Industrially important hydrolytic enzyme diversity explored in stove ash bacterial isolates

Extreme environments merit special attention and significance because of the possible existence of thermophilic microorganisms in such ecological niches. Keeping this in mind indigenous stove ash samples were explored for extremophilic bacteria in term of their biodiversity. Accordingly, this study reports 37 bacterial isolates from the local wood run oven [Tandoor] ash samples. All the isolated strains belong to genus Bacillus on the bases of morphocultural and biochemical considerations. The average temperature tolerance profile was >45[degree]C thereby, indicating towards the thermophilic nature of the isolated strains. The Bacillus isolates were screened for 10 different hydrolytic enzymes [cellulase, xylanase, amylase, pectinase, caseinase, keratinase, lipase, esterase, dextranase and beta-galactosidase] by plate screening method using the medium incorporated with specific substrate[s]. It was found that keratinase was produced by all the isolates while, 36 [97.2%] isolates showed caseinase and esterase production. Amylase was produced by 35[94.6%] isolates and 34 [91.8%] isolates were able to degrade Tween-80 and xylan as substrate for lipase and xylanase respectively. The enzyme, beta-galactosidase was produced by 31 [89.1%] of the isolates. Cellulase and dextranase were produced by 26 [70.2%] and 22 [59.4%] isolates respectively. None of the isolates could [under the existing conditions] produce pectin-hydrolyzing enzyme. According to the tukey's post hoc test, significant difference was found between the mean enzyme index of all the [screened] enzymes. Thus, the isolated bacterial strains with diverse hydrolytic potential may be of great value and relevance for the existing [national] industrial setups

Evolutionary root defines the structural basis of high and low level vancomycin resistance in enterococci

D-alanyl-D-lactate [Dlac] and D-alanyl-D-serine [Dser] ligases respectively mediates high and low level vancomycin resistance among enterococci. To date, the evolutionary relationship of both ligases is largely unaddressed. Also poorly understood are the molecular differences in the magnitude of vancomycin resistance. To address the mention, we constructed the phylogenetic tree of all vancomycin resistance conferring ligases with the wild type ligases [Dala]. Multiple sequence alignment and tertiary structures of the structurally unresolved proteins were constructed by homology modeling. Phylogenetic tree revealed that both Dlac and Dser are profoundly different from Dala as a result of continuous selection pressure. Separate clustering of Dlac and Dser also highlighted the structural basis of molecule in maintaining different level of resistance as exhibited by the bacteria. This notion was further augmented as the functionally key region, omega loop [omega-loop], was found relatively more structured in only Dlac. Moreover, the critically active residue, His-243/244, was also noticed to be restricted in Dlac and found replaced by non polar residues in Dser. The present study not only provides protein structural explanation of the different intensities of vancomycin resistance among enterococci, but also presents yet another example for the scope of evolutionary science in biomedicine

Isolation- drug resistance profile and molecular characterization of indigenous typical and atypical mycobacteria

One hundred and fifty mycobacterial isolates from different pathological Labs, of Karachi were collected and screened as acid fast. On the bases of phenotypic and biochemical results, it was found that, 58.66% isolates were typical mycobacteria while 41.33% belonged to atypical mycobacteria. The individual percentages of different mycobacterial species include: M. xenopi 35%, M. thermoresistible 19%, M. terrae complex 6%, M. marinum 6%, M fortuitum 6%, M. kansasii 25% and M tuberculosis 58.66%. The sensitivity of mycobacterial isolates was determined against 5 first line, 3 second line and 1 third line anti-tuberculosis drugs. The highest number of the isolates [typical and atypical mycobacteria] offered resistance against isoniazid and streptomycin. Clarithromycin was found to be the drug of choice as regards the drug sensitivity in case of atypical mycobacterial isolates. A total of 40 isolates were subjected to PCR based identification and differentiation of 16S rRNA gene[s]. Accordingly, 37.5% isolates were identified as typical mycobacteria while 25% were identified as atypical mycobacteria. These findings carry significance because a detailed research based identification [PCR and Multiplex PCR based] regarding indigenous mycobacteria has been reported for the first time in Pakistan. However, both the approaches [conventional and molecular methods] have experimental importance while identifying these organisms

Comparative study of two bacteriocins produced by representative indigenous soil bacteria

The aim of this research work was to identify and characterized the bacteriocins produced by soil-associated bacteria. Bacillocin from Bacillus brevis Bb and pyocin from Pseudomonas aeruginosa Pa were found bioactive only against gram-positive bacteria tested. Maximum production of both the bacteriocins was observed at 32°C in BHI medium. Production of both the bacteriocins started in the early exponential growth phase while the maximum production was observed during the stationary phase. Bacillocin Bb remained stable during 1-9 pH while pyocin Pa remained stable at pH 1-11. Both of the bacterocins were found resistant to high temperature [100°C for 30 min], detergents [1% solutions of EDTA, Tween 20, Tween 80 and SDS] and organic solvents [1% solutions of Ethanol, Butanol, Methanol, Propanol, Chloroform, and Acetone]. Activity of both was completely lost after proteinase K treatment suggesting their protein nature. Titre of bacillocin Bb was estimated to be 5280 AU/mL while the titre for pyocin Pa was calculated as 640 AU/mL. Both of the bacteriocins showed bacteriolytic mode of action against the indicator Bacillus strain BC31 and were found <10 KDa in their molecular mass

Bacteriocin production by indigenous marine catfish associated vibrio Spp

Fifty strains of genus Vibrio were isolated [identified] from healthy and diseased marine catflsh[es]. The isolates were screened for bacteriocin [vibriocin] production. About 32% isolates were found bacteriocin producers. The best producer was identified as Vibrio anguillarum AVP10. The maximum production of vibiiocin AVP10 was manifested at 29°C at pH 7, after 18-20h of incubation. Vibriocin activity was enhanced in the presence of citrate-phosphate buffer. The vibriocin AVP10 withstands autoclaving temperature and showed activity even after prolonged chloroform treatment. Proteolytic enzymes inhibited its activity, while lipolytic enzyme had no effect, It was found bioactive only against intrageneric bacterial strains. Mode of action of vibriocin AVP10 varies with the indicator [sensitive] culture used i.e. bactericidal effects was exerted against V. anguillarum AVS9 while bacteriostatic effect was shown against entero-toxigenic E. coil

Role of fissure sealants in the reduction of cariogenic bacteria in 6-8 year old children

Dental caries is a multifactorial bacterial disease affecting the teeth of children and young adults. Fissure sealants are used to prevent bacterial ingress into fissures to minimize decay in children. The aim of this study was to find out role of fissure sealents in reduction of cariogenic bacteria in 6-8 years old children. One hundred [6-8 year old] children/volunteers from Jinnah Foundation Trust School Korangi, Karachi were randomly selected. The oral hygiene and dietary patterns of these volunteers were recorded. The volunteer children were divided into two groups [trial/test and control]. The trial group's fissures were sealed with fissure sealants while control group's fissures were not sealed. The first permanent molar was chosen for sealing as this is the first indicator of caries activity in permanent teeth. All first permanent molars were ensured to be caries-free as fissure sealants are preventive dental materials and are not restorative. The saliva samples of all 100 students were collected in sterilized wide mouth vials. It was then spread on TYCSB and on MRS agar plates for the isolation of Streptococcus mutans and lactobacilli respectively. The plates were incubated for 24 hours at 37°C and then CFU/ml was recorded for each sample. The trial group received fissure sealants while the control group did not. Saliva sampling and isolation procedures were repeated after 4 and 12 weeks of fissure sealant application. The isolated organisms were identified by gram staining, growth characteristics [at different temperature ranges], hemolytic patterns, sugar fermentation, arginine and esculin hydrolysis. The results indicated reduction in the count of cariogenic bacteria in the trial group after the application of fissure sealants on first permanent molars whereas the count remained more or less similar in the control group of all three successive saliva samples. The study was to evaluate the role of fissure sealants in reducing bacterial count of Streptococcus mutans and lactobacilli in the saliva of 6-8 year old children which was found to be significant by the statistical means

Enterococci vs coliforms as a possible fecal contamination indicator: Baseline data for Karachi

Fecal contamination of drinking water is the major cause of water borne illnesses. For long time coliforms are exploited as fecal contamination indicator. However, recent studies indicate low survival rate of coliforms in stress conditions, hence it's use as indicator of fecal pollution is being abandoned in many parts of the developed world. Implementation of such strategy demands availability of local data in the cities like Karachi. The present study provides a comparison between coliforms and enterococcal load and its variation in sewage samples collected [June, August and November, 2006] from eighteen towns of Karachi. All the diluted samples were selective media to obtain colony-forming units [CFU] mainly for coliforms and enterococci. The bacteria isolated were identified on the basis of conventional microbiological methods. Observations thus obtained were subjected to rigorous statistical analysis. The total load of enterococci was found in range of 1.27-8.47 X 107 as compared to coliforms [3.03-13.9 X 107]. However, segregation of data reveals greater inter town variability in CFU/ml both in coliforms and enterococci as suggested by their cumulative standard deviation +1.5 X107. Furthermore, CFU/ml of both coliforms and enterococci also varies to variable scale when collected at different time intervals and at intra town level. Conclusively, the studies suggest high survival rate and lower variability of Enterococci compared to escherichia hence indicating its potential advantage to be used as fecal contamination indicator