测定绿脓杆菌冷休克率方法的研究

本研究基于某些革兰氏阴性杆菌当处于不同生长阶段时对冷敏感性表现不同的现象,建立了绿脓杆菌冷休克率的测定技术,可用于测定群体绿脓杆菌的生长情况。应用该技术,实验群体绿脓杆菌生长各期的肉汤培养物,观察冷休克率变化规律,对照群体细菌生长曲线可见两者有一定程度吻合,同时也有差别,后者主要在于细菌冷休克率峰值出现早于细菌活菌数峰值,而当细菌达到稳定期时,其数量居高不下而后才逐渐降低,此时,冷休克率的降低则迅速而明显,可清楚地表明细菌生长状态,较早地预示群体细菌生长势头。对实验动物及少量烧伤病人创面标本检测的结果表明冷休克率测定有助于检出生长的优势菌群,此点将在机会致病菌的微生物病原性确定方面有一定的应用前景。     

细菌L型的厌氧诱导和培养

厌氧条件下以羧卡青霉素诱导金黄色葡萄球菌、大肠杆菌和蜡样芽胞杆菌形成Ｌ型,观察细菌Ｌ型在厌氧条件下的形成、形态、生长及时渗透压的敏感性等特性。结果表明：蜡样芽胞杆菌在厌氧条件下不能形成Ｌ型或其Ｌ型在厌氧条件下亦不能返祖。金黄色葡萄球菌和大肠杆菌在厌氧条件下虽能诱生Ｌ型,但形成丝状体的构成Ｌ型菌落难以传代培养,厌氧培养未见Ｌ型圆球体和典型Ｌ型油煎蛋样菌落。金黄色葡萄球菌Ｌ型在含１％～１０％ＮａＣｌ的Ｌ型培养基上可生长形成Ｌ型菌落或非菌落形式存在的Ｌ型巨形体;大肠杆菌和蜡样芽胞杆菌的Ｌ型在含２％～６％ＮａＣｌ的Ｌ型培养基上可生长形成Ｌ型菌落或非菌落形式存在的Ｌ型巨形体。涂片染色或返祖试验证实细菌Ｌ型在含０．５％ＮａＣｌ的Ｌ型培养基或常规细菌学培养基上亦可生存。非菌落性Ｌ型巨形体和丝形体是细菌Ｌ型在琼脂培养基上广泛的存在形式。     

枯草杆菌BS_(224)菌体外拮抗试验

为了探讨枯草杆菌BS_(224)菌对烧伤创面感染的几种常见致病菌的拮抗效应,我们分别将BS_(224)菌与金黄色葡萄球菌、绿脓杆菌及大肠杆菌的对数生长期的菌,按等量比例混合于肉汤管内,经28℃不同时间的培养和菌落计数后进行统计分析。结果显示BS_(224)菌对金黄色葡萄球菌、绿脓杆菌及大肠杆菌在体外均有极显著拮抗作用。     

影响多粘菌素B对绿脓杆菌杀菌效力的主要环境因素的研究

本文通过改变温度,水活度,气体条件和营养含量等影响绿脓杆菌生长的主要环境因素,测定多粘菌素B对绿脓杆菌的最小杀菌浓度(MBC)。结果表明环境因素导致或显著影响绿脓杆菌对抗生素的生态耐受性。实验表明多粘菌素B对绿脓杆菌的杀菌效力,除药物对细菌特有的药理学作用外,还取决于细菌的生长环境。结合冷休克率试验表明,环境影响细菌群体处于分裂状态的菌数。若分裂状态菌数下降表明生长速度减慢。提示了多粘菌素B对绿脓杆菌的效力指数,定量分析可以作为其综合效力作用的表现。以同步培养法确定在单个细胞周期中的抗生素敏感阶段。同时以冷休克率试验资料证明细菌处于分裂状态和幼龄期是其敏感阶段。初步阐述了生长速度缓慢与药物的生态耐受性密切相关。     

抑菌生的研究总结报告

本文报告一种由枯草杆菌制成的生态制剂,并命名为抑菌生(Subtilobiogen)。该制剂对创、烧伤感染有治疗作用。经过安全试验、急性毒性试验、Ames试验和微核试验证明,该制剂是一种无害、无毒和有致突变作用的活菌制剂。抑菌生有膏剂、乳剂及粉剂3种剂型。抑菌生在试管和体内对金黄色葡萄球菌、绿脓杆菌和大肠杆菌均具有抑菌作用。临床观察证明,将抑菌生喷洒在创面上,对浅Ⅱ°度、深Ⅱ°度及混合型烧伤感染均具有明显疗效。实验组(181例)与对照组(174例)相比较,在统计学上具有显著性差异。抑菌生的作用机制,经过初步试验证明,与营养争夺和占位性保护有关,因为枯草杆菌的生长速度超过金黄色葡萄球菌、绿脓杆菌和大肠杆菌的生长速度。     

对双歧杆菌DM9227株的实验研究——Ⅰ.双歧杆菌DM9227株的试管内生物拮抗作用的研究

本文对由健康婴儿分离的双歧杆菌DM9227株进行了试管内的生物拮抗试验。将双歧杆菌DM9227株分别与金黄色葡萄球菌、产毒性大肠杆菌及侵袭性大肠杆菌以一定的比例等量混合接种于PYG液体培养基中进行厌氧培养。试验证明双歧杆菌DM9227株能明显抑制上述3种细菌的生长繁殖,显示出较强的生物拮抗作用。拮抗机制可能与双歧杆菌DM9227株能产生一定量的醋酸和乳酸,降低培养基的pH,从而抑制该3种菌的生长有关。     

正常菌群对常见致病菌拮抗作用的研究

生物拮抗作用是微生物种群关系研究的一个侧面,实验中我们使用从皮肤上分离的常住菌——痤疮丙酸杆菌(A14-1)和表皮葡萄球菌(F65)对常见致病菌如金黄色葡萄球菌(C189)、绿脓杆菌(C8514)和埃希氏大肠杆菌(C1356)的体外拮抗试验表明,它们具有明显的拮抗作用,拮抗作用一般从48小时开始,72小时明显。而皮肤常住菌间无拮抗作用,相反它们还有协同作用,这对于皮肤的自净以及维持皮肤的微生态平衡具有重要的作用。此外,实验中还进行了乳杆菌和双歧杆菌等正常菌群分离株与上述常见致病菌的体外拮抗试验,结果表明也     

快速检测金黄色葡萄球菌的一种氮源的酶解优化策略

以金黄色葡萄球菌为指示菌,采用复合蛋白酶酶解法优化调整一种牛肉浸粉的成分,为开发快速检测病原菌的培养基配方提供参考。建立液体摇瓶培养OD_(600)值和平板培养法菌落统计相结合的评价方法,筛选蛋白酶的组合和水解控制条件,考察和分析不同酶解产物对金黄色葡萄球菌的生长作用影响的一些规律。在底物浓度12%,自制复合蛋白酶加酶量200 U/g,pH 7.5,50℃,酶解1 h条件下,比较几种不同细菌生长曲线发现,酶解产物对金黄色葡萄球菌有明显的促进生长作用,且对冷冻负伤的该菌有显著性的复原能力。最优酶解条件下的牛肉浸粉,可以使得其中的多肽和游离氨基酸成分分布达一定的状态,这个状态下多肽分子量集中在小于500 Da,其中分子量小于170 Da高达31.79%,其中的支链氨基酸含量较高时,更有利于促进金黄色葡萄球菌的生长。     

氯霉素滴眼液控制菌检验方法及研究

氯霉素 (C11H12 CL2 N2 O5)是化学合成类抗菌药之 [1] ,是高效、低毒的抗菌剂。几十年来 ,一直在临床上应用。由于它对绿脓杆菌、金黄色葡萄球菌有很强的抑制作用。笔者曾用常规法 [2 ]做控制菌检查 ,但绿脓杆菌、金黄色葡萄球菌阳性对照未生长。为此进行了如下检验方法的研究。1　实验材料菌种 :绿脓杆菌 [CMC(B) 1 0 1 0 4 ]、金黄色葡萄菌 [CMCC(B) 2 60 0 3]培养基 :胆盐乳糖增菌液、营养肉汤、十六烷三甲基溴化铵琼脂、甘露醇高盐琼脂 ,均由中国药品生物制品检定所提供。稀释液 :0 .9%无菌氯化钠溶液 (自配 )。药物 :氯霉素滴眼…     

土壤拮抗放线菌的分离筛选及其抑菌物质特性的研究

目的从土壤中筛选拮抗能力强且抑菌特性稳定的放线菌菌株。方法采用双层琼脂法筛选出4株拮抗放线菌菌株,然后采用杯碟法测这4株菌株发酵液提取物的抗菌谱、最小抑菌浓度、热稳定性和酸碱稳定性。结果 4株菌株发酵液提取物都能抑制金黄色葡萄球菌、大肠埃希菌、铜绿假单胞菌和白色念珠菌的生长。以金黄色葡萄球菌为指示菌测发酵液提取物的最小抑制浓度,6#和9#拮抗作用较强,发酵液提取物稀释0.125mg/ml仍有抑菌作用。6#菌株在100℃处理30min后仍有40%的抑菌活性。6#菌株发酵液提取物在碱性环境条件下比在酸性环境条件下稳定。结论 4株菌株中6#菌株发酵液提取物具有拮抗能力强、最小抑菌浓度低和在碱性条件下活性较稳定的特点。     

Detergent-induced cell aggregation in subpopulations of Pseudomonas aeruginosa as a preadaptive survival strategy

During growth of Pseudomonas aeruginosa strain PAO1 with the toxic detergent SDS, a part of the population actively formed macroscopic cell aggregates while the other part grew as freely suspended cells. The physiological function of aggregation for growth with SDS was investigated. Three mutants growing with SDS without aggregation were isolated: the spontaneous mutant strain N and two mutants with transposon insertions in the psl operon for exopolysaccharide synthesis. SDS-induced aggregation in strain N but not in a pslJ mutant was restored by complementation with two genes encoding diguanylate cyclases responsible for synthesis of cyclic-di-guanosine monophosphate (c-di-GMP). By expressing a c-di-GMP-specific phosphodiesterase SDS-induced aggregation of strain PAO1 was reduced. Upon exposure to SDS in the presence of the uncoupler carbonyl cyanide chlorophenylhydrazone, the aggregating strains had ca. 500-fold higher survival rates than the non-aggregating strains. Co-incubation experiments revealed that strain N could integrate into aggregates of strain PAO1 and thereby increase its survival rate more than 1000-fold. These results showed that SDS-induced aggregation involved c-di-GMP signalling with the psl operon as a possible target. Cell aggregation could serve as a pre-adaptive strategy ensuring survival and growth of P. aeruginosa populations in environments with multiple toxic chemicals.     

冷激对高温胁迫下番茄幼苗生长及花芽分化的影响

为了解苗期冷激锻炼对番茄幼苗生长和花芽分化的影响,试验采用人工气候箱模拟夏季设施高温环境,每天对番茄幼苗进行10 ℃、10 min的冷激锻炼,研究冷激处理对高温胁迫下番茄幼苗生长、叶片超微结构和花芽分化进程的影响,并观察定植后开花和坐果情况.结果表明：在4叶期经过冷激锻炼的番茄幼苗茎粗、壮苗指数分别比对照提高了7.2%和55.5%;经过冷激锻炼处理的番茄幼苗叶片中叶绿体和线粒体等细胞器形状及结构正常完整,一定程度上缓解了高温对番茄幼苗叶肉细胞超微结构的破坏;冷激锻炼显著提高了番茄幼苗早期花芽分化的分化进程,但随着苗龄的延长这种差异变得不显著.定植后经冷激处理的番茄幼苗第1、2穗果的坐果数和坐果率显著高于未经冷激处理.表明冷激锻炼不仅能够缓解高温对番茄幼苗细胞超微结构的伤害和生长的胁迫,还有利于早期花芽分化进程的提前,提高番茄坐果数和坐果率.     

The galactophilic lectin, LecA, contributes to biofilm development in Pseudomonas aeruginosa

LecA (PA-IL) is a cytotoxic lectin and adhesin produced by Pseudomonas aeruginosa which binds hydrophobic galactosides with high specificity and affinity. By using a lecA-egfp translation fusion and immunoblot analysis of the biofilm extracellular matrix, we show that lecA is expressed in biofilm-grown cells. In static biofilm assays on both polystyrene and stainless steel, biofilm depth and surface coverage was reduced by mutation of lecA and enhanced in the LecA-overproducing strain PAO-P47. Biofilm surface coverage by the parent strain, PAO-P47 but not the lecA mutant on steel coupons was also inhibited by growth in the presence of either isopropyl-beta-D-thiogalactoside (IPTG) or p-nitrophenyl-alpha-D-galactoside (NPG). Furthermore, mature wild-type biofilms formed in the absence of these hydrophobic galactosides could be dispersed by the addition of IPTG. In contrast, addition of p-nitrophenyl-alpha-L-fucose (NPF) which has a high affinity for the P. aeruginosa LecB (PA-IIL) lectin had no effect on biofilm formation or dispersal. Planktonic growth of P. aeruginosa PAO1 was unaffected by the presence of IPTG, NPG or NPF, nor was the strain able to utilize these sugars as carbon sources, suggesting that the observed effects on biofilm formation were due to the competitive inhibition of LecA-ligand binding. Similar results were also obtained for biofilms grown under dynamic flow conditions on steel coupons, suggesting that LecA contributes to P. aeruginosa biofilm architecture under different environmental conditions.     

Aprotic polar solvents that affect porcine brain tubulin aggregation in vitro induce aneuploidy in yeast cells growing at low temperatures

Seven aprotic polar solvents which had previously been shown to interfere with the aggregation in vitro of porcine brain tubulin have been examined for their ability to induce mitotic aneuploidy in Saccharomyces cerevisiae in relation to temperature during exposure. Induction of aneuploidy was in general considerably enhanced when incubation at 28 degrees C was interrupted by overnight storage at low temperature (cold shock). The optimum cold-shock temperatures for individual chemicals varied over a range of 0-16 degrees C. While storage at reduced temperature enhanced the effect of treatment at 28 degrees C, it was also shown that continuous incubation at reduced temperature could greatly enhance the induction of aneuploidy. Only 2 chemicals, 1-methyl-2-pyrrolidinone and gamma-valerolactone, required cold shock to yield a positive response. The other chemicals did not require cold shock for enhanced induction. The observation that the agents examined also interfere with in vitro tubulin aggregation suggests that there is a temperature component to the interaction of these agents with tubulin in vivo. This temperature component is unusual in that the most effective temperature range for aneuploidy induction can be well below the optimal growth temperature for the test organism.     

一株奇异变形杆菌对铜绿假单胞菌多细胞行为的抑制作用*

摘要目的：抗生素耐药性成为了全球性的健康问题。研究发现病原菌的多细胞行为在抗生素的耐药性中起着至关重要的作用 （尤其是生物膜）,因而通过抑制多细胞行为而控制耐药性成为当务之急。本文以奇异变形杆菌（Proteus Mirabilis ）为研究对象,考 察它的发酵滤液对一种机会致病菌———铜绿假单胞菌（ Pseudomonas aeruginose）多细胞行为的作用,以期得到一株多细胞行为抑 制菌：在不影响 P.aeruginosa 生长的前提下,抑制生物膜形成、EPS 产生以及定向丛集运动,解除保护,减缓扩散,为降低P.aeruginosa 耐药性,增强抗生素作用效果提供可能。方法：采用结晶紫生物膜测定法、蒽酮-硫酸法、平板检测法,探究P.aeruginosa 发酵滤 液对P.aeruginosa 生物膜、胞外多聚物、定向丛集运动和生长的影响。结果： P.aeruginosa 发酵滤液能显著抑制生物膜 量,在体积百分比浓度为1 %时,抑制率可达60.9 %。该菌的发酵滤液还能阻碍的定向丛集运动,减弱它的吸附和扩 散运动;同时,也减少了P.aeruginosa 胞外多聚物的产量,在滤液体积百分比浓度为1 %时,抑制率达到45.9%。更重要的是,固体 平板实验证明该发酵滤液对P.aeruginosa 的生长没有影响。结论： 在不影响病原菌生长的前提下,对病原菌的多细胞 行为有一定的控制作用。其发酵滤液中存在着抑制微生物膜、定向丛集运动等的成分,在治疗细菌感染性疾病和降低抗生素耐药 性方面有潜在应用价值。     

In situ growth rates and biofilm development of Pseudomonas aeruginosa populations in chronic lung infections

The growth dynamics of bacterial pathogens within infected hosts are a fundamental but poorly understood feature of most infections. We have focused on the in situ distribution and growth characteristics of two prevailing and transmissible Pseudomonas aeruginosa clones that have caused chronic lung infections in cystic fibrosis (CF) patients for more than 20 years. We used fluorescence in situ hybridization (FISH) directly on sputum specimens to examine the spatial distribution of the infecting P. aeruginosa cells. Mucoid variants were present in sputum as cell clusters surrounded by an extracellular matrix, whereas nonmucoid variants were present mainly as dispersed cells. To obtain estimates of the growth rates of P. aeruginosa in CF lungs, we used quantitative FISH to indirectly measure growth rates of bacteria in sputum samples (reflecting the in vivo lung conditions). The concentration of rRNA in bacteria isolated from sputa was measured and correlated with the rRNA contents of the same bacteria growing in vitro at defined rates. The results showed that most cells were actively growing with doubling times of between 100 and 200 min, with some growing even faster. Only a small stationary-phase subpopulation seemed to be present in sputa. This was found for both mucoid and nonmucoid variants despite their different organizations in sputum. The results suggest that the bacterial population may be confronted with selection forces that favor optimized growth activities. This scenario constitutes a new perspective on the adaptation and evolution of P. aeruginosa during chronic infections in CF patients in particular and on long-term infections in general.     

一株奇异变形杆菌对铜绿假单胞菌多细胞行为的抑制作用

目的：抗生素耐药性成为了全球性的健康问题。研究发现病原菌的多细胞行为在抗生素的耐药性中起着至关重要的作用（尤其是生物膜）,因而通过抑制多细胞行为而控制耐药性成为当务之急。本文以奇异变形杆菌（Proteus mirabilis）为研究对象,考察它的发酵滤液对一种机会致病菌——铜绿假单胞菌（Pseudomonas aeruginosa）多细胞行为的作用,以期得到一株多细胞行为抑制菌：在不影响Paerugiliosa生长的前提下,抑制生物膜形成、EPS产生以及定向丛集运动,解除保护,减缓扩散,为降低Paemgi—nosa耐药性,增强抗生素作用效果提供可能。方法：采用结晶紫生物膜测定法、蒽酮一硫酸法、平板检测法,探究Pmirabilis发酵滤液对Paemginosa生物膜、胞外多聚物、定向丛集运动和生长的影响。结果：Pmirabilis发酵滤液能显著抑制Paeruginosa生物膜量,在体积百分比浓度为1％时,抑制率可达60．9％。该菌的发酵滤液还能阻碍Paeruginosa的定向丛集运动,减弱它的吸附和扩散运动;同时,也减少了Pacrugillosa胞外多聚物的产量,在滤液体积百分比浓度为1％时,抑制率达到45．9％。更重要的是,固体平板实验证明该发酵滤液对P．aemginosa的生长没有影响。结论：Pmirabilis在不影响病原菌生长的前提下,对病原菌的多细胞行为有一定的控制作用。其发酵滤液中存在着抑制微生物膜、定向丛集运动等的成分,在治疗细菌感染性疾病和降低抗生素耐药性方面有潜在应用价值。     

Efficacy of silver-releasing rubber for the prevention of Pseudomonas aeruginosa biofilm formation in water

The aim of the present study was to evaluate the efficacy of Elastoguard silver-releasing rubber in preventing Pseudomonas aeruginosa biofilm formation in water. Biofilm formation by P. aeruginosa under various conditions in an in vitro model system was compared for silver-releasing and conventional rubber. Under most conditions tested, the numbers of sessile cells attached to silver-releasing rubber were considerably lower with reference to conventional rubber, although the effect diminished with increasing volumes. The release of silver also resulted in a decrease in planktonic cells. By exposing both materials simultaneously to conditions for biofilm growth, it became obvious that the antibiofilm effect was due to a reduction in the number of planktonic cells, rather than to contact-dependent killing of sessile cells. The data demonstrate that the use of silver-releasing rubber reduces P. aeruginosa biofilm in water and reduces the number of planktonic cells present in the surrounding solution.     

Characterization of nutrient-induced dispersion in Pseudomonas aeruginosa PAO1 biofilm

The processes associated with early events in biofilm formation have become a major research focus over the past several years. Events associated with dispersion of cells from late stage biofilms have, however, received little attention. We demonstrate here that dispersal of Pseudomonas aeruginosa PAO1 from biofilms is inducible by a sudden increase in carbon substrate availability. Most efficient at inducing dispersal were sudden increases in availability of succinate > glutamate > glucose that led to approximately 80% reductions in surface-associated biofilm biomass. Nutrient-induced biofilm dispersion was associated with increased expression of flagella (fliC) and correspondingly decreased expression of pilus (pilA) genes in dispersed cells. Changes in gene expression associated with dispersion of P. aeruginosa biofilms were studied by using DNA microarray technology. Results corroborated proteomic data that showed gene expression to be markedly different between biofilms and newly dispersed cells. Gene families that were upregulated in dispersed cells included those for flagellar and ribosomal proteins, kinases, and phage PF1. Within the biofilm, genes encoding a number of denitrification pathways and pilus biosynthesis were also upregulated. Interestingly, nutrient-induced dispersion was associated with an increase in the number of Ser/Thr-phosphorylated proteins within the newly dispersed cells, and inhibition of dephosphorylation reduced the extent of nutrient-induced dispersion. This study is the first to demonstrate that dispersal of P. aeruginosa from biofilms can be induced by the addition of simple carbon sources. This study is also the first to demonstrate that dispersal of P. aeruginosa correlates with a specific dispersal phenotype.     

SadB is required for the transition from reversible to irreversible attachment during biofilm formation by Pseudomonas aeruginosa PA14

Current models of biofilm formation by Pseudomonas aeruginosa propose that (i) planktonic cells become surface associated in a monolayer, (ii) surface-associated cells form microcolonies by clonal growth and/or aggregation, (iii) microcolonies transition to a mature biofilm comprised of exopolysaccharide-encased macrocolonies, and (iv) cells exit the mature biofilm and reenter the planktonic state. Here we report a new class of P. aeruginosa biofilm mutant that defines the transition from reversible to irreversible attachment and is thus required for monolayer formation. The transposon insertion carried by the sadB199 mutant was mapped to open reading frame PA5346 of P. aeruginosa PA14 and encodes a protein of unknown function. Complementation analysis and phage-mediated transduction demonstrated that the transposon insertion in PA5346 was the cause of the biofilm-defective phenotype. Examination of flow cell-grown biofilms showed that the sadB199 mutant could initiate surface attachment but failed to form microcolonies despite being proficient in both twitching and swimming motility. Closer examination of early attachment revealed an increased number of the sadB199 mutant cells arrested at reversible attachment, functionally defined as adherence via the cell pole. A positive correlation among biofilm formation, irreversible attachment, and SadB level was demonstrated, and furthermore, RpoN and FleR appear to negatively affect SadB levels. Fractionation studies showed that the SadB protein is localized to the cytoplasm, and with the use of GPS-linker scanning mutagenesis, the C-terminal portion of SadB was shown to be dispensable for function, whereas the two putative domains of unknown function and the linker region spanning these domains were required for function. We discuss the results presented here in the context of microbial development as it applies to biofilm formation.