氧化压力下沙门氏菌可培养性检测及其活的非可培养状态形成情况

【背景】氧化压力会导致细菌进入活的非可培养(viable but non-culturable,VBNC)状态,菌落形成能力可能受到亚致死损伤的影响。目前对于VBNC态细菌的定量检测是基于活菌数与可培养数的差值,因此可培养数的检测对于VBNC态定量研究很关键,培养基组成不合适可能会造成漏检。【目的】分析培养基组成对氧化压力下亚致死损伤细菌检测的重要影响;探究常见食源性致病菌肠炎沙门氏菌在氧化压力下形成VBNC态的情况。【方法】分别采用Luria-Bertani (LB)、beef peptone yeast (BPY)和Salmonella Shigella (SS)培养基检测并比较肠炎沙门氏菌的可培养数;采用RT-qPCR、荧光染色-激光共聚焦显微镜观测氧化压力下肠炎沙门氏菌形成VBNC态的情况。【结果】非选择性培养基LB和BPY能检出亚致死细菌,SS培养基中牛胆盐导致可培养数减少;肠炎沙门氏菌经53°C过氧化氢处理1.5 h后进入VBNC态的比例显著高于53°C过氧化氢+亚铁离子和过氧化氢+柠檬酸处理(P<0.05)。【结论】在对VBNC态的检测中应选择合适的固体培养基检测可...     

FM4-64和Hoechst染料在活细菌胞膜和拟核标记定位中的条件优化与应用

摘要:【目的】为了观察细菌细胞膜和拟核的形态结构,准确对亚细胞进行定位。【方法】本文利用FM4-64和Hoechst两种染料,分别采用不同浓度和不同时间对大肠杆菌活细胞进行染色和荧光共聚焦显微成像,并对7种细菌的活细胞(金黄色葡萄球菌、铜绿假单胞菌、肺炎克雷伯菌、鼠疫耶尔森菌、军团菌、霍乱弧菌和炭疽芽孢杆菌)进行染色观察。【结果】相同观察条件下,不同染料浓度和不同的染色时间影响了细胞膜、拟核的荧光强度;确定了FM4-64染色通用条件(浓度20 μg/mL 染色1 min)和Hoechst的最佳条件(浓度20 μg/mL染色20min)。上述条件下,Hoechst对8种细菌染色效果均较理想,然而FM4-64对细菌染色效果不同,革兰氏阴性菌(大肠杆菌、肺炎克雷伯菌、鼠疫耶尔森菌、霍乱弧菌、铜绿假单胞菌和军团菌)表现较好的细胞膜轮廓,革兰氏阳性菌(炭疽芽孢杆菌和金黄色葡萄球菌)效果较差。【结论】FM4-64和Hoechst两种染料共染8种细菌,对细胞膜和拟核的染色观察,可为原核细胞结构染色提供借鉴,并为大分子的亚细胞定位研究奠定基础。     

荒漠土壤中两株抗氧化细菌的抗氧化生理生化特征

【背景】微生物在荒漠生态系统中经常面临多重胁迫,包括干旱、高温、UV辐射,这些环境胁迫使得荒漠土壤微生物极易在体内外积累大量的超氧离子或过氧化物,抑制其生长或者直接造成死亡。【目的】荒漠土壤细菌为适应荒漠环境表现出抗氧化特性,作为荒漠生态系统重要组成部分,对其抗氧化特性的研究为荒漠地区抗氧化资源的开发提供科学依据和技术基础,也对荒漠微生物抗氧化机制的挖掘奠定了基础。【方法】利用过氧化氢氧化筛选出两株具有强抗氧化性的荒漠土壤细菌:海床动性微菌AX6(PlanomicrobiumokeanokoitesAX6)和海洋考克氏菌KD4(Kocuriamarina KD4),通过测定其在过氧化氢条件下的生长曲线、细胞受损程度、抗氧化酶活性以及自由基清除能力,探究荒漠土壤微生物的抗氧化生理生化特征。【结果】两株细菌在低浓度过氧化氢中细胞丙二醛含量显著低于阴性对照大肠杆菌,在1.5mmol/L过氧化氢中菌株AX6的谷胱甘肽过氧化物酶活性可达108.33 U/mL,同时DPPH、超氧阴离子自由基的清除能力显著升高;此外,在3 mmol/L过氧化氢中菌株KD4的过氧化氢酶活性升高至1.16 U/mL,显...     

超高压处理对副溶血性弧菌的影响研究

摘要：【目的】探讨超高压致死微生物的机理。【方法】本文以副溶血性弧菌为对象,研究了超高压处理对副溶血性弧菌的灭菌效果、对副溶血性弧菌细胞超微结构、细胞无机盐离子含量以及细胞膜蛋白的影响。【结果】结果表明,在20℃下分别经100、200 MPa高压处理10min后,副溶血性弧菌致死率为40%、84.7%,经300 MPa及以上的压力处理,副溶血性弧菌的致死率为100%。超高压处理对细菌细胞形态结构造成明显的损伤：局部细胞壁遭到破坏,出现缺口;胞质内含物结构紊乱,出现泄漏,细胞中部出现透电子区;细胞结构不完整     

亚致死剂量吡虫啉对意大利蜜蜂中肠细菌群落的影响（英文）

【目的】在实验室条件下,研究亚致死剂量吡虫啉对意大利蜜蜂 Apis mellifera ligustica 中肠细菌群落的影响,以期为杀虫剂的生物安全性评价体系提供理论依据。【方法】分别用3种不同浓度（0.005, 0.015 和0.045 mg/L）的吡虫啉蔗糖溶液以及含丙酮（0.03%）的糖水(空白对照)在实验室内饲喂刚出房的意大利蜜蜂,在饲喂3, 6, 9, 12和15 d时分别取样。提取中肠细菌基因组DNA,采用下一代高通量测序技术对4个处理组蜜蜂中肠细菌群落进行检测分析。【结果】细菌序列分析表明,意大利蜜蜂中肠细菌主要属于变形菌门（Proteobacteria）和厚壁菌门（Firmicutes）,同一时间内各处理组间主要细菌数量差异不显著（P>0.05）,同时细菌群落多样性指数差异也不显著（P>0.05）。【结论】在实验室条件下亚致死剂量吡虫啉对意大利蜜蜂中肠细菌群落结构影响不显著。     

余氯对河流水体微生物灭活效应的评价

【目的】研究不同余氯浓度和暴露时间对细菌的去除效果,分析不同余氯条件对细胞ATP的影响。【方法】以河水中微生物群落为试验对象,利用流式细胞术(Flow cytometry,FCM)评估不同余氯浓度和暴露时间的灭活效果,检测不同余氯浓度时细胞内(外)ATP的变化情况。【结果】不同余氯浓度和暴露时间对细菌的去除效果产生不同的结果。在余氯浓度2 mg/L情况下,延长氯暴露时间可以增加细菌的去除效果,在余氯浓度≥2 mg/L条件下,较短氯暴露时间就可以灭活90%细菌。高核苷酸细菌(HNA)和低核苷酸细菌(LNA)表现出不同氯耐受能力,且HNA细菌相比LNA细菌较容易受到氯的损伤。细胞内ATP随余氯浓度增加而减少,在高浓度余氯条件下(≥2 mg/L)细胞外ATP才会增加。【结论】微生物活性随着余氯作用的增加而降低,FCM法和ATP检测法可以用于评估加氯消毒对微生物稳定性的影响。     

不同压力条件下油藏内源细菌群落激活过程中变性梯度凝胶电泳分析?

摘要：【目的】了解常压（1 MPa）和高压（10 MPa）条件下内源细菌激活中的细菌群落和结构的变化。【方法】利用胜利油田沾3×24井产出液水样,在常压和高压下进行富集培养后,定时取样,样品用变性梯度凝胶电泳（denature gradient gel electrophoresis,DGGE）技术分析。【结果】通过对内源微生物激活前后DGGE条带的数量和亮度变化分析表明,油藏环境中的细菌在种类和数量上并不丰富,激活剂的加入改变了菌群原有的贫营养环境,从而使一些因营养缺乏生长受抑制细菌得以大量繁殖,菌群结     

外源化学物质对副溶血性弧菌药物敏感性的影响

【目的】细菌对抗生素的耐药性已成为全球公共卫生问题关注的热点。有研究表明外源添加化学物质可以增强耐药细菌对抗生素的敏感性。本研究比较了3种化学物质葡萄糖、丙氨酸、甘油对增强副溶血性弧菌抗生素敏感性的作用效果。【方法】在亚抑菌浓度抗生素胁迫条件下,通过比较副溶血性弧菌在添加终浓度为10 mmol/L葡萄糖、丙氨酸、甘油后细菌存活率随时间的变化水平,来观察弧菌对亚抑菌浓度抗生素敏感性作用效果的改变,并采用氧化磷酸化解偶联剂CCCP对实验结果进行验证。【结果】发现3种外源化学物质均能增强亚抑菌浓度氨基糖苷类抗生素对副溶血性弧菌的杀菌能力,其中外源添加葡萄糖对增强亚抑菌浓度卡那霉素的杀菌能力最为显著,而对其他种类抗生素的杀菌能力则无明显增强作用。加入氧化磷酸化解偶联剂CCCP后可消除由外源化学物质引发的弧菌抗生素敏感性作用增强的现象。【结论】通过调节细菌细胞代谢水平可提高耐药副溶血性弧菌对氨基糖苷类抗生素的敏感性,对多重耐药副溶血性弧菌的防控具有一定的实际应用价值。     

冷冻致亚致死损伤的金黄色葡萄球菌修复机制

摘要:【目的】研究冷冻致亚致死损伤的金黄色葡萄球菌修复过程中的细胞修复机制。【方法】本文以冷冻致亚致死损伤的金黄色葡萄球菌(Staphylococcus aureus)为研究对象,探讨了不同修复时间细胞的修复情况;利用透射电子显微镜观察修复启动过程中超微结构的变化;通过实时荧光定量PCR(Real-time PCR)方法测定了修复过程中转录弱化子(msrR)、铁离子ABC转运ATP结合蛋白(fhuC)、细胞色素b(cytB)基因表达量的变化,通过紫外分光光度法测定细胞外泄漏物含量、细胞活性氧(ROS)和超氧化物歧化酶( SOD)活性。【结果】修复3 h后,99%以上冷冻致亚致死损伤的金黄色葡萄球菌完成修复,修复后细胞对高盐胁迫抗性恢复。Real-time PCR分析结果表明,msrR和fhuC基因表达量显著下调,而cytB表达量显著上调。修复过程中冷冻致亚致死损伤的金黄色葡萄球菌细胞表面超微结构变化比较明显,细胞表面从光滑透明变得致密结实,细胞内紫外吸收物质泄漏速度也在逐渐变慢,同时细胞中的ROS含量降低,SOD酶活性减弱。【结论】冷冻致亚致死损伤的金黄色葡萄球菌修复过程中,可能是通过细胞膜完整性的修复,细胞恢复对高盐胁迫的抵抗能力;通过基因调控降低细胞内ROS的含量,降低活性氧(O－2)对细胞的毒害作用。同时通过产能代谢相关基因(cytB)的调控为细胞提供修复所需要的能量,最终冷冻致亚致死损伤的细胞得到修复。     

超高压对单增李斯特菌细胞膜的损伤和致死机理

【目的】研究超高压对病原微生物单增李斯特菌细胞膜损伤的影响。【方法】本文以单增李斯特菌为研究对象,探讨了不同压力处理(100-500 MPa)对单增李斯特菌的灭活作用,利用透射电镜观察高压处理对细菌细胞超微结构的影响,通过紫外分光光度法、原子吸收分光光度法和荧光分光光度法测定高压处理对细菌细胞膜通透性的影响,采用超微量Na+/K+-ATP酶试剂盒测定高压处理对细菌细胞膜Na+/K+-ATP酶活力的影响。【结果】25℃经300、350、400 MPa压力处理15 min后,单增李斯特菌总数由9.00分别降至5.20、3.27、1.35个对数单位,经450MPa及以上的压力处理后,单增李斯特菌的致死率达到100%。超高压处理对单增李斯特菌的细胞超微结构造成明显的损伤,细胞结构不完整,细胞壁局部被破坏,细胞膜通透性增大,细胞内物质聚合,出现透电子区。由于细胞膜的损伤使得细胞内无机盐离子、紫外吸收物质流出,细胞膜上的Na+/K+-ATPase失活。【结论】超高压处理造成单增李斯特菌细胞形态结构明显损伤,改变细胞膜的通透性,降低细胞膜上Na+/K+-ATP酶活力,最终使得细胞内无机盐离子和胞内大分子物质外流而死亡。     

Mesosome formation is accompanied by hydrogen peroxide accumulation in bacteria during the rifampicin effect

Ultrastructural alteration and hydrogen peroxide localization were examined in Xanthomonas campestris pv. phaseoli during rifampicin effect using transmission electron microscopy. Bacterial cells were treated with rifampicin and then were examined by electron microscopy to observe the changes of ultrastructure or hydrogen peroxide accumulation in living cells that took place before lysis. Intriguingly, rifampicin treatment led to presence of an additional location of hydrogen peroxide accumulation within the cells. There was an association between the frequency and size of the additional location of hydrogen peroxide accumulation and the concentration of rifampicin. Furthermore, an additional ultrastructure, mesosomes, was also present in cells during rifampicin effect. The frequency and size of mesosome increased with the increasing concentration of rifampicin. Result of multiple linear regression showed that the size of mesosome plays as a key factor in the quantity of excess hydrogen peroxide accumulation in cells during rifampicin effect. Linear correlation was confirmed between quantity of excess hydrogen peroxide accumulation and the size of mesosome in cells during rifampicin effect. This finding intensely indicated that mesosomes are just the additional location of hydrogen peroxide accumulation in cells under cellular injury caused by rifampicin treatment. The mesosome formation is always accompanied by excess hydrogen peroxide accumulation in X. campestris pv. phaseoli during rifampicin effect.     

Mesosomes associated with hydrogen peroxide in bacteria

Mesosomes are unique membranous structures in bacteria. It is recognized that the mesosomes should be involved in several fundamental processes. The structure and behaviour of mesosomes have been studied and largely identified, while new evidences of mesosome function have been strikingly obtained. Our previous studies confirmed that hydrogen peroxide is involved in mesosomes formation during cell injury and cell division processes. Mesosome formation is always accompanied by excessive H₂O₂ accumulation. Furthermore, our recent data showed that mesosomes could not only enrich the excess H₂O₂, but also bring the H₂O₂ outside of the cells injured by antibiotics. It is a possibility that the enrichment of H₂O₂ in mesosomes might be a mechanism of drug resistance of bacteria. This article describes the bacterial mesosome and its functions as well as the involvement of hydrogen peroxide in mediating these functions.     

Revisiting the Mesosome as a Novel Site of Hydrogen Peroxide Accumulation in Escherichia coli

The major source of endogenous hydrogen peroxide is generally thought to be the respiratory chain of bacteria and mitochondria. In our previous works, mesosome structure was induced in cells during rifampicin effect, and the mesosome formation is always accompanied by excess hydrogen peroxide accumulation in bacterial cells. However, the underlying mechanisms of hydrogen peroxide production and the rationale behind it remain still unknown. Here we report that hydrogen peroxide can specifically accumulate in the mesosome in vitro. Mesosomes were interpreted earlier as artifacts of specific cells under stress through TEM preparation, while, in the current study, mesosomes were shown as intracellular compartments with specific roles and features by using quickly freezing preparation of TEM. Formation of hydrogen peroxide was observed in suspension of mesosomal vesicles by using either a fluorescence-based reporter assay or a histochemical method, respectively. Our investigation provides experimental evidence that mesosomes can be a novel site of hydrogen peroxide accumulation.     

Mesosome Structure in Chromobacterium violaceum

Exponentially growing cells of the gram-negative bacterium Chromobacterium violaceum demonstrate invaginations of the cytoplasmic membrane with a high frequency. These invaginations conform to the ultrastructural appearance of mesosomes of gram-positive bacteria. As many as four mesosomes are observed per cell, each of which may increase the total membrane surface of the cell by 30%. Washing of cells in dilute tris(hydroxymethyl)aminomethane buffer effects a distension of the mesosome "neck" and/or cytoplasmic membrane clarifying the association of the mesosome to the cytoplasmic membrane. Plasmolysis effects an eversion of the mesosome into the plasmolysis vacuole.     

FACTORS INFLUENCING THE FREQUENCY OF MESOSOMES OBSERVED IN FIXED AND UNFIXED CELLS OF STREPTOCOCCUS FAECALIS

Mesosomes of Streptococcus faecalis (American Type Culture Collection 9790) were seen about 92% less frequently in freeze fractures of unfixed cells than in freeze fractures and sections of fixed cells. This difference in frequency was not related to any period of unbalanced macromolecular synthesis induced by chemical fixation. All measured synthetic processes (DNA, RNA, and protein synthesis, and glycerol incorporation) were halted with either osmium tetroxide (OS) or glutaraldehyde fixation. That fewer mesosomes were seen in freeze fractures of unfixed cells was probably due to the difficulty of observing cross-fractured mesosomes in this organism in the unfixed state. Unfortunately, mesosomes probably preferentially cross fracture in the unfixed state and therefore are usually only observed, infrequently, in those cases where the freeze fracture follows the surface layer of a mesosomal membrane. However, the addition of glycerol to unfixed cells, especially in the chilled state, greatly increased the frequency of observation of cytoplasmic mesosomes in freeze fractures. It is thought that glycerol, like chemical fixation, increases the number of surface-fractured mesosomes, which in turn increases the frequency of mesosome observation. It was also observed that cellular autolysis occurring during OS fixation seemingly reduced the number of mesosomes observed in thin sections and freeze fractures of OS-fixed cells.     

Mesosomes in Escherichia coli

When Escherichia coli was grown in a synthetic medium and fixed with osmium, sections of the cells revealed clearly defined mesosomes. These mesosomes appeared to develop, in dividing cells, as coiled infoldings of the cytoplasmic membrane. Mature mesosomes formed a link between the cytoplasmic membrane and the nucleus of the cell. The arrangement of the mesosomes in dividing cells led to the hypothesis that division of the nucleus in these cells is accomplished by two separate polar mesosomes. One mesosome is derived from the parent cell and is present at one pole of the daughter cell. The other is freshly synthesized at or near the newly forming pole of the daughter cell. While the old mesosome remains attached to the chromosome received from the parent cell, the newly synthesized mesosome becomes attached to and initiates replication of the new chromosome. As the cell grows and elongates, the two mesosomes, attached to their respective chromosomes move apart, thus effecting nuclear division.     

Organization of mesosomes in fixed and unfixed cells.

After the addition of glutaraldehyde (GA) to cells incubated at 3 or 37 degrees C, mesosomes were observed with increasing frequencies in freeze fractures of cells. These increases were related to the kinetics with which GA cross-linked adjacent amino acids. Upon the addition of GA, mesosomes were first observed in the periphery of freeze-fractured cells usually attached to septal membranes. However, the time, while the septal attachment sites were maintained, the "bodies" of the mesosomes were observed to move toward the center of the cytoplasm. This centralization process was much more rapid at 37 than at 3 degrees C. It is hypothesized that upon fixation, or receipt of some physical insult, mesosome precursors found in undisturbed cells undergo a change in state that results in their visibility in freeze fractures.     

Relationship between the DNA content and mesosome number in cells of Bacillus.

In cells of Bacillus there is evidence that deoxyribonucleic acid forms an association with some membranous structure within the cell, possibly mesosomes. Cells of varieties of Bacillus cereus and Bacillus subtilis were examined to see if any quantitative relationship existed between the numbers of mesosomes and DNA content. No direct relationship could be domonstrated. However, cells of Bacillus cereus var. alesti A(-) maintained a characteristic and constant DNA content and number of mesosomes regardless of growth conditions. During sporulation, a variant of A(-), termed A(-)3, SEQUESTERS ITS DNA at both ends of the cell, leaving a small amount of DNA but no mesosomes in the center compartment. Since this center compartment is capableof growth and division upon replacement in fresh medium (rejuventation) it was examinedfor mesosome content as DNA synthesis and division were initiated. In most cells, acentral mesosome was formed at the site of cell septum formation; however, the presenceof a mesosome was not an absolute prerequisite for cell division. We propose that atthe onset of cell growth, mesosomes primarily function in the process of cell septum formation. As growth and division proceed, mesosomes are produced in characteristicnumbers and may act as the site of DNA synthesis and (or) segregation.     

Bacterial mesosomes: Real structures of artifacts?

The ultrastructural study of membrane organization in gram-positive bacteria related to the OsO₄ fixation conditions revealed that large, complex mesosomes are observed only when the bacteria are subjected to an initial fixation with 0.1% OsO₄ in the culture broth, as in the prefixation step of the Ryter-Kellenberger procedure. Evidence was obtained suggesting that the large mesosomes are produced by this prefixation. The kinetic study of the membrane morphological alterations occurring during the prefixation of Bacillus cereus with 0.1% OsO₄ in the culture broth showed that the amount of mesosome material increases linearly from zero to a maximum observed at 1.7 min of prefixation and that at about this time a maximum is reached for the number of mesosomes per unity of cell area and for the average individual mesosome area. The large mesosomes observed in gram-positives fixed by the complete Ryter-Kellenberger procedure would be the result of the membrane-damaging action of 0.1% OsO₄. Such damaging action was deduced from the observation that 0.1% OsO₄ quickly lyses protoplasts and induces a quick and extensive leakage of intracellular K⁺ from B. cereus and Streptococcus faeculis. In support of that interpretation is the observation that in bacteria subjected to several membrane-damaging treatments, mesosome-like structures are seen after three different fixation procedures. In bacteria initially fixed with 1% OsO₄, 4% OsO₄ or 2.5% glutaraldehyde, no large, complex mesosomes are observed, small and simple invaginations of the cytoplasmic membrane being present. The size of these minute mesosomes is inversely proportional that causes of fixation. Uranyl acetate was found among the studied fixatives the one to the rate the least damage to bacterial membranes. This fixative satisfactorily preserves protoplasts. In bacteria initially fixed with uranyl acetate no mesosomes were found. The results of the present work throw serious doubts on the existence of mesosomes, both large and small, as real structures of bacterial cells. It is proposed that a continuous cytoplasmic membrane without infoldings (mesosomes) would be the real pattern of membrane organization in gram-positives.     

Nuclear and cell division in Bacillus subtilis: cell development from spore germination.

The changes in the morphology of the nucleoids and the mesosomes in Bacillus subtilis cells during synchronous outgrowth after spore germination were followed in large-scale three-dimensional cell reconstructions. Shortly after outgrowth of the cell begins in Spizizen medium with glucose, the mesosome becomes an elongated structure in close contact with a rounded nucleoid. When nuclear replication reaches full activity, the mesosome develops into a single, complicated versatile system, with tubules that traverse the cytoplasm and have elaborations in and near the nucleoplasm. Later the system may retract to form large rounded mesosomes; the tubules and strings of vesicles within these mesosomes probably have been collected from the cytoplasm. Shortly after the first cell division, both sister cells have two nucleoids, but with longer generation times induced by growth in media containing acetate instead of glucose; these sister cells have only one nucleoid each. In acetate-grown cells rounded nucleoids that have no contact with a mesosome may represent nucleoids in a temporary stage of rest. On the other hand, the nucleoids of cells growing in glucose-containing medium are always penetrated by mesosomal material, superficially or deeply. Since the mesosome appears capable of traversing the nuclear fibrils, and even reaching the last strands connecting the dividing nucleoids, it is suggested that this organelle may play a vital role in the Bacillus division cycle.