Crystal Violet Staining Alone Is Not Adequate to Assess Synergism or Antagonism in Multi-Species Biofilms of Bacteria Associated With Bacterial Vaginosis

Bacterial Vaginosis (BV) involves the presence of a multi-species biofilm adhered to vaginal epithelial cells, but its in-depth study has been limited due to the complexity of the bacterial community, which makes the design of in vitro models challenging. Perhaps the most common experimental technique to quantify biofilms is the crystal violet (CV) staining method. Despite its widespread utilization, the CV method is not without flaws. While biofilm CV quantification within the same strain in different conditions is normally accepted, assessing multi-species biofilms formation by CV staining might provide significant bias. For BV research, determining possible synergism or antagonism between species is a fundamental step for assessing the roles of individual species in BV development. Herein, we provide our perspective on how CV fails to properly quantify an in vitro triple-species biofilm composed of Gardnerella vaginalis, Fannyhessea (Atopobium) vaginae, and Prevotella bivia, three common BV-associated bacteria thought to play key roles in incident BV pathogenesis. We compared the CV method with total colony forming units (CFU) and fluorescence microscopy cell count methods. Not surprisingly, when comparing single-species biofilms, the relationship between biofilm biomass, total number of cells, and total cultivable cells was very different between each tested method, and also varied with the time of incubation. Thus, despite its wide utilization for single-species biofilm quantification, the CV method should not be considered for accurate quantification of multi-species biofilms in BV pathogenesis research.

Adaptation of the Start-Growth-Time Method for High-Throughput Biofilm Quantification

Colony forming unit (CFU) determination by agar plating is still regarded as the gold standard for biofilm quantification despite being time- and resource-consuming. Here, we propose an adaption of the high-throughput Start-Growth-Time (SGT) method from planktonic to biofilm analysis, which indirectly quantifies CFU/mL numbers by evaluating regrowth curves of detached biofilms. For validation, the effect of dalbavancin, rifampicin and gentamicin against mature biofilms of Staphylococcus aureus and Enterococcus faecium was measured by accessing different features of the viability status of the cell, i.e., the cultivability (conventional agar plating), growth behavior (SGT) and metabolic activity (resazurin assay). SGT correlated well with the resazurin assay for all tested antibiotics, but only for gentamicin and rifampicin with conventional agar plating. Dalbavancin treatment-derived growth curves showed a compared to untreated controls significantly slower increase with reduced cell doubling times and reduced metabolic rate, but no change in CFU numbers was observed by conventional agar plating. Here, unspecific binding of dalbavancin to the biofilm interfered with the SGT methodology since the renewed release of dalbavancin during detachment of the biofilms led to an unintended antimicrobial effect. The application of the SGT method for anti-biofilm testing is therefore not suited for antibiotics which stick to the biofilm and/or to the bacterial cell wall. Importantly, the same applies for the well-established resazurin method for anti-biofilm testing. However, for antibiotics which do not bind to the biofilm as seen for gentamicin and rifampicin, the SGT method presents a much less labor-intensive method suited for high-throughput screening of anti-biofilm compounds.

Evaluación in vitro de la formación de biopelículas de bacterias marinas del Caribe colombiano

Biofilm formation is an important process for marine bacterial communities because this mechanism favors adaptation to variations in environmental conditions. The objective of this study was to evaluate the biofilm formation of bacteria isolated from marine sediments under in vitro conditions. For this, biofilm quantification assays were performed using two methods, violet crystal and reduction of the XTT dye; In addition, the viability of the bacteria was evaluated by Live / Dead staining. Subsequently, the strains evaluated were identified using the 16S RNA marker. The results showed that the isolates belong to the genus Bacillus, all in different ranges were able to form filmsand strains B. safensis 64181 and Bacillus sp 64186 were selected which indicated greater production of this. Tests at different temperatures showed that for the strains selected the best temperature was 28 ° C. In addition, a mixed culture was carried out with these isolates, resultingin differences in the density of the biofilm and less changes in its metabolic activity in temperature variation experiments. From the results we can infer that bacterial consortiums can favor resistance to environmental variations in biofilms formed by bacteria of the genus Bacillus.

620. Sub-MIC Concentrations of Levofloxacin and Delafloxacin Enhance Staphylococcus aureus Biofilm Formation: Significance of Maximizing Exposure

Background Fluoroquinolones are utilized in Staphylococcal prosthetic joint infections due to their anti-biofilm activity. When antibiotic dosing is not optimized or antibiotics do not reach the site of infection, additional virulence factors may upregulate. We aimed to determine whether exposure to sub-MIC concentrations of levofloxacin and delafloxacin affect biofilm formation in Staphylococcus aureus. Methods This study utilized 50 diverse methicillin-susceptible S. aureus (MSSA) clinical isolates collected between 2004 and 2018. Sources included blood, skin/tissue, bone, and joint fluid. Minimum inhibitory concentrations and minimum bactericidal concentrations were identified according to CLSI. Biofilm assays were conducted as previously described by our program. Biofilm quantification was categorized as strong (OD570 ≥ 2), moderate (OD570 ≥ 1 and < 2), or weak (OD570 < 1). Prevention assays were conducted with the addition of increasing concentrations of delafloxacin or levofloxacin. We evaluated the amount of isolates that demonstrated increased biofilm formation in the presence of sub-MIC concentrations and extent of biofilm enhancement. Percent change was calculated between OD570 of the isolate growth control without antibiotic exposure and peak biofilm OD570 when exposed to the antibiotic. Results Of the 50 MSSA isolates, 14 (28%) exhibited moderate/strong formation and 36 (32%) exhibited weak biofilm formation. 52% and 58% of the isolates demonstrated a ≥50% increase in formation when exposed to sub-MIC concentrations of delafloxacin and levofloxacin, respectively. None of the strong biofilm formers demonstrated a ≥50% peak increase in formation when exposed to the antibiotics. Of the isolates that demonstrated a ≥50% peak increase, the average percent change was 267% (±29) with levofloxacin and 258% (±33) with delafloxacin. Conclusion Sub-MIC concentrations of delafloxacin and levofloxacin increased biofilm formation in S. aureus isolates that normally exhibit weak or moderate biofilm formation when not in the presence of antibiotics. Maintaining appropriate fluoroquinolone concentrations at the site of action is critical in preventing enhancement of biofilm formation. Further research is needed to identify the mechanism behind this increase. Disclosures All authors: No reported disclosures.

An Improved Crystal Violet Assay for Biofilm Quantification in 96-Well Microtitre Plate

Microplates are essential tools for biofilm research since it allows high throughput screening of biofilm forming strains or in the assay of anti-biofilm drugs. However, 96 well microtitre plate based assays share the issue of “edge effect”. The primary cause of the “edge effect” phenomenon is evaporation. As edge effect causes a significant increase in plate rejection rate by introducing experimental error, we improvised the classical crystal violet assay to reduce water loss from the peripheral wells. The improvised method showed a significant reduction in edge effect and minimised error in crystal violet assay

Antibiofilm and Antioxidant Activity of Propolis and Bud Poplar Resins versusPseudomonas aeruginosa

Pseudomonas aeruginosais a common biofilm-forming bacterial pathogen implicated in lung, skin, and systemic infections. Biofilms are majorly associated with chronic lung infection, which is the most severe complication in cystic fibrosis patients characterized by drug-resistant biofilms in the bronchial mucus with zones, where reactive oxygen species concentration is increased mainly due to neutrophil activity. Aim of this work is to verify the anti-Pseudomonasproperty of propolis or bud poplar resins extracts. The antimicrobial activity of propolis and bud poplar resins extracts was determined by MIC and biofilm quantification. Moreover, we tested the antioxidant activity by DPPH and neutrophil oxidative burst assays. In the end, both propolis and bud poplar resins extracts were able to inhibitP. aeruginosabiofilm formation and to influence both swimming and swarming motility. Moreover, the extracts could inhibit proinflammatory cytokine production by human PBMC and showed both direct and indirect antioxidant activity. This work is the first to demonstrate that propolis and bud poplar resins extracts can influence biofilm formation ofP. aeruginosacontrasting the inflammation and the oxidation state typical of chronic infection suggesting that propolis or bud poplar resins can be used along with antibiotic as adjuvant in the therapy againstP. aeruginosainfections related to biofilm.