一株产低温碱性蛋白酶海洋细菌Pseudoalteromonas flavipulchra HH407的筛选与生长特性

对从连云港海域筛选的一株产低温碱性蛋白酶的海洋适冷菌HH407进行鉴定和生长特性研究结果表明,该菌为革兰氏阴性杆菌,好氧,端生鞭毛,无芽孢及荚膜,大小为0.4～0.7μm×1.0-1.8μm,在酪蛋白培养基的菌落特征为椭圆金黄色、光滑、半透明、四周隆起.根据其生理生化性质、16S rDNA 同源性和系统进化树分析初步鉴定该菌属于 Pseudoalteromonas flavipulchra.该菌的生长温度范围为0～40℃,最适生长温度为20℃;pH范围为5.5～11.0,最适生长pH值为8.5;NaCl质量浓度范围为0.5～13 g/dL,最适生长NaCl质量浓度为4 g/dL,无NaCl时不能生长.该菌能较好利用胰蛋白胨、酵母膏和蛋白胨等蛋白质物质,对糖的利用较差.该菌株所产蛋白酶在pH 10.0和35℃时表现最高酶活.     

产过氧化氢酶菌株培养条件的优化

通过对溶壁微球菌培养基优化,确定最佳发酵培养基,并对发酵工艺条件进行了初步的优化.最适碳源是麦芽糖,最适氮源是蛋白胨和酵母膏,最近无机盐是MnSO4;该菌株产酶的最佳培养基配方为:60 g麦芽糖,13 g蛋白胨,8 g酵母膏,1 g MnSO4,5 g NaCl,2 g NaH2 PO4·2H2O,2 g K2HPO4·3H2O,0.5 g MgSO4·7H2O,0.5 g CaCl2,1 000 mL水.从该菌株发酵过程曲线发现,该菌株大量产酶期在12-14 h.摇瓶发酵最适初始pH值为7.0,250 mL三角瓶装液量为25mL,接种体积分数为5%,培养温度为37℃.     

一株沼泽红假单胞菌的鉴定及产类胡萝卜素的研究

作者通过革兰氏染色、鞭毛染色、扫描电镜对一株光合细茵进行了形态学特征观察,并通过平板培养、液体培养特征、生理生化试验来了解其培养特征和生理生化特征;同时还通过双光束紫外一可见光谱仪分析了其活体细胞和细胞色素的光谱吸收行为.研究了其产类胡萝卜素的培养基和培养条件,并通过高效液相色谱仪对类胡萝卜素成分进行了分析.结果表明,此菌株为革兰氏阴性、有极生鞭毛,细胞为杆形,有集束生长的习惯,不对称出芽分裂,细胞大小为(0.6～2.5)μm×(0.6～5.0)μm;单茵落在固体平板培养基上呈淡黄色,表面光滑湿润,边缘整齐;在液体培养基上的培养物为深红色;对氨基苯甲酸、酵母浸出物对菌株的生长有显著刺激作用;菌体细胞色素含有类胡萝卜素和叶绿素a.根据这些形态学和生理生化特征,初步确定该菌株为沼泽红假单胞菌(Rhod0PsPdomonas palustris).此茵在培养基组成为(g/L):KHz PO4 0.6,K2 HPO4 0.9,醋酸钠2.0,氯化铵1.5,酵母浸膏0.5,NaCl 0.5,硫酸镁0.3,氯化钙0.2.在28℃、1 500 lx,pH6.5～8.5条件下培养能够快速生长繁殖.通过HPLC分析表明:类胡萝卜素提取液中成分至少有10种,其中5种是主要成分.     

一株腐乳生产菌的筛选、鉴定及其生理特征

以家制腐乳毛坯为对象,筛选出一株腐乳生产菌。这株菌产生以中性和碱性蛋白酶为主的蛋白酶系,酶活力高达1100 U/g干曲。根据菌株在PSA上的菌落特征、菌丝形态、产孢结构及其孢子形态等对毛霉鉴定的基础上,结合18S rDNA序列分析表明,该株毛霉菌为雅致放射毛霉（Actinomucor elegans WL3.006）。生产菌能在土豆、麸皮及豆腐坯等固体培养基上茂盛生长,对数生长期在12～36 h,最适生长温度为25～28℃,最适碳源为果糖,最适氮源为牛肉浸膏,适宜pH值为6～8;40 g/L NaCl对其生长有明显抑制;用卤水或石膏点制的豆腐白坯均适于此菌的生长。     

利多卡因对细菌培养影响的实验研究

[目的]探讨利多卡因对细菌培养结果的影响,为临床关节假体周围感染(periprosthetic joint infection,PJI)提高细菌培养阳性率提供理论依据。[方法]制备8种标准菌株,包括金黄色葡萄球菌、表皮葡萄球菌、人葡萄球菌、大肠埃希菌、肺炎克雷伯菌、鲍曼不动杆菌、化脓链球菌和白色念珠菌的菌悬液(3×10~6CFU/ml),模拟临床PJI的关节滑液。各标准菌株的菌悬液分为两组,利多卡因组为2%利多卡因100μl+菌悬液600μl,盐水组为0.45%无菌生理盐水100μl+菌悬液600μl,混匀1 min后,分别接种于5个琼脂平板培养基上进行24 h定量培养,通过菌落计数比较两组培养基上的细菌生长情况。[结果] 24 h定量细菌培养结果表明,利多卡因组金黄色葡萄球菌、表皮葡萄球菌、人葡萄球菌、大肠杆菌、肺炎克雷伯杆菌、鲍曼不动杆菌、白色念珠菌菌株的菌落计数显著低于盐水组,差异均有统计学意义(P0.05),而两组间化脓链球菌菌株的菌落计数差异无统计学意义(P0.05)。[结论] 2%利多卡因注射液对临床中常见的PJI病原菌具有很强的抗菌作用。因此,在关节液取样前使用2%利多卡因注射液进行局部浸润麻醉可能影响细菌培养结果。     

HCV NS4A与钙离子信号调节亲环素配体相互作用位点的确定

目的 明确丙型肝炎病毒非结构蛋白4A(HCV NS4A)与钙离子信号调节亲环素配体(CAML)相互结合的具体位点.方法 构建CAML不同剪接体及缺失突变体的酵母表达载体,转化入酵母Y187菌株,分别与转化了HCV NS4A的酵母AH109菌株进行配合,在含X-α-gal的酵母四缺培养基上培养观察.结果 转化了CAML不同剪接体及CAML缺失突变体pGADT7/CAML-1-211,pGADT7/CAML-1-57的酵母Y187菌株与转化了HCV NS4A的酵母AH109菌株配合后在四缺培养基上均有菌落生长,并且菌落呈现蓝色.转化了CAML缺失突变体pGADT7/CAML-58-211,pGADT7/CAML-234-296的酵母Y187菌株与转化了HCV NS4A的酵母AH109菌株配合后在四缺培养基上未见菌落生长.结论 HCV NS4A与CAML的相互结合位点位于CAML的N-末端1～57位氨基酸残基,CAML不同剪接体与HCV NS4A均可以相互结合.     

快速筛选耐酸性α-淀粉酶生产菌株的平板透明圈法

以可溶性淀粉为底物配制固体培养基,然后低温处理(4℃)平板培养物,产淀粉酶的菌株其菌落周围的培养基由白色变成灰色且透明.该方法不具有破坏性,不需要预先接种菌落,能直接发现和分离分解利用淀粉的微生物.利用淀粉琼脂平板还能快速验证淀粉酶的耐酸性,根据在pH4.2和pH6.0的琼脂平板上透明圈的直径d与淀粉酶活力的对数lnA的关系,求出淀粉酶在pH4.2和pH6.0时的淀粉酶活力.pH4.2时比pH6.0时的比值大,说明该淀粉酶产生菌的耐酸性较强.     

牛肝菌生长与发酵主要影响因子的鉴别

采用Plackett-Burman设计法,分别研究了影响牛肝菌(Boletussp.)ACCC50328菌丝生长和发酵产糖的21个相关因素.结果表明,影响牛肝菌ACCC50328菌株菌丝生长速度的主要因子为葡萄糖、麦芽糖、酵母膏、KH2PO4、(NH4)2SO4、FeSO4、发酵温度、发酵时间及装液量;影响该菌株胞外多糖含量的主要因子为麦芽糖、酵母膏、(NH4)2SO4、CuSO4·5H2O、FeSO4和发酵时间.     

用油料作物加工副产物栽培灵芝研究(Ⅰ)——生产菌种的制备

灵芝菌的子实体作为一种药用植物闻名于中国和日本。本文研究了用油料作物加工副产品作为栽培灵芝培养基主要成分的情况。研究结果表明,这些副产品含有足以保证灵芝菌旺盛生长的全部营养成分。发现培养基的结构及相关的氧浓度是菌丝体生长的重要参数。制定了菌丝生长的最佳培养基。     

耐高渗酵母产赤藓糖醇的影响因素

球拟酵母OS-194是一株单产赤藓糖醇的耐高渗酵母,该菌株高产赤藓糖醇的最佳培养基配方为葡萄糖10g/dL,酵母膏0.5g/dL,尿素0.1g/dL.最适培养条件是在摇瓶转速150r/min的条件下于35℃培养4d.在上述培养条件下，该菌株赤藓糖醇的耗糖转化率高达29.6%.磷是限制OS-194菌株高产赤藓糖醇的主要因素,当培养液中的磷质量浓度低于31.5mg/L时，赤藓糖醇的产量最高;随着磷质量浓度的升高，该菌株赤藓糖醇的产量降低,而酒精的产量和生物量却有明显升高.同时,OS-194菌株还能利用果糖、蔗糖和D-甘露糖产赤藓糖醇.     

In vitro studies on bone resorption in neonatal mouse calvariae using a modified dissection technique giving four samples of bone from each calvaria

Bone resorption in a modified bone culture system, based on incubation of small fragments from neonatal mouse calvarial bones, has been studied. Four bone fragments were dissected out from each mouse calvaria and were thereafter cultured in CMRL 1066 medium in plastic multiwell dishes. Bone resorption was assessed by 45Ca release from prelabeled bones. The rate of bone resorption in response to parathyroid hormone (PTH) was less in the anterior part of the calvaria compared to the posterior part. After removing the anterior region, four parietal bone fragments that showed identical basal and PTH-stimulated release of 45Ca could be dissected out from each mouse. Excretion of lactate dehydrogenase and beta-glucuronidase was the same in bones cultured submerged or on grids. Uptake of [3H]thymidine in bones cultured submerged was 54% of [3H]thymidine uptake in bones cultured on grids. Dose-response curves, established by using parietal bone fragments, showed that the sensitivity and the magnitude of the increase in 45Ca release seen after stimulation with PTH, prostaglandin E2, and 1 alpha-hydroxyvitamin D3 were the same for bones cultured submerged or on grids. The 45Ca release in response to stimulation with PTH, prostaglandin E2, and 1 alpha-OHD3 was the same in calvarial fragments cultured submerged and those previously obtained with calvarial halves cultured on grids. Thus, even though the rate of DNA synthesis was slower in bones cultured submerged, the rate and the magnitude of resorption were the same in bones cultured on grids or submerged. These data show that it is possible to perform studies on bone resorption with small fragments of neonatal mouse parietal bones.     

肝移植术后血行感染中产超广谱β内酰胺酶大肠埃希菌、肺炎克雷伯菌流行病学、细菌学及临床回顾性分析

目的探讨肝移植术后血行感染中产超广谱B内酰胺酶（ESBL）的大肠埃希菌、肺炎克雷伯菌的流行病学、细菌学及临床结果。方法选择1998年1月至2009年12月12年间本院肝移植患者资料。对其移植术后血行感染中产ESBL大肠埃希菌、肺炎克雷伯菌的情况进行回顾性分析。结果768例肝移植患者中,19（2．5％）例出现产ESBL革兰阴性杆菌血行感染,共分离到革兰阴性杆菌菌株共23株,其中大肠埃希菌、肺炎克雷伯菌最为常见。产气大肠杆菌、阴沟大肠杆菌、铜绿假单胞菌较少。产ESBL的大肠埃希菌、肺炎克雷伯菌对碳青霉烯类敏感率高达100％,但产ESBL大肠埃希菌对左旋氧氟沙星、头孢曲松、妥布霉素等多种抗生素出现耐药。产ESBL肺炎克雷伯菌则对头孢曲松出现完全耐药。肝移植术后产ESBL大肠埃希菌、肺炎克雷伯菌血行感染患者与非产ESBL大肠埃希菌、肺炎克雷伯菌血行感染患者之间的15d、30d、1年死亡率差异无统计学意义。结论肝移植术后血行感染中产ESBL大肠埃希菌、肺炎克雷伯菌对多种抗生素耐药,对碳青霉烯类敏感：产ESBL大肠埃希菌、肺炎克雷伯菌血行感染并未明显增加患者死亡率。     

A serological Survey of Brucella spp., Salmonella spp., Toxoplasma gondii and Trichinella spp. in Iberian Fattening Pigs Reared in Free‐Range Systems

Zoonotic agents such as Brucella spp., Salmonella spp., Toxoplasma gondii and Trichinella spp., all considered high‐risk zoonotic pathogens by the European Food Safety Agency (EFSA), may cause no symptoms of infection in free‐range pigs yet still have a significant public health impact. A serological survey was therefore performed to determine the history of occurrence of these pathogens in such pigs in southern Spain. A total of 709 serum samples were collected at abattoir from pigs from 79 farms and analysed for specific antibodies against the above pathogens using commercially available ELISA kits. Encysted Trichinella spp. larvae were also sought following the artificial digestion method of diaphragm pillar muscle. The results showed Salmonella spp. to be widely distributed among the sampled herds [73.42%, 95% confidence interval (CI₉₅) 65.6–81.78] and Toxoplasma gondii to be present in over half (58.23%, CI₉₅ 47.33–69.07). The seroprevalence of Brucella spp. was very low (3.8%, CI₉₅ 0.18–7.42), and antibodies against Trichinella spp. were not detected. No encysted Trichinella spp. larvae were microscopically detected.     

利用高通量宏基因组测序技术检测血液病患者感染性病原体的横断面研究

目的利用高通量宏基因组测序技术对血液病患者感染性病原体进行横断面调查，为此类感染性疾病高危患者的快速诊断及早期针对性用药提供依据。 方法回顾性分析2019年1月至6月华中科技大学同济医学院附属同济医院血液科血液病患者外周血临床标本高通量宏基因组分析数据及患者临床特征，并归纳血液病患者的感染病原体种类特征。 结果共纳入41例患者资料，检出病原体阳性患者24例，其中细菌感染以假单胞菌属感染比例最高（4/24、16.67%），其次为不动杆菌属（2/24、8.33%）、克雷伯菌属(2/24、8.33%）、葡萄球菌属（2/24、8.33%）和黄单胞杆菌属（2/24、8.33%）；而奈瑟菌属、米勒菌属、幽门螺杆菌以及结核分枝杆菌则较罕见。共有6例患者检出混合型感染，其中以病毒和细菌混合型感染最为多见（4/24、16.67%）。真菌感染的病原菌种类较细菌性病原体更为分散，白色念珠菌、青霉菌、肺孢子菌和根霉菌检出1例（占0.04%），无明显聚集性；病原体相对丰度方面，83.33%（5/6）真菌病原体阳性患者的检出序列片段数偏少（< 100），仅1例检出高负荷真菌感染（曲霉菌序列片段数为2 836）。病毒类病原体的检测结果显示，病毒筛查谱限于已知的常见DNA病毒；从测序结果看，24例检测阳性患者中巨细胞病毒、EB病毒和单纯疱疹病毒1型为检出率最高的病毒，均占20.83%（5/24）；较真菌，病毒种类存在一定聚集性，其相对负荷亦较高（平均序列片段数> 200）；而单纯疱疹病毒2型和指环病毒属病毒检出率相对较低，分别占4.17%（1/24）和8.33%（2/24）。 结论本研究创新性地利用高通量宏基因组测序技术检测血液病患者感染性疾病的病原体谱系；利用高通量宏基因组测序技术可快速锁定病原体并予以针对性抗菌药物治疗具有重要的临床价值。     

Carboxy terminus of secreted phosphoprotein‐24 kDa (spp24) is essential for full inhibition of BMP‐2 activity

Secreted phosphoprotein‐24 kDa (spp24) is a bone morphogenetic protein (BMP)‐binding protein isolated from bone. It exists in a number of size forms and is hypothesized to function as a BMP latency protein and/or a “slow release” mechanism for BMPs involved in bone turnover and repair. We have examined the hypothesis that proteolytic modification of the C‐terminus of spp24 affects its BMP‐2–binding properties and bioactivity in the BMP‐2–stimulated ectopic bone forming bioassay. Three different size forms of recombinant spp24 that correspond to predicted 18.1 kDa, 16.0 kDa, and 14.5 kDa proteolytic products were compared to full‐length (fl) spp24. One of these forms (spp18.1) we hypothesize to be the protein which Urist initially, but apparently inaccurately, called “BMP.” Only full‐length spp24 completely inhibited BMP‐2–induced bone formation. The 18.1 kDa truncated isoform of spp24 which we hypothesize to be Urist's protein did not. The inhibitory capacity of the proteins was correlated with their kinetic constants, assessed by surface plasmon resonance. At the highest, inhibitory, dose of spp24 and its derivatives, k_d (“stability”) best predicted the extent of ectopic bone formation whereas at the lowest dose, which was not inhibitory, k_a (“recognition”) best predicted the extent of ectopic bone formation. We conclude that proteolytic processing of spp24 affects the interaction of this protein with BMP‐2 and this affects the function of the protein. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:1200–1207, 2010     

Sensitivity of Enterobacteriaceae strains to the third generation cephalosporins

The aim of this study was to evaluate the in vitro activity of cefotaxime, ceftriaxone, ceftazidime, ceftibuten and aztreonam against the clinical isolates of Enterobacteriaceae. MICROORGANISMS: A total of 973 enterobacteria isolated from clinical material (urine and faeces) were tested. Strains were collected during 1996-1998 period, in some centers of Eastern Romania. SENSITIVITY STUDY: MICs were performed using the agar dilution method following standardized techniques (NCCLS, 1994). Stock solutions of antibacterial agents were prepared from their respective powder forms, in concentrations ranging from 0.03 to 128 mg/ml. Our results show a good activity of the tested antimicrobials on Escherichia coli, Shigella spp and Klebsiella spp tested, and reveal a particular behaviour for Salmonella spp, characterized by a heterogenous susceptibility. In conclusion, cefotaxime, ceftriaxone, ceftazidime, ceftibuten and aztreonam were active at low concentrations against tested isolates. Against Salmonella spp, these agents were less active: percent of resistant strains by beta-lactamase production was significant.     

质谱技术快速鉴定临床分离丝状真菌的应用

目的探讨质谱（MALDI-TOF MS）在临床分离的丝状真菌快速鉴定中的应用。 方法收集首都医科大学宣武医院2017年1月至2019年1月经形态学鉴定为丝状真菌的菌株，分别采用双甲酸夹心法和甲酸乙腈萃取法提取真菌蛋白并行MALDI-TOF MS鉴定，并与聚合酶链反应（PCR）内转录间隔区（ITS）基因序列测定结果比较；分析丝状真菌菌种分布和标本来源。 结果共收集临床分离的丝状真菌90株，其中曲霉菌属70株（77.78%），青霉菌属6株（6.67%），链格孢霉5株（5.56%），镰刀菌属4株（4.44%），赛多孢菌2株（2.22%），米根霉、根毛霉和哈茨木霉各1株（1.11%）；其中曲霉菌以烟曲霉47株（52.22%）、黄曲霉13株（14.44%）和黑曲霉4株（4.44%）为主。标本主要来源于呼吸道标本（90.0%）（包括痰标本和肺泡灌洗液），其次为角膜和耳道分泌物（3.33%），痰标本中分离率最多的为曲霉菌（59株、65.56%）。MALDI-TOF MS鉴定到种水平82株，与基因测序结果一致率为91.11%（82/90），鉴定准确率显著高于形态学鉴定结果（68.89%，62/90），差异有统计学意义（χ² = 12.02、P = 0.01）；质谱鉴定丝状真菌中的曲霉菌、镰刀菌、赛多孢菌和链格孢霉时准确率高（98.57%～100%）；双甲酸夹心法和甲酸乙腈萃取法前处理的质谱鉴定丝状真菌到种水平的比率分别为91.11%（82/90）和90.00%（81/90），鉴定曲霉菌种水平分别为98.57%（69/70）和97.14%（68/70），但鉴定准确率差异无统计学意义（χ² = 0.34、P = 0.95）；两种前处理方法均可100%鉴定烟曲霉、黄曲霉、赛多孢菌和链格孢霉到种水平，其中89.36%（42/47）烟曲霉和76.92%（10/13）黄曲霉鉴定得分≥ 2.0分。 结论MALDI-TOF MS技术鉴定丝状真菌快速准确，双甲酸夹心法前处理操作更简便，适合实验室常规检测，可用于实验室常规鉴定曲霉菌等常见丝状真菌。     

Concurrent pulmonary Aspergillus and non-Aspergillus mold infections in allogeneic hematopoetic cell transplant recipients

Patients with hematologic malignancies and recipients of hematopoietic cell transplantation (HCT) are at high risk for invasive mold infections (IMIs). However, risk factors and clinical manifestations are similar between Aspergillus spp. and non-Aspergillus spp. IMIs. Herein, we describe three HCT recipients who had probable invasive pulmonary Aspergillus and non-Aspergillus co-infections. Antifungal agents were changed to voriconazole in two cases where, ultimately, non-Aspergillus molds were diagnosed after these patients died. Our cases highlight the need for better fungal diagnostics in immunocompromised patients. Clinicians should be aware that Aspergillus spp. and non-Aspergillus spp. IMIs can occur concurrently.     

Sepsis and Its Etiology Among Hospitalized Children Less Than 1 Year of Age With Intestinal Failure on Parenteral Nutrition

Infections accompany intestinal failure (IF) more commonly in children than in adults, with reported incidences of 2% to 29%. Appropriate care of the central venous catheter is the most important factor preventing infections; but in addition, bacteria translocate from the dysmotile gut as a possible source of septicemia. The aim of this retrospective analysis was to investigate the rate and the epidemiologic profile of septicemia in the patient group at greatest risk, namely, children less than 1 year of age with IF on parenteral nutrition (PN). Among 63 children less than 1 year of age who were included over a 2-year period, 55% were boys. The overall median age at the start of PN was 0.3 years, with a mean duration of 80 days. Some 68% of patients had at least one episode of septicemia, experiencing a mean of 1.5 episodes (range, 1-12). Also, 19% of children displayed polymicrobial bloodstream infections. The most common Gram-positive pathogens were Staphylococcus spp and Enterococcus spp; the Gram-negative pathogens were Klebsiella spp followed by Enterobacter spp and E. coli.Infants less than 1 year of age with IF >28 days experienced a high (68%) rate of sepsis. There was no difference in the incidence of catheter-related blood stream infection according to the primary underlying diagnosis. The most common pathogens were Staphylococcus spp and Enterococcus spp, similar to etiologies of sepsis among children in intensive care units.     

Influence of premature exposure of implants on early crestal bone loss: an experimental study in dogs.

OBJECTIVE: Several studies have reported on spontaneous early exposure of submerged implants, suggesting that exposed implants have greater bone loss than nonexposed implants. The purpose of this study was to compare the effects of implant-abutment connections and partial implant exposure on crestal bone loss around submerged implants. STUDY DESIGN: Bilateral, edentulated, flat alveolar ridges were created in the mandible of 6 mongrel dogs. After 3 months of healing, 2 fixtures were placed on each side of the mandible following a commonly accepted 2-stage surgical protocol. The fixtures on each side were randomly assigned to 1 of 2 procedures. In the first, a cover screw was connected to the fixture, and the incised gingiva was partially removed to expose the cover screw (partially exposed group). In the second, a healing abutment was connected to the fixture so that the coronal portion of the abutment remained exposed to the oral cavity (abutment-connected group). After 8 weeks, micro-computed tomography (micro-CT) at the implantation site was performed to measure the bone height in the peri-implant bone. Data were analyzed by Wilcoxon's signed rank test. RESULTS: The average bone height was greater for the abutment-connected fixture (9.8 +/- 0.5 mm) than for the partially exposed fixture (9.3 +/- 0.5 mm; P < .05). CONCLUSION: These results suggest that when implant exposure is detected, the placement of healing abutments may help limit bone loss around the submerged implants.