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.

Non-surgical combined modality treatments for laryngeal organ preservation in advanced hypopharyngeal carcinoma / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To investigate the treatment outcome, laryngeal preservation and side-effect in locally advanced hypopharyngeal carcinoma treated with combined Hilical tomotherapy (HT) or intensity-modulated radiotherapy (IMRT) and chemotherapy and/or EGFR inhibitor (Cetuximab or Nimotuzumab).</p><p><b>METHODS</b>A total of 68 patients (20 cases with T1-2N1-3M0 and 48 cases with T3-4N1-3M0) with locally advanced hypopharyngeal cancer were treated individualy with non-surgical combined modality treatments including induction chemotherapy followed by concurrent chemoradiotherapy, induction chemotherapy followed by concurrent radiotherapy and EGFR inhibitor, concurrent chemoradiotherapy and EGFR inhibitor, and concurrent radiotherapy and EGFR inhibitor. HT was used in 40 patients and IMRT in 28 patients. Side-effects were evaluated with the established Common Terminology Criteria for Adverse Events (CTCAE) version 3.0 criteria.</p><p><b>RESULTS</b>The average follow-up time was 25.7 months (range 3-69 months). All patients completed the planned radiotherapy without treatment breaks, and 66 (97.0%) of 68 patients completed the planned chemotherapy. The 2-year and 3-year overall survival rates were 78.8% and 64.7% respectively, with an organ preservation rate of 84.2%. The most common side-effect greater than or equal to grade 3 was oropharyngeal mucositis. No patient dependent on a percutaneous gastrostomy and tracheostomy tube.</p><p><b>CONCLUSION</b>Hypopharyngeal carcinoma can be treated with non-surgical combined modality treatment including HT or IMRT, with a high laryngeal organ preservation rate and minimal toxicities.</p>

Clinical analysis of in-patients with large vestibular aqueduct syndrome / 临床耳鼻咽喉头颈外科杂志

OBJECTIVE@#This study is to investigate the clinical materials of in-patients with the large vestibular aqueduct syndrome (LVAS), and explore the feature, diagnosis and treatment measures of the disease.@*METHOD@#A retrospective review was conducted including the medical history, audiological examinations, vestibular function examinations, imaging examinations and treatment methods of 44 in patients (87 ears) suffering LVAS admitted to our hospital in the past 4 years(from 2008 to 2012).@*RESULT@#ln the 44 in patients, there were 24 male cases and 20 female cases, and the male-female ratio was 1.2 :1. The average of the onset age was 3.39 years. Five cases (11. 36%) had related familial history. The profound hearing loss was found in 67 ears (77.01%), and the severe hearing loss was found in 20 ears (22.99%). After systemic treatment,the hearing of 38 ears improved effectively,but that of 49 ears did not improve obviously. The analysis found that patients suffering sudden hearing loss got better curative effect than those with progressive hearing loss. Patients received combined drug therapy improving arterial circulation as well as venous reflux got better therapeutic effect. There was a significant difference on effect between the patients with course of treatment more than 7 days and those less than 7 days. There was no significant correlation between therapeutic effect and other factors.@*CONCLUSION@#In part of LVAS patients,the hearing level can be effectively improved through a standard internal medicine treatment. We can improve the personalized and standardized treatment strategy for this disease through analysis of diagnosis and treatment of in-patients with complete clinical data.

A new method of measuring the pulse based on facial video / 生物医学工程学杂志

This paper proposes a new method of non-contact pulse measurement by analyzing a clip of human facial video. The method is based on photo plethysmography (PPG) and independent component analysis (ICA) model. A clip of color facial video shot under normal lighting condition is firstly discomposed into RGB channel sequences. Secondly, by applying ICA to the 3 channel sequences, 3 new independent signals are obtained, among which one signal is close to human pulse wave. Thus the pulse can be measured. In this paper, the principles of PPG and ICA are briefly described and the measurement framework is proposed. The experimental results showed that this novel approach was reasonable and feasible.