Real-time monitoring of biofilm growth identifies andrographolide as a potent antifungal compound eradicating Candida biofilms.

Candida species cause life-threatening infections with high morbidity and mortality rates and their resistance to conventional therapy is closely linked to biofilm formation. Thus, the development of new approaches to study Candida biofilms and the identification of novel therapeutic strategies could yield improved clinical outcomes. In the current study, we have set up an impedance-based in vitro system to study Candida spp. biofilms in real-time and to evaluate their sensitivity to two conventional antifungal groups used in clinical practice - azoles and echinocandins. Both fluconazole and voriconazole were unable to inhibit biofilm formation in most strains tested, while echinocandins showed biofilm inhibitory capacity at relatively low concentrations (starting from 0.625 mg/L). However, assays performed on 24 h Candida albicans and C. glabrata biofilms revealed that micafungin and caspofungin failed to eradicate mature biofilms at all tested concentrations, evidencing that once formed, Candida spp. biofilms are extremely difficult to eliminate using currently available antifungals. We then evaluated the antifungal and anti-biofilm effect of andrographolide, a natural compound isolated from the plant Andrographis paniculata with known antibiofilm activity on Gram-positive and Gram-negative bacteria. Optical density measures, impedance evaluation, CFU counts, and electron microscopy data showed that andrographolide strongly inhibits planktonic Candida spp. growth and halts Candida spp. biofilm formation in a dose-dependent manner in all tested strains. Moreover, andrographolide was capable of eliminating mature biofilms and viable cell numbers by up to 99.9% in the C. albicans and C. glabrata strains tested, suggesting its potential as a new approach to treat multi-resistant Candida spp. biofilm-related infections.

Ficin-Cyclodextrin-Based Docking Nanoarchitectonics of Self-Propelled Nanomotors for Bacterial Biofilm Eradication.

Development of bioinspired nanomotors showing effective propulsion and cargo delivery capabilities has attracted much attention in the last few years due to their potential use in biomedical applications. However, implementation of this technology in realistic settings is still a barely explored field. Herein, we report the design and application of a multifunctional gated Janus platinum-mesoporous silica nanomotor constituted of a propelling element (platinum nanodendrites) and a drug-loaded nanocontainer (mesoporous silica nanoparticle) capped with ficin enzyme modified with ß-cyclodextrins (ß-CD). The engineered nanomotor is designed to effectively disrupt bacterial biofilms via H2O2-induced self-propelled motion, ficin hydrolysis of the extracellular polymeric matrix (EPS) of the biofilm, and controlled pH-triggered cargo (vancomycin) delivery. The effective synergic antimicrobial activity of the nanomotor is demonstrated in the elimination of Staphylococcus aureus biofilms. The nanomotor achieves 82% of EPS biomass disruption and a 96% reduction in cell viability, which contrasts with a remarkably lower reduction in biofilm elimination when the components of the nanomotors are used separately at the same concentrations. Such a large reduction in biofilm biomass in S. aureus has never been achieved previously by any conventional therapy. The strategy proposed suggests that engineered nanomotors have great potential for the elimination of biofilms.

Nitrate and a nitrate-reducing Rothia aeria strain as potential prebiotic or synbiotic treatments for periodontitis.

A few studies indicate that nitrate can reduce dysbiosis from a periodontitis point of view. However, these experiments were performed on samples from healthy individuals, and it is unknown if nitrate will be effective in periodontal patients, where the presence of nitrate-reducing bacteria is clearly reduced. The aim of this study was to test the effect of nitrate and a nitrate-reducing R. aeria (Ra9) on subgingival biofilms of patients with periodontitis. For this, subgingival plaque was incubated with 5 mM nitrate for 7 h (n = 20) or 50 mM nitrate for 12 h (n = 10), achieving a ~50% of nitrate reduction in each case. Additionally, Ra9 was combined with 5 mM nitrate (n = 11), increasing the nitrate reduced and nitrite produced (both p < 0.05). The addition of nitrate to periodontitis communities decreased biofilm mass (50 mM > 5 mM, both p < 0.05). Five millimolar nitrate, 50 mM nitrate and 5 mM nitrate + Ra9 led to 3, 28 and 20 significant changes in species abundance, respectively, which were mostly decreases in periodontitis-associated species. These changes led to a respective 15%, 63% (both p < 0.05) and 6% (not significant) decrease in the dysbiosis index. Using a 10-species biofilm model, decreases in periodontitis-associated species in the presence of nitrate were confirmed by qPCR (all p < 0.05). In conclusion, nitrate metabolism can reduce dysbiosis and biofilm growth of periodontitis communities. Five millimolar nitrate (which can be found in saliva after vegetable intake) was sufficient, while increasing this concentration to 50 mM (which could be achieved by topical applications such as a periodontal gel) increased the positive effects. Ra9 increased the nitrate metabolism of periodontitis communities and should be tested in vivo.

Personalized antibiotic selection in periodontal treatment improves clinical and microbiological outputs.

Introduction: Periodontitis is a biofilm-mediated disease that is usually treated by non-surgical biofilm elimination with or without antibiotics. Antibiotic treatment in periodontal patients is typically selected empirically or using qPCR or DNA hybridization methods. These approaches are directed towards establishing the levels of different periodontal pathogens in periodontal pockets to infer the antibiotic treatment. However, current methods are costly and do not consider the antibiotic susceptibility of the whole subgingival biofilm. Methods: In the current manuscript, we have developed a method to culture subgingival samples ex vivo in a fast, label-free impedance-based system where biofilm growth is monitored in real-time under exposure to different antibiotics, producing results in 4 hours. To test its efficacy, we performed a double-blind, randomized clinical trial where patients were treated with an antibiotic either selected by the hybridization method (n=32) or by the one with the best effect in the ex vivo growth system (n=32). Results: Antibiotic selection was different in over 80% of the cases. Clinical parameters such as periodontal pocket depth, attachment level, and bleeding upon probing improved in both groups. However, dental plaque was significantly reduced only in the group where antibiotics were selected according to the ex vivo growth. In addition, 16S rRNA sequencing showed a larger reduction in periodontal pathogens and a larger increase in health-associated bacteria in the ex vivo growth group. Discussion: The results of clinical and microbiological parameters, together with the reduced cost and low analysis time, support the use of the impedance system for improved individualized antibiotic selection.

Functional mgrA Influences Genetic Changes within a Staphylococcus aureus Cell Population over Time.

Prolonged survival in the host-bacteria microenvironment drives the selection of alternative cell types in Staphylococcus aureus, permitting quasi-dormant sub-populations to develop. These facilitate antibiotic tolerance, long-term growth, and relapse of infection. Small Colony Variants (SCV) are an important cell type associated with persistent infection but are difficult to study in vitro due to the instability of the phenotype and reversion to the normal cell type. We have previously reported that under conditions of growth in continuous culture over a prolonged culture time, SCVs dominated a heterogenous population of cell types and these SCVs harbored a mutation in the DNA binding domain of the gene for the transcription factor, mgrA. To investigate this specific cell type further, S. aureus WCH-SK2-ΔmgrA itself was assessed with continuous culture. Compared to the wild type, the mgrA mutant strain required fewer generations to select for SCVs. There was an increased rate of mutagenesis within the ΔmgrA strain compared to the wild type, which we postulate is the mechanism explaining the increased emergence of SCV selection. The mgrA derived SCVs had impeded metabolism, altered MIC to specific antibiotics and an increased biofilm formation compared to non-SCV strain. Whole genomic sequencing detected single nucleotide polymorphisms (SNP) in phosphoglucosamine mutase glmM and tyrosine recombinase xerC. In addition, several genomic rearrangements were detected which affected genes involved in important functions such as antibiotic and toxic metal resistance and pathogenicity. Thus, we propose a direct link between mgrA and the SCV phenotype. IMPORTANCE Within a bacterial population, a stochastically generated heterogeneity of phenotypes allows continual survival against current and future stressors. The generation of a sub-population of quasi-dormant Small Colony Variants (SCV) in Staphylococcus aureus is such a mechanism, allowing for persistent or relapse of infection despite initial intervention seemingly clearing the infection. The use of continuous culture under clinically relevant conditions has allowed us to introduce time to the growth system and selects SCV within the population. This study provides valuable insights into the generation of SCV which are not addressed in standard laboratory generated models and reveals new pathways for understanding persistent S. aureus infection which can potentially be targeted in future treatments of persistent S. aureus infection.

Real-time monitoring of Pseudomonas aeruginosa biofilm growth dynamics and persister cells' eradication.

Biofilm formation and the appearance of persister cells with low metabolic rates are key factors affecting conventional treatment failure and antibiotic resistance. Using impedance-based measurements, crystal violet staining and traditional culture we have studied the biofilm growth dynamics of 13 Pseudomonas aeruginosa strains under the effect of seven conventional antibiotics. Real-time growth quantifications revealed that the exposure of established P. aeruginosa biofilms to certain concentrations of ciprofloxacin, ceftazidime and tobramycin induced the emergence of persister cells, that showed different morphology and pigmentation, as well increased antibiotic resistance. Whole-genome sequencing of wildtype and persister cells identified several SNPs, a genomic inversion and a genomic duplication in one of the strains. However, these mutations were not uniquely associated with persisters, suggesting that the persistent phenotype may be related to metabolic and transcriptional changes. Given that mannitol has been proposed to activate bacterial metabolism, the synergistic combination of mannitol and ciprofloxacin was evaluated on clinical 48 h P. aeruginosa biofilms. When administered at doses ≥320 mg/L, mannitol was capable of preventing persister cell formation by efficiently activating dormant bacteria and making them susceptible to the antibiotic. These results were confirmed using viable colony counting. As the tested ciprofloxacin-mannitol combination appeared to fully eradicate mature biofilms, we conclude that impedance-based biofilm diagnostics, which permits antibiotic susceptibility testing and the identification of persister cells, is of great potential for the clinical practice and could aid in establishing treatment breakpoints for emerging biofilm-related infections.

Antibiofilm activity of flavonoids on staphylococcal biofilms through targeting BAP amyloids.

The opportunistic pathogen Staphylococcus aureus is responsible for causing infections related to indwelling medical devices, where this pathogen is able to attach and form biofilms. The intrinsic properties given by the self-produced extracellular biofilm matrix confer high resistance to antibiotics, triggering infections difficult to treat. Therefore, novel antibiofilm strategies targeting matrix components are urgently needed. The Biofilm Associated Protein, Bap, expressed by staphylococcal species adopts functional amyloid-like structures as scaffolds of the biofilm matrix. In this work we have focused on identifying agents targeting Bap-related amyloid-like aggregates as a strategy to combat S. aureus biofilm-related infections. We identified that the flavonoids, quercetin, myricetin and scutellarein specifically inhibited Bap-mediated biofilm formation of S. aureus and other staphylococcal species. By using in vitro aggregation assays and the cell-based methodology for generation of amyloid aggregates based on the Curli-Dependent Amyloid Generator system (C-DAG), we demonstrated that these polyphenols prevented the assembly of Bap-related amyloid-like structures. Finally, using an in vivo catheter infection model, we showed that quercetin and myricetin significantly reduced catheter colonization by S. aureus. These results support the use of polyphenols as anti-amyloids molecules that can be used to treat biofilm-related infections.

Effect of Dalbavancin on Staphylococcal Biofilms When Administered Alone or in Combination With Biofilm-Detaching Compounds.

Microorganisms grown in biofilms are more resistant to antimicrobial treatment and immune system attacks compared to their planktonic forms. In fact, infections caused by biofilm-forming Staphylococcus aureus and Staphylococcus epidermidis are a large threat for public health, including patients with medical devices. The aim of the current manuscript was to test the effect of dalbavancin, a recently developed lipoglycopeptide antibiotic, alone or in combination with compounds contributing to bacterial cell disaggregation, on staphylococcal biofilm formation and elimination. We used real-time impedance measurements in microtiter plates to study biofilm growth dynamics of S. aureus and S. epidermidis strains, in the absence or presence of dalbavancin, linezolid, vancomycin, cloxacillin, and rifampicin. Further experiments were undertaken to check whether biofilm-detaching compounds such as N-acetylcysteine (NAC) and ficin could enhance dalbavancin efficiency. Real-time dose-response experiments showed that dalbavancin is a highly effective antimicrobial, preventing staphylococcal biofilm formation at low concentrations. Minimum biofilm inhibitory concentrations were up to 22 higher compared to standard E-test values. Dalbavancin was the only antimicrobial that could halt new biofilm formation on established biofilms compared to the other four antibiotics. The addition of NAC decreased dalbavancin efficacy while the combination of dalbavancin with ficin was more efficient than antibiotic alone in preventing growth once the biofilm was established. Results were confirmed by classical biofilm quantification methods such as crystal violet (CV) staining and viable colony counting. Thus, our data support the use of dalbavancin as a promising antimicrobial to treat biofilm-related infections. Our data also highlight that synergistic and antagonistic effects between antibiotics and biofilm-detaching compounds should be carefully tested in order to achieve an efficient treatment that could prevent both biofilm formation and disruption.

Mobilisation Mechanism of Pathogenicity Islands by Endogenous Phages in Staphylococcus aureus clinical strains.

Staphylococcus aureus pathogenicity islands (SaPIs) are a type of mobile genetic element that play a significant role in the pathogenesis and virulence of this microorganism. SaPIs are integrated in the chromosome under the control of the master repressor Stl, but they can be horizontally transferred at a high frequency due to certain bacteriophages. Thus, a phage protein can bind to the SaPI Stl and induce the SaPI cycle, spreading the SaPI virulence factors to other bacterial populations. We report the dissemination mechanism of SaPIs mediated by endogenous prophages in S. aureus clinical strains. We reveal the induction of SaPIs by a co-resident prophage in seven clinically relevant strains, and we further study this mechanism in MW2, a community-acquired methicillin-resistant S. aureus strain that contains two bacteriophages (ɸSa2mw and ɸSa3mw) and one SaPI (SaPImw2) encoding for three enterotoxins (sec, sel and ear). ɸSa2mw was identified as responsible for SaPImw2 induction, and the specific phage derepressor protein DUF3113 was determined. The Stl-DUF3113 protein interaction was demonstrated, along with the existence of variants of this protein in S. aureus phages with different abilities to induce SaPI. Both Stl and DUF3113 are present in other Staphylococcus species, which indicates that this is a generalised mechanism.