Identification and growth optimization of a Marine Bacillus DK1-SA11 having potential of producing broad spectrum antimicrobial compounds

Control of harmful bacteria in food, aquaculture, pharmaceuticals, agriculture, hospitals and recreation water pools are of great global concern. Marine bacteria are an enormous source of bio-controlling agents. The aim of this study was to identify and optimize the growth conditions including effect of different biotic and abiotic factors on antimicrobial activity of strain DK1-SA11 isolated from Qingdao Bay of China Yellow Sea. Microscopic characterization, API[registered sign] 20E and 50 CHB kit base carbohydrates utilization, 16S rDNA and DNA gyrB gene sequencing studies identified the bacterium as Bacillus subtilis subsp. spizizenii DK1-SA11. Antimicrobial spectrum of cell free supernatant [CFS] has shown antimicrobial activities against all test strains including methicillin-resistant Staphylococcus aureus, E. coli O157:H7, Candida albicans, Klebsiella pneumoniae, Listeria monocytogenes, Vibrio parahaemolyticus, E. coli, Pseudomonas fluorescens, Salmonella typhimurium and Vibrio cholerae. Among all the media tested, Marine Broth 2216 was found to be the best medium for bacterial growth and production of antibacterial compounds. The other optimum conditions for growth were pH:7 and incubation temperature: 25[degree sign] C with >/= 180 rpm for 60-72 h. Out of 49 different carbohydrates tested, D-mannose increases the antibacterial activity by 33.3% while Darabitol decreases it by 44.4%. Crude CFS showed activity even after three months of storage below -20[degree sign] C and boiling for 10 min, whereas it loses 100% of its antimicrobial activity after enzymatic treatments of lipase, trypsin and papain. The production of antimicrobial compounds and broad spectrum of antimicrobial activity against all tested pathogens suggested that the strain DK1-SA11 can be used as a source for probiotics, synbiotics and antibiotics

Broad spectrum anti-microbial compounds producing bacteria from coast of Qingdao bays

Anti-microbial resistance burden and hazard associated with chemical treatment of infections demanded for new anti-microbial natural products. Marine associated microorganisms are the enormous source of bioactive compounds. In this study we have isolated 272 marine bacteria among them 136 [50%] were antagonistic to at least one of the four pathogenic strains Listeria monocytogenes, Vibrio cholerae, E. coli and S. aureus. Only two strains exhibited antibacterial activity against all four test strains, which were identified by 16S rDNA sequencing as Bacillus sp. DK1- SA11 and Vibrio sp. DK6-SH8. Marine isolate DK1-SA11 has potential to resist boiling temperature and pH 2-12. Furthermore cell free extract [CFE] inhibited all test organisms including superbug MRSA and pathogenic yeast Candida albicans. Marine isolate Bacillus sp. DK1-SA11 could be a potential combatant for the battle of drugs and bugs

Genomic organization of a vancomycin-resistant staphylococcus aureus

To study the genomic organization of vancomycin resistance in a local isolate of vancomycin resistant Staphylococcus aureus [VRSA]. Experimental study. Department of Microbiology, University of Karachi, January 2008 through December 2010. A vancomycin-resistant Staphylococcus aureus [VRSA-CP2] isolate [MIC 16 microg/ml] was isolated from a local hospital of Karachi. Species identification was confirmed by Gram staining, standard biochemical tests and PCR amplification of the nuc gene. The vancomycin MIC was re-confirmed by E-test. For the genetic determination of vancomycin resistance, in-vitro amplification of vanA cassette was performed by using plasmid DMA of CP2, CP2's transformant as template on MWG Thermo-Cycler. Amplified products of vanR, vanS, vanH, vanA, vanY, or/2, orfID, orf2E, orf-Rev and IS element genes were subjected to Sanger's electrophoresis based sequence determination using specific primers. The Basic Local Alignment Search Tool [BLAST] algorithm was used to identify sequences in GenBank with similarities to the vanA cassette genes. The vancomycin-resistant isolate CP2 was found to be resistant to oxacillin, chloramphenicol, erythromycin, rifampicin, gentamicin, tetracycline and ciprofloxacin, as well. The isolate CP2 revealed four bands: one of large molecular size -56.4 kb and three of small size -6.5 kb, -6.1 kb and -1.5 kb by agarose gel electrophoresis indicating the presence of 3 plasmids. The plasmid DNA of isolate CP2 was analyzed by PCR for the presence of the van cassettes with each of the vanA, vanB and vanC specific primers. It carried vanA cassette, which comprises of vanR, vanS, vanH, vanA, vanY, and or/2. The vanA cassette of isolate CP2 also carried an insertion element [IS]. However, it did not show the PCR product for orfl. Vancomycin resistance was successfully transferred from the donor CP2 to a vancomycin-sensitive recipient S. aureus. The MIC of vancomycin for the transformant was 16 microg/ml, similar to the parent isolate CP2. Nucleotide sequencing of the PCR product showed similarity with van genes of enterococci and other VRSA reported from different parts of the world. Sequence of vanA cassette of CP2 showed partial homology with vancomycin resistant enterococci, VRSA vanA cassette element recorded in gene bank NCBI

Effect of stress on biofilm formation by icaA positive and negative strains of methicillin resistant Staphylococcus aureus

To determine the effect of physical and chemical stress factors e.g. antibiotics, NaCI, glucose, heat shock, cold shock and sonic waves on biofilm formation by icaA positive and negative strains of Methicillin resistant Staphylococcus [S.] aureus. Experimental study. Microbiological Analytical Centre, Pakistan Council of Scientific and Industrial Research [PCSIR] Laboratories Complex, Karachi, from January to December 2010. One strain of Staphylococcus aureus labelled as FA was isolated from a food sample and the other strain labelled as CL was a clinical strain. Biofilm assays were performed in brain-heart infusion [BHI] medium and in BHI supplemented with 7% NaCI, 5% glucose, or sub-inhibitory concentrations of Vancomycin, Oxacillin, Ampicillin, Tetracycline, Erythromycin, Rifampicin and Ciprofloxacin. Polymerase chain reaction [PCR] was used for screening of the icaA and mecA genes. The FA and CL were identified as MRSA carrying mecA gene. The strain FA showed biofilm formation without any treatment and was found to carry icaA gene contrary to CL, that does not contain this gene therefore, is unable to produce biofilm under normal conditions without any stress. The use of sub-lethal doses of cell wall active antibiotics, exposure to 7% NaCI, sonication, and heat shock were found to augment biofilm quantity in FA, an icaA positive strain and induce biofilm mode of growth in CL, an icaA negative strain. Anti-protein synthesis antibiotics did not show any effect on biofilm formation process in icaA positive or negative strains. There is a role of anti-cell wall factors i.e. sonication, heat shock, NaCI and antibiotics in the induction of biofilm mode of growth in MRSA and Methicillin sensitive S. aureus. The factors which partially damage bacterial cell wall, equally, induce biofilm formation in icaA positive or negative S. aureus

Characterization of a vancomycin intermediate-resistant staphylococcus aureus isolated from a hospital

A Vancomycin intermediate resistant strain of Staphylococcus aureus [S. aureus] labeled as CP2 [MIC 16 micro g/ml] was isolated from an in-patient of local Cardiac Hospital of Karachi. CP2 showed typical characters of Vancomycin intermediate resistant S. aureus [VISA] i.e. high level of oxacillin resistance, thickened cell wall with rough surface and reduced autolytic activities associated with murein hydrolase [MH] enzyme. This strain may have acquired vancomycin resistance due to long term exposure to antibiotic during the treatment of the patient. Therefore, it implicates the importance of monitoring the usage and also to control of the abuse of antibiotics for prevention of any further proliferation of this type of notorious bugs