有氧条件下Klebsiella pneumoniae发酵生产1,3-丙二醇的研究

探讨了有氧条件下利用Klebsiella pneumoniae发酵生产1,3-丙二醇的可能性,研究了通气量、初始底物浓度、pH和3-羟基丙醛等因素对发酵过程的影响.当空气通量为0.25vvm时,1,3-丙二醇的得率和生产强度与厌氧时相近.有氧条件下,当初始甘油质量浓度大于50g/L时,发酵中后期出现3-羟基丙醛的长期积累,甘油不能消耗完全.控制pH为7.75～8.00可以促进3-羟基丙醛的转化使甘油完全消耗.     

肺炎克雷伯菌黏附因子相关研究进展

肺炎克雷伯菌为机会性致病菌,是导致医院获得性尿道感染、肺炎、败血症和伤口感染的重要致病菌。黏附于宿主细胞表面是致病菌感染的第一步,也是关键的一步。本文就肺炎克雷伯菌黏附因子相关研究进展及其前景作一综述。     

鸡源肺炎克雷伯菌菌毛的分型

目的对临床分离的5株鸡源肺炎克雷伯菌进行菌毛类型的鉴定。方法首先利用传统的D-甘露糖血凝特性(MSHA/MRHA)试验对临床分离的5株鸡源肺炎克雷伯菌进行菌毛分型,同时以该菌Ⅰ型和Ⅲ型菌毛的主要结构亚单位(fimA/mrkA)基因序列为靶序列,分别设计一对引物,利用PCR扩增的方法对该5株菌进行菌毛分型。结果MSHA/MRHA方法鉴定其中的4株菌具有Ⅲ型菌毛,另外1株无法鉴别,而PCR方法鉴定5株菌均具有Ⅲ型菌毛。结论该5株鸡源肺炎克雷伯菌均具有Ⅲ型菌毛。MSHA/MRHA具有方便、快速的特点,但不敏感;PCR则具有快速、敏感、准确的优点。研究结果对于肺炎克雷伯菌的流行病学调查及该菌的临床诊断具有指导意义。     

The Acquisition of Colistin Resistance Is Associated to the Amplification of a Large Chromosomal Region in Klebsiella pneumoniae kp52145

The appearance of carbapenem-resistant Klebsiella pneumoniae has increased the use of colistin as a last-resort antibiotic for treating infections by this pathogen. A consequence of its use has been the spread of colistin-resistant strains, in several cases carrying colistin resistance genes. In addition, when susceptible strains are confronted with colistin during treatment, mutation is a major cause of the acquisition of resistance. To analyze the mechanisms of resistance that might be selected during colistin treatment, an experimental evolution assay for 30 days using as a model the clinical K. pneumoniae kp52145 isolate in the presence of increasing amounts of colistin was performed. All evolved populations presented a decreased susceptibility to colistin, without showing cross-resistance to antibiotics belonging to other structural families. We did not find any common mutation in the evolved mutants, neither in already known genes, previously known to be associated with the resistance phenotype, nor in new ones. The only common genetic change observed in the strains that evolved in the presence of colistin was the amplification of a 34 Kb sequence, homologous to a prophage (Enterobacteria phage Fels-2). Our data support that gene amplification can be a driving force in the acquisition of colistin resistance by K. pneumoniae.     

Prophylaxis and Treatment against Klebsiella pneumoniae: Current Insights on This Emerging Anti-Microbial Resistant Global Threat

Klebsiella pneumoniae (Kp) is an opportunistic pathogen and the leading cause of healthcare-associated infections, mostly affecting subjects with compromised immune systems or suffering from concurrent bacterial infections. However, the dramatic increase in hypervirulent strains and the emergence of new multidrug-resistant clones resulted in Kp occurrence among previously healthy people and in increased morbidity and mortality, including neonatal sepsis and death across low- and middle-income countries. As a consequence, carbapenem-resistant and extended spectrum β-lactamase-producing Kp have been prioritized as a critical anti-microbial resistance threat by the World Health Organization and this has renewed the interest of the scientific community in developing a vaccine as well as treatments alternative to the now ineffective antibiotics. Capsule polysaccharide is the most important virulence factor of Kp and plays major roles in the pathogenesis but its high variability (more than 100 different types have been reported) makes the identification of a universal treatment or prevention strategy very challenging. However, less variable virulence factors such as the O-Antigen, outer membrane proteins as fimbriae and siderophores might also be key players in the fight against Kp infections. Here, we review elements of the current status of the epidemiology and the molecular pathogenesis of Kp and explore specific bacterial antigens as potential targets for both prophylactic and therapeutic solutions.     

葡萄糖作辅助碳源对klebsiella pneumoniae发酵生产1,3-丙二醇的影响

在Klebsiella pneumoniae发酵甘油生产1,3 丙二醇的种子培养及发酵实验中,考察了葡萄糖作辅助碳源对菌体生长及1,3 丙二醇生成的影响. 结果表明,在种子培养期,以葡萄糖和甘油为混合碳源可缩短种子培养周期;在批次发酵和流加发酵中,葡萄糖作辅助碳源可使1,3 丙二醇产率及得率明显提高,但不同的葡萄糖加入方式对产率及得率促进的效果不同. 在初始发酵培养基中添加5 g/L葡萄糖、并在4 40 h进行葡萄糖与甘油混合液连续流加的条件下,1,3 丙二醇浓度、产率及得率较单一甘油为底物的流加发酵结果分别提高41.2 , 38.6 和8.3 .     

Evaluation of Chemical Changes in Laboratory-Induced Colistin-Resistant Klebsiella pneumoniae

This study evaluates the electrical potential and chemical alterations in laboratory-induced colistin-resistant Klebsiella pneumoniae, as compared to the susceptible strain using spectroscopic analyses. The minimal inhibitory concentration (MIC) of colistin, ζ-potential and chemical composition analysis of K. pneumoniae strains are determined. The results obtained for the K. pneumoniae^Col-R with induced high-level colistin resistance (MIC = 16.0 ± 0.0 mg/L) are compared with the K. pneumoniae^Col-S strain susceptible to colistin (MIC = 0.25 ± 0.0 mg/L). Fourier transform infrared (FTIR) and Raman spectroscopic studies revealed differences in bacterial cell wall structures and lipopolysaccharide (LPS) of K. pneumoniae^Col-R and K. pneumoniae^Col-S strains. In the beginning, we assumed that the obtained results could relate to a negative charge of the bacterial surface and different electrostatic interactions with cationic antibiotic molecules, reducing the affinity of colistin and leading to its lower penetration into K. pneumoniae^Col-R cell. However, no significant differences in the ζ-potential between the K. pneumoniae^Col-R and K. pneumoniae^Col-S strains are noticed. In conclusion, this mechanism is most probably associated with recognisable changes in the chemical composition of the K. pneumoniae^Col-R cell wall (especially in LPS) when compared to the susceptible strain.     

大肠埃希菌和肺炎克雷伯菌临床分离株的耐药性分析

目的分析临床分离的大肠埃希菌和肺炎克雷伯菌的分布情况及耐药性。方法收集吉林大学中日联谊医院临床标本中分离的221株大肠埃希菌和152株肺炎克雷伯菌,采用纸片扩散法检测抗菌素的敏感性;双纸片协同试验和纸片表型确证试验筛选并确证产超广谱β-内酰胺酶的菌株;按照美国国家临床实验室标准化委员会(NCCLS)2005年版的标准判断结果。结果大肠埃希菌和肺炎克雷伯菌对亚胺培南和美罗培南的敏感性最高,均达100%;对头孢他啶、哌拉西林/他唑巴坦和头孢西丁的敏感性也较高,均大于70%,而对氨苄西林的耐药性均大于90%。共检出产超广谱β-内酰胺酶的大肠埃希菌118株,检出率为53.4%;肺炎克雷伯菌21株,检出率为13.8%;产超广谱β-内酰胺酶的两种菌株与非产超广谱β-内酰胺酶的同种菌株相比,对抗菌素的耐药性均明显增加。结论大肠埃希菌和肺炎克雷伯菌仍是产超广谱的β-内酰胺酶的主要菌株,且对常用抗菌素产生了较高的耐药性。     

A Simple DNAzyme-Based Fluorescent Assay for Klebsiella pneumoniae

Pathogenic bacteria pose a serious threat to public health, and the rapid and cost-effective detection of such bacteria remains a major challenge. Herein, we present a DNAzyme-based fluorescent paper sensor for Klebsiella pneumoniae. The DNAzyme was generated by an in vitro selection technique to cleave a fluorogenic DNA–RNA chimeric substrate in the presence of K. pneumoniae. The DNAzyme was printed on a paper substrate in a 96-well format to serve as mix-and-read fluorescent assay that exhibits a limit of detection (LOD) 10⁵ CFUs mL⁻¹. Evaluated with 20 strains of clinical bacterial isolates, the DNAzyme produced the desired fluorescence signal with the samples of K. pneumoniae, regardless of their source or drug resistance. The assay is simple to use, rapid, inexpensive, and avoids the complex procedures of sample preparation and equipment. We believe that this DNAzyme-based fluorescent assay has potential for practical applications to identify K. pneumoniae.     

1,3-丙二醇分批发酵动力学模型

在分批发酵中，研究了Klebsiella pneumoniae的生长、底物甘油消耗及1,3-丙二醇的产生特性. 基于Logistic方程和Luedeking-Piret方程，得到了描述1,3-丙二醇分批发酵过程的动力学模型及模型参数，该组模型能很好地拟合发酵过程，并在初始甘油浓度变化较大的范围内表现出很好的适用性. 同时，所建立的模型也基本反映了Klebsiella pneumoniae分批发酵过程的动力学特征. 基于分批发酵动力学模型，提出了以甘油为单一碳源时的底物流加策略，通过与其他流加策略条件下的发酵对比实验表明，通过基于动力学模型的流加策略可获得更高的1,3-丙二醇浓度及生产强度.     

Rcs信号系统对克雷伯氏肺炎杆菌荚膜合成的影响

克雷伯氏肺炎杆菌是一种重要工业微生物,菌细胞表面具有荚膜结构,荚膜对于菌株的生理特性和工业应用具有重要的影响.Rcs磷酸化信号系统是一种对菌株荚膜合成具有调控作用的信号系统.利用Red同源重组技术,分别构建了基因rcsA,rcsB缺失的菌株,还构建了基因rcsA,rcsB单独过表达的菌株.这些菌株的生理特性的研究结果表...     

氧对Klebsiella pneumoniae产1,3-丙二醇代谢的影响

考察了氧对Klebsiella pneumoniae发酵产1,3-丙二醇(PDO)代谢的影响. 研究结果表明,在厌氧或供氧条件下,K. pneumoniae都能利用甘油产PDO. 起始甘油浓度20 g/L,发酵时间4 h,在充分供氧条件下,PDO产量仅为1.1 mmol/L;但在微量供氧条件下,PDO产量为16 mmol/L,是厌氧发酵时的1.28倍. 在微量供氧条件下,调控PDO合成的关键酶甘油脱氢酶、甘油脱水酶及1,3-丙二醇氧化还原酶的活性分别为7.28, 1.14, 0.52 U/mg,高于厌氧或充分供氧条件下的相应酶活性. 氧对细胞内NADH的合成也有影响,厌氧及微量供氧条件下菌体内NADH含量分别为3.78及3.72 μmol/g (DCW),高于充分供氧发酵时的0.85 μmol/g (DCW).     

重组甲酸脱氢酶对合成1,3-丙二醇的促进作用

在甘油厌氧发酵生产1,3-丙二醇的过程中,需要消耗还原当量NADH,NADH的有效供给决定了1,3-丙二醇的产量。本文从Candida boidinii基因组DNA中克隆了甲酸脱氢酶基因fdh,利用表达质粒pMAL TM-p2X-fdh转化到1,3-丙二醇生产菌 Klebsiella pneumoniae YMU2中,构建了具有NADH再生系统的重组菌Klebsiella pneumoniae F-1。在5 L发酵罐培养中,F-1合成1,3-丙二醇浓度和产率分别达到了78. 6 g·L-1 和1. 33 g·L-1·h-1,分别比YMU2提高了12. 5% 和41. 2%。根据F-1和YMU2菌株的主要代谢产物的生成情况比较了二者的代谢流分布。     

微胶囊固定化克雷伯杆菌生产1,3-丙二醇

引 言 1,3-丙二醇是合成新型聚酯材料--聚对苯二甲酸丙二酯(PTT)的单体.近几年,利用微生物法生产1,3-丙二醇已成为国内外研究的热点[1-2].     

Klebsiella pneumoniae合成1,3-丙二醇过程中的生长与催化耦联

引　言1,3 丙二醇 (1,3 propanediol ,简称 1,3 PD)是一种重要的化工原料 ,主要用于合成对苯二甲酸丙二醇酯 .目前 1,3 PD的生产主要采用化学法 ,但因环境污染、石油资源紧张等 ,使其进一步发展受到限制 .欧美等国家积极开展通过发酵法生产 1,3 PD的研究[1] .Dupont公司开发的由葡萄糖合成 1,3 PD ,进而合成对苯二甲酸丙二醇酯的工艺 ,被授予2 0 0 3年美国总统绿色化学奖 .本课题组根据K pneumoniae兼性厌氧的特点 ,开发了两阶段双底物发酵的生产工艺[2 ] (twophas es twosubstrates ,简称TPTS) .经过工艺的优化 ,1,3 PD的产量已超过…     

Hydrogen Sulfide and Substance P Levels in Patients with Escherichia coli and Klebsiella pneumoniae Bacteraemia

Hydrogen sulfide (H₂S) and substance P (SP) are known from animal models and in vitro studies as proinflammatory mediators. In this study, peripheral blood concentrations of H₂S and SP were measured in patients with Escherichia coli or Klebsiella pneumoniae bacteraemia. Fifty patients were recruited from general wards at Christchurch Hospital, during 2020–2021. Samples from age- and sex-matched healthy subjects previously recruited as controls for studies of cardiovascular disease were used as controls. The concentrations of H₂S were higher than controls on day 0, day 1, and day 2, and SP was higher than controls on all 4 days. The concentrations of H₂S were highest on day 0, whereas SP concentrations were higher on day 2 than other days. Interleukin-6 and C-reactive protein were significantly higher on day 0 and day 1, respectively. The concentrations of H₂S and SP did not differ between 15 non-septic (SIRS 0-1) and the 35 septic subjects (SIRS ≥ 2). Substance P concentrations were higher in subjects with abdominal infection than urinary tract infections on day 0 (p = 0.0002) and day 1 (p = 0.0091). In conclusion, the peak H₂S concentrations precede the SP peak in patients with Gram-negative bacteraemia, but this response varies with the site of infection.     

代谢副产物对Klebsiella pneumoniae生长及合成1,3-丙二醇的影响

研究了Klebsiella pneumoniae在厌氧摇瓶中的生长代谢特性和基质消耗情况,发现主要副产物乙醇是抑制菌体持续生长及1,3-丙二醇合成的主要因素,外源添加实验表明,8 g/L乙醇可使K. pneumoniae比生长速率、1,3-丙二醇比合成速率、最大菌体浓度及1,3-丙二醇终浓度分别下降21.6%, 22.1%, 59.6%及33.5%;指数生长期加入乙醇对菌体生长代谢的抑制作用更加明显. 其他代谢副产物乙酸、乳酸、2,3-丁二醇对K. pneumoniae生长代谢也有不同程度影响,乙酸浓度仅2 g/L即可对菌体生长产生抑制,乙酸浓度达到5 g/     

两段双底物发酵生产1,3–丙二醇

利用Klebsiella pneumoniae生长与催化耦合的特点，将好氧生长与厌氧转化两个过程耦合，开发了两段双底物发酵生产1,3-丙二醇的新工艺，并对其工艺特点进行了初步研究. 结果表明，采用两段双底物发酵工艺，发酵过程菌体浓度明显高于传统的厌氧转化工艺，OD650最大值达到9.37，发酵60 h, 1,3-丙二醇的浓度达到50.16 g/L，生成速率达0.836 g/(L×h)，比传统工艺提高了45%.     

醛脱氢酶基因敲除对克氏肺炎杆菌合成1，3-丙二醇的影响

利用Klebsiella pneumoniae厌氧发酵甘油生产1,3-丙二醇时，一部分甘油通过氧化代谢途径大量合成副产物乙醇，降低了1,3-丙二醇的得率.醛脱氢酶ALDH是乙醇合成途径的关键酶之一，其催化作用不仅消耗了大量甘油，还将还原型辅酶NADH氧化为NAD⁺，降低了同为NADH依赖型的1,3-PD合成途径的效率.本文以醛脱氢酶ALDH为改造目标，以K.pneumoniae为宿主，通过同源重组技术在K.pneumoniae M5aL的ALDH基因中成功地插入了四环素抗性基因，经抗性筛选和基因水平鉴定，得到两株ALDH基因敲除的重组菌0623-1hb及0623-1hc.本文研究了这两株重组菌的生长代谢特性，结果表明两株重组菌的ALDH酶活基本检测不到，菌体生长受到明显抑制，乙醇合成浓度比出发菌株K.pneumoniae M5aL降低了43%～53%，1,3-PD合成浓度及摩尔得率分别提高了27%～42%和19%～24%.     

3-羟丙醛对Klebsiella pneumoniae发酵产 1,3-丙二醇的影响及其调控

中间产物3-羟丙醛在发酵液中的积累对Klebsiella pneumoniae细胞生长及1,3-丙二醇的合成有显著的抑制作用,而调节发酵的起始甘油浓度及控制发酵pH值可调控发酵液中3-羟丙醛的积累.当起始甘油质量浓度分别为20、30、50、70g/L的批式发酵中,发酵液中3-羟丙醛的积累的高峰分别为4.31、6.87、11.48及13.49mmol/L,当起始甘油质量浓度大于50g/L时,3-羟丙醛在到达积累高峰后不能被菌体有效转化,在发酵后期维持较高浓度,抑制了细胞生长及1,3-丙二醇的合成,发酵不能继续进行.控制发酵pH值为7.75～8.0可促进发酵液堆积的3-羟丙醛被迅速转化.在流加发酵中起始甘油质量浓度采用30g/L,发酵pH值控制为7.75条件下,发酵32 h,1,3-丙二醇质量浓度可达37.16g/L,1,3-丙二醇的生产强度和质量得率分别达到1.16g/(L·h)和52.66%.