Biological activity and pathogenicity of M23 superfamily metalloendopeptidases of Leptospira interro-gans / 中华微生物学和免疫学杂志

Objective To understand and determine the biological activity and pathogenicity of metalloendopeptidases encoded by LA2582 and LA2901 genes of Leptospira interrogans(L.interrogans) sero-group Icterohaemorrhagiaeserovar Lai strain Lai. Methods Structures and functions of LA2582 and LA2901 genes were analyzed by using bioinformatic software. Prokaryotic expression systems for expressing the extra-cellular regions of LA2582 and LA2901 genes were generated. The target recombinant expression products, rLA2582 and rLA2901,were extracted by Ni-NTA affinity chromatography. The Azo-casein-hydrolyzingactiv-ity of rLA2582 and rLA2901 was detected by spectrophotometry. Activities of rLA2582 and rLA2901 in the hydrolysis of Dabsyl-Leu-Gly-Gly-Gly-Ala-Edans, a fluorescence-labeling pentapeptide substrate, were de-tected by fluorospectrophotometry,and then the Km and Kcat values were determined. SDS-PAGE and spec-trophotometry were performed to detect the activities of rLA2582 and rLA2901 in hydrolyzing extracellular matrix molecules such as collagen type-Ⅰ (COL1), fibronectin (FN) and Congo red-labeling elastin (ELN). Real-time fluorescent quantitative RT-PCR(qRT-PCR) and Western blot were respectively used to measure the expression of LA2582 and LA2901 genes at mRNA and protein levels after infecting human um-bilical vein endothelial cells(HUVEC) with L. interrogans strain Lai. Results The gene products of LA2582 and LA2901 genes were identified as the signal peptide and matrix metalloproteinase motif HXH-containing Zn2+-dependent Gly-Gly metalloendopeptidases belonging to the M23 superfamily. rLA2582 and rLA2901 did not hydrolyze Azo-casein (Km=126.54 μmol/L, Kcat=4.67/s), but could hydrolyze the pentapeptide substrate (Km=190. 25 μmol/L, Kcat 4. 86/s). rLA2582 and rLA2901 could hydrolyze COL1, FN and ELN. Expression of LA2582 and LA2901 genes at both mRNA and protein levels was signifi-cantly increased after infection of HUVEC with L.interrogans strain Lai(P<0.05). Conclusion The prod-ucts of LA2582 and LA2901 genes of L.interrogans strain Lai are Zn2+-dependent M23 metalloendopeptidas-es, which can hydrolyze multiple ECM molecules and are closely associated with the leptospiral invasiveness.

Incidencia y fármaco-resistencia de cepas de Staphylococcus spp aisladas de exudados conjuntivales / Incidence and drug-resistance of Staphylococcus spp strains isolated from conjunctival swabs

OBJETIVO: Determinar la incidencia de cepas del género Staphylococcus aisladas de exudados conjuntivales y analizar su resistencia frente a diferentes antimicrobianos. MÉTODOS: Se realizó un estudio observacional descriptivo retrospectivo en el que se revisaron 3554 exudados conjuntivales a pacientes que acudieron en el período comprendido entre enero de 2002 a diciembre de 2003 y desde enero de 2005 hasta diciembre de 2007 al Instituto Cubano de Oftalmología Ramón Pando Ferrer por presentar un diagnóstico de infección ocular externa. RESULTADOS: Se aislaron 874 cepas de Staphylococcus aureus para un 47,5 por ciento y 965 cepas de Staphylococcus spp. coagulasa negativa con prueba de patogenicidad positiva para un 52,4 por ciento. En 69 de esos exudados los cultivos presentaron etiología mixta con dos bacterias diferentes, para el 3,7 por ciento. Se determinaron los porcentajes de resistencia a las cepas aisladas pertenecientes al género Staphylococcus. CONCLUSIONES: Se encontró una alta incidencia de las especies del género Staphylococcus en las infecciones oculares, así como se pudo apreciar que la menor fármaco-resistencia para Staphylococcus aureus y Staphylococcus spp. coagulasa negativa correspondieron a los antimicrobianos ciprofloxacina y amikacina. La mayor fármaco-resistencia de las cepas de Staphylococcus aureus correspondió a eritromicina y tetraciclina y en Staphylococcus spp coagulasa negativa fue frente a la tetraciclina

OBJECTIVE: To determine the incidence of Staphylococcus strains isolated from conjunctival swaps and their resistance to several antimicrobial agents. METHODS: A retrospective, descriptive and observational study was performed to review 3554 conjunctival swabs from patients who went to Ramón Pando Ferrer Institute of Ophthalmology in the period from January 2002 to December 2009 due to a diagnosis of external ocular infection. RESULTS: From the total of conjunctival swabs, 874 Staphylococcus aureus strains (47.5 percent) and 965 (52.4 percent) negative Staphylococcus spp. coagulasa strains were isolated, being pathogenecity test positive in 52.4 percent. In 69 swabs, the cultures presented mixed etiology with two different bacteria accounting for 3.7 percent. Resistance percentages of Staphylococcus strains were estimated. CONCLUSION: The incidence of Staphylococcus species in eye infections was high and the drug resistance for Staphylococcus aureus and Staphylococcus spp. coagulasa negative to Ciprofloxacin and Amikacyn was lower. The greatest drug resistance of S. aureus strains corresponded to erythromycin and tetracycline whereas Staphylococcus spp coagulasa negative were more resistant to tetracycline

DNA Variation of Helicobacter Pylori in the Gastroduodenal Disease / 대한내과학회지

BACKGROUND: The evidence for H. pylori as a gastrointestnal pathogen is now very strong, if not overwhelming. Among the pathogenic factors of H. pylori, flagella and urease are considered to be major factors causing the gastrododenal disease. We observed the gene diversity of H. pylori using the PCR-amplified 1.4Kb fla A gene and 0.9Kb ure B gene and examined the relationship between the gene pattern and the gastroduodenal disease. METHOD: Fifty-one cases of isolated strains were cultured at the Helicobacter-selective blood agar plates. To compare the gene diversity among the isolates of gastroduodenal disease genotypes was analyzed by PCR-based RFLP. 1.4Kb fla A gene and 0.9Kb ure B genes from isolates were amplified by PCR and digested with Hae 3 restriction enzymes to observe the restriction fragment length polymophysm. Protein patterns were also compared to examine the antigenic variations. Total cell proteins, and octyl-glucose extracts from isolates were analyzed by SDS-PAGE gel electrophoresis. RESULTS: 41 cases (80.4%) of H. pylori were isolated in the 51 cases of gastroduodenal diseases. We could classify theses isolates 3 types of PCR-RFLP in the fla A gene, 900+500bp, 500+500+400bp, 600+800bp, and 9 types in the ure B gene. PCR-RFLP in the fla A gene and ure B gene of the isolates was different from the standard strain of Australia and the genetic diversity was not related to the types of the gastroduodenal disease. We demonstrated variations in the protein pattern and antigenic profiles among the isolates by SDS-PAGE analysis. These data also did not show any relationship between protein pattern and types of gastroduodenal diseases. CONCLUSION: Tese studies showed many different gene diversity in the flagella and urease gene without any relationship with the types of gastoduodenal disease. And variable protein pattern were noted among the strains of H. pylori. Further studies to demonstrate the pathgenecity of H. pylori should be continued even if there was no relationship between the genomic diversity of the flagella or urease and the types of gastroduodenal disease.