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。     

绿脓杆菌生长状态的初步测定

本文对细菌生长状态的测定进行了初步的研究,建立了绿脓杆菌的分裂指数、冷休克率及透明晕环形式的检测技术。其中,分裂指数的测定能反映单一绿脓杆菌群体的生长状态;冷休克率的测定适用于有其他细菌同时存在的临床标本及实验动物标本的检验;而透明晕环形成的指标能够直观、迅速地通过形态学特征来比较菌群中不同种类细菌的生长状态。     

细菌药物耐受

细菌药物耐受(Drug tolerance)是指在没有发生耐药突变的情况下细菌耐受抗生素杀菌的能力,表现为细菌群体难以或不能被杀菌型药物清除。细菌药物耐受的调控机制包括群体异质性和压力应答两种途径。药物耐受性的本质是细菌通过调控或遗传突变的方式改变生理代谢状态,从而抵制药物引起的细胞死亡途径。比如,处于缓慢生长或生长停滞生理状态的细菌往往能够抵抗药物的杀菌作用。临床研究发现细菌药物耐受是导致持续性感染疾病迁延难愈、复发率高的病原学机制之一。同时,研究证明耐受性的形成是细菌耐药性(Drug resistance)产生的进化途径之一。因此,揭示细菌药物耐受的机制将有助于人们深入了解抗生素的杀菌机理,以及细菌耐药性形成的适应性进化机制,并为新型杀菌药物以及药物增效剂靶标的发现和抗生素合理使用策略的开发奠定理论基础。     

绿脓杆菌的快速鉴定和耐药谱分型的探讨

本文用绿脓杆菌12群“O”多价血清辅以乙酰胺酶试验对临床分离的118株绿脓杆菌作了快速鉴定,2小时即可获得满意结果,与生物学鉴定的符合率达100%。对10种抗生素的药敏试验结果表明,本菌对丁胺卡那霉素和多粘菌素 B 较敏感,敏感率分别为98.31%和100%。118株菌可用8种抗生素分成20种不同的耐药谱型(A—T),其中属耐药谱 D 型(对多粘菌素、丁胺卡那霉素、新霉素敏感)的菌株在分布上最多,占28.8%(34/118)。     

绿脓杆菌群集生长效应

本文通过群集的绿脓杆菌在高盐、酸性培养基上生长试验,抗金黄色葡萄球菌的拮抗试验及冷休克试验,证明了绿脓杆菌群集生长效应。主要表现为群集的绿脓杆菌可以在含10～20%NaCl 的培养基上生长;pH4环境中也可生长。同样条件下,对照几乎均为阴性。分散成单个细胞分布的绿脓杆菌对冷反应敏感,群集则有抗冷休克作用。实验条件下,密集的金黄色葡萄球菌生长对分散的绿脓杆菌有拮抗作用,群集则可抵抗这种作用而生长。讨论中初步分析了群集生长效应的机理和对细菌种群在自然生境中的稳定作用。     

测定绿脓杆菌分裂指数反映生长势头的初步研究

本文以细胞生物学理论为基础,细菌细胞发育周期形态和群体细菌分裂相数量的变化为依据,研究了测定绿脓杆菌DI方法。建立了数学模型,分析了生长过程中DI动态。结果表明,DI可较准确地反映细菌生长势头。实验采用同龄细菌选择和同步培养技术,免疫荧光染色,显微镜下直接观察分裂相细菌,并计算其占总菌数的比例。在对数期前DI已上升,开始后迅速达高峰,接近稳定期时已下降到较低水平。与生长速度比较,两者动态基本一致,但DI高峰较早出现77分钟。认为DI与生长速度关系密切,是与细菌生命活动物质交换的三流学说相伴随的一种特殊信息流。     

结核分枝杆菌GroEL1蛋白过表达的生理效应分析

为研究结核分枝杆菌热休克蛋白GroEL1在耻垢分枝杆菌中过表达所引发的生理效应,本实验采用透射电子显微镜观察细菌个体形态,并绘制细菌群体生长曲线;药敏试验检测重组耻垢分枝杆菌的药物耐受性;细菌攻毒BALB/c小鼠以检测细菌在体内的存活能力;通过病理切片观察细菌毒力。结果显示,GroEL过表达细菌菌体内核糖体数量显著增加,细菌生长分裂能力及群体适应能力提高,但对常用的一线、二线抗结核药物耐受性无影响。GroEL1过表达有助于延长细菌在宿主体内的生存周期,增强细菌毒力。结果提示,结核分枝杆菌GroEL1蛋白过表达会对细菌体内、体外生长产生明显影响。     

圈养猕猴志贺氏菌的分离鉴定及药敏分析

目的确定猕猴感染志贺氏菌的状况,寻找有效治疗措施。方法采用不同选择培养基对13份病猴粪便样品进行分离培养、细菌革兰氏染色、镜检,并对分离的疑似菌株进行细菌生化鉴定和分子鉴定,经小白鼠致病性实验后,再用纸片扩散法测定分离菌株对23种抗生素的敏感性。结果从13份猕猴粪便样品中共检出12株志贺氏菌,检出率为92.31%,其中痢疾志贺菌(A群)1株、福氏志贺菌(B群)10株、宋内氏志贺菌(D群)1株;致病性试验结果表明,12株菌均能在72h内致死小白鼠,并能回收到注射的菌株;药敏试验结果表明,本实验中分离到的志贺氏菌对头孢噻肟(86.49%)最敏感,对头孢三嗪(75.00%)、头孢他啶(66.67%)次之,对多粘菌素B、羧苄西林、苄唑西林素等抗生素耐药性强。结论初步确定猕猴感染志贺氏菌普遍存在,进而引起腹泻、痢疾的可能性较大,头孢噻肟等为最敏感药物。     

哈尔滨市112株绿脓杆菌的血清学分型和耐药性测定

对从临床分离的112株绿脓杆菌进行系统鉴定后,血清学分型表明:6、2和3型分别占32.14％、15.18％、15.18％,为主要流行型,共占总分离株的62.50％。耐药性测定结果为:对10种抗生素5耐以上者占69.6％。其中对多粘菌素、妥布霉素、丁胺卡那霉素三种抗生素最为敏感,敏感率分别为100％、70.6％、86.5％。     

小肠结肠炎耶尔森菌对多粘菌素B的压力应答

【背景】小肠结肠炎耶尔森菌(Yersinia enterocolitica)是重要的人畜共患食源性病原菌。由于其生存环境与传染性生活方式,小肠结肠炎耶尔森菌暴露在各种生存压力中,而胞膜压力应答能力对维持其环境耐受性和毒力发挥着重要作用。【目的】探究小肠结肠炎耶尔森菌在胞膜压力应答中的调节机制。【方法】通过使用多粘菌素B破坏小肠结肠炎耶尔森菌细胞膜的稳定性,并从生长能力、运动能力、生物被膜形成能力以及相关基因表达的变化探讨Rcs (Regulator of Capsule Synthesis)系统对多粘菌素B产生的胞膜压力的应答。【结果】多粘菌素B引起的细胞胞膜压力抑制了小肠结肠炎耶尔森菌的运动和生物被膜形成能力;而阻断Rcs信号途径后,小肠结肠炎耶尔森菌的运动和生物被膜形成能力有所恢复。对flhC、hmsS、hmsT等关键下游表型基因的表达水平的分析结果表明Rcs双组分系统对由多粘菌素B诱导的胞膜压力作出响应,通过感知胞膜胁迫向胞内传递信号,积极地调控细菌增强对抗菌肽的抗性。【结论】明确了Rcs双组分系统在响应多粘菌素B压力胁迫中的特异性调控作用,加深了对小肠结肠炎耶尔森菌环境应答机制的认识。     

In Vitro Effects of Carbenicillin Combined with Gentamicin or Polymyxin B Against Pseudomonas aeruginosa

Disodium carbenicillin and gentamicin sulfate have both shown promise in the treatment of infections caused by Pseudomonas aeruginosa. This study was designed to explore possible synergistic relationships among the new as well as the established antimicrobial agents used to treat such infections. With an agar dilution technique, minimum inhibitory concentrations of 27 strains of P. aeruginosa were determined in two-dimensional tests. Graphs of equal biological activity (isobolograms) demonstrated moderate synergistic effects of the carbenicillin-gentamicin combination over therapeutically feasible concentration ranges. In contrast, the combination of carbenicillin and polymyxin B showed only additive or slightly antagonistic effects. Tests of bacterial killing confirmed the presence of carbenicillin-gentamicin synergy in 3 of 6 strains of P. aeruginosa, but did not show true antagonism between carbenicillin and polymyxin B. Clinical trials of both drug combinations are advisable to determine whether therapeutic results can be improved, and whether the dosages of gentamicin or polymyxin B can thereby be reduced to lessen their toxic hazards.     

Susceptibility of Recent Isolates of Pseudomonas aeruginosa to Gentamicin, Polymyxin, and Five Penicillins, with Observations on the Pyocin and Immunotypes of the Strains

The susceptibilities of recently isolated strains of Pseudomonas aeruginosa to gentamicin, polymyxin B, carbenicillin, ampicillin, penicillin G, and two newer penicillins were tested with the inocula-replicating technique by using undiluted and 10(-3) dilutions of the cultures. With either inoculum, polymyxin B was the most active agent, and a comparison with previous data from this laboratory showed that the susceptibility of P. aeruginosa to this antibiotic had not changed over the past 20 years. Gentamicin was nearly as active as polymyxin, all but 2 of the 141 strains tested with the diluted inoculum being inhibited by 6.25 mug/ml or less. AB-2288, an agent resembling carbenicillin, was four times more active than carbenicillin or BLP-1654; the last two were equally active against the 10(-3) inoculum. A more marked inoculum effect was noted with the penicillin analogues tested, the increase in minimum inhibiting concentration with the undiluted culture being eight-fold for carbenicillin and at least 16-fold for AB-2288 and BLP-1654. Pyocin typing and serotyping failed to demonstrate any clearly predominating types.     

Influence of Serum and Calcium on the Bactericidal Activity of Gentamicin and Carbenicillin on Pseudomonas aeruginosa

Because calcium was found to be antagonistic in vitro to the activity of colistin and polymyxin B on Pseudomonas aeruginosa, the effects of calcium and serum on gentamicin and carbenicillin were also examined. Serum was antagonistic to gentamicin in antibiotic tube dilution tests on five strains of P. aeruginosa. Serum was not antagonistic to carbenicillin in tube dilution tests. Physiologic concentrations of calcium antagonized the activity of gentamicin but not carbenicillin. The antagonism observed with gentamicin was less than that previously seen with colistin. The antagonistic effect of calcium and serum was removed by a chelating agent. Gentamicin and carbenicillin may be more active in vivo against P. aeruginosa than colistin or polymyxin B.     

The activity of polymyxins against dense populations of Escherichia coli.

The activities of polymyxin B sulphate, colistin (polymyxin E) sulphate and their sulphomethyl derivatives were compared by continuous turbidimetric monitoring of dense cultures of an Escherichia coli strain exposed to these agents. Judged by the concentration of antibiotic which caused a rapid fall in opacity of the culture, polymyxin B sulphate and colistin sulphate had similar activities, but the sulphomethyl compounds differed considerably: sulphomyxin sodium induced lysis of the culture at a concentration four times that of the parent compound, whereas colistin sulphomethate sodium induced a delayed fall in opacity consistent with recruitment of activity as the inactive sulphomethyl derivative was broken down to the parent compound. Durign overnight incubation, regrowth of cultures which had initially succumbed to polymyxin action occurred, apparently due to the selection of phenotypically resistant variants from within the population. In this way cultures could easily be adapted to growth in concentrations of antibiotic well above the conventionally-determined minimum inhibitory concentration. The comparative ease of adaptation was in the order: colistin sulphomethate greater than sulphomyxin greater than colistin sulphate greater than polymyxin B sulphate.     

Chemical Communication of Antibiotic Resistance by a Highly Resistant Subpopulation of Bacterial Cells

The overall antibiotic resistance of a bacterial population results from the combination of a wide range of susceptibilities displayed by subsets of bacterial cells. Bacterial heteroresistance to antibiotics has been documented for several opportunistic Gram-negative bacteria, but the mechanism of heteroresistance is unclear. We use Burkholderia cenocepacia as a model opportunistic bacterium to investigate the implications of heterogeneity in the response to the antimicrobial peptide polymyxin B (PmB) and also other bactericidal antibiotics. Here, we report that B. cenocepacia is heteroresistant to PmB. Population analysis profiling also identified B. cenocepacia subpopulations arising from a seemingly homogenous culture that are resistant to higher levels of polymyxin B than the rest of the cells in the culture, and can protect the more sensitive cells from killing, as well as sensitive bacteria from other species, such as Pseudomonas aeruginosa and Escherichia coli. Communication of resistance depended on upregulation of putrescine synthesis and YceI, a widely conserved low-molecular weight secreted protein. Deletion of genes for the synthesis of putrescine and YceI abrogate protection, while pharmacologic inhibition of putrescine synthesis reduced resistance to polymyxin B. Polyamines and YceI were also required for heteroresistance of B. cenocepacia to various bactericidal antibiotics. We propose that putrescine and YceI resemble "danger" infochemicals whose increased production by a bacterial subpopulation, becoming more resistant to bactericidal antibiotics, communicates higher level of resistance to more sensitive members of the population of the same or different species.     

Use of analogs of primary metabolites in the selection of the producer of polymyxin B]

A number of amino acids were found to have effects on the growth of the polymyxin B-producing culture and biosynthesis of the antibiotic by it. Of special importance was the stimulating effect by methionine. Four selection stages were carried out with using structural analogs of purines and amino acids as selective factors. There were no stable variants with increased antibiotic productivity among the mutants resistant to the analogs of purines and leucine. The levels of polymyxin B accumulation by the variants resistant to 4-fluorophenylalanine were 30 to 50 per cent higher than those in the controls and the variants were characterized by low morphological and antibiotic production variation in the subcultures. The mechanisms of the methionine physiological effect and the prospects of using analogs of the primary metabolites in improvement of the culture producing polymyxin B are discussed.     

Turbidimetric Assays: the Antibiotic Dose-Response Line

Several antibiotics reduced growth of bacteria without killing them when present in the range of concentrations of significance in assaying. The reduction in growth rate was linearly related to concentration of antibiotic. From this fact may be derived an equation of the form log N = A - BC where N is the concentration of bacteria at the end of the incubation period, C is the concentration of antibiotic, and A and B are constants. This equation is used to guide the selection of the best range of concentrations of unknown for assay and to show the large influence of variations of temperature upon an assay.     

Effect of polymyxins on Bacillus polymyxa sporogenesis

Bacillus polymyxa var. Ross. producing polymyxin M and Bacillus polymyxa 153 producing polymyxin B form spores during submerged cultivation when the rate of biosynthesis of antibiotic peptides is low and when the production of antibiotics is over. However, sporogenesis is stimulated if polymyxins are added at the early stage of cultural growth. Inhibition of the synthesis of antibiotics suppresses the formation of spores. Substances other than polymyxins do not exhibit such a specific effect on sporogenesis. The fact that the culture requires endogenous polymyxins which are most effective in the period prior to the appearance of spores in the culture suggests the regulatory action of these peptides at the stage between vegetative growth and spore formation in Bacillus polymyxa.     

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。