RESUMO
Antibiotic resistance is increasing and new strategies are needed to fight infection. Advanced materials are promising tools that can be combined with innovative alternatives to conventional antibiotics to allow more targeted and efficient treatment. In this work, we explored the activity against Staphylococcus epidermidis (S. epidermidis) of the α-helical antimicrobial peptide (AMP) MSI-78(4-20) (KFLKKAKKFGKAFVKIL) when covalently bound to a chitosan coating. The AMP MSI-78(4-20) (17 mer) is an improved version of its parent MSI-78 (22 mer; commercially known as Pexiganan), a cost-effective short AMP, which was demonstrated to be as effective as MSI-78 and less toxic to eukaryotic cells. An MSI-78(4-20)-chitosan coating could be applied in several infection scenarios, ranging from bone implants to wound dressings, as chitosan possesses osteoconductive and hemostatic properties. Cysteine-modified MSI-78(4-20) was covalently immobilized onto the chitosan coating through a succinimidyl-[(N-maleimidopropionamido)-octaethyleneglycol] ester (SM(PEG)8), a heterobifuncional crosslinker, with N-hydroxysuccinimide (NHS) ester and maleimide groups, by its N- and C- termini. The MSI-78(4-20)-chitosan coating demonstrated bactericidal properties independently of the tethering site and an improved performance in the presence of plasma proteins, which mimics conditions that will be encountered in vivo. This AMP-chitosan coating has therefore great potential for applications in medical devices such as implants or even wound dressings.
Assuntos
Antibacterianos/farmacologia , Proteínas Sanguíneas/química , Quitosana/farmacologia , Materiais Revestidos Biocompatíveis/farmacologia , Proteínas Citotóxicas Formadoras de Poros/farmacologia , Staphylococcus epidermidis/efeitos dos fármacos , Antibacterianos/química , Quitosana/química , Materiais Revestidos Biocompatíveis/química , Humanos , Proteínas Citotóxicas Formadoras de Poros/química , Staphylococcus epidermidis/crescimento & desenvolvimentoRESUMO
BACKGROUND Phenylketonuria (PKU) is an inborn error of metabolism caused by mutations in the phenylalanine hydroxylase (PAH) gene. When untreated, PKU leads to a significant intellectual deficiency. Although early initiation of dietary therapy allows normal cognitive development, low adherence to treatment may result in neuropsychological deficits, including attention problems. This study was performed to evaluate emotional and behavioral problems in early-treated children and adolescents with PKU using the Child Behavior Checklist - CBCL/6-18 answered by parents. MATERIAL AND METHODS The study included 36 PKU patients. The mean scores of internalizing, externalizing, and total problems, syndrome scales, and DSM-IV-oriented scales of patients were compared with those of controls. An analysis to evaluate the importance of adherence to treatment and presence of intellectual disability was also performed. RESULTS There were no significant differences between patients and controls for almost all CBCL/6-18 scales, with the exception of the Attention Problem Scale - CBCL-APS. The mean (±SD) of the CBCL-APS scores of patients (7.86±5.33) was considerably higher than the mean of the controls (6.07±4.37; p=0.016), but not different from the mean of a matched control subsample (6.69±4.46; p=0.316). The difference between the mean of the scores of DSM-IV/ADHD scale of patients (6.72±4.07) and controls (5.73±3.56; p=0.102) was not significant. Non-adherence to treatment and intellectual disability had a negative impact on both CBCL-APS and DSM-IV/ADHD scale scores. CONCLUSIONS Our findings indicate a significant prevalence of parents' complaints of attention problems and hyperactivity in non-adherent to treatment and intellectually low performing patients with PKU.
Assuntos
Fenilcetonúrias/metabolismo , Fenilcetonúrias/psicologia , Adolescente , Atenção/fisiologia , Transtorno do Deficit de Atenção com Hiperatividade/fisiopatologia , Brasil , Criança , Comportamento Infantil/psicologia , Cognição/fisiologia , Feminino , Humanos , Masculino , Cooperação do Paciente/psicologiaRESUMO
Antimicrobial peptides (AMPs) are a class of broad-spectrum antibiotics known by their ability to disrupt bacterial membranes and their low tendency to induce bacterial resistance, arising as excellent candidates to fight bacterial infections. In this study we aimed at designing short 12-mer AMPs, derived from a highly effective and broad spectrum synthetic AMP, MSI-78 (22 residues), by truncating this peptide at the N- and/or C-termini while spanning its entire sequence with 1 amino acid (aa) shifts. These designed peptides were evaluated regarding antimicrobial activity against selected gram-positive Staphylococcus strains and the gram-negative Pseudomonas aeruginosa (P. aeruginosa). The short 12-mer peptide CEM1 (GIGKFLKKAKKF) was identified as an excellent candidate to fight P. aeruginosa infections as it displays antimicrobial activity against this strain and selectivity, with negligible toxicity to mammalian cells even at high concentrations. However, in general most of the short 12-mer peptides tested showed a reduction in antimicrobial activity, an effect that was more pronounced for gram-positive Staphylococcus strains. Interestingly, CEM1 and a highly similar peptide differing by only one aa-shift (CEM2: IGKFLKKAKKFG), showed a remarkably contrasting AMP activity. These two peptides were chosen for a more detailed study regarding their mechanism of action, using several biophysical assays and simple membrane models that mimic the mammalian and bacterial lipid composition. We confirmed the correlation between peptide helicity and antimicrobial activity and propose a mechanism of action based on the disruption of the bacterial membrane permeability barrier.
Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Membrana Celular/efeitos dos fármacos , Oligopeptídeos/farmacologia , Fragmentos de Peptídeos/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Antibacterianos/química , Antibacterianos/toxicidade , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/toxicidade , Permeabilidade da Membrana Celular/efeitos dos fármacos , Dicroísmo Circular , Relação Dose-Resposta a Droga , Membrana Eritrocítica/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Humanos , Lipídeos de Membrana/química , Membranas Artificiais , Testes de Sensibilidade Microbiana , Oligopeptídeos/química , Oligopeptídeos/toxicidade , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/toxicidade , Estrutura Secundária de Proteína , Pseudomonas aeruginosa/crescimento & desenvolvimento , Staphylococcus aureus/crescimento & desenvolvimento , Relação Estrutura-AtividadeRESUMO
Antimicrobial peptides are widely recognized as an excellent alternative to conventional antibiotics. MSI-78, a highly effective and broad spectrum AMP, is one of the most promising AMPs for clinical application. In this study, we have designed shorter derivatives of MSI-78 with the aim of improving selectivity while maintaining antimicrobial activity. Shorter 17-mer derivatives were created by truncating MSI-78 at the N- and/or C-termini, while spanning MSI-78 sequence. Despite the truncations made, we found a 17-mer peptide, MSI-78(4-20) (KFLKKAKKFGKAFVKIL), which was demonstrated to be as effective as MSI-78 against the Gram-positive Staphylococcus strains tested and the Gram-negative Pseudomonas aeruginosa. This shorter derivative is more selective toward bacterial cells as it was less toxic to erythrocytes than MSI-78, representing an improved version of the lead peptide. Biophysical studies support a mechanism of action for MSI-78(4-20) based on the disruption of the bacterial membrane permeability barrier, which in turn leads to loss of membrane integrity and ultimately to cell death. These features point to a mechanism of action similar to the one described for the lead peptide MSI-78.
Assuntos
Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Pseudomonas aeruginosa/efeitos dos fármacos , Staphylococcus/efeitos dos fármacos , Anti-Infecciosos/química , Peptídeos Catiônicos Antimicrobianos/química , Membrana Celular/metabolismo , Dicroísmo Circular , Humanos , Testes de Sensibilidade MicrobianaRESUMO
Mycobacterium leprae and Mycobacterium tuberculosis antimicrobial resistance has been followed with great concern during the last years, while the need for new drugs able to control leprosy and tuberculosis, mainly due to extensively drug-resistant tuberculosis (XDR-TB), is pressing. Our group recently showed that M. leprae is able to induce lipid body biogenesis and cholesterol accumulation in macrophages and Schwann cells, facilitating its viability and replication. Considering these previous results, we investigated the efficacies of two statins on the intracellular viability of mycobacteria within the macrophage, as well as the effect of atorvastatin on M. leprae infections in BALB/c mice. We observed that intracellular mycobacteria viability decreased markedly after incubation with both statins, but atorvastatin showed the best inhibitory effect when combined with rifampin. Using Shepard's model, we observed with atorvastatin an efficacy in controlling M. leprae and inflammatory infiltrate in the BALB/c footpad, in a serum cholesterol level-dependent way. We conclude that statins contribute to macrophage-bactericidal activity against Mycobacterium bovis, M. leprae, and M. tuberculosis. It is likely that the association of statins with the actual multidrug therapy effectively reduces mycobacterial viability and tissue lesion in leprosy and tuberculosis patients, although epidemiological studies are still needed for confirmation.
Assuntos
Antituberculosos/uso terapêutico , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Mycobacterium leprae/efeitos dos fármacos , Mycobacterium leprae/patogenicidade , Rifampina/uso terapêutico , Animais , Atorvastatina , Linhagem Celular , Sinergismo Farmacológico , Ácidos Heptanoicos/uso terapêutico , Humanos , Hanseníase/tratamento farmacológico , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/patogenicidade , Pirróis/uso terapêutico , Sinvastatina/uso terapêuticoRESUMO
This descriptive exploratory research aims to analyze the effects of bariatric surgery in the lifestyle of people with class III obesity in the workplace, through individual interviews with patients undergoing gastric bypass in gamma-Rouz. Data collection was conducted in June and July, 2011, by means of individual interviews, yielding three Collective Subject Discourse: "More willingness to work" "Life without comorbidities" and "Other effects of bariatric surgery." 30 patients with mean age 44 +/- 12 years old, 24 (80%) female, 19 (63%) performed paid professional activities, 10 (34%) did not work and one (3.3%) students, 23 (96%) hypertension and eight (33%) with a diagnosis of diabetes mellitus were included in the study. Difficulty handling with physical appearance: 13 (43%) and the emotional aspect: 21 (70%). Bariatric surgery positively affected the lifestyle of obese at work with reduction in comorbidities and physical and emotional problems, favoring social and professional reintegration of the individuals.
Assuntos
Derivação Gástrica , Obesidade/cirurgia , Trabalho , Adulto , Comorbidade , Diabetes Mellitus/epidemiologia , Dislipidemias/epidemiologia , Emoções , Feminino , Derivação Gástrica/psicologia , Humanos , Hipertensão/epidemiologia , Estilo de Vida , Masculino , Pessoa de Meia-Idade , Obesidade/epidemiologia , Obesidade/psicologia , Ocupações/estatística & dados numéricos , Resistência Física , Distância Psicológica , Pesquisa Qualitativa , Autoimagem , Ajustamento SocialRESUMO
Wound infection treatment with antimicrobial peptides (AMPs) is still not a reality, due to the loss of activity in vivo. Unlike the conventional strategy of encapsulating AMPs on nanoparticles (NPs) leaving activity dependent on the release profile, this work explores AMP grafting to poly(D,L-lactide-co-glycolide)-polyethylene glycol NPs (PLGA-PEG NPs), whereby AMP exposition, infection targeting and immediate action are promoted. NPs are functionalized with MSI-78(4-20), an equipotent and more selective derivative of MSI-78, grafted through a thiol-maleimide (Mal) Michael addition. NPs with different ratios of PLGA-PEG/PLGA-PEG-Mal are produced and characterized, with 40%PLGA-PEG-Mal presenting the best colloidal properties and higher amounts of AMP grafted as shown by surface charge (+8.6 ± 1.8 mV) and AMP quantification (326 µg mL-1, corresponding to 16.3 µg of AMP per mg of polymer). NPs maintain the activity of the free AMP with a minimal inhibitory concentration (MIC) of 8-16 µg mL-1 against Pseudomonas aeruginosa, and 16-32 µg mL-1 against Staphylococcus aureus. Moreover, AMP grafting accelerates killing kinetics, from 1-2 h to 15 min for P. aeruginosa and from 6-8 h to 0.5-1 h for S. aureus. NP activity in a simulated wound fluid is maintained for S. aureus and decreases slightly for P. aeruginosa. Furthermore, NPs do not demonstrate signs of cytotoxicity at MIC concentrations. Overall, this promising formulation helps unleash the full potential of AMPs for the management of wound infections.
Assuntos
Peptídeos Antimicrobianos , Nanopartículas , Staphylococcus aureus , Polímeros/química , Polietilenoglicóis/química , Nanopartículas/química , Tamanho da Partícula , Portadores de Fármacos/químicaRESUMO
It is key to fight bacterial adhesion to prevent biofilm establishment on biomaterials. Surface immobilization of antimicrobial peptides (AMP) is a promising strategy to avoid bacterial colonization. This work aimed to investigate whether the direct surface immobilization of Dhvar5, an AMP with head-to-tail amphipathicity, would improve the antimicrobial activity of chitosan ultrathin coatings. The peptide was grafted by copper-catalyzed azide-alkyne cycloaddition (CuAAC) chemistry by either its C- or N- terminus to assess the influence of peptide orientation on surface properties and antimicrobial activity. These features were compared with those of coatings fabricated using previously described Dhvar5-chitosan conjugates (immobilized in bulk). The peptide was chemoselectively immobilized onto the coating by both termini. Moreover, the covalent immobilization of Dhvar5 by either terminus enhanced the antimicrobial effect of the chitosan coating by decreasing colonization by both Gram-positive (Staphylococcus aureus, Staphylococcus epidermidis) and Gram-negative (Escherichia coli, Pseudomonas aeruginosa) bacteria. Relevantly, the antimicrobial performance of the surface on Gram-positive bacteria depended on how Dhvar5-chitosan coatings were produced. An antiadhesive effect was observed when the peptide was grafted onto prefabricated chitosan coatings (film), and a bactericidal effect was exhibited when coatings were prepared from Dhvar5-chitosan conjugates (bulk). This antiadhesive effect was not due to changes in surface wettability or protein adsorption but rather depended on variations in peptide concentration, exposure, and surface roughness. Results reported in this study show that the antibacterial potency and effect of immobilized AMP vary greatly with the immobilization procedure. Overall, independently of the fabrication protocol and mechanism of action, Dhvar5-chitosan coatings are a promising strategy for the development of antimicrobial medical devices, either as an antiadhesive or contact-killing surface.
RESUMO
The RNA chaperone Hfq and its associated small RNAs (sRNAs) regulate a variety of phenotypes in bacteria. In this work, we show that Hfq is a master regulator of biofilm formation in Salmonella enterica serovar Typhimurium. Hfq and two Hfq-dependent sRNAs (ArcZ and SdsR) are required for rdar morphotype expression in S. typhimurium. Hfq controls rdar biofilm formation through the major biofilm regulator CsgD. While csgD mRNA steady-state levels are altered in a sdsR mutant, ArcZ seems to work mainly at the post-transcriptional level. Overexpression of ArcZ complemented rdar morphotype formation of an hfq mutant under plate-grown conditions. Although ArcZ activates rpoS expression, its effect on csgD expression is mainly independent of RpoS. ArcZ does not only regulate rdar morphotype expression, but also the transition between sessility and motility and the timing of type 1 fimbriae vs. curli fimbriae surface-attachment at ambient temperature. Consequently, ArcZ is a major regulator of rdar biofilm development.
Assuntos
Proteínas de Bactérias/fisiologia , Biofilmes , Chaperonas Moleculares/fisiologia , Salmonella typhimurium/fisiologia , Regiões 5' não Traduzidas , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Fímbrias Bacterianas/genética , Fímbrias Bacterianas/metabolismo , Fímbrias Bacterianas/fisiologia , Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Fenótipo , Pequeno RNA não Traduzido , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismoRESUMO
The global health threat imposed by the fast spread of antibiotic-resistant bacteria is directing research not only towards the discovery of new antibacterial molecules but also to the repurposing of old drugs, while improving their efficiency and safety [...].
RESUMO
Following our previous reports on dual-action antibacterial and collagenesis-inducing hybrid peptide constructs based on "pentapeptide-4" (PP4, with amino acid sequence KTTKS), whose N-palmitoyl derivative is the well-known cosmeceutical ingredient Matrixyl, herein we disclose novel ionic liquid/PP4 conjugates (IL-KTTKS). These conjugates present potent activity against either antibiotic-susceptible strains or multidrug resistant clinical isolates of both Gram-positive and Gram-negative bacterial species belonging to the so-called "ESKAPE" group of pathogens. Noteworthy, their antibacterial activity is preserved in simulated wound fluid, which anticipates an effective action in the setting of a real wound bed. Moreover, their collagenesis-inducing effects in vitro are comparable to or stronger than those of Matrixyl. Altogether, IL-KTTKS exert a triple antibacterial, antifungal, and collagenesis-inducing action in vitro. These findings provide solid grounds for us to advance IL-KTTKS conjugates as promising leads for future development of topical treatments for complicated skin and soft tissue infections (cSSTI). Further studies are envisaged to incorporate IL-conjugates into suitable nanoformulations, to reduce toxicity and/or improve resistance to proteolytic degradation. IMPORTANCE As life expectancy increases, diseases causing chronic wound infections become more prevalent. Diabetes, peripheral vascular diseases, and bedridden patients are often associated with non-healing wounds that become infected, resulting in high morbidity and mortality. This is exacerbated by the fact that microbes are becoming increasingly resistant to antibiotics, so efforts must converge toward finding efficient therapeutic alternatives. Recently, our team identified a new type of constructs that combine (i) peptides used in cosmetics to promote collagen formation with (ii) imidazolium-based ionic liquids, which have antimicrobial and skin penetration properties. These constructs have potent wide-spectrum antimicrobial action, including against multidrug-resistant Gram-positive and Gram-negative bacteria, and fungi. Moreover, they can boost collagen formation. Hence, this is an unprecedented class of lead molecules toward development of a new topical medicine for chronically infected wounds.
Assuntos
Anti-Infecciosos , Cosmecêuticos , Líquidos Iônicos , Antibacterianos/farmacologia , Anti-Infecciosos/farmacologia , Colágeno/farmacologia , Cosmecêuticos/farmacologia , Bactérias Gram-Negativas , Bactérias Gram-Positivas , Humanos , Líquidos Iônicos/química , Líquidos Iônicos/farmacologia , Testes de Sensibilidade Microbiana , Peptídeos/química , Peptídeos/farmacologiaRESUMO
INTRODUCTION: The coronavirus disease 2019 (COVID-19) was recognized as a pandemic by the World Health Organization on March 11, 2020. As an infectious disease with no specific treatment, several measures have been established to minimize the outbreak of this disease, including social isolation. OBJECTIVE: To evaluate the behavior of adolescents during the isolation period. METHODS: This is a cross-sectional descriptive study conducted at the Adolescent Health Studies Center. Data were obtained from a questionnaire prepared on Google Forms, sent by a multiplatform instant messaging application, and analyzed using the Stata 14 software. RESULTS: A total of 208 adolescents with a mean age of 15.3 years (SD ± 1.8) answered the questionnaire, 57.7% were female. About 93.3% of adolescents said they were in isolation with a changed routine, 67.3% increased their food consumption, 86.5% were inactive, and 58.7% reported screen time over 8 h/d. There was an association between anxiety and increased food consumption (odds ratio: 3.9; CI 95% 2-7.5; p = 0.00), sleeping difficulty (odds ratio: 3.6; CI 95% 1.9-6.8; p = 0.00), and conflicting family relationship (odds ratio: 5.7; CI 95% 1.6-7.8; p = 0.01). CONCLUSION: The study revealed that social isolation due to an infectious disease was associated with several effects on the behavior and eating behavior of adolescents, which need to be acknowledged to encourage them to lead a healthy lifestyle after the COVID-19 confinement.
Assuntos
COVID-19 , Adolescente , COVID-19/epidemiologia , Estudos Transversais , Comportamento Alimentar , Feminino , Humanos , Masculino , Pandemias , SARS-CoV-2RESUMO
In Salmonella enterica serovar Typhimurium, a biofilm mode of growth known as the rdar morphotype is regulated by several networks which sense multiple environmental signals. The transcriptional regulator CsgD is the major target for these regulatory pathways. In this study, we show that two lytic transglycosylases of family I, MltE and MltC, in combination increase CsgD expression and rdar morphotype. MltE and MltC, which share a highly similar transglycosylase SLT domain, work redundantly to regulate CsgD at the transcriptional and posttranscriptional levels. The effect of MltE and MltC on CsgD levels was independent of the known regulatory pathways that sense cell envelope stress. These findings reveal, for the first time, a specific function of lytic transglycosylases in S. Typhimurium and suggest the existence of a new signaling pathway that links cell wall turnover to biofilm formation.
Assuntos
Proteínas de Bactérias/metabolismo , Biofilmes , Parede Celular/metabolismo , Regulação Bacteriana da Expressão Gênica , Glicosiltransferases/metabolismo , Salmonella typhimurium/enzimologia , Proteínas de Bactérias/genética , Parede Celular/genética , Glicosiltransferases/genética , Salmonella typhimurium/genética , Salmonella typhimurium/fisiologiaRESUMO
Bacterial biofilms are a major health concern, mainly due to their contribution to increased bacterial resistance to well-known antibiotics. The conventional treatment of biofilms represents a challenge, and frequently, eradication is not achieved with long-lasting administration of antibiotics. In this context, the present work proposes an innovative therapeutic approach that is focused on the encapsulation of N-acetyl-l-cysteine (NAC) into lipid nanoparticles (LNPs) functionalized with d-amino acids to target and disrupt bacterial biofilms. The optimized formulations presented a mean hydrodynamic diameter around 200 nm, a low polydispersity index, and a high loading capacity. These formulations were stable under storage conditions up to 6 months. In vitro biocompatibility studies showed a low cytotoxicity effect in fibroblasts and a low hemolytic activity in human red blood cells. Nevertheless, unloaded LNPs showed a higher hemolytic potential than NAC-loaded LNPs, which suggests a safer profile of the latter. The in vitro antibiofilm efficacy of the developed formulations was tested against Staphylococcus epidermidis (Gram-positive) and Pseudomonas aeruginosa (Gram-negative) mature biofilms. The results showed that the NAC-loaded LNPs were ineffective against S. epidermidis biofilms, while a significant reduction of biofilm biomass and bacterial viability in P. aeruginosa biofilms were observed. In a more complex therapeutic approach, the LNPs were further combined with moxifloxacin, revealing a beneficial effect between the LNPs and the antibiotic against P. aeruginosa biofilms. Both alone and in combination with moxifloxacin, unloaded and NAC-loaded LNPs functionalized with d-amino acids showed a great potential to reduce bacterial viability, with no significant differences in the presence or absence of NAC. However, the presence of NAC in NAC-loaded functionalized LNPs shows a safer profile than the unloaded LNPs, which is beneficial for an in vivo application. Overall, the developed formulations present a potential therapeutic approach against P. aeruginosa biofilms, alone or in combination with antibiotics.
Assuntos
Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Portadores de Fármacos/farmacologia , Lipossomos/química , Nanopartículas/química , Pseudomonas aeruginosa/efeitos dos fármacos , Acetilcisteína/química , Acetilcisteína/toxicidade , Animais , Antibacterianos/química , Antibacterianos/toxicidade , Linhagem Celular , Portadores de Fármacos/química , Portadores de Fármacos/toxicidade , Sinergismo Farmacológico , Humanos , Lipossomos/toxicidade , Camundongos , Testes de Sensibilidade Microbiana , Moxifloxacina/farmacologia , Nanopartículas/toxicidade , Palmitatos/química , Palmitatos/toxicidade , Fosfatidiletanolaminas/química , Fosfatidiletanolaminas/toxicidade , Polietilenoglicóis/química , Polietilenoglicóis/toxicidade , Pseudomonas aeruginosa/fisiologiaRESUMO
Efficient antibiotics are being exhausted, which compromises the treatment of infections, including complicated skin and skin structure infections (cSSTI) often associated with multidrug resistant (MDR) bacteria, methicillin-resistant S. aureus (MRSA) being the most prevalent. Antimicrobial peptides (AMP) are being increasingly regarded as the new hope for the post-antibiotic era. Thus, future management of cSSTI may include use of peptides that, on the one hand, behave as AMP and, on the other, are able to promote fast and correct skin rebuilding. As such, we combined the well-known cosmeceutical pentapeptide-4 (PP4), devoid of antimicrobial action but possessing collagenesis-boosting properties, with the AMP 3.1, to afford the chimeric peptide PP4-3.1. We further produced its N-methyl imidazole derivative, MeIm-PP4-3.1. Both peptide-based constructs were evaluated in vitro against Gram-negative bacteria, Gram-positive bacteria, and Candida spp. fungi. Additionally, the antibiofilm activity, the toxicity to human keratinocytes, and the activity against S. aureus in simulated wound fluid (SWF) were assessed. The chimeric peptide PP4-3.1 stood out for its potent activity against Gram-positive and Gram-negative bacteria, including against MDR clinical isolates (0.8 ≤ MIC ≤ 5.7 µM), both in planktonic form and in biofilm matrix. The peptide was also active against three clinically relevant species of Candida fungi, with an overall performance superior to that of fluconazole. Altogether, data reveal that PP4-3.1 is as a promising lead for the future development of new topical treatments for severe skin infections.
RESUMO
Bacterial persistence in the environment and in the infected host is often aided by the formation of exopolymer-enclosed communities known as biofilms. Heterogeneous gene expression takes place in microcompartments formed within the complex biofilm structure. This study describes cell differentiation within an isogenic bacterial cell population based on the example of biofilm formation by Salmonella enterica serovar Typhimurium. We analyzed the expression of the major biofilm regulator CsgD at the single-cell level with a chromosomal CsgD-green fluorescent protein (GFP) translational fusion. In individual cells, CsgD-GFP expression is mostly found in the cytoplasm. Quantitative expression analysis and results from three different models of S. Typhimurium biofilms demonstrated that CsgD is expressed in a bistable manner during biofilm development. CsgD expression is, however, monomodal when CsgD is expressed in larger amounts due to a promoter mutation or elevated levels of the secondary signaling molecule c-di-GMP. High levels of CsgD-GFP are associated with cellular aggregation in all three biofilm models. Furthermore, the subpopulation of cells expressing large amounts of CsgD is engaged in cellulose production during red, dry, and rough (rdar) morphotype development and in microcolony formation under conditions of continuous flow. Consequently, bistability at the level of CsgD expression leads to a corresponding pattern of task distribution in S. Typhimurium biofilms.
Assuntos
Proteínas de Bactérias/fisiologia , Biofilmes/crescimento & desenvolvimento , Salmonella typhimurium/crescimento & desenvolvimento , Salmonella typhimurium/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Western Blotting , Citometria de Fluxo , Regulação Bacteriana da Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Microscopia de Fluorescência , Modelos Biológicos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Salmonella typhimurium/genéticaRESUMO
Cyclic di-GMP (c-di-GMP), a novel secondary signalling molecule present in most bacteria, controls transition between motility and sessility. In Salmonella enterica serovar Typhimurium (S. typhimurium) high c-di-GMP concentrations favour the expression of a biofilm state through expression of the master regulator CsgD. In this work, we investigate the effect of c-di-GMP signalling on virulence phenotypes of S. typhimurium. After saturation of the cell with c-di-GMP by overexpression of a di-guanylate cyclase, we studied invasion and induction of a pro-inflammatory cytokine in epithelial cells, basic phenotypes that are major determinants of S. typhimurium virulence. Elevated c-di-GMP had a profound effect on invasion into and IL-8 production by the gastrointestinal epithelial cell line HT-29. Invasion was mainly inhibited through CsgD and the extracellular matrix component cellulose, while inhibition of the pro-inflammatory response occurred through CsgD, which inhibited the secretion of monomeric flagellin. Our results suggest that transition between biofilm formation and virulence in S. typhimurium at the epithelial cell lining is mediated by c-di-GMP signalling through CsgD and cellulose expression.
Assuntos
GMP Cíclico/análogos & derivados , Células Epiteliais/microbiologia , Salmonella typhimurium/patogenicidade , Virulência , Animais , Aderência Bacteriana , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biofilmes , Linhagem Celular , GMP Cíclico/metabolismo , Células Epiteliais/imunologia , Feminino , Flagelina/metabolismo , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Humanos , Interleucina-8/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Salmonelose Animal/microbiologia , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Transdução de SinaisRESUMO
Bacterial species of the Enterobacteriaceae family produce cellulose and curli fimbriae as extracellular matrix components, and their synthesis is positively regulated by the transcriptional activator CsgD. In this group of bacteria, cellulose biosynthesis is commonly regulated by CsgD via the GGDEF domain protein AdrA, a diguanylate cyclase that produces cyclic-diguanylic acid (c-di-GMP), an allosteric activator of cellulose synthase. In the probiotic Escherichia coli strain Nissle 1917 and its recent clonal isolates, CsgD activates the production of curli fimbriae at 28 degrees C, but neither CsgD nor AdrA is required for the c-di-GMP-dependent biosynthesis of cellulose at 28 degrees C and 37 degrees C. In these strains, the GGDEF domain protein YedQ, a diguanylate cyclase that activates cellulose biosynthesis in certain E. coli strains, is not required for cellulose biosynthesis and it has in fact evolved into a novel protein. Cellulose production in Nissle 1917 is required for adhesion of bacteria to the gastrointestinal epithelial cell line HT-29, to the mouse epithelium in vivo, and for enhanced cytokine production. The role of cellulose in this strain is in contrast to the role of cellulose in the commensal strain E. coli TOB1. Consequently, the role of cellulose in bacterial-host interaction is dependent on the E. coli strain background.
Assuntos
Celulose/biossíntese , Proteínas de Escherichia coli/metabolismo , Escherichia coli/fisiologia , Regulação Bacteriana da Expressão Gênica , Fósforo-Oxigênio Liases/metabolismo , Transativadores/fisiologia , Sequência de Aminoácidos , Animais , Aderência Bacteriana , Proteínas de Bactérias/biossíntese , Linhagem Celular , Escherichia coli/metabolismo , Proteínas de Escherichia coli/fisiologia , Humanos , Mucosa Intestinal/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Alinhamento de SequênciaRESUMO
This study analyzed the occurrence of occupational accidents among hospital workers between 2000 and 2005 and health-related quality of life of a sample of injured workers in 2005. Data obtained through occupational accident reports indicated 286 injured workers. In typical accidents (91.6%), accidents with piercing-cutting instruments affected 68.5% of workers. The results related to health-related quality of life obtained from 61 injured workers in 2005, through the SF-36 Medical Outcomes Study 36 - item short form health survey, evidenced high average values in most of the analyzed domains, while the lowest score observed was Vitality and Bodily Pain. No significant differences in health-related quality of life were found among injured workers from the three studied hospitals.