生物制品微生物质量控制与快速微生物检测的研究进展

微生物作为生物制品质量控制关键的安全性指标,是工艺控制和产品放行的关键参数。经典的微生物检查耗时长,无法实时反馈产品现状和工艺控制情况,会对短货架期或需要快速放行生物制品的放行周期产生不利影响。探索快速微生物检测方法(rapid microbiological method,RMM)替代经典方法可解决上述问题,且符合过程分析技术理念。基于微生物培养的RMM通过分析培养液的理化特性变化来判断是否存在微生物,如BacT/ALERT^®系统等。基于活细胞识别的RMM多采用固相细胞计数和流式细胞仪等进行。基于分子水平的RMM通过分析微生物的特征分子来判定是否存在微生物。美国食品药品监督管理局、美国药典和欧洲药典等均提出了RMM理念,第一款RMM Pallchek在2004年已用于制药行业。从2010年版起,中国药典也纳入了微生物检验替代方法指导原则。本文对生物制品微生物质量控制与快速微生物检测方法进行综述。     

基于16S rDNA序列测序的川楝子肝毒性机制研究

目的 通过观察川楝子对大鼠肠道菌群的影响,初步探讨川楝子肝毒性机制。方法 以大鼠为研究对象,川楝子组灌胃川楝子溶液,连续给予21 d,末次给药后收集粪便样本,抽提粪便DNA,于Illumina MiSeq分析平台进行16S rDNA测序。结果 川楝子显著提高血清谷草转氨酶(aspartate aminotransferase,AST)、谷丙转氨酶(alanine aminotransferase,ALT)含量(P<0.05),降低总胆固醇(total cholesterol,CHO)、总蛋白(total protein,TP)含量(P<0.05)。菌群多样性分析和差异分析结果显示,川楝子降低大鼠肠道菌群多样性,可升高肠道厚壁菌门、放线菌门、杆菌、丹毒丝菌、乳杆菌属丰度;可显著降低梭菌、拟杆菌、瘤胃球菌属丰度。关联性分析显示,ALT和AST含量与双歧杆菌属、瘤胃球菌、粪球菌、脱硫弧菌、罗氏菌、梭菌、拟杆菌丰度显著相关;CHO、TP与普雷沃菌、密螺旋体、气球菌丰度显著相关。结论 川楝子能明显降低肠道菌群的多样性,其肝脏毒性机制可能与增加Allobaculum和丹毒丝菌的表达有关。     

用反向斑点杂交法检测结核杆菌对链霉素和乙胺丁醇的耐药性

目的建立结核分支杆菌对链霉素（SM）和乙胺丁醇（EMB）耐药基因突变的快速检测方法。方法根据结核分支杆菌标准株H37Rv序列,自行设计针对rpsL和embB基因常见耐药突变的系列寡核苷酸探针,制成膜芯片,并对64例结核分支杆菌临床株的基因突变情况进行检测。结果在34株SM耐药菌中,有25株被检出在rpsL基因分析部位发生突变,检出率为73．5％（25／34）,其中23株（67．6％）为第43位密码子AAG→AGG突变,2株（5．9％）为第88位密码子AAG→AGG突变;在32株EMB耐药菌中,有19株在embB基因分析部位发生突变,检出率为59．4％（19／32）,其中12株（37．5％）为ATG→GTG突变;6株（18．8％）为ATG→ATA突变;1株（3．1％）为ATG-＊CTG突变。膜芯片检出的突变与基因测序结果一致。结论用膜芯片检测结核分支杆菌对链霉素和乙胺丁醇的耐药性,具有较高的特异性和敏感性,可作为常规药敏方法的补充,用于指导开展早期、有效的临床化疗。     

高尿酸血症与肠道菌群的相关性

最新研究数据表明,我国人群中高尿酸血症患病率已高达13%,且有不断上升及年轻化趋势。尿酸是人体嘌呤代谢的终产物,嘌呤是核酸的氧化分解代谢产物。体内产生的嘌呤在肝脏中再次氧化为(2,6,8-三氧嘌呤),即尿酸,其中2/3尿酸经肾脏随尿液排出体外,1/3通过粪便和汗液排出。近年大量研究表明,肠道菌群的变化与高尿酸血症乃至痛风的发病密切相关,本文就高尿酸血症的发生与肠道菌群的相互关系,及目前研究进展进行综述,进一步探讨尿酸在肠道的代谢机制,旨在为降低血尿酸及治疗尿酸相关疾病提供新的思路。     

淋球菌对五种抗生素的耐药性连续六年监测结果分析

目的了解6年来广州地区淋球菌对5种抗生素的耐药趋势及产青霉素酶淋球菌(PPNG)和高水平耐四环素淋球菌(TRNG)的流行状况。方法用琼脂稀释法测定最低抑菌浓度(MICs)以及用纸片碘量法检测β内酰胺酶。结果719株淋球菌检出PPNG94株(131%)、TRNG119株(166%),6年来,PPNG、TRNG流行率及环丙沙星耐药率,经μ检验,P<001,差异有显著意义。头孢曲松、壮观霉素未发现耐药菌株,壮观霉素抗菌活性最强。结论壮观霉素在临床上可以作为治疗淋病的首选药物,具有良好疗效。     

Nonlinear relations between house dust mite allergen levels and mite sensitization in farm and nonfarm children

BACKGROUND: Low sensitization rates to common allergens have been observed in farm children, which might be due to high exposure to microbial agents. It is not known how microbial agents modify the association between specific allergen exposure and sensitization. OBJECTIVE: To examine the relations between house dust mite allergen exposure and mite sensitization in farm and nonfarm children and to assess the effects of microbial agents levels on this association. METHODS: Major mite allergens of Dermatophagoides pteronyssinus (Der p 1) and Dermatophagoides farinae (Der f 1), endotoxin, beta(1,3)-glucans and fungal extracellular polysaccharides were measured in mattress dust of 402 children participating in a cross-sectional study in five European countries. Mite allergen (Der p 1 + Der f 1) levels were divided into tertiles with cut-offs 1.4 and 10.4 microg/g. Sensitization was assessed by measurement of allergen-specific immunoglobulin E against house dust mite. RESULTS: Prevalence ratios of mite sensitization for medium and high when compared with low mite allergen levels were 3.1 [1.7-5.7] and 1.4 [0.7-2.8] respectively. Highest mite sensitization rates at intermediate exposure levels were consistently observed across country (except for Sweden) and in both farm and nonfarm children. The shape of the dose-response curve was similar for above and below median mattress microbial agent levels, but the 'sensitization peak' appeared to be lower for above median levels. CONCLUSIONS: Our data suggest a bell-shaped dose-response relationship between mite allergen exposure and sensitization to mite allergens. In populations with high microbial agent levels and low sensitization rates, the curve is shifted down.     

三乌胶及三乌胶丸的微生物检查方法验证

目的建立三乌胶及三乌胶丸微生物限度检查方法。方法通过接种代表性的阳性菌株,采用常规法及稀释法进行方法学验证。结果三乌胶丸常规法接种5株阳性试验菌株回收率均高于70%,三乌胶常规法对金黄色葡萄球菌、大肠埃希菌、白色念珠菌、黑曲霉菌回收率高于70%,三乌胶及三乌胶丸由于剂型不同,所采用的检查方法也不同。结论三乌胶丸细菌数、霉菌数及控制菌均可采用常规法进行检验,三乌胶细菌数需采用稀释法进行检验,霉菌数及控制菌可采用常规法进行检验。     

建立具有药物代谢酶CYP2C9活性的微生物模型

 目的研究短刺小克银汉霉AS 3.910体外模拟人体细胞色素P450(CYP)2C9的能力,建立具有CYP2C9活性的微生物模型。方法选用3种人体CYP2C9代谢的药物格列本脲、双氯芬酸和吲哚美辛为底物,利用液相色谱-质谱联用技术检测药物代谢产物的种类和转化率。结果通过调节转化培养基的种类和初始pH,使转化系统在较高底物浓度下具有良好的转化效果,格列本脲、双氯芬酸和吲哚美辛总转化率分别为90%,100%和83%,而且形成的主要转化产物与人体CYP2C9产生的主要代谢产物相同。结论短刺小克银汉霉AS 3.910具有CYP2C9代谢酶活性,是研究人体CYP2C9药物代谢适宜的体外模型。     

Effects of Jian Pi Qu Shi Formula on intestinal bacterial flora in patients with idiopathic membranous nephropathy: A prospective randomized controlled trial

BackgroundThe incidence of idiopathic membranous nephropathy (IMN) has recently increased remarkably. Immune dysfunction caused by disordered intestinal flora might be an important factor affecting IMN. The Jian Pi Qu Shi Formula (JPQSF) shows promise in treating IMN. Here, we sequenced 16S rRNA genes to compare intestinal flora between patients with IMN and healthy persons. We also conducted a randomized controlled clinical trial to further compare the intestinal flora of patients with IMN treated with traditional Chinese medicine (TCM) and western medicine (WM).MethodsAmong 40 patients with IMN treated at Department of Nephrology in Xiyuan Hospital, Chinese Academy of Traditional Chinese Medicine between July 2016 and December 2018, we compared 30 of them with 10 healthy persons (controls). The IMN group was randomly assigned to receive JPQSF (TCM) or immunosuppressant WM therapy in (n = 15 per group) for 6 months. Intestinal microbiota diversity was analyzed using alpha diversity and beta diversity. Intestinal flora that significantly differed between the groups was analyzed using MetaStat. The effects and safety of the therapies were determined based on the values for plasma albumin, 24-h urine protein excretion, serum creatinine, urea nitrogen, estimate glomerular filtration rate (eGFR), complete blood count, and liver enzymes. All data were statistically analyzed using Statistical Package for the Social Sciences (SPSS) 20.0 statistical software.ResultsBaseline characteristics did not significantly differ between the IMN and healthy groups, or the TCM and WM groups. After six months of treatment, 24-h urinary protein significantly declined in the TCM and WM groups (before and after treatment: 3.24 ± 1.74 vs. 1.73 ± 1.85 g, P < 0.05 and 3.94 ± 1.05 vs. 1.91 ± 1.18 g, P < 0.05, respectively). Plasma albumin was significantly increased in the TCM group (before vs. after treatment: 32.44 ± 9.04 vs. 39.99 ± 7.03 g/L, P < 0.05), but did not significantly change in the WM group (31.55 ± 4.23 vs. 34.83 ± 9.14 g/L, P > 0.05). Values for urea nitrogen, serum creatinine, and eGFR did not significantly change in either group. The alpha diversity index for intestinal flora differed between the IMN and healthy groups, and the TCM and WM groups. Comparisons of multiple samples (beta diversity) revealed differences in intestinal flora between the IMN and healthy groups, and the TCM and WM groups. The Metastat analysis findings showed that the main genera that differed between the IMN group before treatment and the healthy group were Christensenellaceae_R-7_group, Bifidobacterium (77), Dorea, Escherichia-Shigella, Parabacteroides, Bifidobacterium, and Coprococcus_3. After TCM therapy, the main differential genera were Butyricimonas, Bacteroides, Alistipes, and Lachnospira, and after WM therapy, these were Ruminococcus_2, Lachnospiraceae_ND3007_group, Lachnospira, Bifidobacterium, Alistipes, and [Eubacterium]_ventriosum_group.ConclusionPatients with IMN might have disordered intestinal flora, and JPQSF can regulate intestinal flora in patients with IMN.     

微酸性电解水实验室微生物杀灭效果研究

 目的 研究某微酸性电解水实验室微生物杀灭效果。方法 选取某次氯酸水发生器现制微酸性电解水,测定不同有效氯含量、不同作用时间其对细菌繁殖体、芽孢以及真菌的杀灭效果。结果 该微酸性电解水有效氯含量为34.3~118 mg/L,有机干扰物牛血清白蛋白浓度为3.0%和0.3%时,分别作用1.0、3.0、5.0、10.0 min,对金黄色葡萄球菌、大肠埃希菌、铜绿假单胞菌的平均杀灭对数值均>5.00,对白假丝酵母菌的平均杀灭对数值均>4.00;有效氯含量为101~118 mg/L,牛血清白蛋白浓度为0.3%时,分别作用5.0、10.0、30.0 min,对枯草杆菌黑色变种芽孢的平均杀灭对数值均>5.00。结论 在特定实验条件下,该微酸性电解水对细菌的繁殖体、芽孢以及真菌均达到消毒剂消毒合格标准。