Heat stable antimicrobial activity of Burkholderia gladioli OR1 against clinical drug resistant isolates.

Background & objectives: Drug resistant microbes are a serious challenge to human health. During the search for novel antibiotics/inhibitors from the agricultural soil, a bacterial colony was found to inhibit the growth of clinical isolates including Staphylococcus (resistant to amikacin, ciprofloxacin, clindamycin, clinafloxacin, erythromycin, gentamicin and methicillin) and Candida (resistant to fluconazole and itraconazole). The culture was identified as Burkholderia gladioli and produced at least five different antimicrobial compounds which were highly stable at high temperature (121°C) and in the broad pH range (3.0-11.0). We report here the antimicrobial activity of B. gladioli against drug resistant bacterial pathogens. Methods: The bacterial culture was identified using morphological, biochemical and 16S rRNA gene sequencing techniques. The antimicrobial activity of the identified organism against a range of microbial pathogens was checked by Kirby-Bauer's disc diffusion method. The antimicrobial compounds in the cell free supernatant were chloroform-extracted and separated by thin layer chromatography (TLC). Results: B. gladioli OR1 exhibited broad spectrum antimicrobial activity against drug resistant clinical isolates belonging to various genera of bacteria (Staphylococcus, Enterobacter, Enterococcus, Acinetobacter and Citrobacter) and a fungus (Candida). Based on TLC profile and bioautography studies, the chloroform extract of B. gladioli OR1 consisted of at least three anti-staphylococcal and two anti-Candida metabolites. The antimicrobial activity was heat stable (121°C/20 min) as well as pH stable (3.0-11.0). Interpretation & conclusions: The bacterial soil isolate, B. gladioli OR1 possessed the ability to kill various drug resistant bacteria and a fungus. This organism produced many antimicrobial metabolites which might have the potential to be used as antibiotics in future.

Chitinase from Enterobacter sp. NRG4: Its purification, characterization and reaction pattern

Enterobacter sp. NRG4 was shown to excrete chitinase into the culture supernatant when cultivated in medium containing chitin. A 60 kDa extracellular chitinase was purified to homogeneity and characterized. The enzyme hydrolyzed swollen chitin, colloidal chitin, regenerated chitin and glycol chitin but did not hydrolyze chitosan. The chitinase exhibited Km and Vmax values of 1.43 mg ml-1 and 83.33 µM µg-1 h-1 for swollen chitin, 1.41 mg ml-1 and 74.07 µM µg-1 h-1 for colloidal chitin, 1.8 mg ml-1 and 40 µM µg-1 h-1 for regenerated chitin and 2.0 mg ml-1 and 33.33 µM µg-1 h-1 for glycol chitin, respectively. The optimal temperature and pH for activity were 45ºC and pH 5.5, respectively. Mg2+, K+ and Ca2+ stimulated chitinase activity by 13, 16 and 18%, respectively whereas Cu2+, Co2+, Ag+ and Hg2+ inhibited chitinase activity by 9.7, 15, 22 and 72.2%, respectively at 1 mM concentration. N-bromosuccinamide (NBS) at 1 mM and iodoacetamide at 10 mM concentration completely inhibited the enzyme activity. Dithiobisnitrobenzoic acid (DTNB) at 10 mM concentration inhibited chitinase activity by 97.2%. Chitin was hydrolyzed to chitobiose and N-acetyl D-glucosamine when incubated with the purified enzyme. The hydrolysis pattern of the purified enzyme indicated that the chitinase was an endochitinase.