Aims: This study aims to evaluate the effectiveness of bacteriophages isolated from Klang and Penang, Malaysia against Dickeya chrysanthemi that causes soft rot disease. Methodology and results: Basic characterization such as dextrose test, citrate test, lactose fermentation test and ornithine test were carried out on D. chrysanthemi. Activity of bacteriophages against D. chrysanthemi was evaluated using spot test. Double agar overlay assay was performed to purify and enumerate the quantify of bacteriophages.Bacteriophages were also checked for its effectiveness in controlling soft rot on post-harvested vegetables: potato (Solanum tuberosum), cucumber (Cucumis sativus) and apple (Malus domestica). Results showed that D. chrysanthemiable to utilize citrate and dextrose as the source of energy, which indicated that D. chrysanthemi inclined to choose fruits and vegetables containing citrate and dextrose as the target of attack. Clear zone observed on the bacterial lawn (spot test) indicated the ability of the bacteriophages to infect and lyse D. chrysanthemi. All the bacteriophages studied herein reached the highest concentration on day 3 and were monovalent. Conclusion, significance and impact of study All the isolated bacteriophages were able to restrain the spreading of soft rot caused by D. chrysanthemi either work alone or as cocktail. This study provides information for the formulation development of bacteriophage against soft rot disease cause by D. chrysanthemi. Furthermore, this study reveals the potential of locally isolated bacteriophages against the D. chrysanthemi and paving the application of phage treatment on agriculture products that are not limited to potatoes, cucumber and apple.
Bacteriophages--isolation & ; purification ; Dickeya chrysanthemi

Bacteriolysis ; physiology ; Bacteriophages ; genetics ; isolation & purification ; physiology ; England ; Gram-Negative Bacteria ; virology ; Humans ; International Cooperation

OBJECTIVETo isolate the wild-type virulent phage of Helicobacter pylori (Hp) and simulate the treatments in vitro to investigate the methods for oral Hp-assisted penetration of the phage through the gastric barrier and offspring phage release for infection and treatment of gastrointestinal Hp.

METHODSThe Hp strain was cultured with the candle cylinder method and the virulent phage was isolated by single plate or double plate experiment. A simulated gastric juice was applied and the bactericidal effect of the phage was tested with double flats experiment.

RESULTSAfter a 1.5-h treatment in simulated gastric juice, the orally derived Hp-borne phage was still capable of forming plaques while the control phage was not.

CONCLUSIONThe oral Hp can help the phage resist the gastric juice and then infect the gastrointestinal Hp.

Bacteriophages ; isolation & purification ; physiology ; Gastrointestinal Tract ; microbiology ; Helicobacter Infections ; therapy ; Helicobacter pylori ; virology ; Humans ; Virulence

To determine the lysis spectrum of Yersinia enterocolitica bacteriophage phiYe-F10 and to analyze the relationship between the lysis ability of phiYe-F10 and the virulence gene of Yersinia enterocolitica. To observe the lysis ability of the phage phiYe-F10 to the different Yersinia strains with the double-layer technique. The strains used in this study including 213 of Yersinia enterocolitica and 36 of Yersinia pseudotuberculosis and 1 of Yersinia pestis. The virulence genes of these Yersinia enterocolitica (attachment invasion locus (ail) and enterotoxin (ystA, ystB) and yersinia adhesin A (yadA), virulence factor (virF), specific gene for lipopolysaccharide O-side chain of serotype O : 3 (rfbc) were all detected. Among the 213 Yersinia enterocolitica, 84 strains were O : 3 serotype (78 strains with rfbc gene), 10 were serotype O : 5, 13 were serotype O : 8, 34 were serotype O : 9 and 72 were other serotypes. Of these, 77 were typical pathogenic Yersinia enterocolitica harboring with virulence plasmid (ail+, ystA+, ystB-, yadA+, virF+), and 15 were pathogenic bacterial strains deficiency virulence plasmid (ail+, ystA+, ystB-, yadA-, virF-) and the rest 121 were non pathogenic genotype strains. PhiYe-F10 lysed the 71 serotype O : 3 Yersinia enterocolitica strains which were all carried with rfbc+, including 52 pathogenic Yersinia enterocolitica, 19 nonpathogenic Y. enterocolitica. The phiYe-F10 can not lysed serotype O : 5, O : 9 and other serotype Y. enterocolitica, the lysis rate of serotype O : 3 was as high as 84.5%. The phiYe-F10 can not lysed Yersinia pseudotuberculosis and Yersinia pestis. Yersinia phage phiYe-F10 is highly specific for serotype O : 3 Yersinia enterocolitic at 25 degrees C, which showed a typical narrow lysis spectrum. Phage phiYe-F10 can lysed much more pathogenic Y. enterocolitica than nonpathogenic Y. enterocolitica.
Bacterial Proteins ; genetics ; metabolism ; Bacteriophages ; genetics ; isolation & purification ; physiology ; Host Specificity ; Virulence Factors ; genetics ; metabolism ; Yersinia enterocolitica ; genetics ; metabolism ; virology

The lysin gene of Bacillus anthracis-diagnosing bacteriophage, obtained by PCR amplification,was cloned into the Escherichia coli exepression vector pET22b which has been cleaved by EcoR I and Nde I. The recombinant vector pET22b-gamma lysin was verified to be correctly constructed by PCR, sequencing and enzyme digestion, and highly expressed in E. coli BL21 (DE3), which accounted for about 40 percent of total protein in E. coli BL21 (DE3), while in the 5L fermentor the expression level reached 15g/L. After expression, disruption and purification with three-step chromatography, Streamline SP, SP HP and Sephacryl S-100, the recombinant gamma lysin was finally obtained with purity of higher than 95 percent as determined by gel scan. The final yield following SP HP was 19.1 percent, with a greater-than-350-fold increase in specific activity. The pure enzyme has been shown active to Bacillus anthracis, and not to E. coli, Bacillus subtilis and Bacillus cereus. Its specific activity was about 1400 u/mg.
Bacillus anthracis ; virology ; Bacteriophages ; enzymology ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Recombinant Fusion Proteins ; genetics ; isolation & purification ; metabolism ; Viral Proteins ; genetics

U251 cell is a sensitive cell line to serum, which stops at G0 phase of cell cycle in no-serum medium, and recovers growth when the serum is added into no-serum medium. The cell can express corresponding proteins in different phase of cell cycle. Therefore it is very signification for the study of cell cycle regulation mechanism that explores these proteins. In this paper, the mouse antibody phage display library was added into the bottle in which the serum starvation U251 cells had been cultured, and the special antibody phages were absorbed. Then the absorbed antibody phages were amplified by adding E. coli TG1 and helper phage M13K07. Amplified antibody phages were added into bottle in which the serum cultured cell after serum starvation (follow named as serum recovered cells) were incubated, so that the cell absorbed the no-special antibody phages for the serum starvation cell and the special antibody phages were in supernatant. The remaining no-special antibody phages in the supernatant were discarded by repeating above program 3-4 times. The pure special antibody phages were gotten, and amplified by adding the host cell E. coli TG1 and helper phage M13K07. Then the host bacterium infected special antibody phage was spread on the plate medium with ampicillin, and the monoclonal antibody phages were gotten. Using same as above program, the monoclonal antibody phages absorbed specially for serum recovered U251 cells were obtained when the serum recovered cells instead of serum starvation cells and serum starvation cells instead of serum recovered cells. In this study, ninety-six positive monoclonal antibody phages that absorbed specially the serum starvation cells and eighty-two positive monoclonal antibody phages that absorbed specially the serum recovered cells were obtained. By using cell immunochemistry assay, two special signification antibodies were obtained. one (No.11) was the strong response in serum starvation cells, the other (No.2) was the strong response in serum recovered cells. The antibody No.2 had the distinctive response to the serum recovered cells in different incubation time (15min, 30min, 1h, 2h, 4h, 8h, 12h and 48h) after serum starvation. The results showed that No.2 antibody would be useful to research the factors of cell cycle regulation and apply to tumor diagnosis.
Antibodies, Neoplasm ; genetics ; immunology ; isolation & purification ; Bacteriophages ; genetics ; Brain Neoplasms ; immunology ; pathology ; Cell Line, Tumor ; Escherichia coli ; genetics ; metabolism ; Glioma ; immunology ; pathology ; Humans ; Peptide Library

OBJECTIVETo study the change of nucleic acid sequence and the germicidal effect of an E. coli bacteriophage with broad host range isolated from hospital sewage as well as to study the mechanism of phage host specificity and the effect of killed bacteria by phage-disinfectant to the samples from sewage water.

METHODSTo extract the nucleic acid from phage f(2) and phage with broad host range using anti-serum-carbamidine hydrochloride assay. Purity with agarose gel electrophoresis was then evaluated. Differences of nucleic acid sequence between phage f(2) and phage with broad host range with reverse transcription-polymerase chain reaction (RT-PCR) and random amplified polymorphic DNA (RAPD)-PCR were also comparing and analysed. Through observing the germicidal test of phage f(2) and phage with broad host range to samples from environment, different sterilization effects between the two phages were compared.

RESULTSAnalystic test for nucleic acid revealed that the two phages both belonged to 6000 bp, single-stranded RNA bacteriophage. Significant differences in their specificity of RAPD-PCR and RT-PCR were found during the changed of host range; with 26 RAPD-cDNA differential fragments found that in two phages RAPD-PCR products. The RT-PCR product of phage f(2) was 450 bp cDNA fragment, but the phage with broad host range did not show PCR product. Treating the sewage water with phage under broad host range, the germicidal test showed that the cleaning rate of E. coli bacteria and phage f(2) in water samples from environment could reach 36.75% - 56.28%, 30.84% - 47.96%, 19.19% - 35.06% and 13.05% - 27.85%, respectively.

CONCLUSIONThe cleaning rates to E. coli and bacteria by phage with broad host range were obviously higher than phage f(2) (P = 0.000). Analytic test for nucleic acid indicated that host-specific lytic effect of phage with broad host range had been changed at genetic level.

Bacteriophages ; genetics ; isolation & purification ; physiology ; Colony Count, Microbial ; Escherichia coli ; virology ; F Factor ; RNA Phages ; genetics ; Sewage ; microbiology ; virology ; Water Microbiology

OBJECTIVETo isolate human antibodies against hepatitis E virus from phage display library by a new method of panning phage antibody library based on immobilized metal affinity chromatography (IMAC).

METHODSPhage antibody library was allowed to mix with hex-His tagged expressed HEV specific antigen, NE2, in solution for adequate binding before affinity resin for hex-His was added. The non-specific phage antibodies were removed by extensive washing and the specific bound phage antibodies could then be eluted to infect TG1 or repeat the binding process for subsequent rounds of purification. The specificity of the selected human antibodies were tested by antigen competitive ELISA, human sera blocking ELISA, scFv expression, and sequence analysis.

RESULTSHis-NE2 specific recombinant phages were successfully enriched after panning procedure. Two individual phage clones, 126 and 138, showed 50% inhibition in NE2 antigen competition ELISA and obvious blocking effect by HEV positive serum in blocking ELISA. Soluble scFv of 126, 138 bound to NE2 specifically.

CONCLUSIONTwo specific human phage antibodies against hepatitis E virus (HEV) from phage display library were isolated by immobilized metal affinity chromatography. The immobilized metal affinity chromatography applied to phage antibody selection was a helpful supplement to the selection in solution.

Amino Acid Sequence ; Antibodies, Viral ; chemistry ; genetics ; immunology ; isolation & purification ; Bacteriophages ; genetics ; Chromatography, Affinity ; methods ; Enzyme-Linked Immunosorbent Assay ; Hepatitis E virus ; immunology ; Humans ; Imidazoles ; chemistry ; Metals ; Molecular Sequence Data