RESUMO
The emergence and global spread of methicillin-resistant Staphylococcus aureus (MRSA) pose a serious threat to public health, underscoring the urgent need for novel antibacterial interventions. Here, we screened 18 newly synthesized N,N'-diarylurea derivatives to identify compounds with activity against MRSA. Our investigations led to the discovery of a small molecule, SCB-24, which exhibited promising antimicrobial activity against MRSA USA300. Notably, SCB-24 demonstrated high activity even in the presence of 10% fetal bovine serum and showed excellent selectivity for bacterial over mammalian cells. SCB-24 also showed potent activity against various MRSA strains, including those resistant to second- and third-line antibiotics. Importantly, the efficacy of SCB-24 was inferior to that of vancomycin in MRSA-infected Galleria mellonella larvae. Overall, our findings suggest that SCB-24 has great potential as a new therapeutic for multidrug-resistant S. aureus infections.
Assuntos
Antibacterianos , Staphylococcus aureus Resistente à Meticilina , Testes de Sensibilidade Microbiana , Ureia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Antibacterianos/farmacologia , Antibacterianos/síntese química , Antibacterianos/química , Animais , Relação Estrutura-Atividade , Ureia/farmacologia , Ureia/química , Ureia/análogos & derivados , Humanos , Larva/efeitos dos fármacos , Estrutura Molecular , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Mariposas/efeitos dos fármacos , Mariposas/microbiologia , Descoberta de Drogas , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , Relação Dose-Resposta a DrogaRESUMO
The emergence and spread of multidrug-resistant bacteria underscore the critical need for novel antibacterial interventions. In our screening of 12 synthesized thienobenzodiazepines, pyridobenzodiazepines, and dibenzodiazepines, we successfully identified a small molecule compound SW33. Notably, SW33 demonstrated potent inhibitory activity against intracellular multidrug-resistant and fluoroquinolone-resistant strains of S. typhimurium in both macrophages and epithelial cells. Furthermore, SW33 was also effective against intramacrophagic Salmonella typhi, Yersinia enterocolitica, and Listeria monocytogenes. These significant findings suggest that SW33 possesses broad-spectrum activity against intracellular bacteria.
RESUMO
Differentiating Streptococcus pneumoniae among nonpneumococcal viridans group streptococci (VGS) is challenging in conventional laboratories. Therefore, we aimed to evaluate the performance of the latest Bruker Biotyper matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) system in identifying VGS by comparing the results to those of the specific gene sequencing approach. Clinical isolates were initially identified using the BD Phoenix system to identify Streptococcus species. The optochin test was used to distinguish nonpneumococcal VGS from S. pneumoniae. The species of individual reference strains and clinical isolates were determined by comparing the sequences of the 16S rDNA, gyrB, sodA, groESL, or coaE genes with those in the GenBank sequence databases. We evaluated the performance of the Bruker Biotyper MALDI-TOF MS in VGS identification using two different machines with three databases. We collected a total of 103 nonpneumococcal VGS and 29 S. pneumoniae blood isolates at a medical center in northern Taiwan. Among these isolates, only seven could not be identified at the species level by the specific gene sequencing approach. We found that none of the nonpneumococcal VGS isolates were misidentified as pneumococci by the latest Biotyper system, and vice versa. However, certain strains, especially those in the mitis and bovis groups, could still not be correctly identified. The latest Bruker Biotyper 4.1 (DB_10833) showed significant improvement in identifying VGS strains. However, a specific gene sequencing test is still needed to precisely differentiate the species of strains in the mitis and bovis groups.
Assuntos
Streptococcus pneumoniae , Estreptococos Viridans , Humanos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Estreptococos Viridans/genética , Bases de Dados de Ácidos Nucleicos , TaiwanRESUMO
BACKGROUND/PURPOSE: The increasing incidence of infections caused by multidrug-resistant Salmonella enterica has become a serious threat to global public health. Here, we found that the tyrosine kinase inhibitor nilotinib exhibits antibacterial activity against intracellular S. enterica serovar Typhimurium in RAW264.7 macrophages. Thus, we aimed to pharmacologically exploit the anti-intracellular Salmonella activity of nilotinib and to elucidate its mechanism of action. METHODS: The antibacterial activity of the compounds was assessed by high-content analysis (HCA) and intracellular CFU, minimum inhibitory concentration (MIC), and bacterial growth assays. The cytotoxicity of the compounds was evaluated by HCA and a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) cell viability assays. The levels of cellular AMPK, phospho-AMPK, Atg7 and ß-actin were determined by immunoblotting. RESULTS: The screen identified two small molecule compounds (SCT1101 and SCT1104) with potent activity against intracellular S. Typhimurium. Moreover, SCT1101 and SCT1104 enhanced the efficacy of ciprofloxacin and cefixime against intracellular S. Typhimurium. However, only SCT1101 exhibited activity against intracellular MDR and fluoroquinolone-resistant S. Typhimurium isolates. Subsequent mechanistic studies showed that neither of these nilotinib derivatives increased the phospho-AMPK level in RAW264.7 cells. Neither the AMPK inhibitor compound C nor SBI-0206965 reversed the inhibitory effects of SCT1101 and SCT1104 on intracellular Salmonella. Furthermore, neither blockade of autophagy by 3-MA nor shRNA-mediated knockdown of Atg7 protein expression in RAW264.7 cells affected the antibacterial activity of SCT1101 and SCT1104. CONCLUSION: The structure of nilotinib could be used to develop novel therapeutics for controlling MDR S. Typhimurium infections.
Assuntos
Salmonella typhimurium , Humanos , Proteínas Quinases Ativadas por AMP/metabolismo , Proteínas Quinases Ativadas por AMP/farmacologia , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Testes de Sensibilidade MicrobianaRESUMO
OBJECTIVES: The global prevalence of vancomycin-resistant Enterococcus faecium (VREfm) highlights the need for new anti-enterococcal agents. Here, we assessed the molecular epidemiology of clinical VREfm bacteraemic isolates from a medical centre in northern Taiwan in 2019-2020 and to evaluate their susceptibility to last-line antibiotics and a new antimicrobial agent, SC5005. METHODS: The molecular epidemiology of VREfm was investigated using van genotyping, MLST and PFGE. The susceptibilities of VREfm strains to antibiotics and SC5005 were determined using the agar dilution and broth microdilution methods. The capability of E. faecium to develop resistance to antibiotics and SC5005 was evaluated using frequency of resistance and multipassage resistance assays. The mode of action of SC5005 was assessed by time-kill, bacterial membrane integrity and membrane potential assays. RESULTS: All 262 VREfm isolates harboured vanA gene, and the most prevalent sequence type was ST17 (51%, nâ=â134, 84 pulsotypes), followed by ST78 (25%, nâ=â65, 54 pulsotypes). Additionally, we identified four new STs (ST2101, ST2102, ST2135 and ST2136) and observed the arrival of multidrug-resistant ST1885 in Taiwan. Moreover, SC5005 was effective against all VREfm isolates, including those non-susceptible to last-line antibiotics. SC5005 can disrupt and depolarize the bacterial membrane to kill E. faecium without detectable resistance. CONCLUSIONS: The findings provide insights into the latest epidemiology and resistance profiles of bacteraemic-causing VREfm in northern Taiwan. Additionally, SC5005 has the potential for development as a new therapeutic to treat VREfm infections.
Assuntos
Enterococcus faecium , Infecções por Bactérias Gram-Positivas , Enterococos Resistentes à Vancomicina , Humanos , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Infecções por Bactérias Gram-Positivas/epidemiologia , Infecções por Bactérias Gram-Positivas/microbiologia , Epidemiologia Molecular , Tipagem de Sequências Multilocus , Taiwan/epidemiologia , Vancomicina/farmacologia , Resistência a Vancomicina/genéticaRESUMO
The emergence and spread of multidrug-resistant bacteria highlight the need for new antibacterial interventions. A screening of 24 newly synthesized dibenzoxazepines identified a small molecule compound, SW14, with potent inhibitory activity against intracellular multidrug-resistant and fluoroquinolone-resistant strains of S. typhimurium in macrophages and epithelial cells. Moreover, intra-macrophagic Salmonella typhi, Yersinia enterocolitica, and Listeria monocytogenes and methicillin-resistant Staphylococcus aureus are also susceptible to SW14. Overall, our findings suggest that SW14 has a broad-spectrum activity against intracellular bacteria.
RESUMO
Staphylococcus aureus can form persister cells and biofilms, making the treatment difficult and often leading to recurrent infections. In an effort to discover new anti-staphylococcal agents, we observed that oleic acid enhances the activity of a new antibacterial agent, SC5005, against S. aureus and MRSA strains. Subsequent studies showed that saturated or trans-form unsaturated fatty acids did not potentiate SC5005's antibacterial activity. SC5005 only exhibits synergistic bactericidal activity with cis-form unsaturated fatty acids with 16 to 22 carbon atoms. In particular, docosahexaenoic acid (DHA) could reduce the MIC of SC5005 to the subng/mL range against different MRSA strains, including those resistant to second- and third-line antibiotics. However, we did not detect any significant shift in SC5005's cytotoxicity toward four different mammalian cell lines, suggesting that the synergy of DHA and SC5005 is highly selective. Most importantly, this combination demonstrated fast-killing activity, completely eradicating MRSA USA300 planktonic and persister cells within 10 and 30 min, respectively, and removing nearly 98% of MRSA biofilms within 1 min. Together, our findings suggest that the combination of SC5005 and DHA has great potential as a new therapeutic for the treatment of infections caused by multidrug-resistant (MDR) S. aureus biofilms.
Assuntos
Staphylococcus aureus Resistente à Meticilina , Infecções Estafilocócicas , Animais , Humanos , Staphylococcus aureus , Ácidos Docosa-Hexaenoicos/farmacologia , Ácidos Docosa-Hexaenoicos/uso terapêutico , Testes de Sensibilidade Microbiana , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Infecções Estafilocócicas/tratamento farmacológico , Biofilmes , MamíferosRESUMO
OBJECTIVES: This study aimed to provide detailed genetic characterization of Tn6636, a multidrug-resistant and composite mobile element, in clinical isolates of Staphylococcus aureus. METHODS: A total of 112 ermB-positive methicillin-susceptible S. aureus (MSSA) and 224 ermB-positive methicillin-resistant S. aureus (MRSA) isolates collected from 2000 to 2015 were tested for the presence of Tn6636. Detection of the plasmids harboring Tn6636 was performed by S1 nuclease digestion pulsed-field gel electrophoresis (PFGE) analysis, conjugation test, and whole genome sequencing (WGS). RESULTS: Prevalence of Tn6636 in MSSA is higher than that in MRSA. Ten MSSA isolates and 10 MRSA isolates carried Tn6636. The 10 MSSA isolates belonged to three sequence types (ST), including ST7 (n = 6), ST5 (n = 3), and ST59 (n = 1). The 10 MRSA isolates belonged to ST188 (n = 8) and ST965 (n = 2). Analysis of plasmid sequences revealed that Tn6636 was harbored by six different mosaic plasmids. In addition to resistance genes, some plasmids also harbored toxin genes. CONCLUSION: The presence of multi-resistant Tn6636 in plasmids of both MSSA and MRSA with various STs suggests its broad dissemination. Results indicate that Tn6636 has existed for at least 16 years in Taiwan. The mosaic plasmids harboring Tn6636 can be transferred by conjugation. Ongoing surveillance of Tn6636 is essential to avoid continued spreading of resistant plasmids.
Assuntos
Staphylococcus aureus Resistente à Meticilina , Infecções Estafilocócicas , Antibacterianos/farmacologia , Humanos , Staphylococcus aureus Resistente à Meticilina/genética , Testes de Sensibilidade Microbiana , Plasmídeos/genética , Infecções Estafilocócicas/epidemiologia , Staphylococcus aureusRESUMO
Salmonella enterica serovar Typhimurium is the leading cause of invasive nontyphoidal salmonellosis. Additionally, the emergence of multidrug-resistant S. Typhimurium has further increased the difficulty of controlling its infection. Previously, we showed that an antipsychotic drug, loxapine, suppressed intracellular Salmonella in macrophages. To exploit loxapine's antibacterial activity, we simultaneously evaluated the anti-intracellular Salmonella activity and cytotoxicity of newly synthesized loxapine derivatives using an image-based high-content assay. We identified that SW14 exhibits potent suppressive effects on intramacrophagic S. Typhimurium with an 50% effective concentration (EC50) of 0.5 µM. SW14 also sensitized intracellular Salmonella to ciprofloxacin and cefixime and effectively controlled intracellular multidrug- and fluoroquinolone-resistant S. Typhimurium strains. However, SW14 did not affect bacterial growth in standard microbiological broth or minimal medium that mimics the phagosomal environment. Cellular autophagy blockade by 3-methyladenine (3-MA) or shATG7 elevated the susceptibility of intracellular Salmonella to SW14. Finally, reactive oxygen species (ROS) scavengers reduced the antibacterial efficacy of SW14, but the ROS levels in SW14-treated macrophages were not elevated. SW14 decreased the resistance of outer membrane-compromised S. Typhimurium to H2O2. Collectively, our data indicated that the structure of loxapine can be further optimized to develop new antibacterial agents by targeting bacterial resistance to host oxidative-stress defense. IMPORTANCE The incidence of diseases caused by pathogenic bacteria with resistance to common antibiotics is consistently increasing. In addition, Gram-negative bacteria are particularly difficult to treat with antibiotics, especially those that can invade and proliferate intracellularly. In order to find a new antibacterial compound against intracellular Salmonella, we established a cell-based high-content assay and identified SW14 from the derivatives of the antipsychotic drug loxapine. Our data indicate that SW14 has no effect on free bacteria in the medium but can suppress the intracellular proliferation of multidrug-resistant (MDR) S. Typhimurium in macrophages. We also found that SW14 can suppress the resistance of outer membrane compromised Salmonella to H2O2, and its anti-intracellular Salmonella activity can be reversed by reactive oxygen species (ROS) scavengers. Together, the findings suggest that SW14 might act via a virulence-targeted mechanism and that its structure has the potential to be further developed as a new therapeutic against MDR Salmonella.
Assuntos
Antibacterianos/farmacologia , Dibenzoxazepinas/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Salmonella typhimurium/efeitos dos fármacos , Animais , Cefixima , Ciprofloxacina , Farmacorresistência Bacteriana/efeitos dos fármacos , Fluoroquinolonas/farmacologia , Peróxido de Hidrogênio , Loxapina/química , Loxapina/farmacologia , Macrófagos , Camundongos , Testes de Sensibilidade Microbiana , Células RAW 264.7 , Espécies Reativas de Oxigênio , Infecções por Salmonella , SorogrupoRESUMO
Streptococcus pyogenes (group A Streptococcus, GAS) is a strict human pathogen causing a broad spectrum of diseases and a variety of autoimmune sequelae. The pathogenesis of GAS infection mostly relies on the production of an extensive network of cell wall-associated and secreted virulence proteins, such as adhesins, toxins, and exoenzymes. PrsA, the only extracellular parvulin-type peptidyl-prolyl isomerase expressed ubiquitously in Gram-positive bacteria, has been suggested to assist the folding and maturation of newly exported proteins to acquire their native conformation and activity. Two PrsA proteins, PrsA1 and PrsA2, have been identified in GAS, but the respective contribution of each PrsA in GAS pathogenesis remains largely unknown. By combining comparative proteomic and phenotypic analysis approaches, we demonstrate that both PrsA isoforms are required to maintain GAS proteome homeostasis and virulence-associated traits in a unique and overlapping manner. The inactivation of both PrsA in GAS caused remarkable impairment in biofilm formation, host adherence, infection-induced cytotoxicity, and in vivo virulence in a murine soft tissue infection model. The concordance of proteomic and phenotypic data clearly features the essential role of PrsA in GAS full virulence.
Assuntos
Infecções Estreptocócicas , Streptococcus pyogenes , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Humanos , Camundongos , Chaperonas Moleculares , Proteômica , Secretoma , Streptococcus pyogenes/genética , Virulência , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
OBJECTIVES: In the past few decades, multiple-antibiotic-resistant Staphylococcus aureus has emerged and quickly spread in hospitals and communities worldwide. Additionally, the formation of antibiotic-tolerant persisters and biofilms further reduces treatment efficacy. Previously, we identified a sorafenib derivative, SC5005, with bactericidal activity against MRSA in vitro and in vivo. Here, we sought to elucidate the resistance status, mode of action and anti-persister activity of this compound. METHODS: The propensity of S. aureus to develop SC5005 resistance was evaluated by assessment of spontaneous resistance and by multi-passage selection. The mode of action of SC5005 was investigated using macromolecular synthesis, LIVE/DEAD and ATPlite assays and DiOC2(3) staining. The effect of SC5005 on the mammalian cytoplasmic membrane was measured using haemolytic and lactate dehydrogenase (LDH) assays and flow cytometry. RESULTS: SC5005 depolarized and permeabilized the bacterial cytoplasmic membrane, leading to reduced ATP production. Because of this mode of action, no resistance of S. aureus to SC5005 was observed after constant exposure to sub-lethal concentrations for 200 passages. The membrane-perturbing activity of SC5005 was specific to bacteria, as no significant haemolysis or release of LDH from human HT-29 cells was detected. Additionally, compared with other bactericidal antibiotics, SC5005 exhibited superior activity in eradicating both planktonic and biofilm-embedded S. aureus persisters. CONCLUSIONS: Because of its low propensity for resistance development and potent persister-eradicating activity, SC5005 is a promising lead compound for developing new therapies for biofilm-related infections caused by S. aureus.
Assuntos
Staphylococcus aureus Resistente à Meticilina , Infecções Estafilocócicas , Animais , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Biofilmes , Humanos , Potenciais da Membrana , Testes de Sensibilidade Microbiana , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureusRESUMO
The treatment of Staphylococcus aureus infections is impeded by the prevalence of MRSA and the formation of persisters and biofilms. Previously, we identified two celecoxib derivatives, Cpd36 and Cpd46, to eradicate MRSA and other staphylococci. Through whole-genome resequencing, we obtained several lines of evidence that these compounds might act by targeting the membrane protein translocase YidC2. Our data showed that ectopic expression of YidC2 in S. aureus decreased the bacterial susceptibility to Cpd36 and Cpd46, and that the YidC2-mediated tolerance to environmental stresses was suppressed by both compounds. Moreover, the membrane translocation of ATP synthase subunit c, a substrate of YidC2, was blocked by Cpd46, leading to a reduction in bacterial ATP production. Furthermore, we found that the thermal stability of bacterial YidC2 was enhanced, and introducing point mutations into the substrate-interacting cavity of YidC2 had a dramatic effect on Cpd36 binding via surface plasmon resonance assays. Finally, we demonstrated that these YidC2 inhibitors could effectively eradicate MRSA persisters and biofilms. Our findings highlight the potential of impeding YidC2-mediated translocation of membrane proteins as a new strategy for the treatment of bacterial infections.
Assuntos
Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Celecoxib/análogos & derivados , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Antibacterianos/química , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Inibidores de Ciclo-Oxigenase 2/química , Inibidores de Ciclo-Oxigenase 2/farmacologia , Estabilidade Enzimática , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Staphylococcus aureus Resistente à Meticilina/enzimologia , Ligação ProteicaRESUMO
BACKGROUND: The emergence of multiple-antibiotic-resistant (MAR) Salmonella has been a serious threat worldwide. Salmonella can invade into host cells and evade the attacks of host humoral defenses and antibiotics. Thus, a new antibacterial agent capable of inhibiting intracellular Salmonella is highly needed. METHODS: The anti-intracellular activity and cytotoxicity of drugs on intracellular bacteria and macrophages were assayed using intracellular CFU assay and MTT cell viability assay, respectively. The uptake of gentamicin into macrophage and the effect of autophagy inhibitor on loxapine's anti-intracellular Salmonella activity were assessed by using image-based high-content system. The expression of bacterial genes was measured by real-time PCR. The efflux pump activity of bacteria was measured by Hoechst accumulation assays. RESULTS: With our efforts, an antipsychotic drug, loxapine, was identified to exhibit high potency in suppressing intracellular MAR S. Typhimurium, Staphylococcus aureus, Shigella flexneri or Yersinia enterocolitica. Subsequent investigations indicated that loxapine's anti-intracellular bacteria activity was not associated with increased penetration of gentamicin into bacteria and macrophages. Loxapine didn't inhibit bacterial growth in broth at concentration up to 500 µM and has no effect on Salmonella's type III secretion system genes' expression. Blockage of autophagy also didn't reverse loxapine's anti-intracellular activity. Lastly, loxapine suppressed bacterial efflux pump activity in all bacteria tested. CONCLUSION: Altogether, our data suggested that loxapine might suppress intracellular bacteria through inhibiting of bacterial efflux pumps. In light of its unique activity, loxapine represents a promising lead compound with translational potential for the development of a new antibacterial agent against intracellular bacteria.
Assuntos
Antibacterianos/farmacologia , Antipsicóticos/farmacologia , Loxapina/farmacologia , Macrófagos/microbiologia , Salmonella typhimurium/efeitos dos fármacos , Animais , Autofagia/efeitos dos fármacos , Proteínas de Bactérias/genética , Sobrevivência Celular/efeitos dos fármacos , Contagem de Colônia Microbiana , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Fluoroquinolonas/farmacologia , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Gentamicinas/farmacologia , Proteínas de Membrana Transportadoras/efeitos dos fármacos , Proteínas de Membrana Transportadoras/genética , Camundongos , Testes de Sensibilidade Microbiana , Fenotiazinas/farmacologia , Células RAW 264.7 , Salmonella typhimurium/genética , Salmonella typhimurium/crescimento & desenvolvimento , Sorogrupo , Shigella flexneri/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Sistemas de Secreção Tipo III/efeitos dos fármacos , Sistemas de Secreção Tipo III/genética , Yersinia enterocolitica/efeitos dos fármacosRESUMO
The enteric pathogen enterohemorrhagic Escherichia coli (EHEC) is responsible for outbreaks of bloody diarrhea and hemolytic uremic syndrome (HUS) worldwide. Several molecular mechanisms have been described for the pathogenicity of EHEC; however, the role of bacterial metabolism in the virulence of EHEC during infection in vivo remains unclear. Here we show that aerobic metabolism plays an important role in the regulation of EHEC virulence in Caenorhabditis elegans. Our functional genomic analyses showed that disruption of the genes encoding the succinate dehydrogenase complex (Sdh) of EHEC, including the sdhA gene, attenuated its toxicity toward C. elegans animals. Sdh converts succinate to fumarate and links the tricarboxylic acid (TCA) cycle and the electron transport chain (ETC) simultaneously. Succinate accumulation and fumarate depletion in the EHEC sdhA mutant cells were also demonstrated to be concomitant by metabolomic analyses. Moreover, fumarate replenishment to the sdhA mutant significantly increased its virulence toward C. elegans. These results suggest that the TCA cycle, ETC, and alteration in metabolome all account for the attenuated toxicity of the sdhA mutant, and Sdh catabolite fumarate in particular plays a critical role in the regulation of EHEC virulence. In addition, we identified the tryptophanase (TnaA) as a downstream virulence determinant of SdhA using a label-free proteomic method. We demonstrated that expression of tnaA is regulated by fumarate in EHEC. Taken together, our multi-omic analyses demonstrate that sdhA is required for the virulence of EHEC, and aerobic metabolism plays important roles in the pathogenicity of EHEC infection in C. elegans. Moreover, our study highlights the potential targeting of SdhA, if druggable, as alternative preventive or therapeutic strategies by which to combat EHEC infection.
Assuntos
Escherichia coli Êntero-Hemorrágica/efeitos dos fármacos , Escherichia coli Êntero-Hemorrágica/metabolismo , Fumaratos/farmacologia , Animais , Escherichia coli Êntero-Hemorrágica/patogenicidade , Humanos , Espectrometria de Massas , Metabolômica/métodos , Proteômica/métodos , Reação em Cadeia da Polimerase em Tempo Real , VirulênciaRESUMO
Methicillin-resistant Staphylococcus aureus (MRSA) is a serious threat to humans. Most existing antimicrobial drugs, including the ß-lactam and quinoxiline classes, are not effective against MRSA. In this study, we synthesized 24 derivatives of malonamide, a new class of antibacterial agents and potentiators of classic antimicrobials. A derivative that increases bacterial killing and biofilm eradication with low cell toxicity was created.
Assuntos
Antibacterianos/síntese química , Biofilmes/efeitos dos fármacos , Ciclopropanos/química , Ácidos Dicarboxílicos/química , Malonatos/síntese química , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Antibacterianos/farmacologia , Biofilmes/crescimento & desenvolvimento , Desenho de Fármacos , Humanos , Malonatos/farmacologia , Staphylococcus aureus Resistente à Meticilina/crescimento & desenvolvimento , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Testes de Sensibilidade Microbiana , Infecções Estafilocócicas/microbiologia , Relação Estrutura-AtividadeRESUMO
The increasing incidence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) Mycobacterium tuberculosis (MTB) adds further urgency for rapid and multiplex molecular testing to identify the MTB complex and drug susceptibility directly from sputum for disease control. A nucleotide matrix-assisted-laser-desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based assay was developed to identify MTB (MTBID panel) and 45 chromosomal mutations for resistance to eight antibiotics (MTBDR panel). We conducted a 300 case trial from outpatients to evaluate this platform. An MTBID panel specifically identified MTB with as few as 10 chromosome DNA copies. The panel was 100% consistent with an acid-fast stain and culture for MTB, nontuberculous mycobacteria, and non-mycobacteria bacteria. The MTBDR panel was validated using 20 known MDR-MTB isolates. In a 64-case double-blind clinical isolates test, the sensitivity and specificity were 83% and 100%, respectively. In a 300-case raw sputum trial, the MTB identification sensitivity in smear-negative cases using MALDI-TOF MS was better than the COBAS assay (61.9% vs. 46.6%). Importantly, the failure rate of MALDI-TOF MS was better than COBAS (11.3% vs. 26.3%). To the best of our knowledge, the test described herein is the only multiplex test that predicts resistance for up to eight antibiotics with both sensitivity and flexibility.
Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana , Mutação , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Escarro/microbiologia , Tuberculose/diagnóstico , Tuberculose/microbiologia , Técnicas de Tipagem Bacteriana , Sequência de Bases , DNA Girase/química , DNA Girase/genética , Análise Mutacional de DNA , Genes Bacterianos , Humanos , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodosRESUMO
BACKGROUND/OBJECTIVES: Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) was recognized as a leading pathogen and has been shown to be genetically different from the health care-associated MRSA (HA-MRSA). Photodynamic therapy (PDT) is considered a potential alternative method for the treatment of resistant bacterial infections, but the effect of PDT on CA-MRSA is unknown. The purpose of this study was to compare the bactericidal effects of toluidine blue O (TBO) on CA-MRSA and HA-MRSA and investigate the photodynamic inactivation effects of TBO (TBO-PDI) against bacterial virulence factors. MATERIALS AND METHODS: TBO-PDI effects were determined by measuring the survival fractions for four strains and bactericidal activities for 26 CA-MRSA isolates and 26 HA-MRSA isolates. The influences of TBO-PDI on DNA fragmentation and the activities of protease, lipase, staphylococcal α-hemolysin, and enterotoxin were studied. RESULTS: TBO-PDI has effective bactericidal activity against both CA- and HA-MRSA. However, the bactericidal activity of TBO-PDI was significantly higher against HA-MRSA than CA-MRSA isolates. In addition, TBO-PDI treatment using a sublethal TBO concentration led to reduced production of several virulence factors, including protease, lipase, staphylococcal α-hemolysin, and enterotoxin. CONCLUSION: Although TBO-PDI is slightly less effective against CA-MRSA than HA-MRSA isolates, TBO-PDI could reduce the production of virulence factors at a sublethal TBO concentration, which would be beneficial for treating CA-MRSA infections.
Assuntos
Antibacterianos/farmacologia , Infecções Comunitárias Adquiridas/microbiologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Fármacos Fotossensibilizantes/farmacologia , Infecções Estafilocócicas/microbiologia , Cloreto de Tolônio/farmacologia , Infecção Hospitalar/microbiologia , Humanos , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Staphylococcus aureus Resistente à Meticilina/fisiologia , Viabilidade Microbiana/efeitos dos fármacos , Fatores de Virulência/análiseRESUMO
Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important foodborne pathogen causing severe diseases in humans worldwide. Currently, there is no specific treatment available for EHEC infection and the use of conventional antibiotics is contraindicated. Therefore, identification of potential therapeutic targets and development of effective measures to control and treat EHEC infection are needed. Lipopolysaccharides (LPS) are surface glycolipids found on the outer membrane of gram-negative bacteria, including EHEC, and LPS biosynthesis has long been considered as potential anti-bacterial target. Here, we demonstrated that the EHEC rfaD gene that functions in the biosynthesis of the LPS inner core is required for the intestinal colonization and pathogenesis of EHEC in vivo. Disruption of the EHEC rfaD confers attenuated toxicity in Caenorhabditis elegans and less bacterial colonization in the intestine of C. elegans and mouse. Moreover, rfaD is also involved in the control of susceptibility of EHEC to antimicrobial peptides and host intestinal immunity. It is worth noting that rfaD mutation did not interfere with the growth kinetics when compared to the wild-type EHEC cells. Taken together, we demonstrated that mutations of the EHEC rfaD confer hypersusceptibility to host intestinal innate immunity in vivo, and suggested that targeting the RfaD or the core LPS synthesis pathway may provide alternative therapeutic regimens for EHEC infection.
Assuntos
Carboidratos Epimerases/genética , Carboidratos Epimerases/metabolismo , Escherichia coli O157/enzimologia , Escherichia coli O157/genética , Intestinos/imunologia , Lipopolissacarídeos/biossíntese , Deleção de Sequência , Actinas/imunologia , Actinas/metabolismo , Animais , Peptídeos Catiônicos Antimicrobianos/farmacologia , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/imunologia , Proteínas de Caenorhabditis elegans/metabolismo , Carboidratos Epimerases/imunologia , Modelos Animais de Doenças , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/terapia , Proteínas de Escherichia coli/imunologia , Proteínas de Escherichia coli/metabolismo , Feminino , Humanos , Imunidade Inata , Enteropatias/imunologia , Enteropatias/microbiologia , Intestinos/microbiologia , Intestinos/patologia , Lipopolissacarídeos/química , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Virulência/genética , Fatores de Virulência/metabolismo , CatelicidinasRESUMO
Patients with triple-negative breast cancer (TNBC) had an increased likelihood of distant recurrence and death, as compared with those with non-TNBC subtype. Regorafenib is a multi-receptor tyrosine kinase (RTK) inhibitor targeting oncogenesis and has been approved for metastatic colorectal cancer and advanced gastrointestinal stromal tumor. Recent studies suggest regorafenib acts as a SHP-1 phosphatase agonist. Here, we investigated the potential of regorafenib to suppress metastasis of TNBC cells through targeting SHP-1/p-STAT3/VEGF-A axis. We found a significant correlation between cancer cell migration and SHP-1/p-STAT3/VEGF-A expression in human TNBC cells. Clinically, high VEGF-A expression is associated with worse disease-free and distant metastasis-free survival. Regorafenib induced significant anti-migratory effects, in association with downregulation of p-STAT3 and VEGF-A. To exclude the role of RTK inhibition in regorafenib-induced anti-metastasis, we synthesized a regorafenib derivative, SC-78, that had minimal effect on VEGFR2 and PDGFR kinase inhibition, while having more potent effects on SHP-1 activation. SC-78 demonstrated superior in vitro and in vivo anti-migration to regorafenib. Furthermore, VEGF-A dependent autocrine/paracrine loops were disrupted by regorafenib and SC-78. This study implies that SHP-1/p-STAT3/VEGF-A axis is a potential therapeutic target for metastatic TNBC, and the more potent SC-78 may be a promising lead for suppressing metastasis of TNBC.
Assuntos
Compostos de Fenilureia/farmacologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Piridinas/farmacologia , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Fator A de Crescimento do Endotélio Vascular/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Comunicação Autócrina/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Feminino , Humanos , Estimativa de Kaplan-Meier , Camundongos Nus , Metástase Neoplásica , Comunicação Parácrina/efeitos dos fármacos , Fator de Transcrição STAT3/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
OBJECTIVES: The emergence of MRSA strains resistant to most antibiotics is a serious threat to public health. Based on our discovery that the tyrosine kinase inhibitor sorafenib exhibits inhibitory activity against Staphylococcus species, the objective of this study is to exploit this unique antibacterial activity of sorafenib to develop novel antibacterial agents against MRSA. METHODS: A sorafenib-based focused compound library was synthesized by substituting the pyridinyl and phenyl groups with different functional groups. The resulting sorafenib derivatives were screened for growth-suppressive activities against Staphylococcus aureus and Staphylococcus epidermidis following CLSI guidelines and for cytotoxicity towards human cells using MTT cell viability assays. Compounds with high selectivity for bacterial inhibition over cytotoxicity were further evaluated by time-kill assay and Caenorhabditis elegans and mice survival assays to evaluate their efficacy in vitro and in vivo. RESULTS: The screening of sorafenib derivatives led to the identification of compound SC5005 as a lead compound with high potency in killing different clinical strains of MRSA with an MIC90 of 0.5 mg/L and with low cytotoxicity, as demonstrated by IC50-to-MIC ratios of up to 40. In addition, SC5005 showed a significant protective effect in MSSA- or MRSA-infected C. elegans. Intraperitoneal administration of SC5005 at 10 mg/kg significantly improved the survival of MRSA-infected C57BL/6 mice. CONCLUSIONS: In light of its high potency in suppressing MRSA in both in vitro and in vivo models, SC5005 represents a potential lead agent for continued preclinical development as a therapeutic intervention against MRSA.