梁河家鼠鼠疫疫源地鼠疫噬菌体的分离及其流行病学意义

目的 调查梁河县家鼠鼠疫疫源地宿主动物中是否携带鼠疫噬菌体,并探讨其流行病学意义。方法 2017年采集梁河家鼠鼠疫疫源地4个曾流行过鼠疫乡镇的鼠类标本,以鼠疫疫苗株EV76为饲养菌,采用双层平板法分离鼠疫噬菌体,同时挑取部分噬菌体进行电镜扫描。结果 共获得338份标本(黄胸鼠234只,臭鼩鼱43只,其余61只),分离到29株鼠疫噬菌体,总分离率为8.58%(29/338),其中19株分离自黄胸鼠,分离率为8.12%(19/234),8株分离自臭鼩鼱,分离率为18.6%(8/43);4个乡镇全部有分离到鼠疫噬菌体,其中遮岛镇分离率最高为16.13%(5/31);初次分离这些鼠疫噬菌体时,其噬斑在双层平板上表现为大(直径≥2.0 mm)、中(≥1.0 mm,≤2.0 mm)及小(≤1.0 mm)3种噬斑;2株有代表性噬菌体皆为肌尾病毒科噬菌体。结论 梁河家鼠鼠疫疫源地中普遍存在鼠疫噬菌体,黄胸鼠是主要的携带宿主,所分鼠疫噬菌体为肌尾病毒科噬菌体且具有多态性,值得进一步研究。     

鹤庆新发野鼠鼠疫疫源地中鼠疫噬菌体的分离及其裂解谱测定

目的 查明鹤庆新发野鼠鼠疫疫源地内是否存在鼠疫噬菌体,并对所离分鼠疫噬菌体进行形态鉴定及噬菌谱分析。方法 以鹤庆县马厂村为核心,选择5 km范围内的自然村为采样点,采用鼠铗法进行捕鼠,实验室中取鼠盲肠置入改良PBS中,采用双层平皿对样本进行筛选、纯化得到噬菌体,观察噬菌斑形态,电镜检查噬菌体形态,并在22 ℃、24 ℃、28 ℃及37 ℃对21株鼠疫菌与115株非鼠疫菌进行裂解特性分析。结果 采集的354份标本,分离到2株鼠疫噬菌体;2株噬菌体在高于24 ℃温度下可裂解鼠疫菌,低于24 ℃不能裂解鼠疫菌,而非鼠疫菌在4个温度下均不裂解;电镜形态检测2株噬菌体均属于肌尾噬菌体。结论 鹤庆新发野鼠疫疫源地内存在着鼠疫噬菌体,且所分鼠疫噬菌体只有在高于24 ℃时才裂解鼠疫菌,此特性与鼠疫菌在鹤庆县的长期存在相关,且两株噬菌体存在裂解谱较窄,特异性良好,可用于备用诊断噬菌体的筛选株。     

噬菌体Chy5的生物学和基因组学特征

目的 筛选能感染结核分枝杆菌的溶源性噬菌体,了解其生物学及遗传学信息。方法 从土壤中分离纯化噬菌体;电镜观察噬菌体形态;单斑法测定宿主谱;提取噬菌体DNA,鸟枪法随机测序。采用DNAStar软件包分析基因组成分,Glimmer3.0、GeneMark软件预测基因功能,同时做共线性分析。分离溶源菌,通过紫外线诱导试验、超免反应验证噬菌体溶源性。结果 分离到一株新的噬菌体Chy5,其头部直径(61.5±1.4) nm,尾部长度(114.1±2.1) nm,为长尾噬菌体,可感染敏感及耐药结核菌株。Chy5基因组全长51 214 bp,G+C含量63.60%,与噬菌体D29基因组最相似,有88个推定基因,含有编码整合酶和阻遏蛋白的基因以及attP位点。经紫外线诱导Chy5溶源菌可形成噬菌斑,Chy5与其溶源菌共培养会发生超免反应。结论 分离到一株新的能感染敏感及耐药结核菌的溶源性噬菌体Chy5,其基因组与噬菌体D29相似,有望应用于构建荧光报告噬菌体。     

噬菌体TM4、Guo1和D29对静止期结核菌裂解作用初步研究

目的 研究噬菌体TM4、Guo1、D29能否裂解静止期结核菌。方法 采用密闭培养至对数生长期的结核分枝杆菌构建静止期结核菌模型;采用药敏检测、金胺o-尼罗红荧光染色和电镜观察作为检测模型方法;采用most probable number(MPN)法计数作为裂解作用的检测指标。结果 密闭培养11个月的静止期结核菌模型构建成功;经不同处理后,MPN计数结果为对照组1100,Guo1组23,TM4组23,D29组600,RFP组673,INH组887;与对照组相比,Guo1组和TM4组菌量明显减少(P<0.01,P<0.01),D29组菌量减少不明显(P=0.05);与RFP组相比,Guo1组和TM4组菌量亦明显减少(P<0.05,P<0.05),D29组菌量减少不明显(P>0.05);Guo1组和TM4组菌量无差异。结论 噬菌体TM4和Guo1能够裂解静止期结核菌,噬菌体D29不能裂解静止期结核菌,噬菌体TM4和Guo1裂解能力无差别。     

奶牛粪便分离的伊氏李斯特菌伊氏亚种基因组序列测定及其分子特征分析

目的 了解某奶牛场奶牛粪便中李斯特菌的携带情况及其分离株的遗传特征。方法 用ISO 11290方法分离196份奶牛粪便样本中的李斯特菌,对分离的伊氏李斯特菌进行全基因测序,用MEGA6.0构建系统发育树,网站在线比对分析伊氏李斯特菌株的毒力基因、耐药基因及前噬菌体等。结果 196份奶牛场养殖的奶牛粪便样本中有9份样本为李斯特菌阳性,其中3株为伊氏李斯特菌伊氏亚种,6株为英诺克李斯特菌,分离率分别为1.53%和3.06%。3株伊氏李斯特菌伊氏亚种分离株具有包括LIPI-1和LIPI-2在内的绝大部分致病性李斯特菌毒力相关基因,携带一个不完整的前噬菌体及22个耐药相关基因。结论 养殖场奶牛粪便中携带伊氏李斯特菌伊氏亚种。对伊氏李斯特菌伊氏亚种分离株的全基因组、毒力基因、耐药基因、前噬菌体基因等遗传特征分析,为进一步分析其致病性提供了参考。     

利用ssr标记浅析云南登革热重点地区埃及伊蚊种群遗传特征

目的 通过研究云南省不同地理来源的埃及伊蚊自然种群遗传结构,探究不同群体间的遗传特征与联系。方法 捕获景洪、勐腊、勐海、耿马、瑞丽的埃及伊蚊,提取DNA并采用12对荧光标记的微卫星引物进行PCR扩增,用毛细管电泳检测扩增片段,分析5个埃及伊蚊自然种群的遗传相关指标。结果 在5个采集点共采集234个有伊蚊幼虫的容器,采集埃及伊蚊603只,随机选取149只提取DNA,进行扩增分析。12个微卫星位点的平均N_a、N_e、H_o、H_e、PIC分别为18、4.653、0.711、0.728、0.778,12个微卫星位点的PIC指数均>0.25,高度多态。每个种群的平均Na、Ne、Ho、He、PIC分别为7.750~10.583、3.606~5.309、0.661~0.741、0.629~0.774、0.588~0.750。12个微卫星位点的F_st,F_is,N_m的平均值分别为0.078、0.028、3.321。5个自然种群埃及伊蚊的成对指数Fast为0.057~0.148,各种群间遗传分化和地理距离未发现显著相关,Nei’s标准遗传距离在0.162~0.527之间,对应的遗传相似度在0.590~0.850之间。 云南省埃及伊蚊遗传多样性比较丰富,种群间存在一定的遗传分化和基因交流。     

111例肺结核合并下呼吸道感染的菌种分布及药敏分析

目的　统计分析肺结核合并下呼吸道感染的菌种分布及药物敏感情况。方法 统计天津市肺科医院2002年间肺结核合并下呼吸道感染111例以及同期天津市10家医院1239例下呼吸道感染痰标本中分离出菌株的菌种分布。对肺炎克雷伯杆菌、绿脓杆菌、鲍曼不动杆菌、液化沙雷氏菌的药敏进行统计分析,全部数据由SPSS软件进行分析处理。结果 肺结核合并下呼吸道感染细菌种类至少有28种,依其所占比例高低排列前8位依次是:肺炎克雷伯杆菌(19.8%)、绿脓杆菌(13.5%)、鲍曼不动菌(9.9%)、白色念珠菌(7.2%)、液化沙雷氏菌(5.4%)、金黄色葡萄球菌(5.4%)、大肠杆菌(4.5%)、阴沟杆菌(2.7%)。结论 阿米卡星、环丙沙星对感染率最高的四种病原菌都有很高的的敏感性,同时二者还被列为二线抗结核药物,故此可以作为肺结核(特别是复治肺结核)合并下呼吸道感染经验治疗的首选抗生素。     

布氏菌噬菌体Bj-309株的研究 Ⅰ.Bj-309株噬菌体的发现和活性

我们从地方分离的犬种布氏菌传代培养物中,首次在国内发现犬种布氏菌噬菌体,称之为Bj-309株。初步证明其裂解强度稍高于国际同类噬菌体R/C株。它对国内分离的30株犬种布氏菌均能裂解,对犬体分离的另6株菌不能裂解,这6株菌对犬种血清亦为阴性反应。它对S型猪种、羊种和牛种都不裂解;对相应的8株R型羊种和牛种菌能部分裂解,结果与对照株R/C相符。对2株国际标准犬种布氏菌Mex51和RM6/66,能产生融合性噬斑。初步证明:Bj-309株为犬种布氏菌噬菌体,并有较强的特异裂解活性。     

重庆市库蚊标本中宜昌病毒的分离鉴定

目的 对2017年采集自重庆地区蚊虫标本携带的病毒进行分离鉴定。方法 采用组织细胞培养法进行病毒分离,应用高通量测序技术获得病毒分离物的全基因组序列,采用系统发育法进行种类及分子特征分析。结果 库蚊标本来源的病毒阳性分离物 CQFD2017能引起白纹伊蚊细胞C6/36病变,但不能引起金黄地鼠肾细胞BHK-21、非洲绿猴肾细胞Vero病变。 CQFD2017全基因组序列长度为20 893 bp, 包含6个开放阅读框。系统进化分析发现,该毒株位于Nidovirales病毒目Mesoniviridae病毒科的Yichang病毒所在的进化分支,与Yichang病毒(NC_040534)的核苷酸一致性为98.6%,ORF1a和ORF1b区氨基酸一致性分别为99.06%和99.15%,因此将该毒株命名为Yichang病毒CQFD2017株。结论 2017年重庆市库蚊标本分离的CQFD2017株为Yichang病毒。     

小毛莨内酯对结核休眠菌感染病人GLS mRNA表达的影响

目的　研究小毛莨内酯抗人体结核休眠菌感染的分子免疫学机理。方法 采用PCR方法检测结核病人PBLs中结核菌16kDα-晶状体同源蛋白DNA阳性的56例患者为本研究对象;采用反转录聚合酶链反应半定量 (QC-RT-PCR)方法,测定不同剂量的Tern.对以上病人PBL颗粒裂解肽mRNA表达水平的影响。结果 当Tern.在100mg/L,200mg/L浓度时,能诱导结核休眠菌感染病人体外活化的PBLGLSmRNA的表达。结论 Tern.可能通过促进颗粒裂解肽mRNA的表达,增强机体CTL杀菌能力,达到抗结核休眠菌的作用。     

Patient factors contributing to the emergence of gentamicin-resistant Serratia marcescens.

One hundred forty nosocomial Serratia marcescens infections (including 76 cases of bacteremia) were identified by prospective surveillance from 1975 through 1977 and retrospective chart review from 1968 through 1974. Thirty-four cases (24 per cent) involved gentamicin-resistant strains. All gentamicin-resistant strains appeared after 1974. Ninety per cent of the patients had undergone surgery, and 88 per cent had received prior antimicrobial therapy. The emergence of gentamicin-resistant S. marcescens paralleled the increase in usage of gentamicin. Prior use of gentamicin for more than two days in an individual patient was a significant risk factor (P = 0.0002) for being infected with a Serratia that was gentamicin-resistant. Other factors which separated gentamicin-resistant Serratia infections from gentamicin-sensitive Serratia infections were (1) urinary site of infection (P = 0.0005), (2) urinary catheter (P = 0.002), (3) endotracheal tube or tracheotomy (P = 0.03) and (4) increasing duration of hospitalization (P less than 0.05). Thirty-three of 34 (97 per cent) patients with gentamicin-resistant strains had urinary catheters. Specific measures to control infection were effective in decreasing the incidence of infections caused by gentamicin-resistant Serratia.     

Two nosocomial outbreaks of sepsis caused by Serratia marcescens, which occurred in July 1999 and January 2002--Tokyo

Two nosocomial outbreaks of sepsis caused by Serratia marcescens, which occurred in Tokyo were the following cases. CASE A: In July 1999, 10 inpatients admitted to the third floor ward of the General Hospital A, developed sudden onset of high fever, coagulation disorders (disseminated intravascular coagulation), and acute renal failure, of which 5 died. Twenty-one strains of Serratia marcescens were isolated from the inpatient's blood and urine, nurse fingers and environmental samples from floor and cooling tower. Serratia infection was strongly suspected as the cause of sepsis. These cases were defined as "inpatients who developed fever 38 degrees C or more during July 26 to 29 and from whom S. marcescens was isolated by blood culture". Ten isolates were detected from the blood. In order to investigate the background of S. marcescens isolation in the hospital and to compare molecular and biochemical characteristics of S. marcescens, cultures were attempted from samples of other inpatients and staffs and hospital environment. Those were classified into 9 groups by various different typings: biotyping with Api Rapid 20; susceptibility typing of antimicrobial agents tested; pulsed-field gel electrophoresis (PFGE) typing of SpeI- or Xba I-restricted chromosome. All 10 isolates causing sepsis were found to be in the same group. CASE B: In January 2002, 24 inpatients, admitted to Neurosurgical Hospital B, developed sudden onset of high fever, of which 7 died. S. marcescens was isolated from a towel, environmental samples and inpatients. These cases were defined as "inpatients who developed fever of 38.5 degrees C and S. marcescens isolated by blood culture". Twelve strains were isolated from the blood samples in 12 cases. In order to investigate the background of S. marcescens isolation in the hospital, cultures were attempted from other inpatient's urine and environmental samples from medical tape, Tshake and a towel. These isolates were classified into 3 groups by the previous typings; biotyping with Api Rapid 20; susceptibility typing of antimicrobial agents tested; and PFGE typing. All 12 isolates in 12 cases were found to be in the same group. These cases of 2 nosocomial outbreaks of sepsis were defined as "in-patient who developed high fever and S. marcescens isolated by blood culture". However in both cases transmission routes of Serratia infection remain unknown by field investigation.     

Population genetic structure and competence as a vector for dengue type 2 virus of Aedes aegypti and Aedes albopictus from Madagascar.

Starch gel electrophoresis was used to assess the polymorphism of 7 isoenzymes in single mosquitoes (field-collected F0 or F1 generation) for Aedes albopictus (8 strains) from northern Madagascar. Mosquitoes of the F2 generation (3 strains of Aedes aegypti and 10 strains of Ae. albopictus) were tested for oral susceptibility to dengue type 2 virus. Aedes aegypti was less susceptible to viral infection than Ae. albopictus. The genetic differentiation was less high between Ae. albopictus populations collected in agglomerations connected by highly frequented roads, indicating that human ground transportation favors mosquito dispersal. These results have implications for the ecology, pattern of migration, and relative importance in epidemic transmission of dengue viruses between the 2 Aedes species.     

Study on new quinolone-resistant strains isolated from urine--annual changes of its frequency and the relation to drug volume

There is concerned that the new quinolone-resistant strains have increased along with its widespread usage. We analysed the annual changes in frequency of ofloxacin-resistant strains isolated from urine in the past four years at two different types of hospitals, department of urology in Sapporo Medical College and Muroran City Hospital, since the usage of these agents seem to be related to the annual changes of the frequencies. The results were summarised as follows: 1) In the two hospitals, drug volume of the new quinolones had been increased, in particular, the past six years from 1984. 2) The annual changes in frequency of ofloxacin-resistant Staphylococcus aureus have been increasing from 0 to 41.2 percent in Sapporo Medical College and 16.7 to 96.7 percent in Muroran City Hospital. The frequency of ofloxacin-resistant Pseudomonas aeruginosa also have been increasing from 24 to 66.7 percent in Sapporo Medical College and 37.5 to 81.8 percent in Muroran City Hospital. 3) The frequency of ofloxacin-resistant indole positive Proteus spp. and Serratia marcescens for four years at Sapporo Medical College (indole positive Proteus spp.: 0-1.8 percent, Serratia marcescens: 10-43 percent) was very different from that at Muroran City Hospital (indole positive Proteus spp.: 65-82 percent, Serratia marcescens: 71-100 percent). The difference seems to be caused by the hospital acquired infection.     

Clinical characterization of blaIMP positive gram-negative rods isolated cases]

We detected the metallo-beta-lactamase gene blaIMP positive strains of the gram-negative rods (GNR) isolated in Oita Medical University Hospital between 1993 and 1999 and studied the clinical characteristics of patients infected or colonized with blaIMP positive GNR. 25 strains (20 Pseudomonas aeruginosa and 5 Serratia marcescens) were detected and most of them were isolated from urinary samples after 1997. In the studies of antimicrobial susceptibility, some strains had sensitivity to aztreonum or imipenem although most of the strains showed multidrug resistance. When blaIMP positive GNR were isolated from patients, these strains were thought to have caused infection in 88% of the patients. About half of the patients were over 65 years old and had malignant diseases. Most of the patients had inserted urinary tract catheters, intratracheal tube or intravernous catheters. It was suggested that the insertion of the catheters were related to infection of blaIMP positive GNRs. Two patients were not treated with any antibiotics before the isolation of blaIMP positive GNRs although more than half of the patients were administered carbapenems and cephems. Most of strains were isolated in the same department and showed the same genotype by pulsed field gel electrophoresis.     

Potential vectors of Rift Valley fever virus in the Mediterranean region

We evaluated the ability of three mosquito species (Aedes caspius, Aedes detritus, Culex pipiens), collected in southern France and Tunisia, and of different laboratory-established colonies (Aedes aegypti, Aedes albopictus, Aedes vexans, Anopheles gambiae, Culex pipiens, Culex quinquefasciatus) to disseminate two strains of Rift Valley fever virus (RVFV), the virulent ZH548 and the avirulent Clone 13. After feeding on an infectious blood meal at 10(8.5) plaque-forming units/mL, females were maintained at 30 degrees C for 14 days. Surviving females were tested for the presence of virus on head squashes. Disseminated infection rate corresponds to the number of females with disseminated infection among surviving females. Among field-collected mosquitoes, Cx. pipiens was the most susceptible species with disseminated infection rates ranging from 3.9% to 9.1% for French strains and up to 14.7% for Tunisian strains. Among laboratory-established colonies, Ae. aegypti from Tahiti exhibited the highest disseminated infection rates: 90% when infected with ZH548 and 72.6% with Clone 13. The presence of competent Cx. pipiens in southern France and Tunisia indicates the potential for RVFV epizootics to occur if the virus was introduced into countries of the Mediterranean basin.     

Spondylodiscitis associated with recurrent Serratia bacteremia due to a transjugular intrahepatic portosystemic shunt (TIPS): a case report.

We report a case of spondylodiscitis caused by multiresistant Serratia marcescens in a cirrhotic patient who had several Serratia bacteremias after the placement of a transjugular intrahepatic portosystemic shunt (TIPS) device. We concluded that an endovascular stent that can not be removed makes management of recurrent bacteremia difficult. Furthermore, back pain due to bacteremia is indicative of spondylodiscitis. Serratia marcescens can be an aggressive pathogen, causing spinal infection.     

Size alters susceptibility of vectors to dengue virus infection and dissemination

The size of arthropod vectors may affect their ability to transmit pathogens. Here we test the hypothesis that body size alters the susceptibility of Aedes aegypti and Aedes albopictus mosquitoes to dengue virus (DENV) infection and subsequent dissemination throughout the body of the mosquito. After feeding on blood containing known quantities of virus, smaller-sized females were significantly more likely to become infected and to disseminate virus than larger individuals. The effects of size were stronger for Ae. aegypti and independent of rearing conditions. Ae. albopictus was more susceptible to DENV infection and had higher virus titer in the body than Ae. aegypti, yet infected Ae. aegypti disseminated DENV more readily than infected Ae. albopictus. These results are consistent with the concept that Ae. aegypti is a more competent vector of DENV and emphasize the importance of body size in determining adult infection parameters.     

IMP-1 type metalo-beta-lactamase producing Serratia marcescens strains isolated from blood culture between 1991 to 2000

Infection caused by metallo beta-lactamase (MBL)-producing bacteria have been increasingly reported in Japan in recent years. We investigated the prevalence, clinical backgrounds and molecular epidemiology of MBL-producing Serratia marcescens strains isolated from blood cultures at our university hospital between 1991 and 2000. Forty-three S. marcescens strains were isolated in the period, and the incidence reached about 2% total bacteria detection in 1998 and 1999. There were 13 ceftazidime-resistant strains (31%), and five of them produced MBL. MICs of imipenem (IPM) were 4-16 mg/ml, of which one strain was susceptible to IPM. Of the five MBL-producers, four were obtained from a tertiary emergency ward, the underlying diseases being either serious trauma or cerebrovascular diseases. The other was isolated from a patient who underwent kidney transplantation. S. marcescens was no longer isolated from patients after administration of aztreonam (AZT), implying that AZT was clinically effective. When analyzed genetically using the AP-PCR technique, the patterns were identical among strains isolated in 1996, 1997 and 1998 and those isolated in 1999 and 2000 respectively, suggesting that the same strains may have inhabited the hospital wards for prolonged periods. Countermeasures against nosocomial infection was undertaken thereafter, resulting in reduction of isolation frequency. It cannot be excluded that the resistant bacteria spread as the amount of carbapenem consumption increased. Prudent use of antibiotics is mandatory to prevent further dissemination of such MBL producers.     

Variation in susceptibility to oral infection with dengue viruses among geographic strains of Aedes aegypti

The comparative susceptibility of 13 geographic strains of Aedes aegypti to oral infection with dengue viruses was studied by feeding the mosquitoes on a virus-erythrocyte-sugar suspension. Significant variation in susceptibility to four dengue serotypes was observed among the geographic strains tested. Mosquito strains which were more susceptible to one serotype were also more susceptible to the other serotypes, suggesting that the factors controlling susceptibility were the same for all types. The amount of virus required to infect mosquitoes orally varied inversely with the susceptibility of the geographic strain. Thresholds of infection were not the same for dengue types 1, 2, 3 and 4. There was no apparent difference in infectivity between prototype and recently isolated strains of dengue types 1 and 3. Crossing experimentibility as the resistant parent. No difference was observed between resistant and susceptible mosquito strains in the rate or the amount of viral replication after infection by the parenteral route, or in their ability to transmit dengue 2 virus after infection by the oral route.