Biological characteristics and genomic information of a bacteriophage against pan-drug resistant Klebsiella pneumoniae in a burn patient and its effects on bacterial biofilm / 中华烧伤杂志

Objective@#To isolate a bacteriophage against pan-drug resistant Klebsiella pneumoniae in a burn patient, and to study its biological characteristics, genomic information, and effects on bacterial biofilm.@*Methods@#(1) In 2018, pan-drug resistant Klebsiella pneumoniae UA168 (hereinafter referred to as the host bacteria) solution isolated from the blood of a burn patient in Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine (hereinafter referred to as Ruijin Hospital) was used to isolate and purify the bacteriophage against pan-drug resistant Klebsiella pneumoniae from the sewage of Ruijin Hospital with sewage co-culture method, drip plate method, and double-agar plate method. The bacteriophage was named as phage KP168 and the plaque morphology was observed. (2) The phage KP168 solution was taken for cesium chloride density gradient centrifugation and dialysis, and then the morphology of phage KP168 was observed through transmission electron microscope after phosphotungstic acid negative staining. (3) The phage KP168 solution was taken to determine the lytic ability of the phage KP168 against 20 strains of pan-drug resistant Klebsiella pneumoniae isolated from the burned patients′ blood in Ruijin Hospital by the drip plate method, and then the lysis rate was calculated. (4) The phage KP168 solution at a initial titer of 9.3×1011 plaque-forming unit (PFU)/mL (400 μL per tube) and the host bacteria solution at a concentration of 1×109 colony-forming unit (CFU)/mL (4 mL per tube) were conventionally shaking cultured together for 4 hours at multiplicity of infection (MOI) of 10.000, 1.000, 0.100, 0.010, or 0.001, respectively (1 tube per MOI). The titer of phage KP168 was measured by the double-agar plate method (the measurement method was the same below) to select the optimal MOI. The experiment was repeated three times. (5) The host bacteria solution at a concentration of 1×109 CFU/mL (4 mL per tube) and the phage KP168 solution at an adjusted titer of 5×107 PFU/mL (400 μL per tube) were mixed at the MOI of 0.005. The plaques were counted 0 (immediately), 1, 2, 3, 4, 5, 15, and 30 minutes (1 tube at each time point) after mixing by the double-agar plate method (the counting method was the same below), and the percentage of adsorbed phages was calculated to screen for the optimal adsorption time. The experiment was repeated three times. (6) The host bacteria solution at a concentration of 1×109 CFU/mL (300 μL per tube) and the phage KP168 solution at a titer of 5×108 PFU/mL (60 μL per tube) were mixed at MOI of 0.005 and conventionally shaking cultured after standing for the optimal adsorption time. The phage KP168 titer was measured 0 (immediately), 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 minutes after culture, and a one-step growth curve was drawn. The experiment was repeated three times. (7) The phage KP168 solution at a titer of 2.5×1010 PFU/mL was left to stand for 1 hour at 37, 40, 50, 60, or 70 ℃ (3 tubes at each time point, 1 mL per tube) for counting the plaques, and then the thermal stability curve was drawn. SM buffer at a pH values of 5.0, 6.0, 7.0, 7.4, 8.0, 9.0, or 10.0 were added to the phage KP168 solution at a titer of 3.0×1010 PFU/mL, respectively. The mixed solution was left to stand for 1 hour at 37 ℃ (3 tubes of each pH, each tube containing 100 μL phage KP168 solution and 900 μL SM buffer), and then the plaques were counted, and an acid-base stability curve was drawn. (8) The phage KP168 solution was taken for DNA extraction and sequencing after dialysis as in experiment (2). The whole genome was annotated with Prokka to obtain the coding sequence of phage KP168. Nucleotide′s BLAST function was used to proceed nucleic acid sequence alignment for finding a known phage with the highest similarity to the phage KP168 nucleic acid sequence, and Blastx function was used to translate the coding sequence into protein for its function prediction. The comparison with Antibiotic Resistance Genes Database and Virulence Factors Database was proceeded. (9) In a 96-well plate, at a MOI of 1.000, 0.100, 0.010 or 0.001 (3 wells per MOI), 20 μL phage KP168 solution at a initial titer of 5.8×1010 PFU/mL was added to 200 μL host bacteria solution at a concentration of 1.5×108 CFU/mL (the same concentration below) for co-cultivation for 48 hours. After 200 μL host bacteria solution was left to stand for 48 hours, 20 μL phage KP168 solution at a titer of 1×106, 1×107, 1×108, 1×109, or 1×1010 PFU/mL (3 wells per titer) was added respectively for action for 4 hours. In both experiments, 200 μL host bacteria solution added with 20 μL SM buffer (3 wells) acted as a negative control, and 220 μL LB culture medium (3 wells) acted as a blank control. Absorbance values were measured by a microplate reader, and inhibition/destruction rates of biofilm were calculated. The experiments were both repeated three times.@*Results@#(1) The plaques of phage KP168 successfully isolated and purified were transparent and round, and its diameter was approximately 1.5 mm. (2) The phage KP168 has a regular polyhedron structure with a diameter of about 50 nm and without a tail. (3) The phage KP168 could lyse 13 of 20 strains of Klebsiella pneumoniae from burned patients, with a lysis rate of 65.0%. (4) When MOI was 1.000, the titer was the highest after co-culturing the phage KP168 with the host bacteria for 4 hours, which was the optimal MOI. (5) After the mixing of the phage KP168 with the host bacteria for 4 minutes, the percentage of the adsorbed phage reached the highest, which was the optimal adsorption time. (6) The one-step growth curve showed that during the lysis of the host bacteria by phage KP168, the incubation period was about 10 minutes, and the lysis period was about 40 minutes. (7) With the condition of 40 ℃ or pH 7.4, the number of plaques and the activity of phage KP168 reached the highest. (8) The genome of phage KP168 was a linear double-stranded DNA with a length of 40 114 bp. There were 48 possible coding sequences. It had the highest similarity to Klebsiella phage_vB_Kp1. The most similar known proteins corresponding to the translated proteins of coding sequences contained 23 hypothetical proteins and 25 proteins with known functions. No resistance genes or virulence factor genes were found. The GeneBank accession number was KT367885. (9) After 48 hours of co-cultivation of the phage KP168 and the host bacteria at each MOI, the inhibition rates of biofilm were similar, with an average of about 45%. After the phage KP168 with a titer of 1×109 PFU/mL acted on the biofilm formed by the host bacteria for 4 h, the destruction rate of biofilm was the highest, reaching an average of 42%.@*Conclusions@#In this study, a bacteriophage against pan-drug resistant Klebsiella pneumoniae from a burn patient, phage KP168, is isolated from sewage, which belongs to the tailless phage. It has a wide host spectrum, short adsorption time, and short incubation period, with certain thermal and acid-base stability. Its genomic information is clear, and it does not contain resistance genes or virulence factor genes. It also has an inhibitory effect on the formation of bacterial biofilm and a destructive effect on the formed bacterial biofilm.

Anti-tumor activity of HER-specific peptide vaccines-CKL9 and YL20 / 中国药理学通报

Aim To explore whether the polypeptide vaccines CKL9 and YL20 can induce immune response and anti-tumor effect on HER-2 (+) tumors in vitro and in vivo, and to provide suggestions for clinical use.Methods The proliferation of specific lymphocytes and cytotoxic T lymphocyte activity(CTL) stimulated by CKL9 and YL20 were studied with CCK-8 assay and LDH assay, and the antitumor activity of CKL9 and YL20 was evaluated in vivo.Results The lymphocyte proliferation was promoted by incubation with CKL9 and YL20, and the relative increase of cells was 11.1% and 16.7% respectively at the concentration of 50 mg·L-1 of CKL9 and YL20.The LDH assay confirmed the CTL effect induced by CKL9 and YL20 on HER2-positive tumor cells, not on HER2-negtive tumor cells.With an effector-target ratio of 80 ∶1, the inhibition of tumor cell by cytotoxic T lymphocyte stimulated by CKL9 and YL20 could reach 89.8% and 84.3%, respectively.The HER2(+) tumor cell N87 transplanted in Babes mice was inhibited by pre-immune polypeptide CKL9 and YL20.Conclusion The HER2-specific polypeptide vaccines CKL9 and YL20 could induce persistent specific CD4 and CD8 T cell immune and inhibit the growth of HER2 positive tumor cells.

Isolation and characterization of bacteriophage against extended-spectrumβ-lactamase-producing Escherichia coli strains / 上海交通大学学报（医学版）

Objective · To isolate phages which can fight against extended-spectrum β-lactamase (ESBLs)-producing Escherichia coli (E. coli), and provide basic research for establishment of E. coli phage library and treatment of bacterial infection. Methods · Samples collected from sewage were co-cultured with 93 ESBLs-producing E. coli strains. A phage named JDEC001 was isolated by double agar overlay plaque assay. The biological characteristics, complete genome sequence and comparative genome analyses of JDEC001 were studied respectively. Results · JDEC001 belongs to the lytic phage as a member of the Caudovirales order, Podoviridae family. It has high activity at pH from 5 to 11 and with temperature from 0 to 39 ℃ .Whole-genome sequencing of JDEC001 demonstrated double-stranded DNA genome of 38745 bp with GC content of 49.93%, which encoded 46 open reading frames. The comparative genomics also showed that there was no virulent genes or antibiotic resistant genes in its genome. Conclusion · The phage JDEC001 against ESBLs-producing E. coli was isolated and purified, with good stability in a broad range of pH and temperature.

A novel pore-forming hemolysin protein in Leptospira species / 中国人兽共患病学报

Hemolysins of Leptospira interragans have been shown to be the virulence factor in the pathogenesis of leptospirosis and 10 potential hemolysin genes were charecterized by genomic annotation of L.interrogans serovar.Lai strain 56601. In the present study, the LA0202 gene supposed to encode one of the new potential hemolysin was cloned and the protein encoded was purified. The purified protein was shown to have highly hemolytic activity as demonstrated on the sheep blood agar plate. It was also confirmed that the LA0202 protein-mediated hemolysis on sheep erythrocytes was osmotically protected by PEG6000. Meanwhile, this protein could induce pore formation on sheep erythrocytes and cause damages on the membrane of human L-02 liver cells. In addition, it could induce apoptosis of human L-02 liver cells after treatment of cells with this protein for 24 hours. It is evident that LA0202 protein acting as a pore-formong hemolysin can induce cytotoxic damage on mammalian cells.