Surveillance of antibiotic susceptibility pattern among shigella flexneri strain isolated in Nagpur district during three years period, January 2009-January 2012.

OBJECTIVE: In the few cases of childhood dirrhea that require the antimicrobial therapy, the correct choice of the drug depends on detailed previous knowledge of local strains and pattern of antimicrobial resistance. Shigellosis is one of the most improtant examples of this group of intestinal infections. In order to establish such parameters in Nagpur city, this study was carried out to determine the antimcrobial resistance profile of Shigella flexneri isolated from patients suffering from diahhrea admitted to Various hoapitals in Nagpur district, India. MATERIALS AND METHODS: The study included 110 stool samples collected from patients during the 3 year period. All the isolates were characterized and confirmed by VITEK® 2 GN ID cards and antimicrobial susceptibility was tested by VITEK® 2 AST test cards. RESULTS: We received 73 positive cultures of S. flexneri out of 110 stool samples during three year periods of January 2009 to January 2012. S. flexneri strains presented a high resistance rate to Ampicillin (100%), Chloramphenicol (76.71%), Trimethoprime-sulfamethaxazole (TMP-SMZ) (68.49%) and low resistance to third- and fourth-generation Cephalosporin. None of the isolates was found to be resistant to Ciprofloxacin (MIC ≥ 4), Norfloxacin (MIC ≥12), and Nalidixic acid (MIC ≥30). CONCLUSION: Our results provide data on antimicrobial resistance to choose a proper antibiotic for the treatment of Shigellosis in our country. According to current findings, Quinolones and Cephalosporins are the drug of choice for the diarrheic patients. In conclusion, systematic monitoring is needed to identify changes in the antimicrobial resistance.

Inhibition of Mycobacterium tuberculosis secretory serine protease blocks bacterial multiplication both in axenic culture and in human macrophages.

To study the possible importance of mycobacterial ES-31 serine protease for bacterial cell growth, the effect of serine and metalloprotease inhibitors, anti-tubercular drugs such as isoniazid and anti-ES-31 antibody, was evaluated on mycobacterial ES-31 serine protease in vitro and on bacilli in axenic and macrophage cultures. Serine protease inhibitors such as pefabloc, 3,4 dichloroisocoumarin, phenyl methyl sulfonyl fluoride (PMSF) and metalloprotease inhibitors such as ethylene diamine tetracetic acid (EDTA) and 1,10 phenanthroline inhibited 65-92% serine protease activity in vitro. Isoniazid showed 95% inhibition on mycobacterial ES-31 serine protease. These inhibitors also showed decreased bacterial growth in axenic culture and inhibition was further confirmed by a decreased amount of ES-31 serine protease in culture filtrate. In human macrophage culture, highly inhibitory pefabloc, 1,10 phenanthroline and isoniazid inhibited infectivity of virulent as well as avirulent M. tuberculosis bacilli to macrophages. It was observed that addition of mycobacterial ES-31 serine protease to macrophage culture enhanced the entry of bacilli and their multiplication in human macrophages. However, the addition of anti-ES-31 serine protease antibody strongly inhibited the mycobacterial growth as observed by decreased CFU count, showing the importance of mycobacterial ES-31 serine protease for entry of bacilli and their multiplication.

Isolation, characterisation and kinetic studies on SEVA TB ES-31 antigen, a metallo-serine protease of interest in serodiagnosis.

BACKGROUND: SEVA TB Excretory secretory-31 (ES-31) antigen, a glycoprotein isolated from M. tb H37Ra culture filtrate, was found to be useful in the serodiagnosis of pulmonary tuberculosis (TB), extrapulmonary TB and in HIV-TB coinfection. Further, it has been shown to be a zinc containing serine protease. AIM: To isolate and purify SEVA TB ES-31 antigen from M. tb H37Ra culture filtrate and study of its enzyme properties and peptide sequence. METHODS: ES-31 antigen was purified from culture filtrate of M. tuberculosis H37Ra strain by ammonium sulphate precipitation, SDS-PAGE fractionation and FPLC. Protease activity of ES-31 antigen was studied using azocasein as substrate. ES-31 antigen was further fractionated by two dimensional polyacrylamide gel electrophoresis (2D PAGE) followed by LCMS-T analysis. RESULTS: Mycobacterial metallo-serine protease was purified 3096 fold from M. tb H37Ra culture filtrate protein. Purified enzyme showed optimum activity at pH 7.0 at 37 degrees C. Of the four substrates explored, the enzyme has shown maximum activity with azocasein and had a Km value of 0.01 mM with specific activity of 6250 x 10(-6) U/mg protein. Further, analysis of ES-31 antigen by 2D PAGE showed two protein spots (A and B). CONCLUSION: Kinetic studies on SEVA TB ES-31 protein, an immunogen with metallo serine protease activity are reported for the first time. Purified enzyme had a Km value of 0.01 mM with azocasein as substrate. Further, study on structure and biological role of serine protease will be of interest.