Malena Skogman

Combining biofilm matrix measurements with biomass and viability assays in susceptibility assessments of antimicrobials against Staphylococcus aureus biofilms

Despite that three types of assays (measuring biofilm viability, biomass, or matrix) are described to assess anti-biofilm activity, they are rarely used together. As infections can easily reappear if the matrix is not affected after antibiotic treatments, our goal was to explore the simultaneous effects of antibiotics on the viability, biomass and matrix of Staphylococcus aureus biofilms (ATCC 25923). Viability and biomass were quantified using resazurin and crystal violet staining sequentially in the same plate, while matrix staining was conducted with a wheat germ agglutinin-Alexa Fluor 488 fluorescent conjugate. Establishment of the detection limits and linearity ranges allowed concluding that all three methods were able to estimate biofilm formation in a similar fashion. In a susceptibility study with 18-h biofilms, two model compounds (penicillin G and ciprofloxacin) caused a reduction on the viability and biomass accompanied by an increase or not changed levels of the matrix, respectively. This response pattern was also proven for S. aureus Newman, S. epidermidis and E. coli biofilms. A classification of antibiotics based on five categories according to their effects on viability and matrix has been proposed earlier. Our data suggests a sixth group, represented by penicillin, causing decrease in bacterial viability but showing stimulatory effects on the matrix. Further, if effects on the matrix are not taken into account, the long-term chemotherapeutic effect of antibiotics can be jeopardized in spite of the positive effects on biofilms viability and biomass. Thus, measuring all these three endpoints simultaneously provide a more complete and accurate picture.

New derivatives of dehydroabietic acid target planktonic and biofilm bacteria in Staphylococcus aureus and effectively disrupt bacterial membrane integrity.

The combination of the dehydroabietic acid scaffold with different amino acids resulted in the discovery of a new class of hybrid compounds that targets both planktonic and biofilms bacteria in Staphylococcus aureus strains and are far more potent anti-biofilm agents than conventional antibiotics. Unlike dehydroabietic acid, these compounds can disrupt biofilms within a short time period and compromise the integrity of the bacterial membrane. Two of the compounds identified in our study are the most potent abietane-type anti-biofilm agents reported so far and display robust activity against pre-formed biofilms at concentrations only 3-6-fold higher than those required to inhibit biofilm formation. Their easy preparation based on proteolysis-resistant d- and unusual amino acids makes them useful chemical probes to gain a deeper understanding of bacterial biofilms and outstanding candidates for further development into new drugs to fight infections.

Prevention of Staphylococcus aureus biofilm formation by antibiotics in 96-Microtiter Well Plates and Drip Flow Reactors: critical factors influencing outcomes

Biofilm formation leads to the failure of antimicrobial therapy. Thus, biofilm prevention is a desirable goal of antimicrobial research. In this study, the efficacy of antibiotics (doxycycline, oxacillin and rifampicin) in preventing Staphylococcus aureus biofilms was investigated using Microtiter Well Plates (MWP) and Drip Flow Reactors (DFR), two models characterized by the absence and the presence of a continuous flow of nutrients, respectively. Planktonic culture of S. aureus was exposed to antibiotics for one hour followed by 24 hours incubation with fresh nutrients in MWP or continuous flow of nutrients in DFR. The DFR grown biofilms were significantly more tolerant to the antibiotics than those grown in MWP without the continuous flow. The differences in log reductions (LR) between the two models could not be attributed to differences in the cell density, the planktonic inoculum concentration or the surface-area-to-volume ratios. However, eliminating the flow in the DFR significantly restored the antibiotic susceptibility. These findings demonstrate the importance of considering differences between experimental conditions in different model systems, particularly the flow of nutrients, when performing anti-biofilm efficacy evaluations. Biofilm antibiotic efficacy studies should be assessed using various models and more importantly, in a model mimicking conditions of its clinical application.

Flavones as Quorum Sensing Inhibitors Identified by a Newly Optimized Screening Platform Using Chromobacterium violaceum as Reporter Bacteria

Quorum sensing (QS) is the process by which bacteria produce and detect signal molecules to coordinate their collective behavior. This intercellular communication is a relevant target for anti-biofilm therapies. Here we have optimized a screening-applicable assay to search for new quorum sensing inhibitors from natural compound libraries. In this system, QS is correlated with the production of violacein, which is directly controlled by the LuxI/LuxR system in Chromobacterium violaceum ATCC 31532. The parallel use of C. violaceum Tn5-mutant CV026, which depends on auto-inducer addition, allows simultaneous discrimination of compounds that act as quenchers of the AHL signal (quorum quenchers). The incorporation of a redox stain into the platform allowed further distinction between QS inhibitors, quorum quenchers and antibacterial compounds. A pilot screening was performed with 465 natural and synthetic flavonoids. All the most active compounds were flavones and they displayed potencies (IC50) in the range of 3.69 to 23.35 μM. These leads were particularly promising as they inhibited the transition from microcolonies into mature biofilms from Escherichia coli and Pseudomonas aeruginosa strains. This approach can be very effective in identifying new antimicrobials posing lesser risks of resistance.

Bioactive glass combined with bisphosphonates provides protection against biofilms formed by the periodontal pathogen Aggregatibacter actinomycetemcomitans

Biofilms play a pivotal role in the progression of periodontitis and they can be treated with antiseptics (i.e. chlorhexidine) or antibiotics, but these therapeutic alternatives are unable of ameliorating periodontal alveolar bone loss, which has been, on the other hand, successfully treated with bone-preserving agents. The improved bone formation achieved in animal models by the combination of two such agents: bioactive glass (BAG) and bisphosphonates has attracted the interest for further exploring dental applications. However, the antimicrobial effects that may result from combining them have not been yet investigated. Here, our aim was to explore the anti-biofilm effects that could result from combining BAG with bisphosphonates, particularly in a dental biofilm model. The experiments were performed with an oral cavity single-specie (Aggregatibacter actinomycetemcomitans) biofilm assay, which was optimized in this contribution. Risedronate displayed an intrinsic anti-biofilm effect, and all bisphosphonates, except clodronate, reduced biofilm formation when combined with BAG. In particular, the anti-biofilm activity of risedronate was significantly increased by the combination with BAG. Since it has been proposed that some of the antimicrobial effects of BAG are caused by local pH changes, studies of pH variations were performed to gain a mechanistic understanding. However, the observed anti-biofilm effects could not be explained with lowered pHs. Overall, these results do provide further support for the promising use of bisphosphonate-BAG combinations in dental applications. These findings are particularly relevant for patients undergoing cancer chemotherapy, or osteoporotic patients, which are known to be more vulnerable to periodontitis. In such cases, bisphosphonate treatment could play a double positive effect: local treatment of periodontitis (in combination with BAG) and systemic treatment of osteoporosis, prevention of hypercalcemia and metastases.

A Platform of Anti-biofilm Assays Suited to the Exploration of Natural Compound Libraries

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of antibiotic-tolerant infections. Biofilms have displayed an exceptionally high tolerance for chemotherapy, which is thought to be multifactorial. For instance, the matrix provides a physical barrier that decreases the penetration of antibiotics into the biofilm. Also, cells within the biofilms are phenotypically diverse. Likely, biofilm resilience arises from a combination of these and other, yet unknown, mechanisms. All of the currently existing antibiotics have been developed against single-cells (planktonic) bacteria. Therefore, so far, a very limited repertoire of molecules exists that can selectively act on mature biofilms. This situation has driven a progressive paradigm shift in drug discovery, in which searching for anti-biofilms has been urged to occupy a more prominent place. An additional challenge is that there are a very limited number of standardized methods for biofilm research, especially those that can be used for large-throughput screening of chemical libraries. Here, an experimental anti-biofilm platform for chemical screening is presented. It uses three assays to measure biofilm viability (with resazurin staining), total biomass (with crystal violet staining), and biofilm matrix (using a wheat germ agglutinin, WGA-fluorescence-based staining of the poly-N-acetyl-glucosamine, PNAG, fraction). All the assays were developed using Staphylococcus aureus as the model bacteria. Examples of how the platform can be used for primary screening as well as for functional characterization of identified anti-biofilm hits are presented. This experimental sequence further allows for the classification of the hits based upon the measured end-points. It also provides information on their mode of action, especially on long-term versus short-term chemotherapeutic effects. Thus, it is very advantageous for the quick identification of high-quality hit compounds that can serve as starting points for various biomedical applications.

Penicillin G increases the synthesis of a suicidal marker (CidC) and virulence (HlgBC) proteins in Staphylococcus aureus biofilm cells.

The present study reports the effect of Penicillin G (PenG) on the proteome dynamics of the Staphylococcus aureus strain Newman during biofilm mode of growth. The viability of the 18-h-old biofilm cells challenged with PenG at the concentration of 1mgmL(-1) was first assessed by plate counting, resazurin and LIVE/DEAD fluorescence staining, which indicated that the viability was reduced by ∼35% and ∼90% at 2h and 24h, respectively, after the addition of PenG. Subsequent two-dimensional difference gel electrophoresis (2D DIGE) assay of the treated and non-treated biofilm cells at the indicated time points revealed 45 proteins showing time- and treatment-specific change (1.5-fold, p<0.01). The 2D DIGE results suggested that the PenG-induced decrease in viability was accompanied by an increased synthesis of pyruvate oxidase (CidC), a suicidal marker known to potentiate acetate-dependent cell death in S. aureus. Increased abundance was also found for the TCA cycle associated malate-quinone oxidoreductase (Mqo), the ClpC ATPase, the HlgBC toxin and phage-associated proteins, which suggests that surviving cells have induced these activities as a last effort to overcome lethal doses of PenG. Proteomic results also revealed that the surviving cells were likely to strengthen their peptidoglycan due to the increased abundance of cell-wall biogenesis associated proteins, FemA and Pbp2; a phenomenon associated with dormancy in S. aureus.

Unsaturated Fatty Acids Affect Quorum Sensing Communication System and Inhibit Motility and Biofilm Formation of Acinetobacter baumannii

The increasing threat of Acinetobacter baumannii as a nosocomial pathogen is mainly due to the occurrence of multidrug-resistant strains that are associated with the real problem of its eradication from hospital wards. The particular ability of this pathogen to form biofilms contributes to its persistence, increases antibiotic resistance, and promotes persistent/device-related infections. We previously demonstrated that virstatin, which is a small organic compound known to decrease virulence of Vibrio cholera via an inhibition of T4-pili expression, displayed very promising activity to prevent A. baumannii biofilm development. Here, we examined the antibiofilm activity of mono-unsaturated chain fatty acids, palmitoleic (PoA), and myristoleic (MoA) acids, presenting similar action on V. cholerae virulence. We demonstrated that PoA and MoA (at 0.02 mg/mL) were able to decrease A. baumannii ATCC 17978 biofilm formation up to 38% and 24%, respectively, presented a biofilm dispersing effect and drastically reduced motility. We highlighted that these fatty acids decreased the expression of the regulator abaR from the LuxIR-type quorum sensing (QS) communication system AbaIR and consequently reduced the N-acyl-homoserine lactone production (AHL). This effect can be countered by addition of exogenous AHLs. Besides, fatty acids may have additional non-targeted effects, independent from QS. Atomic force microscopy experiments probed indeed that PoA and MoA could also act on the initial adhesion process in modifying the material interface properties. Evaluation of fatty acids effect on 22 clinical isolates showed a strain-dependent antibiofilm activity, which was not correlated to hydrophobicity or pellicle formation ability of the tested strains, and suggested a real diversity in cell-to-cell communication systems involved in A. baumannii biofilm formation.

Exploration of Microbial Communities Using the Thermo Scientific Varioskan LUX Multimode Reader and the Invitrogen EVOS FL Cell Imaging System

Introduction One of the most groundbreaking discoveries over the last 40 years has been the recognition of microbial biofilms as the predominant lifestyle of microorganisms, outside laboratory conditions. Bacterial biofilms are defined as aggregated, often sessile bacteria, which differ from free-floating cells by their slow growth and tolerance to antibiotics and immune cells.1 It is estimated that over 65% of microbial infections are biofilm-mediated, conferring biofilms a high clinical relevance.2 Moreover, in industrial settings, biofilms cause biofouling, corrosion, machinery damage and product contamination that cause delays, add cost and give rise to energy losses.3 In a biofilm, living cells integrate into a structured multicellular community that is enclosed by a self-produced polymeric matrix, composed of polysaccharides, extracellular DNA and proteins. As biofilm cells multiply, they dynamically produce matrix components, which make the 3D-community more stable and tolerant to external threats. Thus, the study of the biofilm matrix provides essential clues into the community’s functional behavior, which are critical for the understanding of biofilms from an industrial, environmental, biomedical, pharmaceutical and even purely microbiological perspective.