Clinical Biofilm Ring Test® Reveals the Potential Role of ß-Lactams in the Induction of Biofilm Formation by P. aeruginosa in Cystic Fibrosis Patients.

Biofilms are characterized by high tolerance to antimicrobials. However, conventional antibiograms are performed on planktonic microorganisms. Through the clinical Biofilm Ring Test® (cBRT), initially aimed to measure the adhesion propensity of bacteria, we discerned a variable distribution of biofilm-producer strains among P. aeruginosa samples isolated from expectorations of cystic fibrosis (CF) patients. Despite a majority of spontaneous adherent isolates, few strains remained planktonic after 5 h of incubation. Their analysis by an adapted protocol of the cBRT revealed an induction of the biofilm early formation by sub-inhibitory doses of ß-lactams. Microscopic observations of bacterial cultures stained with Syto 9/Propidium Iodide (PI) confirmed the ability of antimicrobials to increase either the bacterial biomass or the biovolume occupied by induced sessile cells. Finally, the cBRT and its derivatives enabled to highlight in a few hours the potential inducer property of antibiotics on bacterial adhesion. This phenomenon should be considered carefully in the context of CF since patients are constantly under fluctuating antimicrobial treatments. To conclude, assays derived from the Biofilm Ring Test® (BRT) device, not only define efficient doses preventing biofilm formation, but could be useful for the antimicrobial selection in CF, to avoid inducer molecules of the early biofilm initiation.

Biofilm Formation of Listeria monocytogenes Strains Under Food Processing Environments and Pan-Genome-Wide Association Study.

Concerns about food contamination by Listeria monocytogenes are on the rise with increasing consumption of ready-to-eat foods. Biofilm production of L. monocytogenes is presumed to be one of the ways that confer its increased resistance and persistence in the food chain. In this study, a collection of isolates from foods and food processing environments (FPEs) representing persistent, prevalent, and rarely detected genotypes was evaluated for biofilm forming capacities including adhesion and sessile biomass production under diverse environmental conditions. The quantity of sessile biomass varied according to growth conditions, lineage, serotype as well as genotype but association of clonal complex (CC) 26 genotype with biofilm production was evidenced under cold temperature. In general, relative biofilm productivity of each strain varied inconsistently across growth conditions. Under our experimental conditions, there were no clear associations between biofilm formation efficiency and persistent or prevalent genotypes. Distinct extrinsic factors affected specific steps of biofilm formation. Sudden nutrient deprivation enhanced cellular adhesion while a prolonged nutrient deficiency impeded biofilm maturation. Salt addition increased biofilm production, moreover, nutrient limitation supplemented by salt significantly stimulated biofilm formation. Pan-genome-wide association study (Pan-GWAS) assessed genetic composition with regard to biofilm phenotypes for the first time. The number of reported genes differed depending on the growth conditions and the number of common genes was low. However, a broad overview of the ontology contents revealed similar patterns regardless of the conditions. Functional analysis showed that functions related to transformation/competence and surface proteins including Internalins were highly enriched.

Clinical Impact of Antibiotics for the Treatment of Pseudomonas aeruginosa Biofilm Infections.

Bacterial biofilms are highly recalcitrant to antibiotic therapies due to multiple tolerance mechanisms. The involvement of Pseudomonas aeruginosa in a wide range of biofilm-related infections often leads to treatment failures. Indeed, few current antimicrobial molecules are still effective on tolerant sessile cells. In contrast, studies increasingly showed that conventional antibiotics can, at low concentrations, induce a phenotype change in bacteria and consequently, the biofilm formation. Understanding the clinical effects of antimicrobials on biofilm establishment is essential to avoid the use of inappropriate treatments in the case of biofilm infections. This article reviews the current knowledge about bacterial growth within a biofilm and the preventive or inducer impact of standard antimicrobials on its formation by P. aeruginosa. The effect of antibiotics used to treat biofilms of other bacterial species, as Staphylococcus aureus or Escherichia coli, was also briefly mentioned. Finally, it describes two in vitro devices which could potentially be used as antibiotic susceptibility testing for adherent bacteria.

Association between biofilm formation phenotype and clonal lineage in Staphylococcus aureus strains from bone and joint infections.

Biofilm formation is a critical virulence factor responsible for treatment failure and chronicity in orthopaedic device-related infections (ODIs) caused by Staphylococcus aureus. Clonal lineages differ in terms of their biofilm forming capacities. The aim of this study was to investigate the correlation between the clonal complex (CC) affiliation and biofilm phenotype of 30 clinical S. aureus isolates responsible of ODI based on i) early biofilm formation using BioFilm Ring Test® and mature biofilm formation using crystal violet assays, ii) biofilm composition using DNase and proteinase K activity, and iii) prevention of biofilm formation by cloxacillin, teicoplanin and vancomycin using Antibiofilmogram® (biofilm minimal inhibitory concentration-bMIC). In terms of early biofilm formation, the CC30 strains were significantly slower than the CC5, CC15 and CC45 strains. CC45 strains produced significantly more mature biofilm than other group of strains did. The formation of biofilms was highly dependent on the presence of extracellular DNA in the CC5, CC15 and CC30 strains whereas it was mostly dependent on the presence of proteins in CC45. Finally, the CC30 group highlighted higher proportion of susceptible (bMIC < breakpoints of EUCAST guidelines) for cloxacillin, teicoplanin and vancomycin compared to the other CCs. These results demonstrate that the biofilm phenotype of clinical S. aureus isolates from ODIs is strongly related to their respective CC affiliation.

Tobramycin and Amikacin Delay Adhesion and Microcolony Formation in Pseudomonas aeruginosa Cystic Fibrosis Isolates.

Cystic fibrosis (CF) patients are predisposed to chronic colonization of the major airways by Pseudomonas aeruginosa biofilms. Pulmonary infections, involving sessile bacteria, are the main cause of morbidity and mortality. As the eradication of antibiotic-resistant biofilms remains impossible, one key objective for the treatment of lung infections is to delay the switch of P. aeruginosa to a sessile phenotype. Few tools are currently available in hospital laboratories to evaluate the susceptibility of adherent microorganisms to antimicrobials. In this study, we used the Biofilm Ring Test®, for the achievement of Antibiofilmograms® on CF clinical isolates. In comparison to standard antibiograms, these procedures allow the investigation of antibiotic effects on the biofilm formation by bacteria. To confirm the inter-assay reproducibility, conventional Crystal Violet assays were performed. To mimic the pathologic reality of CF, we also used a model allowing the biofilm growth on CF-derived cells. Results obtained from these three different assays showed that amikacin and tobramycin, the two favored aminoglycosides in CF therapies, were able to prevent the early adhesion of P. aeruginosa isolates. This promising inhibitory effect of antimicrobials confirm that biofilm setting up is governed by adaptive responses and depends on environmental conditions, as opposite processes of biofilm induction by aminoglycosides were previously described in literature. Finally, Antibiofilmograms®, whose given results are in concordance with other in vitro antibiotic susceptibility testing, appear to be useful for the optimisation of CF therapies by the selection of antimicrobials able to delay chronic infection establishment.

Kinetics of biofilm formation by Staphylococcus lugdunensis strains in bone and joint infections.

OBJECTIVE: To describe the clinical presentation and 1-year follow-up of patients with bone and joint infections (BJIs) caused by Staphylococcus lugdunensis and evaluate its biofilm-forming capacities. PATIENTS AND METHODS: Overall, 28 patients with BJIs from VISLISI clinical trials were included. We evaluated 1-year clinical follow-up and analyzed biofilm production kinetics of the 28 strains using the BioFilm Ring Test®. RESULTS: Of all patients, 12 had osteoarticular infections without material and 16 had prosthetic joint infections, of which 9 underwent a 1-stage revision procedure. At the 1-year follow-up, all patients were cured but needed a surgical intervention. Diabetes affected 46.4% of all patients. Of all, 20 strains (71.4%) started biofilm formation within 2 h, but all strains started the formation after 4 h experiment, and 25 strains (89.3%) reached a maximum after 6 h. CONCLUSIONS: This study describes the clinical and surgical management of BJIs caused by S. lugdunensis and shows that 1-stage prosthesis exchange procedures may be efficient. Further, It shows that biofilm production by this strain was not marginal and directly impacted clinical and surgical management.

Preliminary results of a new antibiotic susceptibility test against biofilm installation in device-associated infections: the Antibiofilmogram®.

Biofilms are complex communities of microorganisms embedded in an extracellular matrix and adherent to a surface. The development was described as a four-stage process leading to the formation of a mature biofilm which was resistant to immune system and antibiotic actions. In bone and joint infections (BJIs), the formation of biofilms is a leading cause of treatment failure. Here we study the capacity of 11 antibiotics commonly used in the treatment of BJIs to inhibit the biofilm formation on 29 clinical Staphylococcus aureus isolates by a new test called Antibiofilmogram(®) The minimal inhibitory concentration (MIC) and biofilm MIC (bMIC) were determined in vitro and showed similar values for clindamycin, fusidic acid, linezolid and rifampin. Reversely, daptomycin, fosfomycin, gentamicin and ofloxacin showed a bMIC distribution different from MIC with bMIC above breakpoint. Finally, cloxacillin, teicoplanin and vancomycin revealed an intermediate bMIC distribution with a strain-dependent pattern. A murine in vivo model of catheter-associated S. aureus infection was made and showed a significant reduction, but not total prevention, of catheter colonization with cloxacillin at bMIC, and no or limited reduction with cloxacillin at MIC. Antibiofilmogram(®) could be of great interest after surgical operations on contaminated prostheses and after bacteremia in order to prevent the colonization of the device.

The BioFilm Ring Test: a Rapid Method for Routine Analysis of Pseudomonas aeruginosa Biofilm Formation Kinetics.

Currently, few techniques are available for the evaluation of bacterial biofilm adhesion. These detection tools generally require time for culture and/or arduous handling steps. In this work, the BioFilm Ring Test (BRT), a new technology, was used to estimate the biofilm formation kinetics of 25 strains of Pseudomonas aeruginosa, isolated from the sputum of cystic fibrosis (CF) patients. The principle of the new assay is based on the mobility measurement of magnetic microbeads mixed with a bacterial suspension in a polystyrene microplate. If free to move under the magnetic action, particles gather to a visible central spot in the well bottom. Therefore, the absence of spot formation in the plate reflects the bead immobilization by a biofilm in formation. The BRT device allowed us to classify the bacterial strains into three general adhesion profiles. Group 1 consists of bacteria, which are able to form a solid biofilm in <2 h. Group 2 comprises the strains that progressively set up a biofilm during 24 h. Lastly, group 3 includes the strains that stay in a planktonic form. The grouping of our strains did not differ according to culture conditions, i.e., the use of different sets of beads or culture media. The BRT is shown to be an informative tool for the characterization of biofilm-forming bacteria. Various application perspectives may be investigated for this device, such as the addition of antibiotics to the bacterial suspension to select which would have the ability to inhibit the biofilm formation.