Biofilm is a major virulence determinant in bacterial colonization of chronic skin ulcers independently from the multidrug resistant phenotype

Bacterial biofilm is a major factor in delayed wound healing and high levels of biofilm production have been repeatedly described in multidrug resistant organisms (MDROs). Nevertheless, a quantitative correlation between biofilm production and the profile of antimicrobial drug resistance in delayed wound healing remains to be determined. Microbial identification, antibiotic susceptibility and biofilm production were assessed in 135 clinical isolates from 87 patients. Gram-negative bacteria were the most represented microorganisms (60.8%) with MDROs accounting for 31.8% of the total isolates. Assessment of biofilm production revealed that 80% of the strains were able to form biofilm. A comparable level of biofilm production was found with both MDRO and not-MDRO with no significant differences between groups. All the methicillin-resistant Staphylococcus aureus (MRSA) and 80% of Pseudomonas aeruginosa MDR strains were found as moderate/high biofilm producers. Conversely, less than 17% of Klebsiella pneumoniae extended-spectrum beta-lactamase (ESBL), Escherichia coli-ESBL and Acinetobacter baumannii were moderate/high biofilm producers. Notably, those strains classified as non-biofilm producers, were always associated with biofilm producer bacteria in polymicrobial colonization. This study shows that biofilm producers were present in all chronic skin ulcers, suggesting that biofilm represents a key virulence determinant in promoting bacterial persistence and chronicity of ulcerative lesions independently from the MDRO phenotype

Evaluating Streptococcus mutans strain dependent characteristics in a polymicrobial biofilm community

Aim: The purpose of this study was to investigate strain dependent differences of the cariogenic biofilm forming Streptococcus mutans within both simple and complex communities. Methods: A mono-species containing representative S. mutans clinical isolates (caries and non-caries), and a multispecies in vitro caries biofilm model containing Lactobacillus casei, Veillonella dispar, Fusobacterium nucleatum and Actinomyces naeslundii, and either of two representative S. mutans clinical isolates (caries and non-caries), was developed as a comparison model. Compositional analysis of total and live bacteria within biofilms, and transcriptional analysis of biofilm associated virulence factors were evaluated by live/dead PCR and quantitative PCR, respectively. Scanning electron microscopy (SEM) was used to analyze the architecture of biofilm. One-way analysis of variance and t-tests were used to investigate significant differences between independent groups of data. Results: Within a mono-species biofilm, different S. mutans strains responded similarly to one another during biofilm formation in different carbohydrate sources, with sucrose showing the highest levels of biofilm biomass and galactose showing the lowest. Within the polymicrobial biofilm system, compositional analysis of the bacteria within the biofilm showed that S. mutans derived from a caries-free patient was preferentially composed of both total and viable L. casei, whereas S. mutans derived from a caries patient was dominated by both total and viable S. mutans (p < 0.001). Normalized gene expression analysis of srtA, gtfB, ftf, spaP, gbpB, and luxS, showed a general upregulation within the S. mutans dominant biofilm. Conclusion: We were able to demonstrate that individual strains derived from different patients exhibited altered biofilm characteristics, which were not obvious within a simple mono-species biofilm model. Influencing the environmental conditions changed the composition and functionality S. mutans within the polymicrobial biofilm. The biofilm model described herein provides a novel and reproducible method of assessing the impact on the biofilm microbiome upon different environmental influences

Pharmacokinetic/Pharmacodynamic Correlation of Cefquinome Against Experimental Catheter-Associated Biofilm Infection Due to Staphylococcus aureus.

Biofilm formations play an important role in Staphylococcus aureus pathogenesis and contribute to antibiotic treatment failures in biofilm-associated infections. The aim of this study was to evaluate the pharmacokinetic/pharmacodynamic (PK/PD) profiles of cefquinome against an experimental catheter-related biofilm model due to S. aureus, including three clinical isolates and one non-clinical isolate. The minimal inhibitory concentration (MIC), minimal biofilm inhibitory concentration (MBIC), biofilm bactericidal concentration (BBC), minimal biofilm eradication concentration (MBEC) and biofilm prevention concentration (BPC) and in vitro time-kill curves of cefquinome were studied in both planktonic and biofilm cells of study S. aureus strains. The in vivo post-antibiotic effects (PAEs), PK profiles and efficacy of cefquinome were performed in the catheter-related biofilm infection model in murine. A sigmoid E max model was utilized to determine the PK/PD index that best described the dose-response profiles in the model. The MICs and MBICs of cefquinome for the four S. aureus strains were 0.5 and 16 μg/mL, respectively. The BBCs (32-64 μg/mL) and MBECs (64-256 μg/mL) of these study strains were much higher than their corresponding BPC values (1-2 μg/mL). Cefquinome showed time-dependent killing both on planktonic and biofilm cells, but produced much shorter PAEs in biofilm infections. The best-correlated PK/PD parameters of cefquinome for planktonic and biofilm cells were the duration of time that the free drug level exceeded the MIC (fT > MIC, R (2) = 96.2%) and the MBIC (fT > MBIC, R (2) = 94.7%), respectively. In addition, the AUC24h/MBIC of cefquinome also significantly correlated with the anti-biofilm outcome in this model (R (2) = 93.1%). The values of AUC24h/MBIC for biofilm-static and 1-log10-unit biofilm-cidal activity were 22.8 and 35.6 h; respectively. These results indicate that the PK/PD profiles of cefquinome could be used as valuable guidance for effective dosing regimens treating S. aureus biofilm-related infections

Growth limiting conditions and denitrification govern extent and frequency of volume detachment of biofilms

This study aims at evaluating the mechanisms of biofilm detachment with regard of the physical properties of the biofilm. Biofilms were developed in Couette–Taylor reactor under controlled hydrodynamic conditions and under different environmental growth conditions. Five different conditions were tested and lead to the formation of two aerobic heterotrophic biofilms (aeHB1 and aeHB2), a mixed autotrophic and heterotrophic biofilm (MAHB) and two anoxic heterotrophic biofilms (anHB1 and anHB2). Biofilm detachment was evaluated by monitoring the size of the detached particles (using light-scattering) as well as the biofilm physical properties (using CCD camera and image analysis). Results indicate that volume erosion of large biofilm particles with size ranging from 50 to 500 lm dominated the biomass loss for all biofilms. Surface erosion of small particles with size lower than 20 lm dominates biofilm detachment in number. The extent of the volume detachment events was governed by the size of the biofilm surface heterogeneities (i.e., the absolute biofilm roughness) but never impacted more than 80% of the mean biofilm thickness due to the highly cohesive basal layer. Anoxic biofilms were smoother and thinner than aerobic biofilms and thus associated with the detachment of smaller particles. Our results contradict the simplifying assumption of surface detachment that is considered in many biofilm models and suggest that discrete volume events should be considered

Transcriptomic and proteomic analyses of Desulfovibrio vulgaris biofilms: carbon and energy flow contribute to the distinct biofilm growth state.

BackgroundDesulfovibrio vulgaris Hildenborough is a sulfate-reducing bacterium (SRB) that is intensively studied in the context of metal corrosion and heavy-metal bioremediation, and SRB populations are commonly observed in pipe and subsurface environments as surface-associated populations. In order to elucidate physiological changes associated with biofilm growth at both the transcript and protein level, transcriptomic and proteomic analyses were done on mature biofilm cells and compared to both batch and reactor planktonic populations. The biofilms were cultivated with lactate and sulfate in a continuously fed biofilm reactor, and compared to both batch and reactor planktonic populations.ResultsThe functional genomic analysis demonstrated that biofilm cells were different compared to planktonic cells, and the majority of altered abundances for genes and proteins were annotated as hypothetical (unknown function), energy conservation, amino acid metabolism, and signal transduction. Genes and proteins that showed similar trends in detected levels were particularly involved in energy conservation such as increases in an annotated ech hydrogenase, formate dehydrogenase, pyruvate:ferredoxin oxidoreductase, and rnf oxidoreductase, and the biofilm cells had elevated formate dehydrogenase activity. Several other hydrogenases and formate dehydrogenases also showed an increased protein level, while decreased transcript and protein levels were observed for putative coo hydrogenase as well as a lactate permease and hyp hydrogenases for biofilm cells. Genes annotated for amino acid synthesis and nitrogen utilization were also predominant changers within the biofilm state. Ribosomal transcripts and proteins were notably decreased within the biofilm cells compared to exponential-phase cells but were not as low as levels observed in planktonic, stationary-phase cells. Several putative, extracellular proteins (DVU1012, 1545) were also detected in the extracellular fraction from biofilm cells.ConclusionsEven though both the planktonic and biofilm cells were oxidizing lactate and reducing sulfate, the biofilm cells were physiologically distinct compared to planktonic growth states due to altered abundances of genes/proteins involved in carbon/energy flow and extracellular structures. In addition, average expression values for multiple rRNA transcripts and respiratory activity measurements indicated that biofilm cells were metabolically more similar to exponential-phase cells although biofilm cells are structured differently. The characterization of physiological advantages and constraints of the biofilm growth state for sulfate-reducing bacteria will provide insight into bioremediation applications as well as microbially-induced metal corrosion

Small secreted proteins enable biofilm development in the cyanobacterium Synechococcus elongatus.

Small proteins characterized by a double-glycine (GG) secretion motif, typical of secreted bacterial antibiotics, are encoded by the genomes of diverse cyanobacteria, but their functions have not been investigated to date. Using a biofilm-forming mutant of Synechococcus elongatus PCC 7942 and a mutational approach, we demonstrate the involvement of four small secreted proteins and their GG-secretion motifs in biofilm development. These proteins are denoted EbfG1-4 (enable biofilm formation with a GG-motif). Furthermore, the conserved cysteine of the peptidase domain of the Synpcc7942_1133 gene product (dubbed PteB for peptidase transporter essential for biofilm) is crucial for biofilm development and is required for efficient secretion of the GG-motif containing proteins. Transcriptional profiling of ebfG1-4 indicated elevated transcript levels in the biofilm-forming mutant compared to wild type (WT). However, these transcripts decreased, acutely but transiently, when the mutant was cultured in extracellular fluids from a WT culture, and biofilm formation was inhibited. We propose that WT cells secrete inhibitor(s) that suppress transcription of ebfG1-4, whereas secretion of the inhibitor(s) is impaired in the biofilm-forming mutant, leading to synthesis and secretion of EbfG1-4 and supporting the formation of biofilms

Analysis of the molecular machinery implicated in multicellularity in bacillus cereus

Introduction: Bacillus cereus is a Gram-positive bacterium usually implicated in food poisoning outbreaks and human infections that sometimes result fatal. These events are closely related to the assembly of a biofilm that serves as a reservoir of cells, a nest for sporulation and protection from environmental stresses, host defenses or chemotherapy. Objectives: To perform a comprehensive comparative study of biofilm and planktonic cells to: i) delineate the molecular machinery implicated in the different steps of the biofilm life cycle, and ii) define new genes dedicated to biofilm formation. Materials & methods: Bacteria were grown under biofilm inducing conditions. Biofilm cells were separated from planktonic cells at different times and their whole mRNA was isolated, sequenced and analyzed. Results: Our results reveal a high number of genes associated to biofilm, many of them with unknown function, but highly conserved in others bacterial species. Besides, we found global changes in cell wall synthesis, metabolism and interspecies interaction molecules. ​ Conclusions: The interaction of B. cereus with other bacteria is conditioned by secondary metabolites, which are apparently overexpressed in biofilm. On the other hand, toxins are mainly expressed in planktonic cells, which are more oriented to interact with its hosts. These results reveal the defense and attacking positions of B. cereus in biofilm vs planktonic states.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Exploring the anti-biofilm activity of cinnamic acid derivatives in Candida albicans

Some compounds, characterized by phenylethenyl moiety, such as methyl cinnamate and caffeic acid phenethyl ester, are able to inhibit C. albicans biofilm formation. On these bases, and as a consequence of our previous work, we synthesized a series of cinnamoyl ester and amide derivatives in order to evaluate them for the activity against C. albicans biofilm and planktonically grown cells. The most active compounds 7 and 8 showed ⩾50% biofilm inhibition concentrations (BMIC50) of 2 μg/mL and 4 μg/mL respectively, against C. albicans biofilm formation; otherwise, 7 showed an interesting activity also against mature biofilm, with BMIC50 of 8 μg/m

Bacterial Biofilm and its Clinical Implications

Microbial biofilm created huge burden in treatment of both community and hospital infections. A biofilm is complex communities of bacteria attached to a surface or interface enclosed in an exopolysaccharide matrix and protected from unfavorable conditions such as presence of antibiotics, host defense or oxidative stresses. Biofilms are often considered hot spot for horizontal gene transfer among same or different bacterial species. Furthermore, bacteria with increased hydrophobicity facilitate biofilm formation by reducing repulsion between the extracellular matrix and the bacterium. There is a marked increase in the rate of persons nonresponsive to antibiotic therapy for infections of the Urinary Tract (UTIs), burns and upper respiratory tract due to biofilm formations. It is estimated that 90% of nosocomial infections are mediated by biofilm. The role of biofilm in infections has become so great that the treatment of such antibiotic resistance infections is proving difficult and costly to health care systems. The biofilm related infections varied from dental plaque, destruction of prosthetic valve to death of cystic fibrosis patients. This review aims to provide a summary of role of bacterial biofilm and its clinical implications for the patients