Homogeneous Enzymatic Assay for L-Cysteine with βC-S Lyase

We have developed a new enzymatic assay for determining L-cysteine concentration. The method involves the use of βC-S lyase from Streptococcus anginosus, which catalyzes the α,β-elimination of L-cysteine to hydrogen sulfide, pyruvate, and ammonia. The production of pyruvate is measured by D-lactate dehydrogenase and NADH. The decrease in NADH was proportional to the L-cysteine concentration up to 1.0 mM. When serum samples were used, within-day and day-to-day coefficient variations were below 4%. This method is simple, and can easily and reliably be used for accurate determination of L-cysteine concentration in serum or other samples.     

Cloning,Nucleotide Sequence,and Disruption of Streptococcus mutans Glutathione Reductase Gene (gor)

We cloned and sequenced the glutathione reductase gene (gor) of an oxygen-tolerant Streptococcus mutans, and constructed a gor-disruption mutant by homologous recombination. The gor gene consisted of 1,350 bp, coding for a protein of 450 amino acid residues. The deduced amino acid sequence of the S. mutans gor gene product showed extensive similarity with those of glutathione reductases from prokaryotes and eukaryotes. Although the mutant could grow aerobically, it showed no growth in the presence of 2 mM diamide, a thiol-specific oxidant. In contrast, growth of the wild-type strain was not significantly inhibited by 2 mM diamide, and glutathione reductase activity was increased 2.2-fold under these conditions. In addition, the level of glutathione reductase activity in the wild-type strain was increased 3.6-fold upon exposure to air, and the elevated level of the enzyme was retained throughout the aerobic growth. Thus, glutathione reductase may be important in protection of S. mutans against oxidative stress.     

环介导恒温扩增法检测肺炎链球菌的研究

目的 建立环介导恒温扩增(LAMP)检测肺炎链球菌的方法.方法 用LAMP技术扩增肺炎链球菌菌株,并应用50例临床标本采用传统培养法、PCR法、LAMP法进行检测,比较3种方法的检出率,同时检测方法特异性和灵敏度.结果 所测肺炎链球菌均获扩增产物,对其他非肺炎链球菌无交叉反应.LAMP检测灵敏度可达102 CFU/mL.50例临床标本使用LAMP法检出9例肺炎链球菌阳性(18.0％),使用传统培养法检出阳性4例(8.0％).结论 LAMP法较传统培养检测方法特异性强、灵敏度高、操作方便、快速,适合临床标本的肺炎链球菌检测.     

纳米-TiO2对变异链球菌和白色假丝酵母菌抗菌机制的研究

目的 采用原子力显微镜对应用抗菌剂纳米Ag-Ti02作用后的口腔两种常见致病菌的分子形貌进行观测,为研究其抑菌机制提供有力的直观影像科学依据和可靠、直观的实验方法.方法 选择两种菌种:白色假丝酵母菌、变形链球菌,采用液体稀释法将纳米Ag-TiO2与两种菌相互作用,分别使用光学显微镜、原子力显微镜观察两种菌的细胞微观形态变化.结果 抗菌剂与两种菌作用后,细菌形态均有不同程度的改变,甚至是死亡.结论 原子力显微镜能直观地显示白色假丝酵母菌,变形链球菌的分子结构,通过本实验在研究纳米Ag-TiO2抗菌剂对白色假丝酵母菌,变形链球菌的抑菌机理形态学改变方面做了进一步的完善.     

肺炎链球菌粘附和毒力因子A研究进展

肺炎链球菌（Streptococcus pneumoniae,SP）普遍定植于呼吸道,是人类重要的侵袭性病原菌之一,是社区获得性肺炎、中耳炎、脑膜炎、菌血症、鼻窦炎的主要病原菌。肺炎链球菌粘附和毒力因子A（pneumococcal adherence and virulence factor A,PavA）是肺炎链球菌早期感染和侵袭过程中关键的毒力因子。体外试验表明,缺失PavA的肺炎链球菌的突变株其粘附和侵入上皮细胞和内皮细胞的能力明显下降。作为一种保护性抗原,其诱导的细胞和体液免疫可以有效的抵抗肺炎链球菌的感染,是肺炎链球菌新一代疫苗的候选蛋白。但是,PavA在肺炎链球菌与人肺上皮细胞交互对话中作用机制的研究尚属空白,本文就肺炎链球菌粘附和毒力因子A得最新研究进展作一综述。     

Streptococcus pneumoniae capsule determines disease severity in experimental pneumococcal meningitis

Streptococcus pneumoniae bacteria can be characterized into over 90 serotypes according to the composition of their polysaccharide capsules. Some serotypes are common in nasopharyngeal carriage whereas others are associated with invasive disease, but when carriage serotypes do invade disease is often particularly severe. It is unknown whether disease severity is due directly to the capsule type or to other virulence factors. Here, we used a clinical pneumococcal isolate and its capsule-switch mutants to determine the effect of capsule, in isolation from the genetic background, on severity of meningitis in an infant rat model. We found that possession of a capsule was essential for causing meningitis. Serotype 6B caused significantly more mortality than 7F and this correlated with increased capsule thickness in the cerebrospinal fluid (CSF), a stronger inflammatory cytokine response in the CSF and ultimately more cortical brain damage. We conclude that capsule type has a direct effect on meningitis severity. This is an important consideration in the current era of vaccination targeting a subset of capsule types that causes serotype replacement.     

牛源无乳链球菌长尾噬菌体的特性

【目的】分离鉴定噬菌体,对其生物学特性进行研究,并筛选候选毒株为防控牛源无乳链球菌的感染提供依据。【方法】分别采用从牛奶或环境中分离、溶原菌诱导两种方法分离鉴定无乳链球菌噬菌体,利用双层琼脂平板法纯化。将新分离鉴定毒株与前期已分离鉴定的源自乳腺炎牛奶的无乳链球菌噬菌体JX01进行分析和比较,包括噬菌体透射电镜形态观察、对55株无乳链球菌和其他细菌的宿主谱鉴定、噬菌体基因Eco R I、Sal I、Xba I或Pst I的酶切图谱、最适MOI、吸附曲线和一步生长曲线、不同保存条件下的稳定性等。【结果】分离鉴定的3株噬菌体LYGO9、HZ04和p A11(诱导自牛源菌株HAJL2011070601)与JX01比对分析,结果显示,4株噬菌体均为长尾噬菌体;Eco R I、Sal I、Xba I、Pst I的酶切图谱分获4、3、3或2种带型,显示4株噬菌体为不同毒株;均特异性裂解牛源无乳链球菌,对42株牛源无乳链球菌的裂解率如下:LYGO9为28.6%(12/42)、p A11为31%(13/42)、HZ04为47.6%(20/42)、JX01为54.8%(23/42);同时,LYGO9与p A11、HZ04和JX01分别有共同宿主11、12和11株;HZ04与JX01有共同宿主18株,提示它们具有同源性。LYGO9感染宿主的潜伏期短,仅5 min,平均裂解量为30。分离株在SM液中4°C至少可保存1个月。【结论】分离鉴定的3株牛源无乳链球菌噬菌体均为长尾噬菌体,其中LYGO9潜伏期短、裂解量较大。     

无乳链球菌鱼源株10 kb基因序列对细菌致病力的影响

【目的】在前期比较基因组学分析中,我们发现中国无乳链球菌鱼源株GD201008-001基因组中有一段10 kb基因序列,内含11个未知功能的开放阅读框。为了研究该段基因序列与细菌的致病力的关系,本研究将这段基因进行了全段缺失。【方法】运用链球菌-大肠杆菌穿梭质粒p SET4s,构建了10 kb基因缺失株(Δ10 kb),并通过生物学性状的比较,细胞粘附试验,斑马鱼攻毒试验和缺失前后毒力相关基因转录水平的检测,评价该序列对无乳链球菌毒力的影响。【结果】经测序证明缺失株Δ10 kb构建成功,与亲本株GD201008-001相比较,缺失株Δ10 kb在细菌染色形态、对HEp-2细胞的粘附能力无明显差异,但在培养液中的生长速度略慢;缺失株Δ10 kb对斑马鱼的毒力明显增强,LD_(50)有极其显著的差异(P0.001);编码菌毛骨架蛋白2b的基因(PI-2b)和唾液酸酶基因(neul)在缺失株中的转录水平明显上升。【结论】该序列对无乳链球菌GD201008-001的毒力有显著的影响,可能调控某些毒力基因的转录表达,使细菌的毒力减弱。     

育龄妇女泌尿生殖道无乳链球菌感染情况与耐药性分析

目的探讨育龄妇女泌尿生殖道无乳链球菌感染情况与耐药性。方法选择3 000例在我院就诊的育龄期妇女,所有患者送检泌尿道或生殖道标本,进行无乳链球菌培养及药物敏感性试验,探讨育龄妇女泌尿生殖道无乳链球菌感染情况及耐药性。结果 3 000例育龄妇女中177例感染无乳链球菌,感染率5.90%,其中肾内科患者感染率最高,达到8.97%。2012至2015年育龄期妇女无乳链球菌感染率逐年增加。无乳链球菌对四环素、克林霉素、红霉素、左氧氟沙星耐药性较高,分别达到80.80%、60.45%、56.50%、45.20%。无乳链球菌对头孢曲松、头孢他啶、亚胺培南、万古霉素未产生耐药性。结论育龄妇女无乳链球菌感染率较高,呈逐年上升趋势,防治形势严峻,无乳链球菌对多种常用抗生素耐药率较高,应根据药敏结果合理使用抗生素。     

1型CRISPR位点间区序列分析在嗜热链球菌菌株分型中的应用

为了建立适用于嗜热链球菌菌株资源多样性调查的菌株分型方法,尝试将1型CRISPR位点间区序列分析用于嗜热链球菌的菌株分型,并与常用ERIC-PCR指纹图谱方法进行了比较。结果表明,1型CRISPR位点间区序列分析可以把30株从三个不同样品中分离的嗜热链球菌分成三种差异明显的类型:不同类型菌株之间没有相同的间区序列;而同一类型菌株之间,间区序列的组成和排列则基本一致,并且上述分型的结果与用ERIC-PCR指纹图谱技术获得的结果完全一致。因此,1型CRISPR位点间区序列分析是嗜热链球菌分型鉴定的可靠方法,并适用于大量菌株的分型鉴定和多样性调查。