Endocytosis and vacuolar morphology in Saccharomyces cerevisiae are altered in response to ethanol stress or heat shock

The vital lipophilic dye N‐(3‐triethylammoniumpropyl)‐4‐[6‐(4‐(diethylamino)phenyl]hexatrienyl) pyridinium dibromide (FM 4‐64) was used to study the effect of ethanol stress and heat shock on endocytosis in the yeast Saccharomyces cerevisiae. Yeast cells stained with FM 4‐64 were placed in a culture chamber and the internalization of the dye was monitored by fluorescence microscopy during perfusion of the cells with fresh growth medium. In the absence of ethanol in the perfusion medium, the internalization of FM 4‐64 from the plasma membrane to the vacuolar membrane by yeast cells harvested from the exponential phase of growth was completed in 30 min. The presence of 6% (v/v) ethanol in the perfusion medium had no obvious effect on the internalization of FM 4‐64 from the plasma membrane, but did lead to an accumulation of the dye in endocytic intermediates. Consequently, vacuolar membrane staining was delayed. Cells stained with FM 4‐64 and subjected to heat shock displayed a similar effect, with endocytic intermediates becoming more prominent with the severity of the heat shock. For both ethanol stress and heat shock, vacuolar morphology altered from segregated structures to a single, large organelle. The findings of this study reinforce previous observations that ethanol stress and heat shock induce similar responses in yeast. Copyright © 1999 John Wiley & Sons, Ltd.     

流式细胞术检测单增李斯特菌与酿酒酵母

为探讨流式细胞术对单增李斯特菌和酿酒酵母的活菌与热灭活菌的检出效果,本文采用荧光染色试剂SYTO-9和碘化乙锭(PI)对单增李斯特菌和酿酒酵母的活菌与热灭活菌的细胞悬液进行染色,采用流式细胞仪同时测量红色荧光与绿色荧光从而得出细胞悬液中的细菌和酵母的含量。结果表明经核酸荧光染料染色后,再结合流式细胞术对细菌与酵母菌进行检测,步骤简单、耗时短。该法不仅简化了测量步骤且分辨率高,对单增李斯特菌和酿酒酵母均具有良好的检出结果,能分辨同一体系中同一菌种的活细胞与热灭活细胞和同一体系中的细菌与酵母活细胞;该法检出限低,将单增李斯特菌稀释后,最低检出限可达1.2×104 cells/mL,将酿酒酵母稀释后,最低检出限可达6×103 cells/mL,因此能大大缩短增菌时间或者避免繁复的增菌步骤。     

蒲公英脂溶性成分对沙门氏菌抑菌作用机理

目的:研究蒲公英脂溶性成分对沙门氏菌的抑菌活性及其作用机理。方法:采用索氏提取法提取蒲公英脂溶性成分,纸片抑菌法分析蒲公英脂溶性成分对常见食源性致病菌沙门氏菌的抑菌作用,紫外分光光度法研究了蒲公英脂溶性成分的最低抑菌浓度,通过紫外核酸物质测定、倒置荧光显微镜染色观察、电子显微镜细菌形态分析细胞膜完整性。结果:当蒲公英脂溶性成分浓度为10 mg/mL时,抑菌圈直径为11.3 mm;紫外分光光度测定最小抑菌浓度为8 mg/mL;紫外核酸物质测定、荧光染色观察及电镜细胞形态分析均显示蒲公英脂溶性成分对细胞膜有明显破坏作用。结论:蒲公英脂溶性成分对沙门氏菌有很好抑菌活性,初步确定其抑菌作用与对菌体细胞膜的破坏作用有关。     

红豆皮多酚提取物对两种致病菌的抑菌活性及作用机理

多酚类化合物具有一定的抑菌活性，为探究红豆皮多酚对两种致病菌的抑菌机理，本实验采用高效液相色谱法确定红豆皮多酚的组成。通过最小抑菌浓度（minimum inhibitory concentration，MIC）和抑菌生长曲线分析红豆皮多酚提取物对李斯特菌ATCC19119和沙门氏菌ATCC14028的抑菌效果，并从菌体细胞蛋白和核酸含量、细胞膜电位、细胞内ATP含量、细胞外碱性磷酸酶活力和菌体细胞形态变化的角度来揭示其抑菌机理。结果表明，红豆皮多酚组成成分中含有13 种酚类化合物，红豆皮多酚提取物对李斯特菌ATCC19119和沙门氏菌ATCC14028的MIC分别为625 μg/mL和2 500 μg/mL，红豆皮多酚提取物的添加对菌体细胞蛋白和核酸含量、细胞膜电位、细胞内ATP含量、细胞外碱性磷酸酶含量和菌体细胞形态变化等有明显影响。综上，本研究结果可为粮食源天然抑菌产品的开发和粮食加工副产物的综合利用提供理论参考。     

Enrichment and DNA extraction protocols for the simultaneous detection of Salmonella and Listeria monocytogenes in raw sausage meat with multiplex real-time PCR

A novel method of DNA extraction and purification was developed and was used in conjunction with a multiplex real-time PCR assay for the simultaneous detection of Salmonella and Listeria monocytogenes in a raw meat sample. The PCR used primers targeting the invA gene of Salmonella and the hlyA gene of L. monocytogenes, and PCR products were detected with a LightCycler on the basis of fluorescence from SYBR Green and melting temperature. The assay allowed the detection of 3 Listeria cells and 4 Salmonella cells per g of the original sausage within 10 h, including an enrichment period of 6 to 8 h.     

Peptide nucleic acid fluorescence in situ hybridization for identification of Listeria genus, Listeria monocytogenes and Listeria ivanovii

A fluorescent in situ hybridization (FISH) method in conjunction with fluorescin-labeled peptide nucleic acid (PNA) probes (PNA-FISH) for detection of Listeria species was developed. In silico analysis showed that three PNA probes Lis-16S-1, Lm-16S-2 and Liv-16S-5 were suitable for specific identification of Listeria genus, Listeria monocytogenes and Listeria ivanovii, respectively. These probes were experimentally verified by their reactivity against 19 strains of six Listeria species (excluding newly described species Listeria marthii and Listeria rocourtiae) and eight other bacterial species. The PNA-FISH method was optimized as 30 min of hybridization with 0.2% Triton X-100 in the solution and used to identify 85 Listeria strains from individual putative Listeria colonies on PALCAM agar plates streaked from selectively enriched cultures of 780 food or food-related samples. Of the 85 Listeria strains, thirty-seven were identified as L. monocytogenes with the probe Lm-16S-2 and two as L. ivanovii with the probe Liv-16S-5 which was in agreement with the results obtained by the API LISTERIA method. Thus, the PNA-FISH protocol has the potential for identification of pathogenic Listeria spp. from food or food-related samples.     

恒温荧光核酸检测仪检测2种食源性致病菌的方法验证

目的以传统国标培养法作为参考比对方法,考察恒温荧光核酸检测仪(constant temperature fluorescent nucleic acid detector)检测食品(自然食品和加标食品)中食源性致病菌如阪崎肠杆菌和单核细胞增生李斯特氏菌的一致性。方法通过对食品中阪崎肠杆菌和单核细胞增生李斯特氏菌国标培养法和恒温荧光核酸检测仪方法检测结果的比对,考量后者方法的灵敏度、特异性、假阳性率、假阴性率和准确度。结果恒温荧光核酸检测仪检测食品中阪崎肠杆菌和单核细胞增生李斯特氏菌的灵敏度和特异性都是100%,假阴性率为0;假阳性率为0。结论恒温荧光核酸检测仪方法特异性强、准确度高、无一例漏检。     

Specific interaction of mannosylated glycopolymers with macrophage cells mediated by mannose receptor

Poly[N-p-vinylbenzyl-O-beta-mannopyranosyl-(1-4)-D-gluconamide] (PV-Man) is a polystyrene derivative that contains mannose moieties and interacts with the mannose-receptor-carrying macrophage cell line. To clarify the specific interaction between the PV-Man and the macrophage cell line J774A1, PV-Man polymer labeled with fluorescent fluorescein isothiocyanate (FITC) was used to amonitor the specific interaction, which was visualized by confocal laser microscopy. We found that PV-Man strongly binds to macrophage cells, probably due to a specific interaction mediated by the presence of mannose receptors on the cell membrane. The fluorescence intensity of PV-Man and macrophage cells was up to 7-fold that of any other glycopolymers bound to macrophage cells. Moreover, cellular fluorescence intensity increased significantly with increasing incubation time and polymer concentration. Many macrophage cells strongly express mannose and mannose receptor-related receptors, and receptor-mediated gene transfer via the mannose receptor using a PV-Man glycopolymer is a versatile means of targeted gene delivery into these cells.     

The deletion of CaVPS34 in the human pathogenic yeast Candida albicans causes defects in vesicle-mediated protein sorting and nuclear segregation

A Candida albicans null mutant of the phosphatidylinositol (PI) 3-kinase gene (CaVPS34) involved in virulence was examined by different microscopical techniques. We observed that vacuoles of the Cavps34 null mutant were considerably enlarged and electron-transparent. An interesting result obtained by transmission electron microscopy analysis of Cavps34 mutant cells was the aberrant patch-like accumulation of vesicles, which were localized in the periplasm close to the plasma membrane. We assume that the vesicles result from missorted prevacuolar compartments. In contrast to the accumulations of the specific endocytic dye FM4-64 in the vacuole membrane in C. albicans wild-type strains (ring staining pattern), the Cavps34 mutant strain showed a staining of punctuate structures, possibly multivesicular bodies (MVB), that are scattered all over the cell. This defect indicates a late block in endocytic vesicle transport. Measurement of the total activity of carboxypeptidase Y revealed significantly lower activity in Cavps34 mutant cells. This may indicate that carboxypeptidase Y is not properly activated as a result of mislocalization due to the lack of Vps34p. The deletion of the CaVPS34 gene caused disturbance of normal nuclear migration, which suggests that in the Cavps34 mutant the cell-size mediated control process of cell division is affected.     

基于CRISPR-Cas13的单增李斯特菌RNA快速检测方法的建立

为实现食品中单增李斯特菌污染的快速检测,本研究构建了一种基于CRISPR-Cas系统和Broccoli适配体的RNA均相检测技术。利用Cas 13与cr RNA锚定序列结合形成识别元件cr RNA-Cas13复合物,靶标RNA存在时可激活Cas 13的非特异性RNase活性,并利用点亮型RNA适配体Broccoli作为信号探针,监测cr RNA此-Cas13的活化状态。荧光值的变化与单增李斯特菌浓度存在线性关系,利用来检测单增李斯特菌。本研究所构建的检测可在30min内完成对于单增李斯特菌的的识别与检测,检出限为148CFU/m L,对细菌具有良好的检测特异性,可区分大肠杆菌、鼠伤寒沙门氏菌和蜡样芽孢杆菌。在牛奶模型中单增李斯特菌的加标回收率为95.15%～97.99%。该方法具有较好的灵敏度、特异性,可直接靶向检测致病菌RNA,无需逆转录、PCR扩增和核酸标记,简化了实验流程,对于实现食品中单增李斯特菌的现场检测及生物安全控制具有重要意义。     

Differential fluorescent staining of Listeria monocytogenes and a whey food soil for quantitative analysis of surface hygiene

The accurate monitoring of surface cleanliness in terms of bacterial contamination is usually carried out using methods such as plate counts or replica plating. However these methods take at least eighteen hours to obtain results and do not determine the presence or amount of residual organic material on a surface, which may interfere with cleaning and disinfection. This work describes the application of fluorescent stains to cells (Listeria monocytogenes) and food soil (solubilized whey) to optimize a dual staining method that can be used in the quantitative analysis of surface cleanability. Seven different stains were tested at a range of concentrations (0.3%–0.001 mg/ml) and application methods. The best stain combination for differential staining of L. monocytogenes and whey food soil was 0.1 mg/ml rhodamine B with 0.1 g/ml DAPI. Differential staining of the cells and soil occurred regardless of the application method. This method has been successfully used to demonstrate the hygienic status of surfaces in an industrial situation. This novel work enables quantitative assessment of soils and cells on surfaces.     

青钱柳多糖抑菌活性及及作用机理

为研究青钱柳多糖的抑菌活性和作用机制，用不同质量浓度的青钱柳多糖处理细菌，通过最小抑菌浓度（minimum inhibitory concentration, MIC）、抑菌生长曲线分析青钱柳多糖对细菌的抑菌效果，并从抑制效果最佳的受试菌菌体电导率、细胞蛋白和核酸含量、还原糖的释放、呼吸链脱氢酶活性、脂质过氧化程度、细胞内ATP含量、细胞外碱性磷酸酶活力的角度来揭示其抑菌机理。结果表明，青钱柳多糖对金黄色葡萄球菌（Staphylococcus aureus）、大肠杆菌（Escherichia coil）、单增李斯特氏菌（Listeria monocytogenes）和鼠伤寒沙门氏菌（Salmonella typhimurium）有明显的抑制作用，MIC分别为2、4、6、6 mg/mL，对枯草芽孢杆菌（Bacillus subtilis）的抑菌性不明显；对指示菌的电导率、蛋白质和核酸渗漏、还原糖释放、呼吸链脱氢酶活性、细菌脂质过氧化程度、菌体胞内ATP和胞外碱性磷酸酶（AKP）含量均有影响。以上现象表明，青钱柳多糖的抑菌性主要是破坏菌体细胞壁和细胞膜完整性，改变其通透性，使得胞内大小分子物质外泄，抑制了细胞的呼吸代谢和引起氧化损失，进而影响细胞的生长代谢或造成细胞死亡，达到抑菌效果。     

Spectrofluorimetric assessment of bacterial cell membrane damage by pulsed electric field

A rapid fluorescence staining technique was used to assess cell membrane damage and ensuing injury and death caused by pulsed electric field (PEF) treatment. Cell suspensions of Lactobacillus leichmannii ATCC 4797, Listeria monocytogenes Scott A and Escherichia coli O157:H7 ATCC 35150 were subjected to PEF for145.6 μs at field strengths of 5–20 kV/cm. Immediately after PEF treatment, cells were stained with propidium iodide (PI), and changes in fluorescence intensity were measured with a spectrofluorimeter. Increase in field strength decreased the count of survivors and proportionally increased the fluorescence intensity, confirming that cell inactivation by PEF is caused by membrane damage. Cells of E. coli O157:H7 were incubated with or without EDTA before exposure to PEF, but similar inactivation was observed, regardless of the EDTA pre-treatment. Increase in the fluorescence intensity, however, was appreciable in the EDTA-PEF-treated cells. The fluorescence staining technique, therefore, revealed membrane-related injury when EDTA pre-treated cells were PEF-treated. In conclusion, the fluorescence staining technique can be used to assess membrane damage associated with PEF treatments and is potentially useful in determining the relative sensitivity of microorganisms to PEF or monitoring the efficacy of such treatments.     

Induction of Vesicle Formation by Exposing Apple Tissue to Vacuum Impregnation

This paper demonstrates that vacuum impregnation of mature apple tissue in the presence of different sugars results in the formation of membrane vesicles inside the cells. Vesiculation is regarded to be a metabolic consequence of the impregnation process. Vesiculation is shown when the endocytic marker FM4-64 was impregnated into the apple tissue together with the sugar solutions. Vesicles were formed at the plasma membrane already 30 min after impregnation and remained inside the cells for at least 24 h, a metabolic process that was inhibited in the presence of chloroquine, a specific endosomic inhibitor. This phenomenon was not dependent on the osmotic strength when sucrose was used for impregnation. However, the vesiculation drastically dropped when a hypertonic trehalose solution was impregnated. We suggest that the impregnated sugars may not totally remain in the extracellular space between the cells, as normally believed, but at least a fraction might be incorporated into the cells.     

Quantification of Plesiomonas Shigelloides Using PCR Based on 23S rRNA Gene

A rapid and efficient procedure for quantitative detection of Plesiomonas shigelloides in pure culture was developed, which is the first report for quantitative detection of P. shigelloides by the polymerase chain reaction (PCR). A minimum of 4 CFU per PCR reaction could be detected. There was a linear relationship between the relative fluorescent intensities of the DNA bands and the log of the CFU from 4 to 1.2 × 10³ CFU per PCR reaction. We compared the effects of two different nucleic acid dyes, GelStar™ stain and ethidium bromide (EB), and evaluated the effects of two different staining methods with each dye. Adding the dye to the agarose solution before gel formation proved to yield superior results compared to staining the DNA bands after electrophoresis. The GelStar™ stain was found superior to EB with minimum detection levels of 4 CFU and 12 CFU per PCR reaction, respectively.     

Pulsed electric fields cause bacterial envelopes permeabilization depending on the treatment intensity, the treatment medium pH and the microorganism investigated

The relationship between membrane permeabilization and loss of viability by pulsed electric fields (PEF) depending on the treatment intensity and the treatment media pH in two gram-positive (Lactobacillus plantarum, Listeria monocytogenes) and two gram-negative (Escherichia coli, Salmonella senftenberg 775W) bacterial species has been investigated. Loss of membrane integrity was measured as increased uptake of the fluorescent dye propidium iodide (PI). Non-permanent/reversible permeabilization was detected when cells stained with PI during PEF resulted in higher fluorescence than that measured in cells stained after PEF. Whereas loss of viability of the two gram-negative bacteria was correlated with the sum of non-permanent and permanent membrane permeabilization when treated at pH 7.0, in the case of the two gram-positives, loss of viability was correlated with a permanent loss of membrane integrity. At pH 7.0, the four bacteria exhibited reversible permeabilization. However, whereas the gram-positives capable of reversing permeabilization survived, the gram-negative cells died, despite their capacity to reverse permeabilization immediately after PEF. Thus, resealing is not necessarily related to the survival of PEF-treated cells. In contrast, when cells were PEF-treated at pH 4.0 a more complicated picture emerged. Whereas loss of viability was correlated with a permanent loss of membrane integrity in L. monocytogenes cells, in L. plantarum the degree of permeabilization was higher, and in the gram-negative strains, much lower than the percentage of inactivated cells. These results support the view that membrane permeabilization is involved in the mechanism of bacterial inactivation by PEF, but the nature of membrane damage and its relationship with cell death depends on the bacterial species and the treatment medium pH.     

TaqMan实时荧光PCR对沙门氏菌能力验证样品的快速检测与鉴定

本研究依据GB 4789.4-2016标准对沙门氏菌ACAS-PT526能力验证样品进行常规培养法检测,同时使用TaqMan实时荧光聚合酶链反应（polymerase chain reaction,PCR）技术对预增菌培养物进行快速检测和鉴定。本研究首先以沙门氏菌特异性基因hut基因为靶基因,设计合成特异性引物和探针,提取各类食源性菌种的核酸DNA进行实时荧光PCR反应,仅沙门氏菌属出现阳性扩增,非沙门氏菌属、阴性对照和空白对照均无扩增信号,验证设计合成的引物探针具有较高的特异性。其次将能力验证样品和加标样品经预增菌、增菌、分离、纯化、生化试验和血清学鉴定,同时将预增菌培养物经实时荧光PCR测定后,18-D319和加标样品有显著的S型扩增曲线,Ct值分别为24.34和26.21,为沙门氏菌阳性,18-M906无显著荧光信号,Ct值>40.00,为沙门氏菌阴性。经API20E试剂条鉴定,18-D319为猪霍乱沙门菌亚利桑那亚种,鉴定百分率为99.90%,T值为0.97,18-M906为大肠埃希氏菌,鉴定百分率为99.80%,T值为0.94。实时荧光PCR检测结果与常规培养法检测结果一致,且更为简单快速,从预增菌到结果判定仅需12 h,结果准确度高,一批次可检测多个样品,可用于大量样品中沙门氏菌的快速筛查和对能力验证样品的检测验证。     

Quantification of Plesiomonas Shigelloides Using PCR Based on 23S rRNA Gene

A rapid and efficient procedure for quantitative detection of Plesiomonas shigelloides in pure culture was developed, which is the first report for quantitative detection of P. shigelloides by the polymerase chain reaction (PCR). A minimum of 4 CFU per PCR reaction could be detected. There was a linear relationship between the relative fluorescent intensities of the DNA bands and the log of the CFU from 4 to 1.2 × 10³ CFU per PCR reaction. We compared the effects of two different nucleic acid dyes, GelStar? stain and ethidium bromide (EB), and evaluated the effects of two different staining methods with each dye. Adding the dye to the agarose solution before gel formation proved to yield superior results compared to staining the DNA bands after electrophoresis. The GelStar? stain was found superior to EB with minimum detection levels of 4 CFU and 12 CFU per PCR reaction, respectively.     

基于纳米探针检测食源性肠炎沙门氏菌技术初探

目的建立纳米探针快速、准确检测食源性肠炎沙门氏菌的方法。方法利用二氧化硅磁纳米材料与肠炎沙门氏菌抗体制备纳米探针,将制备后的探针与经过荧光染色的肠炎沙门氏菌结合,并通过荧光显微镜和流式细胞仪对探针捕获的细菌进行检测,并比较2种方法的优劣。结果本方法设计合成了肠炎沙门氏菌纳米探针,通过荧光显微镜可以观察到复合纳米探针结合的5′106 CFU/mL和5′107 CFU/mL浓度的肠炎沙门氏菌,在放大400倍的显微镜视野中成倍递增,该探针结合流式细胞术可以检测到5′105 CFU/mL浓度的肠炎沙门氏菌。结论纳米探针技术结合荧光显微镜、流式细胞术可以更加快速、准确、灵敏地用于食源性肠炎沙门氏菌的定性检测,并有望通过进一步实验实现食源性致病菌的定量检测。     

Inactivation of Salmonella and Listeria in ground chicken breast meat during thermal processing.

Thermal inactivation of six Salmonella spp. and Listeria innocua was evaluated in ground chicken breast and liquid medium. Survival of Salmonella and Listeria was affected by the medium composition. Under the same thermal process condition, significantly more Salmonella and Listeria survived in chicken breast meat than in 0.1% peptone-agar solution. The thermal lethality of six tested Salmonella spp. was additive in chicken meat. Survival of Listeria in chicken meat during thermal processing was not affected by the presence of the six Salmonella spp. Sample size and shape affected the inactivation of Salmonella and Listeria in chicken meat during thermal processing.