RESUMO
We have established several models of infectious diseases in silkworms to explore disease-causing mechanisms and identify new antimicrobial substances. These models involve injecting laboratory-cultured pathogens into silkworms and monitoring their survival over a period of days. The use of silkworms is advantageous because they are cost-effective and raise fewer ethical concerns than mammalian subjects, allowing for larger experimental group sizes. To capitalize on these benefits, there is a growing importance in mechanizing and automating the experimental processes that currently require manual labor. This paper discusses the future of laboratory automation, specifically through the mechanization and automation of silkworm-based experimental procedures.
Assuntos
Automação Laboratorial , Bombyx , Descoberta de Drogas , Animais , Humanos , Modelos Animais de Doenças , Descoberta de Drogas/métodosRESUMO
In this present study, we investigated the phenol-soluble modulin (psm-mec) mutations, the staphylococcal cassette chromosome mec (SCCmec) types, and toxin production in 102 methicillin-resistant Staphylococcus aureus (MRSA) isolates from the northeast and central regions of Thailand. The MRSA isolates carrying -7T>C psm-mec in Type II SCCmec (n = 18) and the MRSA isolates carrying no psm-mec in Type IV (n = 8) or Type IX SCCmec (n = 4) had higher hemolytic activity against sheep erythrocytes than MRSA isolates carrying intact psm-mec in Type III SCCmec (n = 34), but MRSA isolates carrying no psm-mec in Type I SCCmec (n = 27) did not.
Assuntos
Toxinas Bacterianas , Staphylococcus aureus Resistente à Meticilina , Infecções Estafilocócicas/epidemiologia , Animais , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Eritrócitos/patologia , Hemolíticos/metabolismo , Humanos , Resistência a Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/metabolismo , Staphylococcus aureus Resistente à Meticilina/patogenicidade , Mutação , Ovinos , Infecções Estafilocócicas/tratamento farmacológico , Tailândia/epidemiologiaRESUMO
Vancomycin is a widely used clinical drug to treat for infection by methicillin-resistant Staphylococcus aureus. Some patients show a weak response to vancomycin treatment. We previously reported that ß-lactams increase the susceptibility to vancomycin by vancomycin-highly resistant S. aureus (VRSA) strains obtained following repeated in vitro mutagenesis and vancomycin selection. Here we found that the susceptibility of the VRSA strains to vancomycin was remarkably increased by combined treatment with D-cycloserine. On the other hand, VRSA did not show increased susceptibility to vancomycin in combination with bacitracin, fosfomycin, erythromycin, lincomycin, gentamicin, levofloxacin or nisin. Furthermore, in an in vivo infection model with silkworms, combined treatment with vancomycin and D-cycloserine exhibited therapeutic effects, whereas treatment with each compound alone did not. These findings suggest that combined treatment with vancomycin and D-cycloserine could be therapeutically effective against infectious diseases caused by VRSA.
Assuntos
Antibacterianos/farmacologia , Ciclosserina/farmacologia , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/efeitos dos fármacos , Vancomicina/farmacologia , Animais , Antibacterianos/administração & dosagem , Bombyx , Ciclosserina/administração & dosagem , Modelos Animais de Doenças , Sinergismo Farmacológico , Testes de Sensibilidade Microbiana , Infecções Estafilocócicas/microbiologia , Vancomicina/administração & dosagem , Resistência a VancomicinaRESUMO
We previously reported isolating vancomycin (VAN) highly resistant Staphylococcus aureus (VRSA) strains from clinical methicillin-resistant S. aureus strains by repeating steps of in vitro mutagenesis and VAN selection. Here we describe that the in vitro susceptibility of these VRSA strains to VAN was markedly increased by combined treatment with ß-lactams such as ceftriaxone and oxacillin. Furthermore, in an in vivo silkworm infection model with VRSA, a combination of VAN and ceftriaxone exhibited therapeutic effects, whereas a combination of VAN and oxacillin did not. These findings suggest that combining VAN with an appropriate ß-lactam, such as ceftriaxone, is therapeutically effective against infectious diseases caused by VRSA.
Assuntos
Antibacterianos/farmacologia , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/efeitos dos fármacos , Vancomicina/farmacologia , beta-Lactamas/farmacologia , Animais , Antibacterianos/administração & dosagem , Bombyx , Ceftriaxona/administração & dosagem , Ceftriaxona/farmacologia , Modelos Animais de Doenças , Sinergismo Farmacológico , Testes de Sensibilidade Microbiana , Oxacilina/administração & dosagem , Oxacilina/farmacologia , Infecções Estafilocócicas/microbiologia , Vancomicina/administração & dosagem , Resistência a Vancomicina , beta-Lactamas/administração & dosagemRESUMO
Staphylococcus aureus produces phenol-soluble modulins (PSMs), which are amphipathic small peptides with lytic activity against mammalian cells. We previously reported that PSMα1-4 stimulate S. aureus colony spreading, the phenomenon of S. aureus colony expansion on the surface of soft agar plates, whereas δ-toxin (Hld, PSMγ) inhibits colony-spreading activity. In this study, we revealed the underlying mechanism of the opposing effects of PSMα1-4 and δ-toxin in S. aureus colony spreading. PSMα1-4 and δ-toxin are abundant on the S. aureus cell surface, and account for 18% and 8.5% of the total amount of PSMα1-4 and δ-toxin, respectively, in S. aureus overnight cultures. Knockout of PSMα1-4 did not affect the amount of cell surface δ-toxin. In contrast, knockout of δ-toxin increased the amount of cell surface PSMα1-4, and decreased the amount of culture supernatant PSMα1-4. The δ-toxin inhibited PSMα3 and PSMα2 binding to the S. aureus cell surface in vitro. A double knockout strain of PSMα1-4 and δ-toxin exhibited decreased colony spreading compared with the parent strain. Expression of cell surface PSMα1-4, but not culture supernatant PSMα1-4, restored the colony-spreading activity of the PSMα1-4/δ-toxin double knockout strain. Expression of δ-toxin on the cell surface or in the culture supernatant did not restore the colony-spreading activity of the PSMα1-4/δ-toxin double knockout strain. These findings suggest that cell surface PSMα1-4 promote S. aureus colony spreading, whereas δ-toxin suppresses colony-spreading activity by inhibiting PSMα1-4 binding to the S. aureus cell surface.
Assuntos
Toxinas Bacterianas/biossíntese , Regulação Bacteriana da Expressão Gênica/fisiologia , Fenol , Staphylococcus aureus/crescimento & desenvolvimento , Toxinas Bacterianas/genética , Técnicas de Silenciamento de Genes , Staphylococcus aureus/genéticaRESUMO
The development of vancomycin (VCM) resistance in Staphylococcus aureus threatens global health. Studies of the VCM-resistance mechanism and alternative therapeutic strategies are urgently needed. We mutagenized S. aureus laboratory strains and methicillin-resistant S. aureus (MRSA) with ethyl methanesulfonate, and isolated mutants that exhibited high resistance to VCM (minimum inhibitory concentration = 32 µg/ml). These VCM-resistant strains were sensitive to linezolid and rifampicin, and partly to arbekacin and daptomycin. Beta-lactams had synergistic effects with VCM against these mutants. VCM-resistant strains exhibited a 2-fold increase in the cell wall thickness. Several genes were commonly mutated among the highly VCM-resistant mutants. These findings suggest that MRSA has a potential to develop high VCM resistance with cell wall thickening by the accumulation of mutations.
Assuntos
Antibacterianos/farmacologia , Genômica , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus/genética , Resistência a Vancomicina/genética , Vancomicina/farmacologia , Parede Celular/química , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Metanossulfonato de Etila/toxicidade , Humanos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Testes de Sensibilidade Microbiana , Microscopia Eletrônica de Transmissão , Mutagênese , Mutagênicos/toxicidade , Fenótipo , Análise de Sequência de DNA , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/patologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/isolamento & purificaçãoRESUMO
Lon protease, a member of the ATP-dependent protease family, regulates numerous cellular systems by degrading specific substrates. Here, we demonstrate that Lon is involved in the regulation of quorum-sensing (QS) signaling systems in Pseudomonas aeruginosa, an opportunistic human pathogen. The organism has two acyl-homoserine lactone (HSL)-mediated QS systems, LasR/LasI and RhlR/RhlI. Many reports have demonstrated that these two systems are regulated and interconnected by global regulators. We found that lon-disrupted cells overproduce pyocyanin, the biosynthesis of which depends on the RhlR/RhlI system, and show increased levels of a transcriptional regulator, RhlR. The QS systems are organized hierarchically: the RhlR/RhlI system is subordinate to LasR/LasI. To elucidate the mechanism by which Lon negatively regulates RhlR/RhlI, we examined the effect of lon disruption on the LasR/LasI system. We found that Lon represses the expression of LasR/LasI by degrading LasI, an HSL synthase, leading to negative regulation of the RhlR/RhlI system. RhlR/RhlI was also shown to be regulated by Lon independently of LasR/LasI via regulation of RhlI, an HSL synthase. In view of these findings, it is suggested that Lon protease is a powerful negative regulator of both HSL-mediated QS systems in P. aeruginosa.