空肠弯曲菌在环境压力条件下的生存机制和可培养性研究进展

空肠弯曲菌是一种重要的革兰氏阴性致病菌,可以产生几种肠毒素能侵袭小肠和大肠黏膜引起急性肠炎,据统计其导致肠炎比例是沙门氏菌的3倍。该菌污染肉类食品后,经冷冻等环境的变化会进入活的非可培养状态(viable but non-culturable state,VBNC),不能用常规方法检测到。空肠弯曲菌在pH值、湿度、温度、营养成分等条件发生极端改变的情况下可以进入VBNC状态。本研究更深入地了解空肠弯曲菌是如何抵御外力影响的,防范食品安全的潜在危害。     

米面制品中微生物的活但不可培养状态检测研究进展

米面制品是我国主要的食品,含有丰富的营养物质与充足的水分,适合微生物生长繁殖。腐败微生物会引起食品货架期缩短或在保质期内腐败变质,而致病微生物的存在则可引起食物中毒事件。近年来对食品微生物的快速检测技术主要包括传统法与核酸扩增法,其中聚合酶螺旋反应(PSR)技术具有较高的灵敏度、特异性与重现性,具有较大应用前景。此外,活但不可培养状态(VBNC)因具有不可培养特性,会造成微生物检测"金标准"培养法获得"假阴性"结果,是食品微生物安全中的重要威胁。通过研究分析污染米面制品的主要微生物和相应快速检测方法,以及活但不可培养状态的生物学特性和检测方法,为进一步实现对米面制品中微生物安全控制提供重要依据。     

德氏乳杆菌保加利亚亚种ND02活的非可培养态诱导和复苏

为有效提高乳酸菌发酵剂菌种在生产和应用过程中的活性,对一株具有优良发酵特性的德氏乳杆菌保加利亚亚种ND02(保加利亚乳杆菌ND02)进行了活的非可培养(viable but non-culturable,VBNC)态诱导和复苏的初步研究。将活化的保加利亚乳杆菌ND02在不同复合条件下进行诱导,同时采用平板计数法和荧光显微镜两种计数方法对其进行检测,将诱导成功的样本通过改变温度及改变培养基成分进行复苏实验。结果表明:保加利亚乳杆菌ND02在液体MRS中4℃诱导190 d便可以进入VBNC态,并发现10%脱脂乳+0.1%酵母粉是有效的复苏方法。为保加利亚乳杆菌ND02在应用过程中避免VBNC态的产生及活性的提高提供一定的参考依据。     

Changes in culturability and virulence of Salmonella typhimurium during long-term starvation under desiccating conditions

The survival of Salmonella typhimurium under desiccation and starvation conditions commonly associated with farm buildings was investigated in a desiccation model system: filtration onto polycarbonate membranes placed in a sealed desiccator with 0.0067 g/m³ absolute humidity. Heterogeneities within bacterial populations in relation to time of desiccation were investigated on a single-cell basis by epifluorescence microscopy coupled with an image analysis system in conjunction with fluorescent dyes Chemchrome V6 and DAPI. Changes in cellular states were compared to the results of plate counts (colony forming units, CFU) on selective (modified semi-solid Rappaport Vassiliadis (MSRV)) and non-selective (nutrient agar (NA) and R2A agar) media, and to the measurements of infectivity and virulence using two animal models (chicks and mice). During 9 weeks of experimental desiccation, total cell counts (DAPI) of starved S. typhimurium remained stable, as did esterase activity (Chemchrome V6), but DAPI fluorescence intensity decreased slowly. Bacterial cells entered gradually into non-culturable states (decrease of CFU counts on MSRV, NA and R2A agar media) and the total loss of culturability on NA (defined as probability of presence of 1 CFU on the membrane inferior to 10⁻⁶) was obtained after 9 weeks. Loss of chick infectivity and mice virulence in animal models occurred more rapidly, within three weeks of experimental desiccation.     

热辅助超声波处理中VBNC态鼠伤寒沙门氏菌产生及其机制

为了研究热辅助超声波(TS)处理中鼠伤寒沙门氏菌进入活的非可培养态(VBNC)的情况及可能机制,本文采用RT-qPCR与平板计数法检测其VBNC态发生情况,采用ESR方法定性、定量检测TS处理中产生自由基并用数学模型分析其与VBNC态发生率指数之间的关系。结果表明:TS处理强度与VBNC态鼠伤寒沙门氏菌产生及自由基强度有重要影响。经TS处理,在BPY培养基中主要产生3种自由基,包括以碳为中心自由基、羟基自由基与氢质子自由基。自由基强度与VBNC态发生率指数之间高度相关,可用Boltzmann模型进行拟合和验证。以丙酮酸钠作为自由基清除剂,验证了自由基对鼠伤寒沙门氏菌进入VBNC态具有重要作用。本研究结果对于TS处理技术的应用具有指导意义。     

次氯酸钠诱导肠炎沙门氏菌活的非可培养状态形成及复苏过程研究

目的 研究不同有效氯浓度次氯酸钠溶液诱导肠炎沙门氏菌进入活的非可培养状态（viable but non-culturable state: VBNC）及复苏条件研究。方法 以不同有效氯浓度次氯酸钠处理肠炎沙门氏菌,分析肠炎沙门氏菌活的非可培养状态菌数及VBNC发生率的变化,结合扫描电镜观察肠炎沙门氏菌VBNC态细胞与正常细胞的形态差异,而后比较不同复苏条件的复苏效果,同时对复苏后的细胞生理活性进行评价。结果 40 mg/L有效氯浓度处理20 min可使活菌全部进入VBNC状态,细胞表面出现褶皱塌陷;高于60 mg/L有效氯浓度可全部杀灭,细胞出现断裂、破损;进入VBNC态的肠炎沙门氏菌在5种复苏处理下,均可恢复可培养性;在20 mmol/L丙酮酸钠×热激条件下恢复效果较好。pHi和胞内ATP显示复苏后细胞活性较正常细胞有所降低。结论 在食品生产、环境清洁中应注意次氯酸钠使用浓度,否则极易造成肠炎沙门氏菌VBNC态的形成和复苏,导致食品污染,从而增加食品安全风险。     

VBNC Legionella pneumophila cells are still able to produce virulence proteins

Legionella pneumophila is the agent responsible for legionellosis. Numerous bacteria, including L. pneumophila, can enter into a viable but not culturable (VBNC) state under unfavorable environmental conditions. In this state, cells are unable to form colonies on standard medium but are still alive. Here we show that VBNC L. pneumophila cells, obtained by monochloramine treatment, were still able to synthesize proteins, some of which are involved in virulence. Protein synthesis was measured using ³⁵S-labeling and the proteomes of VBNC and culturable cells then compared. This analysis allowed the identification of nine proteins that were accumulated in the VBNC state. Among them, four were involved in virulence, i.e., the macrophage infectivity potentiator protein, the hypothetical protein lpl2247, the ClpP protease proteolytic subunit and the 27 kDa outer membrane protein. Others, i.e., the enoyl reductase, the electron transfer flavoprotein (alpha and beta subunits), the 50S ribosomal proteins (L1 and L25) are involved in metabolic and energy production pathways. However, resuscitation experiments performed with Acanthamoeba castellanii failed, suggesting that the accumulation of virulence factors by VBNC cells is not sufficient to maintain their virulence.     

Changes in culturability and virulence of Salmonella typhimurium during long-term starvation under desiccating conditions

The survival of Salmonella typhimurium under desiccation and starvation conditions commonly associated with farm buildings was investigated in a desiccation model system: filtration onto polycarbonate membranes placed in a sealed desiccator with 0.0067 g/m³ absolute humidity. Heterogeneities within bacterial populations in relation to time of desiccation were investigated on a single-cell basis by epifluorescence microscopy coupled with an image analysis system in conjunction with fluorescent dyes Chemchrome V6 and DAPI. Changes in cellular states were compared to the results of plate counts (colony forming units, CFU) on selective (modified semi-solid Rappaport Vassiliadis (MSRV)) and non-selective (nutrient agar (NA) and R2A agar) media, and to the measurements of infectivity and virulence using two animal models (chicks and mice). During 9 weeks of experimental desiccation, total cell counts (DAPI) of starved S. typhimurium remained stable, as did esterase activity (Chemchrome V6), but DAPI fluorescence intensity decreased slowly. Bacterial cells entered gradually into non-culturable states (decrease of CFU counts on MSRV, NA and R2A agar media) and the total loss of culturability on NA (defined as probability of presence of 1 CFU on the membrane inferior to 10⁻⁶) was obtained after 9 weeks. Loss of chick infectivity and mice virulence in animal models occurred more rapidly, within three weeks of experimental desiccation.     

Extremophiles: developments of their special functions and potential resources

Extremophilles are valuable resources in biotechnology. Enzymes from extremophiles are expected to fill the gap between biological and chemical processes due to their unusual properties. Especially enzymes from hyperthermophiles that can grow at above 90 degrees C were devoted owing to its extraordinary thermostability and denaturant tolerance. Screening trials of hyperthermophilic microorganisms were performed by a number of microbiologists and various unique strains were isolated from natural environments. One of the most successful uses of thermostable enzymes was DNA polymerase in the polymerase chain reaction (PCR). Thermostable enzymes are used in the chemical, food, pharmaceutical, paper and textile industries. Recombinant forms of thermostable enzymes that have been expressed in Escherichia coli are commonly utilized in industrial applications however their enzymatic characteristics and tertiary structure are different from the native ones produced in the original strains. In vitro heat treatment induces a structural conversion of the recombinant protein to its natural form. High temperature itself plays an important role in determining the specific characteristics and tertiary structure of the enzyme. Recent studies have revealed that hyperthermophiles can grow under numerous conditions not only in geothermal or deep-sea thermal environments. Technological advances have allowed DNA to be isolated from natural environments. Now genes could be isolated from microorganisms that have not been cultured. In this review, innovative approaches to hunt genes from natural environments without pure culturing of microorganisms are also discussed.     

牛奶发酵过程中阪崎克罗诺杆菌可形成活的非可培养状态

该研究以保加利亚乳杆菌为牛奶发酵剂,研究了牛奶发酵过程中阪崎克罗诺杆菌与保加利亚乳杆菌的相互作用以及阪崎克罗诺杆菌存活量的变化情况,并结合16S rDNA高通量测序技术和PMA-qPCR方法分析阪崎克罗诺杆菌在牛奶发酵过程中是否形成“活的非可培养”状态。结果显示,保加利亚乳杆菌在牛奶中发酵48 h后pH值可达到3.40,酸度值可达212.00。在发酵前期接种阪崎克罗诺杆菌,48 h后pH值和酸度值分别为3.50和193.30;在发酵中期接种阪崎克罗诺杆菌,48 h后pH值和酸度值分别为3.47和214.30。发酵前期阪崎克罗诺杆菌的污染对保加利亚乳杆菌发酵结果的影响更大。阪崎克罗诺杆菌在牛奶中生长48 h后菌浓度可稳定在9.08 lg(CFU/mL);不论在牛奶经发酵前期或中期接种阪崎克罗诺杆菌,发酵后期平板计数法均检测不到阪崎克罗诺杆菌,但16s rDNA和PMA-qPCR技术均可检测到阪崎克罗诺杆菌活菌的存在。该研究结果说明,阪崎克罗诺杆菌在牛奶发酵后进入了“活的非可培养”状态,该状态的存在会导致该菌检测时活菌数量的低估,从而引发乳及乳制品的安全隐患。