The role of Psl in the failure to eradicate Pseudomonas aeruginosa biofilms in children with cystic fibrosis

The exopolysaccharide Psl contributes to biofilm structure and antibiotic tolerance and may play a role in the failure to eradicate Pseudomonas aeruginosa from cystic fibrosis (CF) airways. The study objective was to determine whether there were any differences in Psl in P. aeruginosa isolates that were successfully eradicated compared to those that persisted, despite inhaled tobramycin treatment, in children with CF. Initial P. aeruginosa isolates were collected from children with CF undergoing eradication treatment, grown as biofilms and labeled with 3 anti-Psl monoclonal antibodies (Cam003/Psl0096, WapR001, WapR016) before confocal microscopy visualization. When grown as biofilms, P. aeruginosa isolates from children who failed antibiotic eradication therapy, had significantly increased Psl0096 binding compared to isolates from those who cleared P. aeruginosa. This was confirmed in P. aeruginosa isolates from the SickKids Eradication Cohort as well as the Early Pseudomonas Infection Control (EPIC) trial. Increased anti-Psl antibody binding was associated with bacterial aggregation and tobramycin tolerance. The biofilm matrix represents a potential therapeutic target to improve P. aeruginosa eradication treatment.

UV-C LED Irradiation Reduces Salmonella on Chicken and Food Contact Surfaces

Ultraviolet (UV-C) light-emitting diode (LED) light at a wavelength of 250–280 nm was used to disinfect skinless chicken breast (CB), stainless steel (SS) and high-density polyethylene (HD) inoculated with Salmonella enterica. Irradiances of 2 mW/cm2 (50%) or 4 mW/cm2 (100%) were used to treat samples at different exposure times. Chicken samples had the lowest Salmonella reduction with 1.02 and 1.78 Log CFU/cm2 (p ≤ 0.05) after 60 and 900 s, respectively at 50% irradiance. Higher reductions on CB were obtained with 100% illumination after 900 s (>3.0 Log CFU/cm2). Salmonella on SS was reduced by 1.97 and 3.48 Log CFU/cm2 after 60 s of treatment with 50% and 100% irradiance, respectively. HD showed a lower decrease of Salmonella, but still statistically significant (p ≤ 0.05), with 1.25 and 1.77 Log CFU/cm2 destruction for 50 and 100% irradiance after 60 s, respectively. Longer exposure times of HD to UV-C yielded up to 99.999% (5.0 Log CFU/cm2) reduction of Salmonella with both irradiance levels. While UV-C LED treatment was found effective to control Salmonella on chicken and food contact surfaces, we propose three mechanisms contributing to reduced efficacy of disinfection: bacterial aggregation, harboring in food and work surface pores and light absorption by fluids associated with CB.

Thermonucleases Contribute to Staphylococcus aureus Biofilm Formation in Implant-Associated Infections–A Redundant and Complementary Story

Biofilms formed by Staphylococcus aureus are one of the predominant causes of implant-associated infections (IAIs). Previous studies have found that S. aureus nucleases nuc1 and nuc2 modulate biofilm formation. In this study, we found low nuc1/nuc2 expression and high biofilm-forming ability among IAI isolates. Furthermore, in a mouse model of exogenous IAIs, Δnuc1/2 exhibited higher bacterial load on the surface of the implant than that exhibited by the other groups (WT, Δnuc1, and Δnuc2). Survival analysis of the hematogenous IAI mouse model indicated that nuc1 is a virulence factor related to mortality. We then detected the influence of nuc1 and nuc2 on biofilm formation and immune evasion in vitro. Observation of in vitro biofilm structures with scanning electron microscopy and evaluation of bacterial aggregation with flow cytometry revealed that both nuc1 and nuc2 are involved in biofilm structuring and bacterial aggregation. Unlike nuc1, which is reported to participate in immune evasion, nuc2 cannot degrade neutrophil extracellular traps. Moreover, we found that nuc1/nuc2 transcription is negatively correlated during S. aureus growth, and a possible complementary relationship has been proposed. In conclusion, nuc1/nuc2 are complementary genes involved in biofilm formation in exogenous IAIs. However, nuc2 contributes less to virulence and is not involved in immune evasion.

The depletion mechanism can actuate bacterial aggregation by self-produced exopolysaccharides and determine species distribution and composition in bacterial aggregates

Bacteria causing chronic infections are often found in cell aggregates suspended in polymer secretions, and aggregation may be a factor in infection persistence. One aggregation mechanism, called depletion aggregation, is driven by physical forces between bacteria and polymers. Here we investigated whether the depletion mechanism can actuate the aggregating effects of P. aeruginosa exopolysaccharides for suspended (i.e. not surface attached) bacteria, and how depletion affects bacterial inter-species interactions. We found cells overexpressing the exopolysaccharides Pel and Psl, but not alginate remained aggregated after depletion-mediating conditions were reversed. In co-culture, d epletion aggregation had contrasting effects on P. aeruginosa’s interactions with coccus- and rod-shaped bacteria. Depletion caused S. aureus (cocci) and P. aeruginosa (rods) to segregate from each other, S. aureus to resist secreted P. aeruginosa antimicrobial factors, and the species to co-exist. In contrast , depletion aggregation caused P. aeruginosa and Burkholderia sp. to intermix, enhancing type VI secretion inhibition of Burkholderia by P. aeruginosa , leading to P. aeruginosa dominance . These results show that in addition to being a primary cause of aggregation in polymer-rich suspensions, physical forces inherent to the depletion mechanism can actuate the aggregating effects of self-produced exopolysaccharides and determine species distribution and composition of bacterial communities.

Staphylococcus aureus Floating Biofilm Formation and Phenotype in Synovial Fluid Depends on Albumin, Fibrinogen, and Hyaluronic Acid

Biofilms are typically studied in bacterial media that allow the study of important properties such as bacterial growth. However, the results obtained in such media cannot take into account the bacterial localization/clustering caused by bacteria–protein interactions in vivo and the accompanying alterations in phenotype, virulence factor production, and ultimately antibiotic tolerance. We and others have reported that methicillin-resistant or methicillin-susceptible Staphylococcus aureus (MRSA or MSSA, respectively) and other pathogens assemble a proteinaceous matrix in synovial fluid. This proteinaceous bacterial aggregate is coated by a polysaccharide matrix as is characteristic of biofilms. In this study, we identify proteins important for this aggregation and determine the concentration ranges of these proteins that can reproduce bacterial aggregation. We then test this protein combination for its ability to cause marked aggregation, antibacterial tolerance, preservation of morphology, and expression of the phenol-soluble modulin (PSM) virulence factors. In the process, we create a viscous fluid that models bacterial behavior in synovial fluid. We suggest that our findings and, by extension, use of this fluid can help to better model bacterial behavior of new antimicrobial therapies, as well as serve as a starting point to study host protein–bacteria interactions characteristic of physiological fluids.

Fine tuning the mechanism of Antigen 43 self-association to modulate aggregation levels of Escherichia coli pathogens

Bacterial aggregates and biofilms allow bacteria to colonise a diverse array of surfaces that can ultimately lead to infections, where the protection they afford permits bacteria to resist anti-microbials and host immune factors. Despite these advantages there is a trade-off, whereby bacterial spread is reduced. As such, biofilm development needs to be regulated appropriately to suit the required niche. Here we investigate members from one of largest groups of bacterial adhesins, the autotransporters, for their critical role in the formation of bacterial aggregates and biofilms. We describe the structural and functional characterisation of autotransporter Ag43-homologues from diverse pathogenic Escherichia strains. We reveal a common mode of trans-association that leads to cell clumping and show that subtle variations in these interactions governs their aggregation kinetics. Our in depth investigation reveals an underlying molecular basis for the 'tuning' of bacterial aggregation.

Biofilm Formation on Dental Implant Biomaterials by Staphylococcus aureus Strains Isolated from Patients with Cystic Fibrosis

Implants made of ceramic and metallic elements, which are used in dentistry, may either promote or hinder the colonization and adhesion of bacteria to the surface of the biomaterial to varying degrees. The increased interest in the use of dental implants, especially in patients with chronic systemic diseases such as cystic fibrosis (CF), is caused by an increase in disease complications. In this study, we evaluated the differences in the in vitro biofilm formation on the surface of biomaterials commonly used in dentistry (Ti-6Al-4V, cobalt-chromium alloy (CoCr), and zirconia) by Staphylococcus aureus isolated from patients with CF. We demonstrated that S. aureus adherence and growth depends on the type of material used and its surface topography. Weaker bacterial biofilm formation was observed on zirconia surfaces compared to titanium and cobalt-chromium alloy surfaces. Moreover, scanning electron microscopy showed clear differences in bacterial aggregation, depending on the type of biomaterial used. Over the past several decades, S. aureus strains have developed several mechanisms of resistance, especially in patients on chronic antibiotic treatment such as CF. Therefore, the selection of an appropriate implant biomaterial with limited microorganism adhesion characteristics can affect the occurrence and progression of oral cavity infections, particularly in patients with chronic systemic diseases.