RESUMO
AIMS: We present a dynamic typodont biofilm model (DTBM) incorporating (1) human dentition anatomy, (2) fluid flow over intermittently fluid bathed tooth surfaces and (3) an oxic headspace to allow aerobic and anaerobic niches to develop naturally, as a screening tool to assess the effect of stannous fluoride (SnF2 ) toothpaste against a simulated human plaque biofilm (SPB). METHODS AND RESULTS: First, hydroxyapatite (HA) coupons were inoculated with human saliva/plaque and cultured at 37°C under air. Selected species representative of common commensal and anaerobic pathogens were quantified for relative abundance changes over 4 days by PCR densitometry to confirm the culture conditions allowed the proliferation of these species. A continuous culture DTBM reactor on a rocker table was inoculated with saliva/plaque and incubated at 37°C for 24 h. Tooth shear stress was estimated by particle tracking. A SnF2 toothpaste solution, or a sham rise was administered twice daily for 3 days to mimic routine oral hygiene. SPB biomass was assessed by total bacterial DNA and methylene blue (MB) staining. Early colonizer aerobes and late colonizer anaerobes species were detected in the HA and DTBM, and the trends in changing abundance were consistent with those seen clinically. CONCLUSIONS: Treatment with the SnF2 solution showed significant reductions of 53.05% and 54.4% in the SPB by MB staining and DNA, respectively. SIGNIFICANCE AND IMPACT OF STUDY: The model has potential for assessing dentition anatomy and fluid flow on the efficacy of antimicrobial efficacy against localized SPB and may be amenable to the plaque index clinical evaluation.
Assuntos
Fluoretos de Estanho , Cremes Dentais , Biofilmes , Humanos , Saliva , Fluoretos de Estanho/uso terapêutico , Cremes Dentais/farmacologia , Cremes Dentais/uso terapêuticoRESUMO
In recent years, Acinetobacter baumannii has emerged as a major cause of nosocomial infections, including infections of implanted medical devices. The treatment of infections caused by A. baumannii has been severely hampered due to their frequent resistance to currently available antibiotics, and most importantly the ability of A. baumannii to form biofilms, which plays a significant role in both persistence and antibiotic resistance. The inherent resistance of A. baumannii biofilms to host defenses and antimicrobial agents necessitates the search for novel approaches to deter biofilm formation. Here, we report our findings on nine compounds identified from structure-activity relationship (SAR) studies on an antibiofilm compound LP3134 that was reported earlier by Biofouling2014, 30, 17. Compounds were evaluated for antibiofilm and anti-adherence activities against A. baumannii. The ability of the compounds to prevent biofilm development on urinary catheters was studied. Growth curve experiments indicated that compounds did not affect the planktonic growth of A. baumannii. All compounds inhibited A. baumannii biofilm development as well as impacting early adhesion on abiotic surfaces. Seven compounds were able to deter biofilm development on silicone catheters. Due to the continued rise of emerging multidrug-resistant A. baumannii, results from this study provide foundation for further development of these molecules to treat A. baumannii infections in wounds and medical devices.
Assuntos
Acinetobacter baumannii/efeitos dos fármacos , Biofilmes/efeitos dos fármacos , Hidrazinas/síntese química , Animais , Antibacterianos/síntese química , Antibacterianos/farmacologia , Linhagem Celular , Humanos , Hidrazinas/farmacologia , Camundongos , Células RAW 264.7 , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Biosurfactants (BS) are amphiphilic compounds produced by microbes, either on the cell surface or secreted extracellularly. BS exhibit strong antimicrobial and anti-adhesive properties, making them good candidates for applications used to combat infections. In this study, our goal was to assess the in vitro antimicrobial, anti-adhesive and anti-biofilm abilities of BS produced by Lactobacillus jensenii and Lactobacillus rhamnosus against clinical Multidrug Resistant (MDR) strains of Acinetobacter baumannii, Escherichia coli, and Staphylococcus aureus (MRSA). Cell-bound BS from both L. jensenii and L. rhamnosus were extracted and isolated. The surface activities of crude BS samples were evaluated using an oil spreading assay. The antimicrobial, anti-adhesive and anti-biofilm activities of both BS against the above mentioned MDR pathogens were determined. RESULTS: Surface activities for both BS ranged from 6.25 to 25 mg/ml with clear zones observed between 7 and 11 cm. BS of both L. jensenii and L. rhamnosus showed antimicrobial activities against A. baumannii, E. coli and S. aureus at 25-50 mg/ml. Anti-adhesive and anti-biofilm activities were also observed for the aforementioned pathogens between 25 and 50 mg/ml. Finally, analysis by electron microscope indicated that the BS caused membrane damage for A. baumannii and pronounced cell wall damage in S. aureus. CONCLUSION: Our results indicate that BS isolated from two Lactobacilli strains has antibacterial properties against MDR strains of A. baumannii, E. coli and MRSA. Both BS also displayed anti-adhesive and anti-biofilm abilities against A. baumannii, E. coli and S. aureus. Together, these capabilities may open up possibilities for BS as an alternative therapeutic approach for the prevention and/or treatment of hospital-acquired infections.
Assuntos
Acinetobacter baumannii/efeitos dos fármacos , Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Lactobacillus/química , Staphylococcus aureus/efeitos dos fármacos , Tensoativos/farmacologia , Acinetobacter baumannii/fisiologia , Antibacterianos/isolamento & purificação , Aderência Bacteriana/efeitos dos fármacos , Escherichia coli/fisiologia , Staphylococcus aureus/fisiologia , Tensoativos/isolamento & purificaçãoRESUMO
Extracellular DNA (eDNA) is an important component of the extracellular polymeric substance matrix and is important in the establishment and persistence of Staphylococcus aureus UAMS-1 biofilms. The aim of the study was to determine the temporal expression of genes involved in early biofilm formation and eDNA production. We used qPCR to investigate expression of agrB, which is associated with secreted virulence factors and biofilm dispersal, cidA, which is associated with biofilm adherence and genomic DNA release, and alsS, which is associated with cell lysis, eDNA release and acid tolerance. The contribution of eDNA to the stability of the biofilm matrix was assessed by digesting with DNase I (Pulmozyme) and quantifying structure by confocal microscopy and comstat image analysis. AgrB expression initially increased at 24 h but then dramatically decreased at 72 h in an inverse relationship to biomass, supporting its role in regulating biofilm dispersal. cidA and alsS expression steadily increased over 72 h, suggesting that eDNA was an important component of early biofilm development. DNase I had no effect on biomass, but did cause the biofilms to become more heterogeneous. Carbohydrates in the matrix appeared to play an important role in structural stability.
Assuntos
Proteínas de Bactérias/genética , Biofilmes/crescimento & desenvolvimento , Carboidratos , DNA Bacteriano , Regulação Bacteriana da Expressão Gênica , Staphylococcus aureus/fisiologia , Desoxirribonuclease I/metabolismo , Espaço Extracelular/metabolismo , Humanos , Staphylococcus aureus/isolamento & purificaçãoRESUMO
Biofilm formation by pathogenic bacteria is an important virulence factor in the development of numerous chronic infections, thereby causing a severe health burden. Many of these infections cannot be resolved, as bacteria in biofilms are resistant to the host's immune defenses and antibiotic therapy. An urgent need for new strategies to treat biofilm-based infections is critically needed. Cyclic di-GMP (c-di-GMP) is a widely conserved second-messenger signal essential for biofilm formation. The absence of this signalling system in higher eukaryotes makes it an attractive target for the development of new anti-biofilm agents. In this study, the results of an in silico pharmacophore-based screen to identify small-molecule inhibitors of diguanylate cyclase (DGC) enzymes that synthesize c-di-GMP are described. Four small molecules, LP 3134, LP 3145, LP 4010 and LP 1062 that antagonize these enzymes and inhibit biofilm formation by Pseudomonas aeruginosa and Acinetobacter baumannii in a continuous-flow system are reported. All four molecules dispersed P. aeruginosa biofilms and inhibited biofilm development on urinary catheters. One molecule dispersed A. baumannii biofilms. Two molecules displayed no toxic effects on eukaryotic cells. These molecules represent the first compounds identified from an in silico screen that are able to inhibit DGC activity to prevent biofilm formation.
Assuntos
Biofilmes/efeitos dos fármacos , Proteínas de Escherichia coli/antagonistas & inibidores , Fósforo-Oxigênio Liases/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/fisiologia , Simulação por Computador , GMP Cíclico/metabolismo , GMP Cíclico/fisiologia , Células HEK293 , Humanos , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/fisiologia , Bibliotecas de Moléculas PequenasRESUMO
Acinetobacter baumannii infections are difficult to treat due to multidrug resistance. Biofilm formation by A. baumannii is an additional factor in its ability to resist antimicrobial therapy. The antibacterial and antibiofilm activities of the human antimicrobial peptide LL-37 and its fragments KS-30, KR-20 and KR-12 against clinical isolates of multidrug-resistant (MDR) A. baumannii were evaluated. The minimal inhibitory concentration (MIC) of LL-37 against MDR A. baumannii isolates ranged from 16 to 32 µg/mL. The MIC of KS-30 fragment varied from 8.0 to 16 µg/mL and the KR-20 fragment MIC ranged from 16 to 64 µg/mL. LL-37 and KS-30 fragment exhibited 100% bactericidal activity against five A. baumannii strains, including four MDR clinical isolates, within 30 min at concentrations of 0.25-1 µg/mL. By 0.5h, the fragments KR-20 and KR-12 eliminated all tested strains at 8 and 64 µg/mL respectively. LL-37 and its fragments displayed anti-adherence activities between 32-128 µg/mL. A minimum biofilm eradication concentration (MBEC) biofilm assay demonstrated that LL-37 inhibited and dispersed A. baumannii biofilms at 32 µg/mL respectively. Truncated fragments of LL-37 inhibited biofilms at concentrations of 64-128 µg/mL. KS-30, the truncated variant of LL-37, effectively dispersed biofilms at 64 µg/mL. At 24h, no detectable toxicity was observed at the efficacious doses when cytotoxicity assays were performed. Thus, LL-37, KS-30 and KR-20 exhibit significant antimicrobial activity against MDR A. baumannii. The prevention of biofilm formation in vitro by LL-37, KS-30 and KR-20 adds significance to their efficacy. These peptides can be potential therapeutics against MDR A. baumannii infections.
Assuntos
Acinetobacter baumannii/efeitos dos fármacos , Biofilmes/efeitos dos fármacos , Catelicidinas/farmacologia , Resistência Microbiana a Medicamentos , Resistência a Múltiplos Medicamentos , Sequência de Aminoácidos , Peptídeos Catiônicos Antimicrobianos , Catelicidinas/química , Testes de Sensibilidade Microbiana , Dados de Sequência MolecularRESUMO
Bacterial biofilm formation is responsible for numerous chronic infections, causing a severe health burden. Many of these infections cannot be resolved, as bacteria in biofilms are resistant to the host's immune defenses and antibiotic therapy. New strategies to treat biofilm-based infections are critically needed. Cyclic di-GMP (c-di-GMP) is a widely conserved second-messenger signal essential for biofilm formation. As this signaling system is found only in bacteria, it is an attractive target for the development of new antibiofilm interventions. Here, we describe the results of a high-throughput screen to identify small-molecule inhibitors of diguanylate cyclase (DGC) enzymes that synthesize c-di-GMP. We report seven small molecules that antagonize these enzymes and inhibit biofilm formation by Vibrio cholerae. Moreover, two of these compounds significantly reduce the total concentration of c-di-GMP in V. cholerae, one of which also inhibits biofilm formation by Pseudomonas aeruginosa in a continuous-flow system. These molecules represent the first compounds described that are able to inhibit DGC activity to prevent biofilm formation.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Biofilmes/efeitos dos fármacos , Proteínas de Escherichia coli/antagonistas & inibidores , Fósforo-Oxigênio Liases/antagonistas & inibidores , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/metabolismo , Vibrio cholerae/efeitos dos fármacos , Vibrio cholerae/metabolismoRESUMO
Bacterial biofilm formation causes significant industrial economic loss and high morbidity and mortality in medical settings. Biofilms are defined as multicellular communities of bacteria encased in a matrix of protective extracellular polymers. Because biofilms have a high tolerance for treatment with antimicrobials, protect bacteria from immune defense, and resist clearance with standard sanitation protocols, it is critical to develop new approaches to prevent biofilm formation. Here, a novel benzimidazole molecule, named antibiofilm compound 1 (ABC-1), identified in a small-molecule screen, was found to prevent bacterial biofilm formation in multiple Gram-negative and Gram-positive bacterial pathogens, including Pseudomonas aeruginosa and Staphylococcus aureus, on a variety of different surface types. Importantly, ABC-1 itself does not inhibit the growth of bacteria, and it is effective at nanomolar concentrations. Also, coating a polystyrene surface with ABC-1 reduces biofilm formation. These data suggest ABC-1 is a new chemical scaffold for the development of antibiofilm compounds.
Assuntos
Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Benzimidazóis/farmacologia , Biofilmes/efeitos dos fármacos , Pseudomonas aeruginosa/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacosRESUMO
BACKGROUND: Staphylococcus aureus is an important pathogen that forms biofilms. The global regulator sarA is essential for biofilm formation. Since the modulator of sarA (msa) is required for full expression of sarA and regulates several virulence factors, we examined the capacity of the msa mutant to form biofilm. RESULTS: We found that mutation of msa results in reduced expression of sarA in biofilm and that the msa mutant formed a weak and unstable biofilm. The msa mutant is able to adhere to surfaces and begins to form biofilm but fails to mature indicating that the defect of the msa mutant biofilm is in the accumulation stage but not in primary adhesion. CONCLUSION: The msa gene plays an important role in biofilm development which is likely due to its role in modulating the expression of sarA. This finding is significant because it identifies a new gene that plays a role in the development of biofilm.
Assuntos
Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus aureus/genética , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Mutação/genética , Staphylococcus aureus/metabolismo , Transativadores/metabolismoRESUMO
BACKGROUND: Streptococcus pneumoniae is a common respiratory pathogen and a major causative agent of respiratory infections, including otitis media (OM). Pneumococcal biofilms have been demonstrated on biopsies of the middle ear mucosa in children receiving tympanostomy tubes, supporting the hypothesis that chronic OM may involve biofilm development by pathogenic bacteria as part of the infectious process. To better understand pneumococcal biofilm formation six low-passage encapsulated nasopharyngeal isolates of S. pneumoniae were assessed over a six-eight day period in vitro. RESULTS: Multiparametric analysis divided the strains into two groups. Those with a high biofilm forming index (BFI) were structurally complex, exhibited greater lectin colocalization and were more resistant to azithromycin. Those with a low BFI developed less extensive biofilms and were more susceptible to azithromycin. dsDNA was present in the S. pneumoniae biofilm matrix in all strains and treatment with DNase I significantly reduced biofilm biomass. Since capsule expression has been hypothesized to be associated with decreased biofilm development, we also examined expression of cpsA, the first gene in the pneumococcal capsule operon. Interestingly, cpsA was downregulated in biofilms in both high and low BFI strains. CONCLUSION: All pneumococcal strains developed biofilms that exhibited extracellular dsDNA in the biofilm matrix, however strains with a high BFI correlated with greater carbohydrate-associated structural complexity and antibiotic resistance. Furthermore, all strains of S. pneumoniae showed downregulation of the cpsA gene during biofilm growth compared to planktonic culture, regardless of BFI ranking, suggesting downregulation of capsule expression occurs generally during adherent growth.
Assuntos
Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , DNA Bacteriano/metabolismo , Desoxirribonuclease I/metabolismo , Streptococcus pneumoniae/fisiologia , Antibacterianos/farmacologia , Azitromicina/farmacologia , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Criança , Contagem de Colônia Microbiana , DNA Bacteriano/isolamento & purificação , Perfilação da Expressão Gênica , Humanos , Nasofaringe/microbiologia , Streptococcus pneumoniae/isolamento & purificaçãoRESUMO
The staphylococcal accessory regulator (sarA) plays a central role in the regulation of virulence in Staphylococcus aureus. To date, studies involving sarA have focused on its activity as a global regulator that modulates transcription of a wide variety of genes (>100) and its role in virulence. However, there is also evidence to suggest the existence of accessory elements that modulate SarA production and/or function. A reporter system was developed to identify such elements, and a new gene, msa (SA1233), mutation of which results in reduced expression of SarA, was identified and characterized. Additionally, it was shown that mutation of msa resulted in altered transcription of the accessory gene regulator (agr) and the genes encoding several virulence factors including alpha toxin (hla) and protein A (spa). However, the impact of mutating msa was different in the laboratory strain RN6390 and the clinical isolate UAMS-1. For instance, mutation of msa caused a decrease in spa and hla transcription in RN6390 but had a different effect in UAMS-1. The strain-dependent effects of the msa mutation were similar to those observed previously, which suggests that msa may modulate the production of specific virulence factors through its impact on sarA. Interestingly, sequence analysis of Msa suggests that it is a putative membrane protein with three membrane-spanning regions, indicating that Msa might interact with the environment. The findings show that msa is involved in the expression of SarA and several virulence factors.