Bacitracin is a broad-spectrum antibiotics mainly produced by Bacillus, and is used as veterinary medicine in the fields of livestock and poultry breeding. Insufficient supply of precursor amino acids might be an important factor that hinders high-level microbial production of bacitracin. We investigated the effect of strengthening L-cysteine supply on bacitracin production by an industrial bacitracin producer, Bacillus licheniformis DW2. Overexpression of cysK encoding L-cysteine synthase led to a 9.17% increase of the bacitracin titer. Moreover, overexpression of cysE encoding L-serine acetyltransferase and cysP encoding thiosulfate/sulfate intracellular transporter increased the bacitracin titers by 7.23% and 8.52%, respectively. Moreover, overexpression of a putative cystine importer TcyP led to a 29.19% increase of intracellular L-cysteine, and bacitracin titer was increased by 7.79%. Subsequently, the strong promoter PbacA was used to replace the promoters of genes cysP, cysE and tcyP in strain DW2::ysK, respectively. The resulted strain CYS4 (DW2::cysK-PbacA-(cysP)-PbacA(cysE)- PbacA(tcyP) produced 910.02 U/mL bacitracin, which was 21.10% higher than that of the original strain DW2 (747.71 U/mL). Together with the experiments in 3 L fermenters, this research demonstrated that enhancing intracellular L-cysteine supply is an effective strategy to increase bacitracin production of B. licheniformis.
Amino Acids ; Bacillus licheniformis/genetics* ; Bacitracin ; Cysteine ; Metabolic Engineering

Bacitracin is a broad-spectrum cyclic peptide antibiotic, and mainly produced by Bacillus. Energy metabolism plays as a critical role in high-level production of target metabolites. In this study, Bacillus licheniformis DW2, an industrial strain for bacitracin production, was served as the original strain. First, our results confirmed that elimination of cytochrome bd oxidase branch via deleting gene cydB benefited bacitracin synthesis. Bacitracin titer and ATP content were increased by 10.97% and 22.96%, compared with those of original strain, respectively. Then, strengthening cytochrome aa3 oxidase branch via overexpressing gene qoxA was conducive to bacitracin production. Bacitracin titer and ATP content were increased by 18.97% and 34.00%, respectively. In addition, strengthening ADP synthesis supply is also proven as an effective strategy to promote intracellular ATP accumulation, overexpression of adenosine kinase DcK and adenylate kinase AdK could all improve bacitracin titers, among which, dck overexpression strain showed the better performance, and bacitracin titer was increased by 16.78%. Based on the above individual methods, a method of combining the deletion of gene cydB and overexpression of genes qoxA, dck were used to enhance ATP content of cells to 39.54 nmol/L, increased by 49.32% compared to original strain, and bacitracin titer produced by the final strain DW2-CQD (DW2ΔcydB::qoxA::dck) was 954.25 U/mL, increased by 21.66%. The bacitracin titer produced per cell was 2.11 U/CFU, increased by 11.05%. Collectively, this study demonstrates that improving ATP content was an efficient strategy to improve bacitracin production, and a promising strain B. licheniformis DW2-CQD was attained for industrial production of bacitracin.
Bacillus licheniformis ; metabolism ; Bacitracin ; biosynthesis ; Energy Metabolism ; genetics ; Industrial Microbiology ; methods

OBJECTIVE: Protein disulfide isomerase (PDI) acts as a chaperone on the cell surface, and it has been reported that PDI is associated with the tumor cell migration and invasion. The aims of this study are to investigate the anti-migration effect of bacitracin, which is an inhibitor of PDI, and the associated factor in this process. METHODS: U87-MG glioma cells were treated with bacitracin in 1.25, 2.5, 3.75, and 5.0 mM concentrations. Western blot with caspase-3 was applied to evaluate the cytotoxicity of bacitracin. Adhesion, morphology, migration assays, and organotypic brain-slice culture were performed to evaluate the effect of bacitracin to the tumor cell. Western blot, PCR, and gelatin zymography were performed to investigate the associated factors. Thirty glioma tissues were collected following immunohistochemistry and Western blot. RESULTS: Bacitracin showed a cytotoxicity in 3rd (p<0.05) and 4th (p<0.001) days, in 5.0 Mm concentration. The cell adhesion significantly decreased and the cells became a round shape after treated with bacitracin. The migration ability, the expression of phosphorylated focal adhesion kinase (p-FAK) and matrix metalloproteinase-2 (MMP-2) decreased in a bacitracin dose- and time-dependent manner. The U87-MG cells exhibited low-invasiveness in the 2.5 mM, compared with the untreated in organotypic brain-slice culture. PDI was expressed in the tumor margin, and significantly increased with histological glioma grades (p<0.001). CONCLUSION: Bacitracin, as a functional inhibitor of PDI, decreased the phosphorylated FAK and the secreted MMP-2, which are the downstream of integrin and play a major role in cell migration and invasion, might become one of the feasible therapeutic strategies for glioblastoma.
Bacitracin* ; Blotting, Western ; Caspase 3 ; Cell Adhesion ; Cell Movement ; Focal Adhesion Protein-Tyrosine Kinases ; Gelatin ; Glioblastoma ; Glioma* ; Immunohistochemistry ; Matrix Metalloproteinase 2 ; Polymerase Chain Reaction ; Protein Disulfide-Isomerases

BACKGROUND: The aim of this study was to evaluate the antibiotic susceptibility of Group A streptococcus (GAS) to antibiotics usually used in Iran for treatment of GAS pharyngitis in children. MATERIALS AND METHODS: From 2011 to 2013, children 3-15 years of age with acute tonsillopharyngitis who attended Mofid Children's Hospital clinics and emergency ward and did not meet the exclusion criteria were enrolled in a prospective study in a sequential manner. The isolates strains from throat culture were identified as GAS by colony morphology, gram staining, beta hemolysis on blood agar, sensitivity to bacitracin, a positive pyrrolidonyl aminopeptidase (PYR) test result, and the presence of Lancefield A antigen determined by agglutination test. Antimicrobial susceptibility was identified by both disk diffusion and broth dilution methods. RESULTS: From 200 children enrolled in this study, 59 (30%) cases were culture positive for GAS. All isolates were sensitive to penicillin G. The prevalence of erythromycin, azithromycin, and clarithromycin resistance by broth dilution method was 33.9%, 57.6%, and 33.9%, respectively. Surprisingly, 8.4% of GAS strains were resistant to rifampin. In this study, 13.5% and 32.2% of the strains were resistant to clindamycin and ofloxacin, respectively. CONCLUSION: The high rate of resistance of GAS to some antibiotics in this study should warn physicians, especially in Iran, to use antibiotics restrictedly and logically to prevent the rising of resistance rates in future. It also seems that continuous local surveillance is necessary to achieve the best therapeutic option for GAS treatment.
Agar ; Agglutination Tests ; Anti-Bacterial Agents ; Azithromycin ; Bacitracin ; Child* ; Clarithromycin ; Clindamycin ; Diffusion ; Drug Resistance, Microbial ; Emergency Service, Hospital ; Erythromycin ; Hemolysis ; Humans ; Iran* ; Logic ; Ofloxacin ; Penicillin G ; Pharyngitis* ; Pharynx ; Prevalence ; Prospective Studies ; Rifampin ; Streptococcus pyogenes ; Streptococcus* ; Tonsillitis

OBJECTIVES: This study evaluated the antibacterial effect and mechanical properties of composite resins (LCR, MCR, HCR) incorporating chitosan with three different molecular weights (L, Low; M, Medium; H, High). MATERIALS AND METHODS: Streptococcus (S). mutans 100 mL and each chitosan powder were inoculated in sterilized 10 mL Brain-Heart Infusion (BHI) solution, and was centrifuged for 12 hr. Absorbance of the supernatent was measured at OD660 to estimate the antibacterial activities of chitosan. After S. mutans was inoculated in the disc shaped chitosan-containing composite resins, the disc was cleansed with BHI and diluted with serial dilution method. S. mutans was spread on Mitis-salivarius bacitracin agar. After then, colony forming unit (CFU) was measured to verify the inhibitory effect on S. mutans biofilm. To ascertain the effect on the mechanical properties of composite resin, 3-point bending and Vickers hardness tests were done after 1 and 3 wk water storage, respectively. Using 2-way analysis of variance (ANOVA) and Scheffe test, statistical analysis was done with 95% significance level. RESULTS: All chitosan powder showed inhibition effect against S. mutans. CFU number in chitosan-containing composite resins was smaller than that of control resin without chitosan. The chitosan containing composite resins did not show any significant difference in flexural strength and Vickers hardness in comparison with the control resin. However, the composite resin, MCR showed a slightly decreased flexural strength and the maximum load than those of control and the other composite resins HCR and LCR. CONCLUSIONS: LCR and HCR would be recommended as a feasible antibacterial restorative due to its antibacterial nature and mechanical properties.
Agar ; Bacitracin ; Biofilms ; Chitosan ; Composite Resins ; Hardness ; Hardness Tests ; Molecular Weight ; Stem Cells ; Streptococcus ; Streptococcus mutans ; Water

OBJECTIVES: Streptococcus mutans (S. mutans) is the major causative bacteria in human dental decay. Most findings have shown that S. mutans are widely distributed in populations with moderate to high caries prevalence. However, observations have shown that S. mutans are also frequent in populations with low caries prevalence, as well. This study evaluated the relationship between the dental caries experience and the virulence factors of S. mutans, which are isolated from caries-free and high-caries individuals. METHODS: The dental caries experience states with the WHO diagnostic criteria, which were examined for children (aged 8 to 9 years) from two schools that are located in Daegu, Korea. A total of 22 caries-free (dfs+DFS=0) and 12 high-caries (dfs+DFS> or =14, ds+DS> or =9) were selected for this study. Dental plaque samples were obtained and incubated in MSB plate to culture S. mutans. After isolation and identification, the level of S. mutans, attachment ability, and streomicroscopy were analyzed. RESULTS: The S. mutans counts and attachment ability were higher in the high-caries group than in the caries-free group (P<0.05). In addition, the colonies isolated from the caries-free group adhered less strongly to the Mitis Salivarius Bacitracin agar. There were less of the sticky substances from the surface of the S. mutans colonies in the caries-free group. CONCLUSIONS: These results indicate that caries activity affected by the virulence not only the number of S. mutans.
Agar ; Bacitracin ; Bacteria ; Child ; Culture ; Dental Caries ; Dental Plaque ; Humans ; Korea ; Prevalence ; Streptococcus ; Streptococcus mutans ; Virulence Factors

OBJECTIVE: To estimate the effects of bracket material type on enamel decalcification during orthodontic treatment, this study analyzed the adhesion level of mutans streptococci (MS) to orthodontic bracket materials in vivo. METHODS: Three different types of orthodontic bracket materials were used: stainless steel, monocrystalline sapphire, and polycrystalline alumina. A balanced complete block design was used to exclude the effect of positional variation of bracket materials in the oral cavity. Three types of plastic individual trays were made and one subject placed the tray in the mouth for 12 hours. Then, the attached bacteria were isolated and incubated on a mitis salivarius media containing bacitracin for 48 hours. Finally, the number of colony forming units of MS was counted. The experiments were independently performed 5 times with each of the 3 trays, resulting in a total of 15 times. Mixed model ANOVA was used to compare the adhesion amount of MS. RESULTS: There was no difference in colony forming units among the bracket materials irrespective of jaw and tooth position. CONCLUSIONS: This study suggested that the result of quantitative analysis of MS adhesion to various orthodontic bracket materials in vivo may differ from that of the condition in vitro.
Aluminum Oxide ; Bacitracin ; Bacteria ; Bacterial Adhesion ; Dental Enamel ; Jaw ; Mouth ; Orthodontic Brackets ; Plastics ; Stainless Steel ; Stem Cells ; Tooth

BACKGROUND: Group A streptococci (GAS) are the most common cause of pharyngitis in children. The streptococci in throat cultures from healthy elementary school children in Jinju were compared with previous results. METHODS: Throat cultures were taken from 1,402 healthy school children in 2006. beta-hemolytic streptococci (BHS) were identified with a bacitracin disk (0.04 U) and latex agglutination test (Seroiden Strepto Kit, Eiken, Tokyo, Japan). RESULTS: Two-hundred sixteen (15.4%) and 149 (10.6%) cultures grew BHS and GAS, respectively. The isolation rate of GAS was significantly lower than in 2004 (16.0%) or 2002 (16.9%) (P<0.05). Among BHS, the prevalence of group A strains (69.0%) decreased significantly compared with 2004 (84.9%) and 2002 (83.8%) (P<0.05). None of the 1st-grade children yielded BHS or GAS. CONCLUSION: The isolation rates of BHS and GAS from healthy school children were lower in 2006 than in previous years. Natural immunization against the common serotypes or improvement in individual hygiene might have played roles in the reduction of isolations of GAS.
Bacitracin ; Child ; Humans ; Hygiene ; Immunization ; Latex Fixation Tests ; Pharyngitis ; Pharynx ; Prevalence ; Streptococcus pyogenes ; Tokyo

AIMTo investigate the degradation of recombinant hirudin-2 (rHV2) in nasal mucosa of rabbit.

METHODSThe specific and accurate HPLC method was developed for analyzing rHV2; The degrading ratios of rHV2 at different concentrations and at pH conditions in rabbit nasal mucosa homogenate were determined; The results in nasal mucosa homogenate were compared with that in small intestinal mucosa homogenate of rabbits. The stability of rHV2 in the enzyme extract of nasal mucosa surface and the effect of proteolysis inhibitor bacitracin on the degradation of rHV2 in nasal mucosa homogenate were also estimated.

RESULTSThe degradation of rHV2 in rabbit nasal mucosa homogenate showed concentration- and pH-dependence; rHV2 in nasal mucosa homogenate was more stable than that in intestinal mucosa homogenate. Also rHV2 was more stable in the enzyme extracts of nasal serosal surface than that of mucosa surface. Addition of bacitracin was able to inhibit the degradation to some degree.

CONCLUSIONComparing with oral administration, rHV2 nasal delivery was a more tolerant route.

Animals ; Bacitracin ; pharmacology ; Chromatography, High Pressure Liquid ; Female ; Hirudins ; genetics ; metabolism ; pharmacokinetics ; Hydrogen-Ion Concentration ; In Vitro Techniques ; Intestinal Mucosa ; drug effects ; metabolism ; Kinetics ; Nasal Mucosa ; drug effects ; metabolism ; Rabbits ; Recombinant Proteins ; metabolism ; pharmacokinetics

Objective: To investigate the effect of three preoperative anti-infective regimens on the sterility of anterior-chamber aspirates (ACA) in eyes undergoing extracapsular cataract extraction (ECCE). Methods: Ninety eyes scheduled to undergo ECCE were randomly assigned to receive one of the following preoperative anti-infective regimens: Group 1 (Control) - no additional preoperative preparation; Group 2 (Eye drop) - neomycin/ polymixin B/gramicidin eye drops applied 3 times daily for 3 days prior to surgery; and Group 3 (Lid scrub) - neomycin/polymixin B/ bacitracin ointment lid scrub applied 3 times prior to surgery. ACAs were obtained from all eyes at the conclusion of surgery and cultured. The patients were followed up for 3 months after the surgery. Results: Positive cultures developed from ACAs in 5 (16.6 percent) of 30 eyes from Group 1, in 0 of 30 from Group 2, and in 3 (10 percent) of 30 from Group 3. No eye developed endophthalmitis. Compared with the control group, preoperative neomycin/polymixin B/gramicidin eye drops significantly reduced the ACA contamination rate (p= 0.03). Conclusion: Preoperative neomycin/polymixin B/bacitracin eyedrops can improve the sterility of the anterior chamber during ECCE.
Human ; CATARACT ; ANTI-INFECTIVE AGENTS ; ENDOPHTHALMITIS ; NEOMYCIN ; GRAMICIDIN ; BACITRACIN ; CATARACT EXTRACTION ; EYE DISEASES ;