RESUMO
The high incidence of Mycobacterium infection, notably multidrug-resistant M. tuberculosis infection, has become a significant public health concern worldwide. In this study, we isolate and analyze a mycobacteriophage, BTCU-1, and a foundational study was performed to evaluate the antimycobacterial activity of BTCU-1 and its cloned lytic endolysins. Using Mycobacterium smegmatis as host, a mycobacteriophage, BTCU-1, was isolated from soil in eastern Taiwan. The electron microscopy images revealed that BTCU-1 displayed morphology resembling the Siphoviridae family. In the genome of BTCU-1, two putative lytic genes, BTCU-1_ORF7 and BTCU-1_ORF8 (termed lysA and lysB, respectively), were identified, and further subcloned and expressed in Escherichia coli. When applied exogenously, both LysA and LysB were active against M. smegmatis tested. Scanning electron microscopy revealed that LysA and LysB caused a remarkable modification of the cell shape of M. smegmatis. Intracellular bactericidal activity assay showed that treatment of M. smegmatis-infected RAW 264.7 macrophages with LysA or LysB resulted in a significant reduction in the number of viable intracellular bacilli. These results indicate that the endolysins derived from BTCU-1 have antimycobacterial activity, and suggest that they are good candidates for therapeutic/disinfectant agents to control mycobacterial infections.
Assuntos
Antituberculosos/farmacologia , Endopeptidases/farmacologia , Mycobacterium smegmatis/efeitos dos fármacos , Proteínas Virais/farmacologia , Sequência de Aminoácidos , Animais , Antituberculosos/química , Antituberculosos/isolamento & purificação , Bacteriófagos/enzimologia , Bacteriófagos/ultraestrutura , Sequência Conservada , Endopeptidases/química , Endopeptidases/isolamento & purificação , Camundongos , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Dados de Sequência Molecular , Mycobacterium smegmatis/virologia , Células RAW 264.7 , Proteínas Virais/química , Proteínas Virais/isolamento & purificaçãoRESUMO
Mixed infection with Beijing and non-Beijing strains of Mycobacterium tuberculosis has been reported and has been suggested to mediate elevation of the reinfection rate in regions with a high incidence of tuberculosis (TB). To evaluate the prevalence of infection with both Beijing and non-Beijing strains of M. tuberculosis in eastern Taiwan, the region with the highest TB incidence in Taiwan, 185 active pulmonary TB patients were enrolled at Tzu Chi General Hospital from October 2007 to September 2008. A modified multiplex PCR method was developed to distinguish Beijing and non-Beijing strains directly using the sputum of patients. Of the 185 patients, 46.5% were infected with a Beijing strain, 42.2% were infected with a non-Beijing strain, and 11.3% were infected with both strain types. Notably, mixed infection with both strain types was not associated with TB treatment history or the high-incidence race group, aborigines. In addition, the incidence rate of mixed infection before treatment with anti-TB medication was as high as that in patients with a history of anti-TB treatment. Further analysis of antibiotic susceptibility revealed that Beijing strains alone had the highest multidrug resistance rate (17.5%), mixed infection had the highest rate of resistance to at least one drug (23.8%), and non-Beijing strains had the highest rate of sensitivity to all drugs (79.5%), implying that Beijing strains are predominant in the development of drug resistance in tuberculosis.
Assuntos
Antituberculosos/farmacologia , Farmacorresistência Bacteriana , Mycobacterium tuberculosis/classificação , Mycobacterium tuberculosis/efeitos dos fármacos , Tuberculose Pulmonar/epidemiologia , Tuberculose Pulmonar/microbiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Técnicas Bacteriológicas/métodos , Criança , Pré-Escolar , DNA Bacteriano/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Tipagem Molecular , Mycobacterium tuberculosis/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Prevalência , Taiwan/epidemiologia , Adulto JovemRESUMO
Innate immunity involves a cascade of inflammatory events, resulting in the secretion of chemokines and cytokines to recruit mediator cells in adaptive immunity. To study epithelial inflammatory responses initiated by Streptococcus pyogenes infection, we investigated chemotaxis ability in the supernatant of infected human respiratory epithelial HEp-2 cells. Our results showed that these supernatants showed significantly increased ability to attract monocytes, implying the release of inflammatory chemoattractants into the medium. Expression of interleukin (IL)-8 and IL-6 in HEp-2 cells was significantly increased at both the mRNA and protein levels after infection with S. pyogenes. Electrophoretic mobility shift and reporter-gene assays demonstrated that the transcription factors NF-kappaB and AP-1, regulated by mitogen-activated protein (MAP) kinase, were activated after streptococcal infection. The increases in mRNAs for IL-8 and IL-6 were abrogated by addition of NF-kappaB and MAP kinase inhibitors, suggesting that the upregulation of IL-8 and IL-6 is mediated through NF-kappaB and MAP kinase signaling pathways. Taken together, our results indicate that S. pyogenes infection of epithelial cells induces the secretion of pro-inflammatory chemokines/cytokines through activation of NF-kappaB and MAP kinase signaling pathways. These early innate responses initiated by S. pyogenes-infected respiratory epithelial cells may recruit immune cells to the airway and induce inflammation.
Assuntos
Interleucina-6/imunologia , Interleucina-8/imunologia , Proteínas Quinases Ativadas por Mitógeno/imunologia , NF-kappa B/imunologia , Infecções Estreptocócicas/imunologia , Streptococcus pyogenes/fisiologia , Apigenina/farmacologia , Autoanticorpos/imunologia , Movimento Celular/imunologia , Quimiotaxia/imunologia , Ensaio de Desvio de Mobilidade Eletroforética , Ensaio de Imunoadsorção Enzimática , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Humanos , Imidazóis/farmacologia , Interleucina-6/biossíntese , Interleucina-6/genética , Interleucina-8/biossíntese , Interleucina-8/genética , Sistema de Sinalização das MAP Quinases/imunologia , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Infecções Estreptocócicas/enzimologia , Fator de Transcrição AP-1/imunologia , Fator de Transcrição AP-1/metabolismo , Células U937RESUMO
Spread of multidrug-resistant tuberculosis (MDR-TB) strains in the general population presents a serious threat to public health and severely threatens existing control efforts. Techniques such as spoligotyping and Mycobacterium interspersed repetitive units-variable-number tandem-repeat typing of mycobacterial isolates have been employed to confirm familial outbreaks of MDR-TB. We diagnosed and traced four MDR-TB cases in a family via genotyping. Despite aggressive treatment, the index case remained culture positive, but the other patients were cured. This is the first documentation of a familial MDR-TB outbreak affecting human immunodeficiency virus-seronegative patients in eastern Taiwan. Molecular techniques are important in the identification of sources of MDR-TB infections. The adult index case in our study developed MDR-TB due to poor compliance with the drug regimen (acquired resistance), followed by transmission of MDR-TB to his children in close household contact. This emphasizes the importance of an effective drug delivery program, such as directly observed treatment, to improve drug compliance and prevent the emergence of drug-resistant cases.