concerning the identity of the NO sensor and mechanism of signal transduction. NO has been reported to disperse bacterial biofilms through regulation of intracellular cyclic-di-guanosine monophosphate concentrations. C-di-GMP is a tightly regulated second messenger-signaling molecule that is tightly correlated with biofilm formation. H-NOX proteins are well known NO sensors conserved in many bacteria. Indeed, we have shown that NO/H-NOX signaling disperses bacterial biofilms through a mechanism consistent with c-di-GMP signaling. However, H-NOX proteins are not conserved in most human pathogens. Therefore, an alternate NO sensor must also exist. We have identified a potential alternate NO sensor, a novel hemoprotein we named NosP (nitric oxide sensing protein). NosP domains are conserved in many bacterial genomes, they bind NO, but not molecular oxygen, as expected for a NO-specific sensor, and they are encoded as fusions with, or in close chromosomal proximity to, proteins annotated as c-di-GMP synthesis or hydrolysis enyzmes. We hypothesize that NO generally disperses bacterial biofilms through regulation of intracellular c-di-GMP concentrations, but the sensor varies; both NosP and H-NOX can fill this role.

How to cite: Boon, E.: Discovery and characterization of NO-responsive hemoproteins that regulate bacterial biofilms, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-20, https://doi.org/10.5194/biofilms9-20, 2020.