To elucidate iron utilization patterns of Staphylococcus aureus according to the growth, we checked the residual iron concentration and the production of siderophores at the indicated times while culturing S. aureus ATCC 6538 and 25923 strains in brain heart infusion broth. By using streptonigrin susceptibility test and investigating growth curves in three culture media of which iron concentration is 0.2, 20, 45 uM, respectively, we found out that iron metabolism of 6538 strain was more active than that of 25923 strain. In point of tendency of iron consumption, 6538 strain steeply consumed iron just before the onset of stationary phase, but 25923 strain did gradually iron throughout the growth phase. Nevertheless, total amount of iron consumed by each strain during the growth was almost no difference between the strains. CAS diffusion assay in detecting siderophores showed that siderophore production followed iron consumption. These results suggest that the siderophores play significant role in iron utilization in vitro.
Brain ; Culture Media ; Diffusion ; Heart ; Iron* ; Metabolism ; Siderophores ; Staphylococcus aureus* ; Staphylococcus* ; Streptonigrin

BACKGROUND: We previously reported that activity of iron-uptake systems (IUS) influenced on the growth of Staphylococcus aureus in laboratory medium and body fluids according to the iron and oxygen concentrations, which they are closely related each other in several microbial metabolism. In the present study, we tried to investigate the profiles of cell wall proteins of S. aureus according to the change of iron and oxygen concentrations. METHODS: SR-1 strain, whose IUS are defective, was isolated from the standard strain ATCC 6538 by repeated exposure against streptonigrin. These two strains were cultured under the aerobic, microaerobic and anaerobic conditions in the iron-sufficient BHI and iron-depleted BHI, respectively. Cell wall proteins were visualized by Coomassie staining after SDS-PAGE. RESULTS: Cell wall proteins of the both strains were expressed more than under the aerobic condition than under the anaerobic condition in the iron-sufficient medium as well as in the iron-deficient medium. However, expression of cell wall proteins of SR-1 strain was markedly inhibited compared to that of parental ATCC 6538 strain, especially in the iron-deficient medium. Among the proteins more expressed under the aerobic culture condition in the iron-deficient medium, about 88, 55, 39, 36, 35 and 33 kDa of proteins were iron-repressible and oxygen-inducible, and corresponded to the iron-repressible proteins which other researchers reported. CONCLUSION: Expression of cell wall proteins of S. aureus was affected by simultaneous and respective change of iron and oxygen concentrations. Activity of IUS influenced more on the expression of cell wall proteins of S. aureus in the iron-deficient environment than in the iron-sufficient environment. These results suggest the possibility that the iron-repressible and oxygen-inducible proteins mimic those (antigens) found commonly in clinical infections.
Body Fluids ; Cell Wall* ; Electrophoresis, Polyacrylamide Gel ; Humans ; Iron* ; Metabolism ; Oxygen* ; Parents ; Staphylococcus aureus* ; Staphylococcus* ; Streptonigrin

BACKGROUND: We previously reported that activity of iron-uptake systems (IUS) influenced on the growth of Staphylococcus aureus in body fluids which are relatively iron-restrictive conditions. Iron and oxygen are closely related each other in several microbial metabolism. In the present study, we tried to investigate the effect of IUS on the growth of S. aureus according to the iron concentration and oxygen tension. METHODS: SR-1 strain, whose IUS are defective, was isolated from the standard strain, ATCC 6538. These two strains were cultured under the aerobic, microaerobic and anaerobic condition in the iron-sufficient BHI and iron-depleted BHI, respectively. Bacterial growth was measured by optical density. RESULTS: Growth of both strains was inhibited in the iron-depleted BHI. Growth of parental strain was more active in the iron-sufficient BHI as well as in the ion-depleted BHI than that of SR-1 strain. Growth of both strains was more active under the aerobic condition than under the microaerobic or anaerobic condition. In the iron-depleted BHI, parental strain showed a striking difference of growth according to the oxygen tension. In the iron-depleted BHI, growth of SR-1 strain was markedly inhibited regardless of oxygen tension. CONCLUSION: IUS influenced more on the growth of S. aureus in the iron-depleted environments than in the iron-sufficient environments, and under the aerobic condition than under the microaerobic or anaerobic condition in the iron-depleted environments. These results indicated the possibility that oxygen as well as iron regulate activity and expression of IUS.
Body Fluids ; Humans ; Iron* ; Metabolism ; Oxygen* ; Parents ; Staphylococcus aureus* ; Staphylococcus* ; Strikes, Employee

Aims: To characterize the plantaricin IIA-1A5 crude extract that biosynthesized by Lactobacillus plantarum IIA-1A5 using corn steep liquor (CSL) based medium. Methodology and results: Lactobacillus plantarum IIA-1A5 was grown in several media containing different components including corn steep liquor (CSL), molasses and MRS (de Man Rogosa Sharpe) as control medium for 24 h at 37 °C. Antibacterial activities of the cell-free supernatant were expressed as diameter of inhibition zones observed by paper disc method. The results showed that CSL medium produced cell-free supernatant of L. plantarum IIA-1A5 with significantly higher antibacterial activity againts Staphylococcus aureus ATCC 25923 (9.81 mm), Lactobacillus monocytogenes ATCC 7644 (9.61 mm), Bacillus cereus (8.97 mm) and Escherichia coli ATCC 25922 (9.23 mm) were not significantly different compared to control MRS broth media (9.59 mm). CSL medium added only with 3% yeast extract and Tween 80 produced supernatant which showed similar antibacterial activity either to 10% molasses or control medium (Medium K and B). The CSL medium was considered more efficient and low cost, therefore this medium was selected for production and characterization of plantaricin IIA-1A5 crude extract. Further characterization performed by SDS PAGE analysis showed that crude plantaricin had molecular weight of approximately 9.9 kDa, higher than that produced in control medium (8.0 kDa). However, both plantaricins were categorized under the same class for small bacteriocin (class II). This study also revealed the plantaricin IIA-1A5 produced in CSL medium was stable to heat and pH and not significantly different compared to control MRS broth media. The antibacterial activity of plantaricin IIA-1A5 crude extract against S. aureus ATCC 25923 (10.09 mm) was not significantly different with 1000 ppm sodium benzoate (9.70 mm) and 300 ppm sodium nitrite (9.82 mm). Conclusion, significance and impact of study The CSL medium produced cell-free supernatant of L. plantarum IIA 1A5 had significant antibacterial activity characterization againts S. aureus ATCC 25923, L. monocytogenes ATCC 7644, B. cereus and E. coli ATCC 25922. Comparison of the inhibition activity of plantaricin IIA-1A5 crude extract against pathogen with synthetic preservatives indicated that plantaricin IIA-1A5 crude extract have the potency to replace synthetic preservatives. CSL based medium is potential to be used for low-cost plantaricin IIA-1A5 production.
Anti-Bacterial Agents--metabolism ; Bacteriocins--metabolism ; Lactobacillus plantarum ; Microbial Viability--drug effects ; Staphylococcus aureus

In order to characterize the immunogenicity and immunoprotection of the Staphylococcus aureus (S. aureus) surface protein Clumping factor A (ClfA), we amplified clfa genes from S. aureus Newman strain, Wood46 strain and HLJ23-1. The clfa gene from Newman strain was subsequently inserted into pQE-30 vector and the recombinant plasmid was transformed into Escherichia coli strain M15 (pREP4). The recombinant ClfA protein was expressed and purified. Then, we immunized mice with the purified recombinant protein. The antibody level and the concentration of cytokines were measured by enzyme-linked immunosorbent assay. Finally, immunized mice were challenged with S. aureus Newman, Wood46 and HLJ23-1. These results suggested that clfa gene sequences were highly conserved, and the recombinant ClfA was expressed correctly with good antigenicity. The antibody titer and the concentration of cytokines in the immunized groups increased significantly (P < 0.05) compared with control, and the mice in the immunized groups were protected against the challenge strains to some extent. These results showed that the ClfA had high immunogenicity and immunoprotective potential.
Animals ; Coagulase ; genetics ; immunology ; metabolism ; Escherichia coli ; genetics ; metabolism ; Immunization ; Mice ; Recombinant Proteins ; genetics ; immunology ; metabolism ; Staphylococcus aureus ; metabolism ; pathogenicity

Treatment of bovine mastitis caused by Staphylococcus (S.) aureus is becoming very difficult due to the emergence of multidrug-resistant strains. Hence, the search for novel therapeutic alternatives has become of great importance. Consequently, bacteriophages and their endolysins have been identified as potential therapeutic alternatives to antibiotic therapy against S. aureus. In the present study, the gene encoding lysin (LysSA4) in S. aureus phage SA4 was cloned and the nucleotide sequence was determined. Sequence analysis of the recombinant clone revealed a single 802-bp open reading frame encoding a partial protein with a calculated mass of 30 kDa. Results of this analysis also indicated that the LysSA4 sequence shared a high homology with endolysin of the GH15 phage and other reported phages. The LysSA4 gene of the SA4 phage was subsequently expressed in Escherichia coli. Recombinant LysSA4 induced the lysis of host bacteria in a spot inoculation test, indicating that the protein was expressed and functionally active. Furthermore, recombinant lysin was found to have lytic activity, albeit a low level, against mastitogenic Staphylococcus isolates of bovine origin. Data from the current study can be used to develop therapeutic tools for treating diseases caused by drug-resistant S. aureus strains.
Animals ; Base Sequence ; Cloning, Molecular ; Gene Expression Regulation, Viral/physiology ; Mucoproteins/genetics/*metabolism ; Phylogeny ; Polymerase Chain Reaction/methods ; Recombinant Proteins ; Staphylococcus Phages/genetics/*metabolism/physiology ; Staphylococcus aureus/*virology

OBJECTIVETo study the effects of inhibitory peptide of Staphylococcus epidermidis (SE) biofilm (briefly referred to as inhibitory peptide) on adhesion and biofilm formation of SE at early stage.

METHODSBy using peptide synthesizer, the inhibitory peptide was synthesized with purity of 96.8% and relative molecular mass of 874.4. (1) Solution of SE ATCC 35984 (the same below) was cultivated with inhibitory peptide in the final concentrations of 1-256 µg/mL, and the M-H broth without bacteria solution was used as blank control. The MIC of the inhibitory peptide against SE was determined (n=3). (2) Solution of SE was cultivated with trypticase soy broth (TSB) culture solution containing inhibitory peptide in the final concentrations of 16, 32, 64, 128, and 256 µg/mL (set as inhibitory peptide groups in corresponding concentration), and solution of SE being cultivated with TSB culture medium was used as negative control group. Growth of SE was observed every one hour from immediately after cultivation (denoted as absorbance value), and the growth curve of SE during the 24 hours of cultivation was drawn, with 3 samples in each group at each time point. (3) Solution of SE was cultivated with TSB culture solution containing inhibitory peptide in the final concentrations of 16, 32, 64, 128, and 256 µg/mL (set as inhibitory peptide groups in corresponding concentration), and solution of SE being cultivated with TSB culture medium was used as negative control group. Adhesive property of SE was observed after cultivation for 4 hours (denoted as absorbance value, n=10); biofilm formation of SE was observed after cultivation for 20 hours (denoted as absorbance value, n=10). (4) Solution of SE was cultivated with TSB culture solution containing inhibitory peptide in the final concentration of 128 µg/mL (set as 128 µg/mL inhibitory peptide group), and solution of SE being cultivated with TSB culture medium was used as negative control group. Adhesive property of SE and its biofilm formation were observed with confocal laser scanning microscope (CLSM), and the sample numbers were both 3. Data were processed with one-way analysis of variance, LSD test, and Dunnett T3 test.

RESULTS(1) The MIC of inhibitory peptide against SE exceeded 256 µg/mL. (2) There was no significant difference in the growth curve of SE between inhibitory peptide groups in different concentrations and negative control group. (3) After 4 hours of cultivation, the absorbance values of adhesive property of SE in 256, 128, 64, and 32 µg/mL inhibitory peptide groups were respectively 0.20 ± 0.04, 0.27 ± 0.03, 0.35 ± 0.04, and 0.40 ± 0.04, which were significantly lower than the absorbance value in negative control group (0.53 ± 0.10, P<0.05 or P<0.01); the absorbance value of adhesive property of SE in 16 µg/mL inhibitory peptide group was 0.47 ± 0.09, which was close to the absorbance value in negative control group (P>0.05). After 20 hours of cultivation, the absorbance values of biofilm formation of SE in 256, 128, and 64 µg/mL inhibitory peptide groups were respectively 0.49 ± 0.10, 0.68 ± 0.06, and 0.93 ± 0.13, which were significantly less than the absorbance value in negative control group (1.21 ± 0.18, P<0.05 or P<0.01); the absorbance values of biofilm formation in 32 and 16 µg/mL inhibitory peptide groups were respectively 1.18 ± 0.22 and 1.15 ± 0.26, which were close to the absorbance value in negative control group (with P values above 0.05). (4) CLSM showed that more adhering bacteria and compact structure of biofilm were observed in negative control group, but less adhering bacteria and loose structure of biofilm were observed in 128 µg/mL inhibitory peptide group.

CONCLUSIONSThe inhibitory peptide can inhibit adhesion and biofilm formation of SE at early stage, but its structure still needs to be further modified.

Bacterial Adhesion ; Biofilms ; growth & development ; Humans ; Microscopy, Confocal ; Peptides ; Staphylococcus epidermidis ; genetics ; metabolism ; physiology

Peptide cyclization, a pivotal approach to modifying linear precursors of proteins and pepticles, has been used to enhance their biological activities and serum stabilities. Recently, sortase A (SrtA) from Staphyloccus aureus becomes a promising new technology for efficiently incorporating site specific modifications into proteins, conjugating the cell surface and cyclizing the linear peptides. In this study, we constructed two recombinant expression systems, one with chitin binding domain and the other with six-histidine tag and chitin binding domain on the N-terminal of SrtA, separately. The results of enzymatic kinetics indicate that the two recombinant tags do not impair the transpeptidase activity of SrtA compared with the standard reaction reported under the same reaction condition. The two synthesized peptides with N-ternimal three glycines and C-terminal penta-amino acid motif, LPETG, were cyclized using immobilized and recycled SrtA. The SrtA-based cyclization promises to represent a simple method for easy and efficient enzymatic synthesis of large cyclic peptides.
Aminoacyltransferases ; metabolism ; Bacterial Proteins ; metabolism ; Cyclization ; Cysteine Endopeptidases ; metabolism ; Enzymes, Immobilized ; metabolism ; Kinetics ; Peptides ; metabolism ; Peptides, Cyclic ; biosynthesis ; Staphylococcus aureus ; enzymology

OBJECTIVETo observe the biochemical characters and antibiotic susceptibility of isolated Staphylococcus aureus (S. auerus) strains against some conventional and traditional antibiotics.

METHODSThirty post operative pathogenic isolated S. aureus strains were used in this study. Bacterial culture was done in Mueller-Hinton broth at 37 °C. Characters of these strains were determined by traditional biochemical tests such as hydrolysis test of gelatin, urea, galactose, starch and protein, and fermentation of lactose and sucrose. Antibiotic susceptibility were carried out by minimum inhibitory concentration test, minium bactericidal concentration test, disc agar diffusion test and brain heart infusion oxacillin screening agar.

RESULTSFrom this study, it was observed that 100% S. aureus isolates showed positive results in gelatin, urea and galactose hydrolysis test, 50% isolates were positive in starch hydrolysis test, 35% in protein hydrolysis test, 100% isolates in lactose fermenting test, but no isolate was positive in sucrose fermenting test. Antibiotic susceptibility testing suggested that 20% of isolates were resistant to kanamycin and 46.67% were resistant to oxacillin.

CONCLUSIONSThese findings show that all these isolates have gelatin, urea, galactose hydrolysis and lactose fermenting activity. 20% of these isolates were resistant to kanamycin and 46.67% were resistant to oxacillin.

Anti-Bacterial Agents ; pharmacology ; Disk Diffusion Antimicrobial Tests ; Galactose ; metabolism ; Gelatin ; metabolism ; Hydrolysis ; Microbial Sensitivity Tests ; Staphylococcus aureus ; drug effects ; isolation & purification ; metabolism ; Starch ; metabolism ; Urea ; metabolism

Although activity of iron uptake system (IUS) was thought to play an important role in staphylococcal growth in human peritoneal dialysate (HPD) solution, siderophore production, one of the well-known IUS, was not yet detected directly in HPD solution. Therefore, we tried to detect siderophore production directly in HPD solution by using a newly developed chrome azurol S (CAS) agar diffusion assay and to investigate the effect of IUS activity on bacterial growth in HPD solution. According to the susceptibility test for streptonigrin and the productivity of siderophore in the iron-deficient (ID) medium, Staphylococcus aureus ATCC 6538 strain and Staphylococcus epidermidis clinical isolate had higher IUS activity and grew better than S. aureus ATCC 25923 strain in the ID medium. These bacteria did not grow and produce siderophore in the unused chronic ambulatory peritoneal dialysis solution. However, these bacteria grew and produced siderophore in the HPD solution. Moreover, S. aureus ATCC 25923 strain with lower activity of IUS grew poorly and produced smaller amount of siderophore in HPD compared to S. aureus ATCC 6538 strain and S. epidermidis clinical isolate with higher activity of IUS like in the ID medium. To the best of our knowledge, this is the first report that sidero-phore production is directly detected in the HPD by CAS agar diffusion assay. These results indicated that activity of IUS plays an important role in bacterial growth in the HPD solution and pathogenesis of continuous ambulatory peritoneal dialysis peritonitis.
Biological Assay ; Dialysis Solutions/chemistry* ; Drug Contamination ; Human ; Peritoneal Dialysis, Continuous Ambulatory/adverse effects* ; Siderophores/metabolism* ; Staphylococcus/metabolism*