Evaluation of epifluorescence image analysis of biofilm growth on stainless steel surfaces

Biofilm growth of Bacillus subtilis, Pseudomonas fragi, Pediococcus inopinatus and Listeria monocytogenes was studied on stainless steel surfaces at room and low temperatures to evaluate the results of traditional hygiene measures. The results were compared with those of image analysis of stainless steel surfaces in an epifluorescence microscope. Statistical analyses were carried out to determine the variations between the conventional cultivation swab method, the glycocalyx amount obtained using swabbing, and the values of the areas of the biofilm, slime and cells. As a general rule, old biofilms showed total counts at approximately the same levels as the young biofilm. The results showed that temperature affected the results for all strains except B. subtilis. The strains of Pe. inopinatus and Ps. fragi showed increased attachment at 6°C and L. monocytogenes at 25°C. The biofilm slime was more easily detached than the cells. The results indicated that the traditional swab method is not reliable for the measurement of biofilm formation on surfaces.     

基于显微共聚焦拉曼技术对三种食源性致病菌的快速鉴别研究

采用显微共聚焦拉曼技术,建立了对三种常见食源性致病菌快速鉴别的检测方法。使用XploRA PLUS共聚焦拉曼光谱仪,在激光功率为5 mW、积分时间为30s、积分次数为1次的条件下,对德尔卑沙门氏菌、副溶血性弧菌和金黄色葡萄球菌进行了拉曼光谱数据的采集。对拉曼光谱采用多项式平滑算法和荧光背底扣除后,采用主成分分析法(PCA)对预处理后的数据进行降维,提取出前三个主成分的累计方差贡献率达到了95.4％,样本明显的聚为了3类。同时结合Fisher判别分析法(FLD)构建分类模型,对三种样本进行交叉验证,分类准确率达到了100％。结果表明,采用显微共聚焦拉曼技术与PCA-FLD方法结合可实现对三种食源性致病菌的快速准确鉴别且模型检测精度高,方法具有一定的实用性及参考价值。     

Inactivation of foodborne pathogens based on synergistic effects of ultrasound and natural compounds during fresh produce washing

Ultrasound has potential to be used for disinfection, and its antimicrobial effectiveness can be enhanced in presence of natural compounds. In this study, we compared the antimicrobial effects of ultrasound at 20 kHz (US 20 kHz) or 1 MHz (US 1 MHz) in combination with carvacrol, citral, cinnamic acid, geraniol, gallic acid, lactic acid, or limonene against E. coli K12 and Listeria innocua at a constant power density in water. Compared to the cumulative effect of the individual treatments, the combined treatment of US 1 MHz and 10 mM citral generated >1.5 log CFU/mL additional inactivation of E. coli K12. Similarly, combined treatments of US 1 MHz and 2 mM carvacrol (30 min), US 20 kHz and 2 mM carvacrol, 10 mM citral, or 5 mM geraniol (15 min) generated >0.5–2.0 log CFU/mL additional inactivation in L. innocua. The synergistic effect of citral, as a presentative compound, and US 20 kHz treatment was determined to be a result of enhanced dispersion of insoluble citral droplets in combination with physical impact on bacterial membrane structures, whereas the inactivation by US 1 MHz was likely due to generation of oxidative stress within the bacteria. Combined ultrasound and citral treatments improved the bacterial inactivation in simulated wash water in presence of organic matter or during washing of inoculated blueberries but only additive antimicrobial effects were observed. Findings in this study will be useful to enhance fresh produce safety and shelf-life and design other alternative ultrasound based sanitation processes.     

基于硒化锌薄膜法的食源性致病菌原位红外光谱检测

食源性致病菌是引发和威胁公众健康的主要因素之一.由于食源性致病菌种类繁多,常规检测方法复杂耗时要求高,因此迫切需要一种更加快速精确的致病菌检测技术.在传统红外光谱检测致病菌的流程中,如经典的溴化钾压片法,除了压片本身的操作之外通常还需对样品进行冷冻干燥(约需2 d)等耗时前处理过程,因而不利于高通量快速检测.本研究利用...     

Electrochemical genosensing of Salmonella, Listeria and Escherichia coli on silica magnetic particles

A magneto-genosensing approach for the detection of the three most common pathogenic bacteria in food safety, such as Salmonella, Listeria and Escherichia coli is presented. The methodology is based on the detection of the tagged amplified DNA obtained by single-tagging PCR with a set of specific primers for each pathogen, followed by electrochemical magneto-genosensing on silica magnetic particles. A set of primers were selected for the amplification of the invA (278 bp), prfA (217 bp) and eaeA (151 bp) being one of the primers for each set tagged with fluorescein, biotin and digoxigenin coding for Salmonella enterica, Listeria monocytogenes and E. coli, respectively. The single-tagged amplicons were then immobilized on silica MPs based on the nucleic acid-binding properties of silica particles in the presence of the chaotropic agent as guanidinium thiocyanate. The assessment of the silica MPs as a platform for electrochemical magneto-genosensing is described, including the main parameters to selectively attach longer dsDNA fragments instead of shorter ssDNA primers based on their negative charge density of the sugar-phosphate backbone. This approach resulted to be a promising detection tool with sensing features of rapidity and sensitivity very suitable to be implemented on DNA biosensors and microfluidic platforms.     

Simultaneous detection of Escherichia coli O157:H7, Salmonella spp. and Listeria monocytogenes with an array-based immunosorbent assay using universal protein G-liposomal nanovesicles

A novel universal reagent for immunoassays, protein G-liposomal nanovesicles has been developed and successfully used in an immunomagnetic bead sandwich assay for the detection of Escherichia coli O157:H7 [C.-S. Chen, A.J. Baeumner, R.A. Durst, Talanta 67 (2005) 205]. To demonstrate the universal capability of protein G-liposomal nanovesicles, this reagent was used to develop an array-based immunosorbent assay for the simultaneous detection of E. coli O157:H7, Salmonella, and Listeria monocytogenes. Both direct and competitive immunoassay formats were used to demonstrate the feasibility of detecting multiple analytes in a single test by using universal protein G-liposomal nanovesicles. Both pure and mixed cultures were examined in the direct immunoassay format. Results indicate that the limits of detection (LODs) of the direct assay for E. coli O157:H7, Salmonella enterica serovar Typhimurium and L. monocytogenes in pure cultures were approximately 100, 500 and 1.5 × 10⁴ CFU/ml, respectively. In mixed cultures, the LODs were approximately 3.1 × 10³, 7.8 × 10⁴, and 7.9 × 10⁵ CFU/ml. In the competitive assay format, the LODs for E. coli O157:H7, S. enterica serovar Typhimurium, and L. monocytogenes were approximately 1.5 × 10⁴, 5 × 10⁴, and 1.2 × 10⁵ CFU/ml for the pure cultures. These results showed that protein G-liposomal nanovesicles can be successfully used in a simultaneous immunoassay for several food-borne pathogens, thereby demonstrating that they are effective universal reagents for use in immunoassays.     

多响应曲面优化-毛细管电泳-激光诱导荧光快速检测食源性致病菌

建立了食品中常见致病菌: 沙门菌的invA基因、大肠杆菌O157∶H7的rfbO157基因、志贺菌的ipaH基因及副溶血性弧菌Vpara(16S-23S rDNA IGS)基因的多重PCR产物-毛细管电泳快速检测方法. 根据沙门菌、大肠杆菌O157∶H7、志贺菌及副溶血性弧菌的特异性基因保守序列设计出多重PCR引物, 优化PCR 扩增反应体系. 采用多响应曲面法优化毛细管电泳的分离条件, 以含有DNA荧光染料SYBR Green Ⅰ的1.0％甲基纤维素为筛分介质, 通过毛细管电泳-激光诱导荧光同时检测4种常见致病菌的PCR 扩增产物. 在优化的多重PCR反应体系和毛细管筛分电泳条件下, 此方法可以同时检测出沙门菌的invA基因、大肠杆菌O157∶H7的rfbO157基因、志贺菌的ipaH基因及副溶血性弧菌Vpara(16S-23S rDNA IGS)基因的多重PCR扩增产物, 25 min内即可完成检测. 迁移时间的日内相对标准偏差为0.92%~1.58%. 通过多响应曲面的优化, 有效改善了毛细管电泳对DNA分子的分离能力.     

Garlic essential oil in water nanoemulsion prepared by high-power ultrasound: Properties,stability and its antibacterial mechanism against MRSA isolated from pork

Food-borne methicillin-resistance Staphylococcus aureus (MRSA) has caused significant health threats and economic loss in livestock and poultry products. Garlic essential oil (GEO) is an effective antibacterial agent but presents strong instability and hydrophobicity. In this study, GEO in water nanoemulsion (GEON) with good stability was produced by emulsification technique of high-power ultrasound. Its antibacterial activity and underlying mechanism against MRSA isolated from retailed pork were investigated. Results showed that ultrasonic treatment significantly reduced the particle size of GENO from 820.3 to 215.0 nm as time increased from 0 to 10 min. Comparatively, GEON of 10 min ultrasound was more stable than other GEONs (0, 1, 5 min) during 30 d storage. It also displayed good thermal stability and relatively good ion stability (NaCl, MgCl₂, and glucose). Antibacterial analysis showed that GEON (10 min) exhibited the best anti-MRSA activity among all GEONs, and the minimum inhibitory concentration of GEO in this nanoemulsion was 0.125 % (1.25 mg/mL). Treatment of GEON (10 min) significantly suppressed the cell proliferation of MRSA, which was mainly achieved by damaging the cell membrane as evidenced by membrane depolarization and considerable leakage of intracellular nucleic acids and protein. Laser scanning confocal microscope and scanning electron microscopy showed that treatment of GEON (10 min) significantly altered the membrane integrity and severely damaged the cellular membrane and structure. The present work illustrated that GEON produced by ultrasonic emulsification is a promising alternative to inhibit the contamination and spread of MRSA in livestock and poultry products.     

Segmented continuous-flow multiplex polymerase chain reaction microfluidics for high-throughput and rapid foodborne pathogen detection

High-throughput and rapid identification of multiple foodborne bacterial pathogens is vital in global public health and food industry. To fulfill this need, we propose a segmented continuous-flow multiplex polymerase chain reaction (SCF-MPCR) on a spiral-channel microfluidic device. The device consists of a disposable polytetrafluoroethylene (PTFE) capillary microchannel coiled on three isothermal blocks. Within the channel, n segmented flow regimes are sequentially generated, and m-plex PCR is individually performed in each regime when each mixture is driven to pass three temperature zones, thus providing a rapid analysis throughput of m × n. To characterize the performance of the microfluidic device, continuous-flow multiplex PCR in a single segmented flow has been evaluated by investigating the effect of key reaction parameters, including annealing temperatures, flow rates, polymerase concentration and amount of input DNA. With the optimized parameters, the genomic DNAs from Salmonella enterica, Listeria monocytogenes, Escherichia coli O157:H7 and Staphylococcus aureus could be amplified simultaneously in 19 min, and the limit of detection was low, down to 10² copies μL⁻¹. As proof of principle, the spiral-channel SCF-MPCR was applied to sequentially amplify four different bacterial pathogens from banana, milk, and sausage, displaying a throughput of 4 × 3 with no detectable cross-contamination.