Antimicrobial Mechanisms of Leucocyte- and Platelet Rich Fibrin Exudate Against Planktonic Porphyromonas gingivalis and Within Multi-Species Biofilm: A Pilot Study

Leucocyte- and platelet rich fibrin (L-PRF) is an autologous biomaterial used in regenerative procedures. It has an antimicrobial activity against P. gingivalis although the mechanism is not fully understood. It was hypothesized that L-PRF exudate releases hydrogen peroxide and antimicrobial peptides that inhibit P. gingivalis growth. Agar plate and planktonic culture experiments showed that the antimicrobial effect of L-PRF exudate against P. gingivalis was supressed by peroxidase or pepsin exposure. In developing multi-species biofilms, the antimicrobial effect of L-PRF exudate was blocked only by peroxidase, increasing P. gingivalis growth with 1.3 log genome equivalents. However, no effect was shown on other bacteria. Pre-formed multi-species biofilm trials showed no antimicrobial effect of L-PRF exudate against P. gingivalis or other species. Our findings showed that L-PRF exudate may release peroxide and peptides, which may be responsible for its antimicrobial effect against P. gingivalis. In addition, L-PRF exudate had an antimicrobial effect against P. gingivalis in an in vitro developing multi-species biofilm.

Correction to: Differential production and secretion of potentially toxigenic extracellular proteins from hypervirulent Aeromonas hydrophila under biofilm and planktonic culture

An amendment to this paper has been published and can be accessed via the original article.

Protein interactions between the oral bacteria Fusobacterium nucleatum and Porphyromonas gingivalis in biofilm and planktonic culture

The opportunistic pathogens Fusobacterium nucleatum and Porphyromonas gingivalis are Gram-negative bacteria associated with oral biofilm and periodontal disease. Although liquid cultures are often the preferred cultivation method in microbiology, bacterial cells in biofilm adopt a profoundly different phenotype reflecting the close cell-to-cell contact compare to their planktonic counterparts. To investigate F. nucleatum and P. gingivalis interactions relevant in biofilm formation, we applied liquid chromatography-tandem mass spectrometry to determine the expressed proteome of F. nucleatum and P. gingivalis cells that were grown either as biofilm or in planktonic culture, and individually or together.The proteomic analyses detected 1,322 F. nucleatum and 966 P. gingivalis proteins. We statistically compared the proteins label-free quantitative (LFQ) intensities between biofilm and planktonic culture and identified significant changes (p-value ≤0.05) in 0,4% F. nucleatum proteins, 7% P. gingivalis proteins, and more than 14% of all proteins in the dual-species model. For both species, proteins involved in vitamin B2 (riboflavin) metabolic process had significantly increased levels in the biofilm condition. In both mono- and dual-species biofilm models, P. gingivalis increased the production of proteins functional in translation, oxidation-reduction, and amino acid metabolism, when compared to planktonic cultures. However, when we compared LFQ intensities between mono- and dual-species models, over 90% of the significantly changed P. gingivalis proteins had their levels reduced in biofilm and planktonic settings of the dual-species model.Our findings suggest that the two bacteria interact with each other at the protein level and indicate that P. gingivalis reduces the production of multiple proteins because of more favourable growth conditions provided by F. nucleatum presence. The results highlight the complex interactions of bacteria contributing to oral biofilm, which need to be considered in the design of future prevention strategies.

The Application of Impedance Spectroscopy for Pseudomonas Biofilm Monitoring during Phage Infection

Bacterial biofilm prevention and eradication are common treatment problems, hence there is a need for advanced and precise experimental methods for its monitoring. Bacterial resistance to antibiotics has resulted in an interest in using a natural bacterial enemy—bacteriophages. In this study, we present the application of quartz tuning forks (QTF) as impedance sensors to determine in real-time the direct changes in Pseudomonas aeruginosa PAO1 biofilm growth dynamics during Pseudomonas phage LUZ 19 treatment at different multiplicities of infection (MOI). The impedance of the electric equivalent circuit (EEC) allowed us to measure the series resistance (Rs) corresponding to the growth-medium resistance (planktonic culture changes) and the conductance (G) corresponding to the level of QTF sensor surface coverage by bacterial cells and the extracellular polymer structure (EPS) matrix. It was shown that phage impacts on sessile cells (G dynamics) was very similar in the 10-day biofilm development regardless of applied MOI (0.1, 1 or 10). The application of phages at an early stage (at the sixth h) and on three-day biofilm caused a significant slowdown in biofilm dynamics, whereas the two-day biofilm turned out to be insensitive to phage infection. We observed an inhibitory effect of phage infection on the planktonic culture (Rs dynamics) regardless of the MOI applied and the time point of infection. Moreover, the Rs parameter made it possible to detect PAO1 population regrowth at the latest time points of incubation. The number of phage-insensitive forms reached the level of untreated culture at around the sixth day of infection. We conclude that the proposed impedance spectroscopy technique can be used to measure the physiological changes in the biofilm matrix composition, as well as the condition of planktonic cultures in order to evaluate the activity of anti-biofilm compounds.

2606. A Divergent Ferrichrome Receptor Associated with an Insertional Element (IS3) Identified on Novel Locus in Clinical Strain of Pseudomonas aeruginosa

Background Pseudomonas aeruginosa is an aerobic Gram-negative bacterium that causes life-threatening acute and chronic infections in humans. Genotypic mutations and phenotypic variations are key features of its antimicrobial resistance and adaptation to the host environment. Pyoverdine associated genes and divergent receptors play a key role in acute Pseudomonas infections. This study seeks to address the heterogeneity of ferrichrome-iron receptor (fpvA) expression, its effect on pathogenicity and its propensity to cause acute infections clinically. Genetic and phenotypic variation of a clinical isolates of P. aeruginosa (PA097 and PA115) were identified by complete genome sequencing method. Methods An IRB-approved prospective study collected 38 P. aeruginosa clinical isolates and stored at Carilion Medical Center. Two genetically unrelated clinical strains were selected from tracheal aspirates: PA097 and PA115. These isolates were characterized by pyoverdine (pvd) quantification in planktonic culture filtrate at OD405 nm. Multiplex PCR was carried out using primers for fpvA receptors. Quantification of iron acquisition was done on chrome azurol S (CAS) agar. Genomes for PA115 and PA097 were sequenced by Illumina Next-generation DNA sequencing. Results Genome assembly shows a 6.3 Mb genome size in PA115 with G+C content of 66.4%. Seven insertion sequence elements were located. We found a 101 kb locus for pvD and a highly diversified fpvA associated with an insertional element (IS3). PA115, exhibits rich green pigment of pvd followed by PAO1 and PA097 in LB media (Figure 1A) and also in Planktonic culture filtrate (Fig 1B) for quantitative estimation of pvd (Figure 1C). On CAS agar, PA115 showed high uptake of iron by orange pigment compared with lower pigmentation in PAO1 and PA097 (Fig 1D). We confirmed the ferrichrome-iron receptor as fpvAIIb in PA115 by Multiplex PCR seen in sequencing of PA115 (Figure 2). Conclusion We found high genetic and phenotypic variation in clinical isolate of P. aeruginosa (PA115) from an acute pneumonia patient. The novel IS element found in its receptor gene locus suggests an increased role in pvd expression and iron uptake from the host. Increased pvd expression and diversified fpvAIIb association with an IS3 element may indicate higher virulence in the PA115 strain. Disclosures All authors: No reported disclosures.

Antimicrobial Activity of Selected Antimicrobial Peptides Against Planktonic Culture and Biofilm of Acinetobacter baumannii

Acinetobacter baumannii is one of the most challenging pathogens, on account of its predisposition to develop resistance leading to severe, difficult-to-treat infections. As these bacteria are more usually isolated from nosocomial infections, the new therapeutic options are demanded. Antimicrobial peptides (AMPs) are compounds likely to find application in the treatment of A. baumannii. These compounds exhibit a wide spectrum of antimicrobial activity and were found to be effective against biofilm. In this study, eight AMPs, namely aurein 1.2, CAMEL, citropin 1.1., LL-37, omiganan, r-omiganan, pexiganan, and temporin A, were tested for their antimicrobial activity. A reference strain of A. baumannii ATCC 19606 was used. Antimicrobial assays included determination of the minimum inhibitory concentration and the minimum biofilm eradication concentration. Considering the fact that the majority of A. baumannii infections are associated with mechanical ventilation and the use of indwelling devices, the activity against biofilm was assessed on both a polystyrene surface and tracheal tube fragments. In addition, cytotoxicity (HaCaT) was determined and in vitro selectivity index was calculated.