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1.
Nat Commun ; 12(1): 5845, 2021 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-34615859

RESUMO

Conjugation has classically been considered the main mechanism driving plasmid transfer in nature. Yet bacteria frequently carry so-called non-transmissible plasmids, raising questions about how these plasmids spread. Interestingly, the size of many mobilisable and non-transmissible plasmids coincides with the average size of phages (~40 kb) or that of a family of pathogenicity islands, the phage-inducible chromosomal islands (PICIs, ~11 kb). Here, we show that phages and PICIs from Staphylococcus aureus can mediate intra- and inter-species plasmid transfer via generalised transduction, potentially contributing to non-transmissible plasmid spread in nature. Further, staphylococcal PICIs enhance plasmid packaging efficiency, and phages and PICIs exert selective pressures on plasmids via the physical capacity of their capsids, explaining the bimodal size distribution observed for non-conjugative plasmids. Our results highlight that transducing agents (phages, PICIs) have important roles in bacterial plasmid evolution and, potentially, in antimicrobial resistance transmission.


Assuntos
Ilhas Genômicas/genética , Plasmídeos/genética , Fagos de Staphylococcus/genética , Infecções Estafilocócicas/genética , Infecções Estafilocócicas/metabolismo , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidade
2.
Molecules ; 26(15)2021 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-34361812

RESUMO

Metal oxide nanoparticles synthesized by the biological method represent the most recent research in nanotechnology. This study reports the rapid and ecofriendly approach for the synthesis of CeO2 nanoparticles mediated using the Abelmoschus esculentus extract. The medicinal plant extract acts as both a reducing and stabilizing agent. The characterization of CeO2 NPs was performed by scanning electron microscopy (SEM), X-ray diffraction (XRD), ultraviolet-visible spectroscopy (UV-Vis), and Fourier transform infrared spectroscopy (FTIR). The in vitro cytotoxicity of green synthesized CeO2 was assessed against cervical cancerous cells (HeLa). The exposure of CeO2 to HeLa cells at 10-125 µg/mL caused a loss in cellular viability against cervical cancerous cells in a dose-dependent manner. The antibacterial activity of the CeO2 was assessed against S. aureus and K. pneumonia. A significant improvement in wound-healing progression was observed when cerium oxide nanoparticles were incorporated into the chitosan hydrogel membrane as a wound dressing.


Assuntos
Abelmoschus/química , Antioxidantes/síntese química , Extratos Vegetais/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Antibacterianos/química , Antibacterianos/farmacologia , Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Antioxidantes/química , Antioxidantes/farmacologia , Proliferação de Células/efeitos dos fármacos , Cério/química , Química Verde/tendências , Células HeLa , Humanos , Nanopartículas Metálicas/química , Microscopia Eletrônica de Varredura , Extratos Vegetais/química , Espectroscopia de Infravermelho com Transformada de Fourier , Staphylococcus aureus/patogenicidade , Cicatrização/efeitos dos fármacos
4.
Molecules ; 26(16)2021 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-34443349

RESUMO

Ulva sp. is known to be a source of bioactive compounds such as ulvans, but to date, their biological activity on skin commensal and/or opportunistic pathogen bacteria has not been reported. In this study, the effects of poly- and oligosaccharide fractions produced by enzyme-assisted extraction and depolymerization were investigated, for the first time in vitro, on cutaneous bacteria: Staphylococcus aureus, Staphylococcus epidermidis, and Cutibacterium acnes. At 1000 µg/mL, poly- and oligosaccharide fractions did not affect the growth of the bacteria regarding their generation time. Polysaccharide Ulva sp. fractions at 1000 µg/mL did not alter the bacterial biofilm formation, while oligosaccharide fractions modified S. epidermidis and C. acnes biofilm structures. None of the fractions at 1000 µg/mL significantly modified the cytotoxic potential of S. epidermidis and S. aureus towards keratinocytes. However, poly- and oligosaccharide fractions at 1000 µg/mL induced a decrease in the inflammatory potential of both acneic and non-acneic C. acnes strains on keratinocytes of up to 39.8%; the strongest and most significant effect occurred when the bacteria were grown in the presence of polysaccharide fractions. Our research shows that poly- and oligosaccharide Ulva sp. fractions present notable biological activities on cutaneous bacteria, especially towards C. acnes acneic and non-acneic strains, which supports their potential use for dermo-cosmetic applications.


Assuntos
Bactérias/efeitos dos fármacos , Bactérias/crescimento & desenvolvimento , Microbiota/efeitos dos fármacos , Extratos Vegetais/farmacologia , Pele/microbiologia , Ulva/química , Bactérias/patogenicidade , Relação Dose-Resposta a Droga , Propionibacteriaceae/efeitos dos fármacos , Propionibacteriaceae/crescimento & desenvolvimento , Propionibacteriaceae/patogenicidade , Propionibacteriaceae/fisiologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus aureus/patogenicidade , Staphylococcus aureus/fisiologia , Staphylococcus epidermidis/efeitos dos fármacos , Staphylococcus epidermidis/crescimento & desenvolvimento , Staphylococcus epidermidis/patogenicidade , Staphylococcus epidermidis/fisiologia , Virulência/efeitos dos fármacos
5.
Molecules ; 26(16)2021 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-34443523

RESUMO

Newly designed and synthesized cyano, amidino and acrylonitrile 2,5-disubstituted furane derivatives with either benzimidazole/benzothiazole nuclei have been evaluated for antitumor and antimicrobial activity. For potential antitumor activity, the compounds were tested in 2D and 3D cell culture methods on three human lung cancer cell lines, A549, HCC827 and NCI-H358, with MTS cytotoxicity and BrdU proliferation assays in vitro. Compounds 5, 6, 8, 9 and 15 have been proven to be compounds with potential antitumor activity with high potential to stop the proliferation of cells. In general, benzothiazole derivatives were more active in comparison to benzimidazole derivatives. Antimicrobial activity was evaluated with Broth microdilution testing (according to CLSI (Clinical Laboratory Standards Institute) guidelines) on Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus. Additionally, Saccharomyces cerevisiae was included in testing as a eukaryotic model organism. Compounds 5, 6, 8, 9 and 15 showed the most promising antibacterial activity. In general, the compounds showed antitumor activity, higher in 2D assays in comparison with 3D assays, on all three cell lines in both assays. In natural conditions, compounds with such an activity profile (less toxic but still effective against tumor growth) could be promising new antitumor drugs. Some of the tested compounds showed antimicrobial activity. In contrast to ctDNA, the presence of nitro group or chlorine in selected furane-benzothiazole structures did not influence the binding mode with AT-DNA. All compounds dominantly bound inside the minor groove of AT-DNA either in form of monomers or dimer and higher-order aggregates.


Assuntos
Anti-Infecciosos/farmacologia , Benzimidazóis/farmacologia , Benzotiazóis/farmacologia , Neoplasias/tratamento farmacológico , Anti-Infecciosos/síntese química , Anti-Infecciosos/química , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Benzimidazóis/síntese química , Benzimidazóis/química , Benzotiazóis/síntese química , Benzotiazóis/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Estrutura Molecular , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/patogenicidade , Relação Estrutura-Atividade
6.
Int J Mol Sci ; 22(16)2021 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-34445684

RESUMO

The shape and transparency of the cornea are essential for clear vision. However, its location at the ocular surface renders the cornea vulnerable to pathogenic microorganisms in the external environment. Pseudomonas aeruginosa and Staphylococcus aureus are two such microorganisms and are responsible for most cases of bacterial keratitis. The development of antimicrobial agents has allowed the successful treatment of bacterial keratitis if the infection is diagnosed promptly. However, no effective medical treatment is available after progression to corneal ulcer, which is characterized by excessive degradation of collagen in the corneal stroma and can lead to corneal perforation and corneal blindness. This collagen degradation is mediated by both infecting bacteria and corneal fibroblasts themselves, with a urokinase-type plasminogen activator (uPA)-plasmin-matrix metalloproteinase (MMP) cascade playing a central role in collagen destruction by the host cells. Bacterial factors stimulate the production by corneal fibroblasts of both uPA and pro-MMPs, released uPA mediates the conversion of plasminogen in the extracellular environment to plasmin, and plasmin mediates the conversion of secreted pro-MMPs to the active form of these enzymes, which then degrade stromal collagen. Bacterial factors also stimulate expression by corneal fibroblasts of the chemokine interleukin-8 and the adhesion molecule ICAM-1, both of which contribute to recruitment and activation of polymorphonuclear neutrophils, and these cells then further stimulate corneal fibroblasts via the secretion of interleukin-1. At this stage of the disease, bacteria are no longer necessary for collagen degradation. In this review, we discuss the pivotal role of corneal fibroblasts in corneal ulcer associated with infection by P. aeruginosa or S. aureus as well as the development of potential new modes of treatment for this condition.


Assuntos
Úlcera da Córnea/metabolismo , Fibroblastos/metabolismo , Ceratite/microbiologia , Animais , Colágeno/metabolismo , Córnea/metabolismo , Córnea/fisiologia , Substância Própria/metabolismo , Úlcera da Córnea/etiologia , Úlcera da Córnea/microbiologia , Infecções Oculares Bacterianas/microbiologia , Infecções Oculares Bacterianas/fisiopatologia , Fibrinolisina/metabolismo , Humanos , Metaloproteinases da Matriz/metabolismo , Plasminogênio/metabolismo , Ativadores de Plasminogênio/metabolismo , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/patogenicidade , Staphylococcus aureus/metabolismo , Staphylococcus aureus/patogenicidade , Ativador de Plasminogênio Tipo Uroquinase/metabolismo
7.
J Med Microbiol ; 70(8)2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34338626

RESUMO

Introduction. Biofilm formation is a major virulence factor associated with Staphylococcus aureus infections. However, the influence of plasma proteins on biofilm formation of clinical isolates in vitro remains unclear.Hypotheses. We hypothesized that coating surfaces with plasma proteins might induce biofilm formation by S. aureus of different clonal lineages.Aim. To evaluate biofilm production by clinical S. aureus isolates of different clonal lineages isolated in Rio de Janeiro hospitals and investigated the presence of biofilm-associated genes.Methodology. This study assessed biofilm production of 60 S. aureus isolates in polystyrene microtitre plates with and without fibrinogen or fibronectin. The biochemical composition of the biofilm matrices was determined and the biofilm formation on fibrinogen-coated surfaces was also evaluated by confocal laser scanning microscopy. The presence of biofilm-related genes was detected by PCR, and the typing and functionality of agr operon was also evaluated.Results. Most of the isolates (45 %) were weak biofilm producers or non-producers. However, most of them presented a significant increase in biofilm production on plates covered with plasma proteins. There was no significant difference in biofilm formation between methicillin-resistant and -susceptible S. aureus isolates, or between different clonal lineages, except for ST30-IV (weak producers) and ST239-III (strong producers). The fnbB gene was associated with higher biofilm production.Conclusion. An increase in biofilm production in the presence of plasma proteins highlights the importance of investigating biofilm formation by S. aureus clinical isolates under different conditions since this virulence factor contributes to persistent infections and increased resistance to antimicrobials.


Assuntos
Biofilmes/crescimento & desenvolvimento , Fibrinogênio , Fibronectinas , Staphylococcus aureus Resistente à Meticilina/patogenicidade , Staphylococcus aureus/patogenicidade , Adesinas Bacterianas/genética , Aderência Bacteriana/genética , Proteínas de Bactérias/genética , Genes Bacterianos , Genótipo , Humanos , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Staphylococcus aureus Resistente à Meticilina/fisiologia , Óperon , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/genética , Staphylococcus aureus/isolamento & purificação , Staphylococcus aureus/fisiologia , Transativadores/genética
8.
Biomed Res Int ; 2021: 6637617, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34395621

RESUMO

Staphylococcus aureus is a major human pathogen present on a third of the healthy population. The bacterium possesses an extensive arsenal of virulence factors. The pathogenicity is linked with S. aureus high plasticity and its exceptional ability to incorporate foreign genetic material. The aim of the present study was to perform molecular characterization of Staphylococcus aureus strains isolated from the clinical environment of the CHU-Z Abomey-Calavi/Sô-Ava. Isolation of Staphylococcus aureus bacterium was performed on Chapman agar. Toxin production by isolated S. aureus strains was investigated using the radial immunoprecipitation technique. A colorimetric assay was used to evaluate Staphylococcus aureus lipase (SA-Lipase) production. Finally, the expression of antibiotic resistance genes and genes encoding toxins production was investigated. Our data suggest that none of the isolated Staphylococcus aureus strains expressed the investigated toxin genes. Interestingly, SA-Lipase was produced by 14.28% of our isolated S. aureus strains. The mecA gene was present in 57.14% of the isolated strains, while PVL and TSST-1 genes were identified in 2.85 and 7.14% of S. aureus, respectively. Significant genetic diversity was observed along the hospital environment S. aureus strains. The present study reveals the level of virulence of S. aureus strains isolated in the different units of CHU-Z Abomey Calavi/Sô-Ava through the production of lipase, PVL, and epidermolysins. The molecular study has favored a genetic characterization within the isolated strains.


Assuntos
Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/isolamento & purificação , Staphylococcus aureus/patogenicidade , Fatores de Virulência/genética , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Benin , Enterotoxinas/genética , Exotoxinas/genética , Hospitais Universitários , Humanos , Leucocidinas/genética , Lipase/genética , Proteínas de Ligação às Penicilinas/genética , RNA Bacteriano/genética , RNA Ribossômico/genética , RNA Ribossômico 16S/genética , Staphylococcus aureus/genética , Superantígenos/genética , Virulência
9.
Int J Mol Sci ; 22(16)2021 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-34445550

RESUMO

Within-host adaptation is a typical feature of chronic, persistent Staphylococcus aureus infections. Research projects addressing adaptive changes due to bacterial in-host evolution increase our understanding of the pathogen's strategies to survive and persist for a long time in various hosts such as human and bovine. In this study, we investigated the adaptive processes of S. aureus during chronic, persistent bovine mastitis using a previously isolated isogenic strain pair from a dairy cow with chronic, subclinical mastitis, in which the last variant (host-adapted, Sigma factor SigB-deficient) quickly replaced the initial, dominant variant. The strain pair was cultivated under specific in vitro infection-relevant growth-limiting conditions (iron-depleted RPMI under oxygen limitation). We used a combinatory approach of surfaceomics, molecular spectroscopic fingerprinting and in vitro phenotypic assays. Cellular cytotoxicity assays using red blood cells and bovine mammary epithelial cells (MAC-T) revealed changes towards a more cytotoxic phenotype in the host-adapted isolate with an increased alpha-hemolysin (α-toxin) secretion, suggesting an improved capacity to penetrate and disseminate the udder tissue. Our results foster the hypothesis that within-host evolved SigB-deficiency favours extracellular persistence in S. aureus infections. Here, we provide new insights into one possible adaptive strategy employed by S. aureus during chronic, bovine mastitis, and we emphasise the need to analyse genotype-phenotype associations under different infection-relevant growth conditions.


Assuntos
Adaptação Fisiológica , Hemólise , Adaptação ao Hospedeiro , Glândulas Mamárias Animais/patologia , Mastite Bovina/patologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/patogenicidade , Animais , Apoptose , Bovinos , Feminino , Glândulas Mamárias Animais/microbiologia , Mastite Bovina/microbiologia , Fenótipo
10.
Nucleic Acids Res ; 49(14): 8324-8338, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34302475

RESUMO

Bacterial replication is a fast and accurate process, with the bulk of genome duplication being catalyzed by the α subunit of DNA polymerase III within the bacterial replisome. Structural and biochemical studies have elucidated the overall properties of these polymerases, including how they interact with other components of the replisome, but have only begun to define the enzymatic mechanism of nucleotide incorporation. Using transient-state methods, we have determined the kinetic mechanism of accurate replication by PolC, the replicative polymerase from the Gram-positive pathogen Staphylococcus aureus. Remarkably, PolC can recognize the presence of the next correct nucleotide prior to completing the addition of the current nucleotide. By modulating the rate of pyrophosphate byproduct release, PolC can tune the speed of DNA synthesis in response to the concentration of the next incoming nucleotide. The kinetic mechanism described here would allow PolC to perform high fidelity replication in response to diverse cellular environments.


Assuntos
Proteínas de Bactérias/genética , Replicação do DNA/genética , DNA Polimerase Dirigida por DNA/genética , Infecções Estafilocócicas/genética , Staphylococcus aureus/genética , Difosfatos/metabolismo , Humanos , Cinética , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/patogenicidade
11.
Mol Immunol ; 137: 134-144, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34247099

RESUMO

Mastitis is one of the most serious diseases in humans and animals, especially in the modern dairy industry. Seeking safe and effective mastitis prevention strategies is urgent since food safety and drug residues in milk remain an enormous concern, despite the contribution of antibiotics to control mastitis. Kynurenic acid (KYNA), derived from the kynurenine pathway of tryptophan metabolism, has been shown to exhibit anti-inflammatory and immunomodulatory effects in many diseases. Recently, it was reported that impaired KYNA levels were associated with mastitis. However, the physiological role of KYNA in mastitis has not yet been elucidated. Therefore, the aim of this study was to investigate the protective role of KYNA in pathogen-induced mastitis in mice, as well as the underlying mechanism of this effect. We first evaluated the effects of KYNA on LPS-induced mastitis in mice. Additionally, the underlying anti-inflammatory mechanism of KYNA was investigated in mammary epithelial cells (MMECs). Furthermore, we examined the effects of KYNA on S. aureus and E. coli induced mastitis in mice. Our results demonstrated that KYNA alleviated LPS-induced mastitis by reducing inflammatory responses and enhancing blood-milk barrier integrity. The fundamental mechanisms involved the inhibition of NF-κB and activation of Nrf2/Ho-1, which is probably mediated by G protein-coupled receptor 35 but not aryl hydrocarbon receptor. Notably, KYNA also protected against S. aureus and E. coli induced mastitis in mice. In conclusion, our results highlight the role of KYNA in mastitis and serve as a basis for using endogenous metabolite as a novel preventative or therapeutic strategy for disease intervention.


Assuntos
Inflamação/tratamento farmacológico , Ácido Cinurênico/farmacologia , Mastite/tratamento farmacológico , Leite/metabolismo , Animais , Anti-Inflamatórios/farmacologia , Escherichia coli/patogenicidade , Infecções por Escherichia coli/tratamento farmacológico , Infecções por Escherichia coli/metabolismo , Infecções por Escherichia coli/microbiologia , Feminino , Heme Oxigenase-1/metabolismo , Humanos , Inflamação/metabolismo , Inflamação/microbiologia , Glândulas Mamárias Animais/efeitos dos fármacos , Glândulas Mamárias Animais/metabolismo , Glândulas Mamárias Animais/microbiologia , Mastite/metabolismo , Mastite/microbiologia , Camundongos , Fator 2 Relacionado a NF-E2/metabolismo , NF-kappa B/metabolismo , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/metabolismo , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/patogenicidade
12.
Methods Mol Biol ; 2341: 17-24, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34264456

RESUMO

Staphylococcal secreted nuclease contributes to S. aureus virulence by degrading neutrophil extracellular traps (NETs), which allows the bacterium to evade the host immune system and has also been shown to promote biofilm dispersal. In this chapter, two methods for detecting nuclease activity are described, both of which have increased sensitivity compared to the traditional nuclease agar method.


Assuntos
Proteínas de Bactérias/análise , Nuclease do Micrococo/análise , Salmão/genética , Staphylococcus aureus/enzimologia , Animais , Proteínas de Bactérias/metabolismo , Técnicas Bacteriológicas , Armadilhas Extracelulares/metabolismo , Evasão da Resposta Imune , Masculino , Nuclease do Micrococo/metabolismo , Espermatozoides/química , Staphylococcus aureus/patogenicidade , Fatores de Virulência/análise , Fatores de Virulência/metabolismo
13.
Methods Mol Biol ; 2341: 25-30, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34264457

RESUMO

Many strains of Staphylococcus aureus produce a variety of cytolysins that target many different cell types to both fight the immune system and acquire nutrients. This includes hemolysins which destroy erythrocytes and are well studied virulence factors. Traditionally, hemolysin activity is measured on blood agar plates due to the simplicity of the assay. While this is telling, it cannot encapsulate the full story because S. aureus is known to behave differently in broth and on agar. Furthermore, plate-based assays are primarily semiquantitative and often a more accurate determination of hemolytic potential is needed to discern differences between strains. Here, we describe a method to quantify hemolysin activity from broth or similarly grown cells.


Assuntos
Eritrócitos/fisiologia , Proteínas Hemolisinas/análise , Staphylococcus aureus/crescimento & desenvolvimento , Animais , Proteínas de Bactérias/análise , Proteínas de Bactérias/metabolismo , Meios de Cultura/química , Proteínas Hemolisinas/metabolismo , Hemólise , Humanos , Staphylococcus aureus/metabolismo , Staphylococcus aureus/patogenicidade , Fatores de Virulência/análise , Fatores de Virulência/metabolismo
14.
Methods Mol Biol ; 2341: 127-131, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34264468

RESUMO

Staphylococcus aureus is an important human pathogen that causes a plethora of diverse infections within the human host that range in severity from the relatively minor to the severe. Of note, bloodstream infections caused by this organism result in high mortality rates, often following failed rounds of surgical and antibiotic intervention. The capacity for S. aureus to exist in blood is driven by myriad virulence factors that engage in a manipulation of various host responses to evade destruction and ensure survival. These include both secreted elements, such as coagulase and von Willebrand factor protein, as well as surface displayed factors, including clumping factor A and fibronectin binding protein A. In addition to this, there are a number of other loci within the S. aureus genome whose products have been shown to contribute to blood survival by more indirect means. Accordingly, ex vivo whole human blood survival assays are often used as a preliminary study to investigate host-bacterial interactions in an effort to delineate the pathogenicity of S. aureus strains. Herein we provide a detailed assessment of the protocol required to perform such studies.


Assuntos
Sangue/microbiologia , Staphylococcus aureus/crescimento & desenvolvimento , Fatores de Virulência/metabolismo , Proteínas de Bactérias/metabolismo , Sangue/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Viabilidade Microbiana , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/metabolismo , Staphylococcus aureus/patogenicidade
15.
Methods Mol Biol ; 2341: 141-152, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34264470

RESUMO

Staphylococcus aureus is a Gram-positive bacterium that is capable of infecting and inducing tissue pathology in nearly every organ system. The pathogenesis of staphylococcal infection is dictated, in part, through the production of toxins that induce cellular death through receptor-dependent and -independent mechanisms, thereby contributing to tissue injury. One common manifestation of invasive staphylococcal infection is osteomyelitis, or infection of bone. Osteomyelitis triggers extreme bone loss, in part, through production of secreted toxins. Cytotoxicity assays, therefore, can be instrumental in elucidating how S. aureus triggers bone loss, and such assays are rapidly adaptable to study of tissue damage across multiple cell types and organ systems. Additionally, in conjunction with proteomic approaches, cytotoxicity studies may help identify toxins capable of inducing host cell death. Here, a protocol is described for the isolation and stimulation of primary osteoblasts with S. aureus supernatants for rapid detection of cytotoxicity. This assay provides an excellent in vitro system to better understand how staphylococcal secreted toxins impact skeletal cell biology to induce changes in bone homeostasis.


Assuntos
Toxinas Bacterianas/toxicidade , Osteoblastos/citologia , Staphylococcus aureus/patogenicidade , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Camundongos , Osteoblastos/efeitos dos fármacos , Cultura Primária de Células , Staphylococcus aureus/metabolismo
16.
Methods Mol Biol ; 2341: 153-159, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34264471

RESUMO

Biofilms provide an environment in which bacteria can survive adverse conditions such as nutrient or oxygen deficiencies, and antibiotic treatments. Bacterial survival of antibiotic treatments can often result in antimicrobial resistance, which can make treating infections substantially more difficult, increase the burden of healthcare costs, and hinder the healing of infected wounds. As Staphylococcus aureus is a bacterium that commonly causes skin infections, can be found in infected skin wounds, and is prone to developing antimicrobial resistance-especially within a biofilm microenvironment, the study and development of methodologies to treat infected wounds have become an important topic of research. To study the development of bacterial biofilm in a skin wound, this chapter discusses an in vitro model to access biofilm growth in an environment that mimics a human skin wound. This model serves as a tool to study the biofilm growth and efficacy of antibiotic use in an in vitro system that more closely resembles human skin tissue, rather than a polystyrene plate.


Assuntos
Biofilmes/crescimento & desenvolvimento , Staphylococcus aureus/patogenicidade , Infecção dos Ferimentos/microbiologia , Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Farmacorresistência Bacteriana , Humanos , Viabilidade Microbiana , Modelos Biológicos , Pele/microbiologia , Staphylococcus aureus/efeitos dos fármacos
17.
Int J Nanomedicine ; 16: 4239-4250, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34194227

RESUMO

Purpose: Pore-forming toxins (PFTs) perform important functions during bacterial infections. Among various virulence-targeting therapies, nanosponges (NSs) have excellent neutralization effects on multiple PFTs. To enhance treatment efficacy, NSs tend to be incorporated into other biomaterials, such as hydrogels. Methods: In the present work, red blood cell (RBC) vesicles were harvested to wrap polymer nanoparticles, leading to the formation of NSs, and the optimal Pluronic F127 hydrogel concentration was determined for gelation. Then, a novel detoxification system was constructed by incorporating NSs into an optimized Pluronic F127 hydrogel (NS-pGel). Next, the system was characterized by rheological and sustained release behavior as well as micromorphology. Then, the in vitro neutralization effect of NS-pGel on various PFTs was examined by a hemolysis protocol. Finally, therapeutic and prophylactic detoxification efficiency was evaluated in a mouse subcutaneous infection model in vivo. Results: A thermosensitive, injectable detoxification system was successfully constructed by loading NSs into a 30% Pluronic F127 hydrogel. Characterization results demonstrated that the NS-pGel hybrid system sustained an ideal fluidity and viscosity at lower temperatures but exhibited a quick sol-gel transition capacity near body temperature. In addition, this hybrid system had a sustained release behavior accompanied by good biocompatibility and biodegradability. Finally, the NS-pGel system showed neutralization effects similar to those of NSs both in vitro and in vivo, indicating a good preservation of NS functionality. Conclusion: In conclusion, we constructed a novel temperature-sensitive detoxification system with good biocompatibility and biodegradability, which may be applied to the clinical treatment of PFT-induced local lesions and infections.


Assuntos
Antibacterianos/administração & dosagem , Antibacterianos/química , Hidrogéis/administração & dosagem , Hidrogéis/química , Poloxâmero/química , Animais , Proteínas de Bactérias , Materiais Biocompatíveis , Eritrócitos/química , Proteínas Hemolisinas , Hemólise/efeitos dos fármacos , Masculino , Teste de Materiais , Camundongos Endogâmicos ICR , Nanopartículas/química , Testes de Neutralização , Reologia , Staphylococcus aureus/patogenicidade , Temperatura , Vibrio vulnificus/patogenicidade , Viscosidade
18.
BMC Infect Dis ; 21(1): 627, 2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-34210263

RESUMO

BACKGROUND AND OBJECTIVE: Carriage of virulence factors confers some evolutionary benefit to bacteria, which favors the resistant strains. We aimed to analyze whether antibiotic susceptibility of Staphylococcus aureus strains is affected by agr typing, biofilm formation ability, and virulence profiles. METHODS: A total of 123 S. aureus clinical isolates were subjected to antimicrobial susceptibility testing by disk diffusion method, biofilm formation by microtiter plate method, as well as polymerase chain reaction screening to identify virulence genes and the accessory gene regulator (agr) types I-IV. A P value < 0.05 was considered significant. RESULTS: The most prevalent virulence gene was staphyloxanthin crtN, followed by hemolysin genes, capsular cap8H, toxic shock toxin tst, and enterotoxin sea, respectively. Resistant isolates were more commonly found in the agr-negative group than in the agr-positive group. Isolates of agr type III were more virulent than agr I isolates. Strong biofilm producers showed more antibiotic susceptibility and carried more virulence genes than non-strong biofilm producers. Associations were found between the presence of virulence genes and susceptibility to antibiotics. Carriage of the virulence genes and agr was higher in the inpatients; while, resistance and strong biofilms were more prevalent in the outpatients. CONCLUSION: These findings indicated the presence of several virulence factors, biofilm production capacity, agr types and resistance to antibiotics in clinical S. aureus isolates. Considering the importance of S. aureus for human medicine, an understanding of virulence and resistance relationships would help to reduce the impact of S. aureus infections.


Assuntos
Proteínas de Bactérias , Biofilmes , Staphylococcus aureus/genética , Staphylococcus aureus/isolamento & purificação , Staphylococcus aureus/fisiologia , Staphylococcus aureus/patogenicidade , Transativadores , Fatores de Virulência/genética , Toxinas Bacterianas/genética , Farmacorresistência Bacteriana , Enterotoxinas/genética , Exfoliatinas/genética , Feminino , Proteínas Hemolisinas/genética , Humanos , Masculino , Testes de Sensibilidade Microbiana , Fenótipo , Reação em Cadeia da Polimerase , Superantígenos/genética , Xantofilas
19.
Int J Mol Sci ; 22(13)2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-34281154

RESUMO

Staphylococcal aureus (S. aureus), a Gram-positive bacteria, is known to cause various infections. Extracellular vesicles (EVs) are a heterogeneous array of membranous structures secreted by cells from all three domains of life, i.e., eukaryotes, bacteria, and archaea. Bacterial EVs are implied to be involved in both bacteria-bacteria and bacteria-host interactions during infections. It is still unclear how S. aureus EVs interact with host cells and induce inflammatory responses. In this study, EVs were isolated from S. aureus and mutant strains deficient in either prelipoprotein lipidation (Δlgt) or major surface proteins (ΔsrtAB). Their immunostimulatory capacities were assessed both in vitro and in vivo. We found that S. aureus EVs induced pro-inflammatory responses both in vitro and in vivo. However, this activity was dependent on lipidated lipoproteins (Lpp), since EVs isolated from the Δlgt showed no stimulation. On the other hand, EVs isolated from the ΔsrtAB mutant showed full immune stimulation, indicating the cell wall anchoring of surface proteins did not play a role in immune stimulation. The immune stimulation of S. aureus EVs was mediated mainly by monocytes/macrophages and was TLR2 dependent. In this study, we demonstrated that not only free Lpp but also EV-imbedded Lpp had high pro-inflammatory activity.


Assuntos
Vesículas Extracelulares/imunologia , Lipoproteínas/metabolismo , Staphylococcus aureus/metabolismo , Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Vesículas Extracelulares/metabolismo , Humanos , Inflamação/imunologia , Lipoproteínas/fisiologia , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/patogenicidade , Receptor 2 Toll-Like/metabolismo
20.
Int J Mol Sci ; 22(10)2021 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-34069497

RESUMO

The epidemiological success of Staphylococcus aureus as a versatile pathogen in mammals is largely attributed to its virulence factor repertoire and the sophisticated regulatory network controlling this virulon. Here we demonstrate that the low-molecular-weight protein arginine phosphatase PtpB contributes to this regulatory network by affecting the growth phase-dependent transcription of the virulence factor encoding genes/operons aur, nuc, and psmα, and that of the small regulatory RNA RNAIII. Inactivation of ptpB in S. aureus SA564 also significantly decreased the capacity of the mutant to degrade extracellular DNA, to hydrolyze proteins in the extracellular milieu, and to withstand Triton X-100 induced autolysis. SA564 ΔptpB mutant cells were additionally ingested faster by polymorphonuclear leukocytes in a whole blood phagocytosis assay, suggesting that PtpB contributes by several ways positively to the ability of S. aureus to evade host innate immunity.


Assuntos
Neutrófilos/metabolismo , Neutrófilos/microbiologia , Infecções Estafilocócicas/imunologia , Arginina/análogos & derivados , Arginina/química , Arginina/metabolismo , Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Humanos , Peso Molecular , Compostos Organofosforados/química , Compostos Organofosforados/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , RNA Bacteriano/metabolismo , Staphylococcus aureus/metabolismo , Staphylococcus aureus/patogenicidade , Fatores de Virulência/metabolismo
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