Growth of Salmonella enteritidis and Salmonella typhimurium in the presence of quorum sensing signalling compounds produced by spoilage and pathogenic bacteria

The effect of acylated homoserine lactones (AHLs) and autoinducer-2 (AI-2) signalling compounds present in the cell-free culture supernatants (CFS), of Pseudomonas aeruginosa, Yersinia enterocolitica-like GTE 112, Serratia proteamaculans 00612, Y. enterocolitica CITY650 and Y. enterocolitica CITY844, on the growth of two Salmonella Enteritidis and two S. Typhimurium strains was assessed though monitoring of changes in conductance of the medium. Detection times (T_det), area and slope of conductance curves were recorded. Except for P. aeruginosa 108928, which was not found to produce AI-2, all other strains produced both AHLs and AI-2. Thereafter, aliquots (20% in the final volume) of these CFS were transferred into NZ Amine broth inoculated with ca. 10³ CFU/ml of stationary phase cultures of each Salmonella strain. While the CFS of P. aeruginosa induced a shorter detection time, i.e. acceleration of the metabolic activity, the CFS of the other microorganisms increased the detection time of Salmonella strains compared to control samples (i.e. without CFS). Results indicate that the growth of Salmonella may be affected by the presence of Quorum sensing (QS) signalling compounds and/or other novel signals existing in CFS, produced by other bacterial species and confirm the complexity of bacterial communication.     

Magnetoelastic biosensor for the detection of Salmonella typhimurium in food products

In this article, a magnetoelastic sensor immobilized with polyclonal antibody for the detection of Salmonella typhimurium in food products is described. The remote query nature of magnetoelastic sensors enables the detection of bacterial species in sealed and opaque containers. Bacterial binding to the antibody on the sensor surfaces changed the resonance parameters, and these changes were quantified by the shift in the sensor’s resonance frequency. Response of the sensors to increasing concentrations (5 × 10¹–5 × 10⁸ cfu/ml) of S. typhimurium in three different food products (water, fat-free milk and apple juice) was studied and similar responses were observed. These results were also further ascertained by Scanning Electron Microscopy (SEM) studies. A detection limit of 5 × 10³ cfu/ml, with a sensitivity of 139 Hz/decade was obtained for the sensors tested in water samples, as compared to 129 Hz/decade in apple juice and 127 Hz/decade in fat free milk. A 2 × 0.4 × 0.015 mm sensor was employed in all the investigations. The dissociation constant K _d and the binding valencies for S. typhimurium spiked in water samples was 435 cfu/ml and 2.33 respectively; as compared to 309 cfu/ml and 2.38 for apple juice; and 1389 cfu/ml and 1.85 for fat free milk samples. Bacterial binding was specific and a divalent binding was observed.     

Effect of sodium chlorate on Salmonella typhimurium concentrations in the weaned pig gut

Salmonella cause economic losses to the swine industry due to disease and compromised food safety. Since the gut is a major reservoir for Salmonella, strategies are sought to reduce their concentration in pigs immediately before processing. Respiratory nitrate reductase activity possessed by Salmonella also catalyzes the intracellular reduction of chlorate (an analog of nitrate) to chlorite, which is lethal to the microbe. Since most gastrointestinal anaerobes lack respiratory nitrate reductase, we conducted a study to determine if chlorate may selectively kill Salmonella within the pig gut. Weaned pigs orally infected with 8 x 10(7) CFU of a novobiocin- and nalidixic acid-resistant strain of Salmonella Typhimurium were treated 8 and 16 h later via oral gavage (10 ml) with 0 or 100 mM sodium chlorate. Pigs were euthanized at 8-h intervals after receiving the last treatment. Samples collected by necropsy were cultured qualitatively and quantitatively for Salmonella and for most probable numbers of total culturable anaerobes. A significant (P < 0.05) chlorate treatment effect was observed on cecal concentrations of Salmonella, with the largest reductions occurring 16 h after receiving the last chlorate treatment. An observed treatment by time after treatment interaction suggests the chlorate effect was concentration dependent. Chlorate treatment may provide a means to reduce foodborne pathogens immediately before harvest.     

食品检测用鼠伤寒沙门氏菌标准物质的研制

目的 研制均匀稳定的鼠伤寒沙门氏菌标准物质。方法 采用冷冻干燥技术制备含量为1.5-2.0×103 CFU /样品的菌球, 参照CNAS—GL29: 2010《标准物质/标准样品定值的一般原则和统计方法》, 随机抽取22件样品进行均匀性检验, 采用单因素方差分析对结果进行统计分析, 将样品分别于-20、4、25、37 ℃条件下保藏, 对其储藏稳定性和运输稳定性进行评价, 并组织3家实验室进行协同标定, 再使用45件食品作为基质, 按照国标法检验鼠伤寒沙门氏菌标准物质的适用性。结果 对22件标准物质的均匀性检测结果进行单因素方差分析, F=1.986, 符合标准物质的要求。标准物质在?20 ℃保藏28 d, 复苏率为103.1%; 在4 ℃保藏28 d, 复苏率为102.0%; 在25 ℃保藏14 d或者37℃保藏7 d, 样品中菌含量仍保持在103 CFU/样品的水平, 说明样品的短期储藏稳定性、长期储藏稳定性和运输稳定性都符合要求。经3家实验室协同标定, 样品活菌含量均在103 CFU/样品水平, 生化鉴定结果均符合沙门氏菌的特征; 标准物质加入到45种食品基质中, 均可以检出沙门氏菌。结论 本研究所制备的鼠伤寒沙门氏菌标准物质的均匀性、储藏稳定性和运输稳定性均符合要求, 适用性良好, 可用于食品检测实验室的质量控制和食品中沙门氏菌检测结果的评价。     

基于pH响应聚合物的比色法检测牛奶中的鼠伤寒沙门氏菌

目的 基于pH响应聚合物构建一种用于检测牛奶中鼠伤寒沙门氏菌的比色传感器。方法 通过溶剂诱导法,使用酚酞、适配体、牛血清白蛋白制备pH响应聚合物,基于适配体对鼠伤寒沙门氏菌的特异性结合与pH响应聚合物遇碱释放酚酞的比色反应建立鼠伤寒沙门氏菌比色检测方法,优化反应条件,并将其用于检测牛奶。结果 该传感器在鼠伤寒沙门氏菌菌液浓度10²～10⁷ CFU/mL范围内与吸光度呈现良好的线性关系,线性系数为0.982,检出限可达52 CFU/mL。将此法用于牛奶中鼠伤寒沙门氏菌的检测,加标回收率为83.2%～102.0%。结论 该方法操作简便、结果可视化,为牛奶中鼠伤寒沙门氏菌的快速检测提供了一种新思路。     

Evaluation of a capillary immunoassay system for detection of Salmonella typhimurium in poultry products

A capillary immunoassay system was constructed and optimized for detection of Salmonella Typhimurium. The system consisted of a capillary bioseparator-bioreactor and a flow-injection electrochemical detector. Three methods were compared for immobilizing antibodies on the inner surface of silica capillary columns; these methods were based on the use of a homobifunctional cross-linker glutaraldehyde, a heterobifunctional cross-linker N-succinimidyl-4-maleimidobutyrate, and biotin-streptavidin chemistry, respectively. The glutaraldehyde method gave the best reproducibility with a relative standard deviation of 1 to 6% for detection of Salmonella Typhimurium. The optimized immunoassay system could detect Salmonella Typhimurium in chicken breast and ground turkey meats with a detection limit of 2.4 x 10(3) and 2.4 x 10(4) CFU/ml, respectively. The total detection time was less than 2.5 h without any preenrichment. When stored at 4 degrees C, the immunocolumns could retain their activities for at least 3 months.     

Rapid detection of Salmonella typhimurium in chicken carcass wash water using an immunoelectrochemical method

An immunoelectrochemical method coupled with immunomagnetic separation was developed for rapid detection of Salmonella Typhimurium in chicken carcass wash water. Samples of chicken carcass wash water were inoculated with Salmonella Typhimurium at different cell numbers. Possible nonspecified inhibitors in the wash water were minimized by filtration and centrifugation. An approximately 9.4% loss of Salmonella cells was found after filtration (P < 0.01). The samples were mixed with anti-Salmonella-coated magnetic beads (ASCMB) and alkaline phosphatase-labeled anti-Salmonella (APLAS) to form ASCMB-Salmonella-APLAS conjugates. The conjugates were separated from the solution using a magnetic separator and then incubated with phenylphosphate substrate to produce phenol. The number of Salmonella was determined by measuring the phenol concentration using an amperometric tyrosinase carbon paste electrode in a flow injection analysis system. Under optimized parameters (1 mM MgCl2, 0.2 microg/ml APLAS, and 1 mM phenylphosphate in pH 7.0 Tris buffer solution), Salmonella Typhimurium in chicken carcass wash water could be identified and enumerated within 2.5 h with a detection limit of 5 x 10(3) CFU/ml. A linear relationship on a log-log scale was found between Salmonella cell number and the peak current ratio for Salmonella concentrations ranging from 10(3) to 10(7) CFU/ml (R2 = 0.963). The peak currents of multibacteria samples, containing Salmonella Typhimurium, Listeria monocytogenes, and Campylobacter jejuni, were not significantly different from Salmonella-only samples (P > 0.01).     

Inactivation of Salmonella typhimurium DT 104 in UHT whole milk by high hydrostatic pressure

Cell suspensions of Salmonella typhimurium DT 104 in ultra-high temperature (UHT) whole milk were exposed to high hydrostatic pressure at 350, 400, 450, 500, 550, and 600 MPa at ambient temperature (ca. 21 degrees C). Tailing was observed in all survival curves, and sigmoidal survival curves were observed at relatively high pressure (500-600 MPa). Four modeling methods (linear and nonlinear including Weibull, modified Gompertz, and log-logistic models) were fitted to these data at 500, 550, and 600 MPa. Performances of the modeling methods were compared using mean square error (MSE). The linear regression model at these three pressure levels had a mean square error (MSE) of 1.260-2.263. Nonlinear regressions using Weibull, modified Gompertz, and log-logistic models had MSE values in the range of 0.334-0.764, 0.601-1.479, and 0.359-0.523, respectively. Modeling results indicated that first-order kinetics could not accurately describe pressure inactivation of S. typhimurium DT 104 in UHT milk; the log-logistic model produced the best fit to data.     

Distribution of Salmonella typhimurium in romaine lettuce leaves

Leafy greens are occasionally involved in outbreaks of enteric pathogens. In order to control the plant contamination it is necessary to understand the factors that influence enteric pathogen-plant interactions. Attachment of Salmonella enterica serovar typhimurium to lettuce leaves has been demonstrated before; however, only limited information is available regarding the localization and distribution of immigrant Salmonella on the leaf surface. To extend our knowledge regarding initial pathogen-leaf interactions, the distribution of green-fluorescent protein-labeled Salmonella typhimurium on artificially contaminated romaine lettuce leaves was analyzed. We demonstrate that attachment of Salmonella to different leaf regions is highly variable; yet a higher attachment level was observed on leaf regions localized close to the petiole (7.7 log CFU g⁻¹) compared to surfaces at the far-end region of the leaf blade (6.2 log CFU g⁻¹). Attachment to surfaces located at a central leaf region demonstrated intermediate attachment level (7.0 log CFU g⁻¹). Salmonella displayed higher affinity toward the abaxial side compared to the adaxial side of the same leaf region. Rarely, Salmonella cells were also visualized underneath stomata within the parenchymal tissue, supporting the notion that this pathogen can also internalize romaine lettuce leaves. Comparison of attachment to leaves of different ages showed that Salmonella displayed higher affinity to older compared to younger leaves (1.5 log). Scanning electron microscopy revealed a more complex topography on the surface of older leaves, as well as on the abaxial side of the examined leaf tissue supporting the notion that a higher attachment level might be correlated with a more composite leaf landscape. Our findings indicate that initial attachment of Salmonella to romaine lettuce leaf depends on multiple plant factors pertaining to the specific localization on the leaf tissue and to the developmental stage of the leaf.     

Quantum dots as fluorescent labels for quantitative detection of Salmonella typhimurium in chicken carcass wash water

Fluorescent semiconductor quantum dots have recently emerged as a novel and promising class of fluorescent labels for biological detection. In this study, quantum dots were used as fluorescent labels in immunoassays for quantitative detection of foodborne pathogenic bacteria. Salmonella Typhimurium cells were separated from chicken carcass wash water using anti-Salmonella antibody coated magnetic beads and reacted to secondary biotin-labeled anti-Salmonella antibody. Quantum dots coated with streptavidin were added to react with biotin on the secondary antibody. Measurement of the intensity of fluorescence produced by quantum dots provided a quantitative method for microbial detection. A linear relationship between Salmonella Typhimurium cell number (log N) in the samples of chicken carcass wash water and the fluorescence intensity (FI) was found for the cell numbers ranging from 10(3) to 10(7) CFU/ml. The regression model can be expressed as FI = 198.6 Log N - 639.03 with R2 = 0.96. The detection limit of this method was 10(3) CFU/ml.     

生物传感器在食品检测领域的应用研究进展

近年来食品安全事故频繁发生,食品的安全问题被广泛关注。现阶段,谷物、蔬菜、水果等农产品的种植及采摘后的加工处理过程虽均在相对安全卫生的条件下进行,但因环境污染及使用化学药品处理,处理后的食品是否依旧安全可靠存在较大的不确定性。传统食品检测技术操作复杂、专业性强、仪器设备贵且无法实现现场操作,而生物传感器操作简单、可重复性好、易于实现快速的现场检测且检测成本低,近年来在食品安全检测方面被广泛研究利用。本文主要阐述了生物传感器在食品检测中的重金属离子、H2O2、生物毒素、食源性致病菌、有机磷农药残留、动物源药物残留等方面的应用,并简述了生物传感器在建立过程中所存在的问题,可为日后生物传感器在食品安全检测方面的进一步研发利用提供参考。     

Development of an isothermal amplification-based assay for the rapid visual detection of Salmonella bacteria

The efficient and timely detection of pathogens is a major concern worldwide. The aim of this study was to establish a rapid detection method for Salmonella bacteria in food samples to facilitate timely treatment. Widely used detection methods currently include culture-based methods and PCR-based methods. The former are time consuming, requiring 2 to 3 d, whereas the latter have higher accuracy but are typically complicated, requiring expertise and expensive instruments. In this study, a sensitive and rapid approach for the visual and point-of-use detection of Salmonella bacteria based on recombinase polymerase amplification (RPA) and a lateral-flow (LF) nucleic acid strip was established. We designed a pair of primers according to the invA gene of Salmonella bacteria: one was modified with digoxin, and the other was modified with biotin. In the presence of the biotin- and digoxin-modified primers and target DNA, the RPA produced a substantial amount of duplex DNA attached to biotin and digoxin. The products were detected using LF strips through immunoreaction: anti-digoxin antibodies on the gold nanoparticles, digoxin on the duplex, streptavidin on the LF test line, and biotin on the duplex. The developed RPA-LF assay allowed detection of Salmonella genomic DNA in less than 20 min with simple water bath equipment or portable thermal equipment. In addition, the RPA-LF assay was highly sensitive, with a detection limit as low as 20 fg of target DNA or 1.05 × 10¹ cfu of bacteria in pure culture, and highly specific, exhibiting no cross-reaction with Staphylococcus aureus, Escherichia coli, Listeria monocytogenes, Shigella, Enterobacter aerogenes, or Campylobacter jejuni. Importantly, Salmonella could be detected in milk and chicken breast at concentrations as low as 1.05 × 10⁰ cfu/mL or 1.05 × 10⁰ cfu/g after enrichment for 2 h and in eggs at 1.05 × 10⁰ cfu/g after enrichment for 4 h. Furthermore, RPA was more sensitive than PCR, which requires a thermal cycling device. In summary, this study describes a sensitive, simple, and point-of-use detection method for Salmonella bacteria.     

Growth response of Salmonella typhimurium in the presence of natural and synthetic antimicrobials: estimation of MICs from three different models

The aim of this study was to determine the MICs of 14 antimicrobials for Salmonella Typhimurium with three methods and to check the influence of experiment duration on the estimation of MICs. The growth of Salmonella Typhimurium in a brain heart infusion medium containing various concentrations of natural aromatic compounds, organic acids, or salts was monitored by absorbance measurements for 24 or 72 h. Three different ways of analyzing optical density (OD) curves were tested for the determination of MICs. Both quantitative methods gave similar MICs for most of the compounds. The semiquantitative method does not allow estimating the MIC for all compounds. Noticeable differences were found between MICs obtained for 24- or 72-h experiments, whatever the method used. The proposed methods and models can be used for the estimation of MICs from OD data. MICs could be used for a quantitative approach to Salmonella Typhimurium growth.     

PCR法检测肉鸭屠宰加工生产链中沙门氏菌的污染

为建立肉鸭加工中快速、准确的沙门氏菌PCR 检测方法,并应用于肉鸭屠宰生产加工链沙门氏菌的实时监测。采用Primer Premier 5.0 软件针对沙门氏菌特有的fimY 基因设计合成一对引物5′- GCATTCCGCTCATTAGAT-3′和5′-TGGAGGCTGATAACAAGG-3′,并对沙门氏菌DNA 扩增PCR 体系的退火温度、引物浓度、Mg2+ 浓度和聚合酶浓度进行优化。结果表明：成功扩增出沙门氏菌标准株和分离株的2 7 5b p 目的片段,灵敏度达到了1.2pg;应用建立的PCR 方法对国内某典型肉鸭屠宰厂的肉鸭屠宰链环节进行沙门氏菌污染的检测,发现在屠宰环境、拔毛浸烫水、浸蜡冷却水、清洗池水、宰前毛、食道、粪便和沥水体腔内共有25 个样品中扩增出了275bp大小的特异性片段,而空白对照无扩增条带。     

Mutagenicity evaluation of nitroanilines and nitroaminophenols in Salmonella typhimurium

In our studies of structure-activity relationships, three nitroanilines and nine nitroaminophenols were tested for their ability to induce mutations in Salmonella typhimurium strains TA1535, TA100, TA1537, TA1538 and TA98. The compounds m -nitroaniline, 4-nitro-2-aminophenol, and 3-nitro-6-aminophenol were active in two or more of these strains. The compounds o -nitroaniline, 2-nitro-3-aminophenol, 3-nitro-4-aminophenol, 4-nitro-3-aminophenol, 3-nitro-2-aminophenol, 2-nitro-6-aminophenol, 2-nitro-4-aminophenol and 2-nitro-5-aminophenol were inactive, both in the presence and in the absence of S9 mix. The compound p -nitroaniline was also inactive in all tests with the possible exception of that in strain TA98 in the presence of S9 mix, where it was either very weakly mutagenic or non-mutagenic. The mutagenic activity or inactivity of these compounds appears to be dependent on the positions of the amino, nitro, and hydroxy groups in their chemical structures.
Etude en mutagénèse des nitroanilines et nitroaminophénols sur Salmonella typhimurium .     

Simultaneous detection by PCR of Escherichia coli, Listeria monocytogenes and Salmonella typhimurium in artificially inoculated wheat grain

A multiplex PCR procedure was established to detect Escherichia coli, Listeria monocytogenes and Salmonella typhimurium in artificially inoculated wheat grain. The PCR protocol with an enrichment step successfully detected all three organisms inoculated together in non-autoclaved wheat grain. After a one day enrichment, E. coli, L. monocytogenes and S. typhimurium were detected at levels of 56, 1800 and <54 CFU/mL, respectively, in the initial sample. For L. monocytogenes, an improved detection limit of <62 CFU/mL was achieved using singleplex PCR. For autoclaved wheat grain inoculated with the three bacterial strains individually, a detection limit of 3 CFU/mL was achieved after an enrichment step. The ability to test for the three bacteria simultaneously will save time and increase the ability to assure grain quality.     

沙拉沙星体外诱导鼠伤寒沙门菌耐药实验

目的为获得体外鼠伤寒沙门菌(Salmonella typhimurium)对盐酸沙拉沙星(sarafloxacin hydrochloride)的活性耐药菌株,研究诱导其产生耐药的盐酸沙拉沙星最低浓度,为评价食品中沙拉沙星残留导致的微生物耐药提供依据。方法设置盐酸沙拉沙星0.001、0.002 5、0.005、0.025、0.05、0.1μg/ml实验组和空白对照组、NaOH溶剂对照组,采用NCCLS法测试最小抑菌浓度,对鼠伤寒沙门菌进行耐药诱导,耐药判断为≥8×MIC(0.25μg/ml)。对产生耐药的鼠伤寒沙门菌的gyrA基因片段进行PCR扩增,焦磷酸凝胶测序法鉴定gyrA基因常见5个位点观察突变。结果盐酸沙拉沙星在0.005μg/ml水平传到第10代时,MIC增大32倍,抑菌浓度增加到1μg/ml,耐药菌增殖停止;传到第25代时,鼠伤寒沙门菌gyrA基因Ser83位点发生突变,获得多株稳定、有生物活性的耐药菌株。沙拉沙星浓度≥0.025μg/ml时,细菌不生长;沙拉沙星浓度≤0.002 5μg/ml时细菌生长,不产生耐药。结论沙拉沙星浓度为0.005μg/ml时可体外诱导鼠伤寒沙门菌产生耐药,经传代后获得具生物活性的耐药菌株。     

鼠伤寒沙门菌多重耐药性机制研究进展

AcrAB外流泵系统的过度表达和拓扑异构酶基因突变是导致鼠伤寒沙门菌产生多重耐药性 (MAR)的重要原因。AcrAB外流泵系统的合成有多重耐药调节子 (marRAB)调控 ,AcrAB与多种抗生素的耐药有关。拓扑异构酶 (topoisomerase)的突变主要与氟喹诺酮类药物的耐药性有关 ,突变主要发生在gyrA ,parC基因上     

基于核酸适配体的生物传感技术检测食源性致病菌研究进展

食品供应的全球化导致食源性疾病传播快、分布广,严重威胁人类健康。病原检测需要特异性强和灵敏度高的方法。核酸适配体是可以识别并与多种类型靶标分子的单链DNA或RNA。基于核酸适配体的生物传感器特异性好、灵敏度高、易储存。为食源性致病菌快速检测提供了新的方法。本文介绍了核酸适配体的特点和筛选技术,综述了基于适配体的电化学、比色、荧光、表面增强拉曼散射和质量生物传感器技术的基本原理及其在食源性致病菌检测中的应用,以期为开发高效、精准检测食源致病菌技术提供参考。     

Adhesion of Salmonella typhimurium var. copenhagen in the intestines of pigeons.

The attachment of Salmonella typhimurium var. copenhagen to the intestinal epithelium of pigeons was studied. After experimental infection, the intestines of both young and adult pigeons were examined in vivo and in vitro. Investigations were carried out by electron and immunofluorescence microscopy. Attachment of the Salmonella strain to duodenal epithelium was established. Following repeated washing after inoculation, bacterial cells could be seen by SEM and TEM in association with the surface structure of the tissue. The adhesive properties of fimbriae, which are also involved in haemagglutination, could be demonstrated.