肺炎克雷伯菌ATCC700603裂解性噬菌体的分离鉴定和基因组学研究

摘要：目的 从医院污水中分离裂解性肺炎克雷伯菌噬菌体，对其进行生物学特性研究与遗传信息分析，为研发抗生素替代疗法提供依据。方法 以产超广谱β-内酰胺酶(ESBLs)的标准菌株肺炎克雷伯菌ATCC700603为宿主菌，采用双层平板法分离鉴定出1株裂解性噬菌体P1。对噬菌体P1的生物学特性进行了测试和研究，并对噬菌体P1进行了全基因组测序和分析。结果 分离获得的裂解性噬菌体P1，形成的噬菌斑有明显的晕环；裂解谱窄，具有强特异性；最佳感染复数(MOI)为10；在50℃及以下活性稳定，在≥60℃时稳定性显著降低；电镜观察噬菌体属于长尾噬菌体科，有一直径约为60 nm的二十面体头部和 155 nm的不可伸缩尾部。全基因组测序结果显示基因组大小为50675 bp，序列上与噬菌体Klebsiella phage KOX1最为相近，具有97.18%的一致性和89%的覆盖度；预测到82个编码序列(CDS)，其中32个具有已知功能。结论 P1为1株全新的肺炎克雷伯菌裂解性噬菌体，具有对宿主菌的强裂解性，且形成噬菌斑时所呈现的晕环值得进一步研究，为噬菌体疗法治疗肺炎克雷伯耐药菌株感染提供了新的选择。     

绛红色小单孢菌烈性噬菌体Mph1的分离及其特性

从庆大霉素产生菌——绛红色小单孢72~#异常发酵液中分离到一株烈性噬菌体,能裂解绛红色小单孢菌,形成清晰的噬菌斑,直径2mm左右,侵染指示菌的潜伏期为2小时,增殖期为1.5小时,当pH小于3或大于10及温度高于60℃时明显失活.用限制性内切酶酶解噬菌体发现有EcoRⅠ、HindⅡ、BamHⅠ、SaIⅠ切割位点,DNA分子量约为30kb,电镜观察表明该噬菌体有一六角形头部和一短尾部。     

铜绿假单胞菌噬菌体vB＿PaeM-YQ78生物学与基因组特征及其裂解酶的改造

目的 从医院废水中筛选并鉴定铜绿假单胞菌烈性噬菌体，表达和优化其裂解酶。方法 以收集的临床铜绿假单胞菌为宿主菌，分离纯化烈性噬菌体，并深入鉴定其中一株裂解能力强的广谱噬菌体的生物学特性，分析其基因组特征和进化特征，表达和改造噬菌体裂解酶，测定噬菌体与抗生素的协同杀菌作用以及不同穿膜辅助剂对于裂解酶杀菌活性的影响。结果噬菌体vB＿PaeM-YQ78是一株新的铜绿假单胞菌肌尾噬菌体，潜伏期为10 min，裂解量为43；最佳感染复数为0.00001；在40℃以下和pH 5～10范围内保持稳定。噬菌体与环丙沙星具有较强的协同作用，在24 h内无突变菌株生长。不使用穿膜辅助剂时，裂解酶LysYQ78在1 h内能将多重耐药铜绿假单胞菌降低1个log单位，在添加5 mmol/L EDTA时，LysYQ78可以将细菌浓度降低6个log单位。将ApoE蛋白N端穿膜肽融合到LysYQ78的N端后，Lys1410-YQ78的杀菌活性得到显著提高，可以在1 h内将宿主菌浓度降低2个log单位。结论 本实验分离鉴定了一株新的铜绿假单胞菌烈性噬菌体，其具有较宽的宿主谱，与环丙沙星具有较强的协同作用。并且，其裂解酶...     

铜绿假单胞菌噬菌体的分离筛选和体内外抗菌活性研究

目的:分离筛选针对铜绿假单胞茵的强裂解性噬菌体,并研究其生物学特性和体内外抗菌活性.方法:药敏纸片法筛选铜绿假单胞菌的耐药菌株,以此为宿主菌,采用双层平板法和斑点法分离筛选噬菌体并分析其生物学特性,透射电镜观察噬菌体的微观形态,检测噬菌体和细菌共培养液的OD600值分析体外抗菌活性,建立小鼠全身感染模型评估噬菌体疗效.结果:分离获得3株铜绿假单胞菌噬菌体,从中筛选出一株宽谱的强裂解活性噬菌体PPa24.PPa24属于长尾噬菌体科,其在55℃以下、pH6～10条件下活性稳定,最佳感染复数为0.1,一步生长曲线显示PPa24感染宿主菌的潜伏期为20 min、裂解期为70 min、裂解量为62 PFU· cell-1.噬菌体PPa24可以有效降低细菌培养物的OD600值,用来治疗小鼠全身感染可显著降低血液载菌量(P＜0.01),并使存活率提高至75％ (P＜0.001).结论:筛选得到的噬菌体PPa24在抗击铜绿假单胞菌感染方面初步显示了良好的体内外活性,可能成为治疗铜绿假单胞菌感染的潜在抗菌剂.     

结肠弯曲菌噬菌体的分离及其特性的研究

本文报导从12份猪粪标本中,分离到结肠弯曲菌噬菌体4株,并对其部分生物学特性,如温度、PH值及氯仿对存活率的影响,寄主范围以及噬菌体形态等进行了观察。分离的4株噬菌体较不耐热,其PH值的稳定范围甚窄(pH7.5～8.0),但耐氯仿,常见的肠道杆菌对其均不敏感,对种内细菌的感染能力则不一。试验表明,此类噬菌体的增殖条件,与其寄主菌的生长条件相似,对今后进一步分离及筛选分型用的空肠(结肠)弯曲菌噬菌体,有一定参考价值。     

由鸡引起的空肠弯曲菌肠炎

1983年5月～1984年3月我们发现5例由鸡感染的空肠弯曲菌肠炎,5例发病前均有宰鸡、养鸡等与鸡的接触史。其中例1和例2为家庭内爆发。例1女性,49岁,教师,于1984年1月31日宰鸡,10天后出现腹痛、腹泻、粪便培养空肠弯曲菌阳性。例2为例1之■,于宰鸡后5天出现腹痛、腹泻。当时未引起注意,后因其母培养到变曲菌时,再作肛拭培养,也获阳性。例1的爱人在例1发病时也曾腹痛、腹泻,因外出而未能及时作大便培养。从该2例及鸡分离到的菌株、血清型别均为2/18,说明例1、2空肠弯曲菌感染来自宰杀的鸡。例3女性,40岁,教师,家中养鸡2只,每天由例3喂养及清扫鸡粪。于1984年2月10日起,全家4口人突然先后发生腹泻,水样便。仅例3及鸡分离到2型空肠弯曲菌。例4女婴,55天,1983年5月9日腹泻,其粪便及家中饲养的母鸡粪标术均分离到11型空肠弯曲菌。例5男孩,1■岁,1984年3月9日腹泻。家中养鸡两只,放养于院内,病孩     

应用选择增菌法从腹泻病人粪便中检出空肠弯曲菌

弯曲菌以前棱称为胎儿弧苗，一般认为引起家畜流产病原菌，是引起腹泻的重要致病菌之一。1972年比利时Butzler等人从腹泻患粪便中检出空肠弯曲菌，此后英国人skirrow(1977)报告了同样事实，空肠弯曲菌引起肠道内感染，危害性日趋严重，对其分离方法多是直接分离，较少用增菌法。为此，我们从1992年6月以来选用在直接分离前先增苗方法，提高对空肠弯曲菌的检出率，现分析如下。     

噬菌体培养与鉴定方法研究进展

摘要：噬菌体是地球上数量最多的生命体，不仅广泛存在于自然界，也大量存在于人体之中。但是，目前分离、鉴定的 噬菌体数量却仅有数万个。虽然宏基因组技术的出现为探究噬菌体提供了高通量的方法，但是宏基因组测序结果中，大量噬菌 体序列功能未知，也不知道其来源于何宿主菌，是一个亟须研究的“黑匣子”。微生物的分离培养是研究微生物的起点，噬菌 体的研究亦是如此。本文首先对噬菌体分离、培养与鉴定研究进行总结，然后对裂解性和溶原性噬菌体分离方法进行归纳和分 析，为分离新的噬菌体提供较为全面的方法学综述。     

婴幼儿空肠弯曲菌肠炎25例报告

我院自1982年6月至10月,对儿科3岁以下腹泻患儿进行了粪便培养,检出空肠弯曲菌25例,阳性率为10.6%,现报告如下: 一、病原菌的检测对236例3岁以上,病程在7天以内(仅1例是半月)的急性腹泻患儿的新鲜粪便或肛拭子标本,全部作常规检查及其他肠道病原菌和空肠弯曲菌的培养。空肠弯曲菌检查系将粪便标本保存于Cary-Blair,氏运送管中,并采用简易微需氧封闭袋(由上海第一医学院药学系物理化学教研室研制)法进行分离培养。结果检出空肠弯曲菌25例,阳性率为10.6%,其中2例同时培养出致病性大肠杆菌O126B_6。共检出致病性大肠杆菌8例(3.4%),志贺氏痢疾杆菌属18例(7.5%),说明空肠弯曲菌亦是腹泻的主要病原菌。21株空肠弯曲菌在42℃微需氧环镜下进行纸片法药物敏     

马鞍山市铜绿假单胞菌血清学和噬菌体分型比较

目的　了解马鞍山市铜绿假单胞菌流行状况。方法　采集腹泻患者与健康人群粪便、外环境地面水、医院科室环境与住院人标本增菌后用十六烷基三甲溴化铵营养琼脂培养按常规方法分离鉴定 ,并将分离出的 36 0株铜绿假单胞菌进行血清学和噬菌体分型。结果　血清学分型率 98.33% (35 1 / 36 0 ) ,噬菌体分型率 96 .1 9% (346 / 36 0 ) ,且主要血清型的噬菌体裂解符合率均在 82 %以上。结论　外环境地面水、医院科室环境分别是引起人群和医院感染该菌的主要因素     

New born chicks can serve as an experimental animal model for human campylobacteriosis

Campylobacter enteritis is an emerging food borne zoonotic disease. Improperly cooked chicken serve as a potential source for this infection. Diarrheogenic potential of Campylobacter jejuni is tested either by in-vivo rat ileal loop (RIL) test or by molecular methods. This study reveals that 3-day-old chicks can serve as an animal model for toxigenic C. jejuni.     

UV-Sensitivity of Shiga Toxin-Converting Bacteriophage Virions Φ24B, 933W,P22, P27 and P32

Shiga toxin-converting bacteriophages (Stx phages) are present as prophages in Shiga toxin-producing Escherichia coli (STEC) strains. Theses phages can be transmitted to previously non-pathogenic E. coli cells making them potential producers of Shiga toxins, as they bear genes for these toxins in their genomes. Therefore, sensitivity of Stx phage virions to various conditions is important in both natural processes of spreading of these viruses and potential prophylactic control of appearance of novel pathogenic E. coli strains. In this report we provide evidence that virions of Stx phages are significantly more sensitive to UV irradiation than bacteriophage λ. Following UV irradiation of Stx virions at the dose of 50 J/m², their infectivity dropped by 1–3 log₁₀, depending on the kind of phage. Under these conditions, a considerable release of phage DNA from virions was observed, and electron microscopy analyses indicated a large proportion of partially damaged virions. Infection of E. coli cells with UV-irradiated Stx phages resulted in significantly decreased levels of expression of N and cro genes, crucial for lytic development. We conclude that inactivation of Stx virions caused by relatively low dose of UV light is due to damage of capsids that prevents effective infection of the host cells.     

高效液相色谱法同时测定苦碟子中四种黄酮的含量

目的:建立RP-HPLC法同时测定苦碟子药材中木犀草素7-O-β-D葡萄糖1→2葡萄糖苷（Ⅰ）、木犀草素-7-O-β-D-葡萄糖苷（Ⅱ）、木犀草素7-O-β-D-葡萄糖醛酸苷（Ⅲ）、芹菜素-7-O-β-D葡萄糖醛酸苷（Ⅳ）的含量。方法:采用C₁₈色谱柱（250mm×4.6 mm,5μm）,以乙腈-0.4%磷酸溶液为流动相,梯度洗脱;检测波长348 nm。结果:8批次苦碟子药材中Ⅰ含量在0.30～1.20 mg·g^-1,Ⅱ含量在0.30～1.10 mg·g^-1,Ⅲ含量在2.40～6.00 mg·g^-1,Ⅳ含量在0.90～1.70 mg·g^-1;平均回收率为Ⅰ:97.32%（RSD=0.91%）;Ⅱ:98.87%（RSD=0.86%）;Ⅲ:98.55%（RSD=1.62%）;Ⅳ:96.40%（RSD=0.85%）。结论:该方法简便,准确,灵敏,可用于苦碟子药材的质量控制。     

高效液相色谱法测定清炒白术中白术内酯Ⅰ、Ⅱ、Ⅲ

目的测定清炒白术中白术内酯的含量。方法采用外标法,Dikma Kromasil 100AC18（4．6mm×250mm,5μm）色谱柱,乙腈-水梯度洗脱（60％～100％乙腈）,流速为1．0mL·min,柱温25℃,检测波长为220nm（Ⅰ、Ⅲ）、276nm（Ⅱ）。结果测得4批清炒白术中白术内酯Ⅰ、Ⅱ、Ⅲ含量合计分别是：1．3261、1．5910、1．5348和1．7380mg·g^-1结论白术清炒后对所含白术内酯Ⅰ、Ⅱ、Ⅲ的含量有一定影响。     

Recent advances in bacteriophage therapy: how delivery routes, formulation, concentration and timing influence the success of phage therapy

Objectives Bacteriophages are bacteria‐specific viruses that infect and, in the case of obligately lytic phages, destroy their host bacteria. Phage therapy has been used therapeutically to combat bacterial infections since their discovery. This paper reviewed recent in‐vivo phage therapy studies, with a distinct focus on the effect of delivery routes, phage concentration and timing of administration on the success of the therapy. Key findings It was found that the most successful route of administration for the treatment of systemic infections was via the parenteral route. Oral delivery is mainly used to treat gastrointestinal infections. However, in some cases phages can also reach the systemic circulation. Local delivery (skin, ears, teeth) has proved extremely successful in the treatment of topical infections, as has the inhalation of phages for the treatment of lung infections. The ability of phages to prevent biofilm formation on medical devices has received much attention, mainly in the area of catheter coatings. This review also highlights areas in which phage therapy needs substantial development. Many papers were lacking in formulation details, with crude phage stocks being used in most cases. No phage stability data were included in any of the papers. Summary The review concluded that although phage therapy is an excellent alternative for the treatment of bacterial infections, optimisation of formulations and long‐term stability data is required before it can be widely used within a clinical setting.     

Endotoxic activity and enterotoxigenicity of human strains of Campylobacter jejuni isolated from patients in a Nigerian hospital

Limulus gelation assay and dermal Schwartzman reaction provided a sensitive and reproducible means of testing the endotoxic activity of hospital strains of Campylobacter jejuni in Lagos, Nigeria. All the 22 isolates of Campylobacter jejuni tested for the limulus gelation assay were positive for the production of endotoxin. Furthermore, the Campylobacter suspensions caused a positive dermal Schwartzman reaction in rabbits. The area of skin reaction was less extensive than that produced by Escherichia coli 01114B and E7539/77 which served as positive controls. Five local strains of Campylobacter jejuni tested for enterotoxin production showed negative reaction in the infant mouse test whereas enterotoxin production was observed in Campylobacter jejuni strain 11168 and Escherichia coli E7539/77. Consequently, the infant mouse test may not be suitable for enterogenicity testing of our local isolates of Campylobacter jejuni.     

Feasibility of spray drying bacteriophages into respirable powders to combat pulmonary bacterial infections

The use of bacterial viruses for antibacterial treatment (bacteriophage therapy) is currently being reevaluated. In this study, we analyze the potential of processing bacteriophages in a dry powder formulation, using a laboratory spray dryer. The phages were dried in the presence of lactose, trehalose or dextran 35, serving as an excipient to give the resulting powder the necessary bulk mass and offer protection to the delicate phage structure. Out of the three excipients tested, trehalose was found to be the most efficient in protecting the phages from temperature and shear stress throughout the spray drying process. A low inlet air temperature and atomizing force appeared to be the best parameter conditions for phage survival. Pseudomonas podovirus LUZ19 was remarkably stable, suffering less than 1 logarithmic unit reduction in phage titer. The phage titer of Staphyloccus phage Romulus-containing powders, a member of the Myoviridae family, showed more than 2.5 logarithmic units reduction. On the other hand, Romulus-containing powders showed more favorable characteristics for pulmonary delivery, with a high percentage of dry powder particles in the pulmonary deposition fraction (1–5 μm particle diameter). Even though the parameters were not optimized for spray drying all phages, it was demonstrated that spray drying phages with this industrial relevant and scalable set up was possible. The resulting powders had desirable size ranges for pulmonary delivery of phages with dry powder inhalers (DPIs).     

慢性心衰患者血清GDF-15与PⅠCP、 PⅢNP的相关性分析

目的 检测心力衰竭（心衰）患者血清生长分化因子15（GDF-15）与Ⅰ型前胶原羧基端肽（PⅠCP）、Ⅲ型前 胶原氨基端肽（PⅢNP）的变化并分析其相关性,探究GDF-15对心衰患者心室重构的指导作用。方法 纳入219例 心衰患者,按照美国纽约心脏病协会（NYHA）心功能分级分为NYHAⅠ级组58例、Ⅱ级组39例、Ⅲ级组47例、Ⅳ级组 75例;按左室射血分数（LVEF）分为射血分数减低的心衰组（LVEF＜0.45,HFrEF组,69例）和射血分数保留的心衰组 （LVEF≥0.45,HFpEF组,150例）;选取32例同期健康体检者作为对照组。采用ELISA法检测血清GDF-15、PⅠCP、P ⅢNP 水平,超声心动图测量左室舒张末期内径（LVEDD）、左心房内径（LA）、室间隔厚度（IVST）、左室后壁厚度 （LVPWT）以及 LVEF,计算左室质量指数（LVMI）,并分析心衰患者血清 GDF-15 与 PⅠCP、PⅢNP、NT-proBNP、 LVEDD、LA、LVMI及LVEF的相关性。结果 心衰患者血清NT-proBNP、GDF-15、PⅠCP、PⅢNP水平随NYHA分级 增加而升高（P＜0.05）。对照组、HFpEF组和HFrEF组LA、LVEDD、LVMI、NT-proBNP、GDF-15、PⅠCP及PⅢNP依 次升高（P＜0.05）。心衰患者血清GDF-15水平与PⅠCP、PⅢNP、NT-proBNP、LA、LVEDD、LVMI呈正相关（rs分别为 0.549、0.533、0.539、0.393、0.403、0.485,均 P＜0.01）,与 LVEF 呈负相关（rs=-0.568,P＜0.01）。结论 心衰患者血清 GDF-15与心衰严重程度相关,并可反映心衰患者心室重构情况。     

基于指纹图谱分析和多成分同时定量的玉屏风丸质量评价

目的建立玉屏风丸的高效液相色谱(HPLC)指纹图谱,测定7种主要成分(毛蕊异黄酮葡萄糖苷、芒柄花素、升麻素苷、升麻素、白术内酯Ⅲ、白术内酯Ⅱ、白术内酯Ⅰ)的含量,为玉屏风丸的质量控制提供可靠方法。方法色谱柱为Shim-pack VP-ODS柱(250 mm×4.6 mm,5μm);流动相为乙腈(A)-0.3%磷酸溶液(B),梯度洗脱(0~20 min时12%A,20~40 min时12%A→35%A,40~46 min时35%A→68%A,46~50 min时68%A→80%A,50~60 min时80%A→12%A);流速为0.85 m L/min,检测波长升麻素苷为300 nm(0~20 min),毛蕊异黄酮葡萄糖苷和芒柄花素及升麻素为254 nm(20~40 min),白术内酯Ⅲ及白术内酯Ⅱ为220 nm(40~46 min),白术内酯Ⅰ为275 nm(46~60 min);柱温为35℃;流速为0.85 m L/min;进样量为10μL。结果玉屏风丸HPLC指纹图谱有18个共有峰,通过比较,指认其中7个指标成分,分别为4号(升麻素苷)、9号(毛蕊异黄酮葡萄糖苷)、10号(升麻素)、11号(芒柄花素)、16号(白术内酯Ⅲ)、17号(白术内酯Ⅱ)和18号(白术内酯Ⅰ),利用相似度软件对14批样品的指纹图谱进行分析,各批样品相似度均大于0.97。在建立的色谱条件下,7组分分离度良好,精密度和重复性试验的RSD均小于1.5%(n=6),供试品溶液在24 h内稳定,各成分具有良好的线性关系和较宽的线性范围。14批玉屏风丸中毛蕊异黄酮葡萄糖苷、芒柄花素、升麻素苷、升麻素、白术内酯Ⅲ、白术内酯Ⅱ、白术内酯Ⅰ质量浓度范围分别为1.5490~1.5640 mg/g,0.7015~0.7029 mg/g,0.7960~0.7977 mg/g,0.5905~0.5922 mg/g,0.6745~0.6762 mg/g,0.5210~0.5229 mg/g,0.3992~0.4015 mg/g。结论该建立的玉屏风丸HPLC指纹图谱检测和定量测定分析方法快速、准确,可有效评价玉屏风丸的质量。