ATP bioluminescence rapid detection of total viable count in soy sauce

The adenosine triphosphate (ATP) bioluminescence rapid determination method may be useful for enumerating the total viable count (TVC) in soy sauce, as it has been previously used in food and beverages for sanitation with good precision. However, many factors interfere with the correlation between total aerobic plate counts and ATP bioluminescence. This study investigated these interfering factors, including ingredients of soy sauce and bacteria at different physiological stages. Using the ATP bioluminescence method, TVC was obtained within 4 h, compared to 48 h required for the conventional aerobic plate count (APC) method. Our results also indicated a high correlation coefficient (r = 0.90) between total aerobic plate counts and ATP bioluminescence after filtration and resuscitation with special medium. The limit of quantification of the novel detection method is 100 CFU/mL; there is a good linear correlation between the bioluminescence intensity and TVC in soy sauce in the range 1 × 10²–3 × 10⁴ CFU/mL and even wider. The method employed a luminescence recorder (Tristar LB‐941) and 96‐well plates and could analyse 50–100 samples simultaneously at low cost. In this study, we evaluated and eliminated the interfering factors and made the ATP bioluminescence rapid method available for enumerating TVC in soy sauce. Copyright © 2011 John Wiley & Sons, Ltd.     

生物发光及化学发光在生物医学领域中应用的进展

生物发光和化学发光在生物医学领域内的应用主要包括细胞学检测,分子生物学、卫生学检测,生物传感器、脂质过氧化检测和药物筛选等六个方面,其中细胞学检测主要是利用细胞内ＡＴＰ导致的虫荧光素酶发光进行活细胞计数,目前已实现快速、动态、单细胞分析;同时发现了一些新的与生物或化学发光有关的细胞学指标。分子生物学领域内的应用主要为报告基因和分子杂交,近年来又有人推出了生物发光实时ＤＮＡ测序技术。卫生学检测则主要     

Characterization of Bioluminescent Derivatives of Assimilable Organic Carbon Test Bacteria

The assimilable organic carbon (AOC) test is a standardized measure of the bacterial growth potential of treated water. We describe the design and initial development of an AOC assay that uses bioluminescent derivatives of AOC test bacteria. Our assay is based on the observation that bioluminescence peaks at full cell yield just prior to the onset of the stationary phase during growth in a water sample. Pseudomonas fluorescens P-17 and Spirillum sp. strain NOX bacteria were mutagenized with luxCDABE operon fusion and inducible transposons and were selected on minimal medium. Independent mutants were screened for high luminescence activity and predicted AOC assay sensitivity. All mutants tested were able to grow in tap water under AOC assay conditions. Strains P-17 I5 (with p-aminosalicylate inducer) and NOX I3 were chosen for use in the bioluminescence AOC test. Peak bioluminescence and plate count AOC were linearly related for both test bacteria, though data suggest that the P-17 bioluminescence assay requires more consistent luminescence monitoring. Bioluminescence results were obtained 2 or 3 days postinoculation, compared with 5 days for the ATP luminescence AOC assay and 8 days for the plate count assay. Plate count AOC assay results for nonmutant and bioluminescent bacteria from 36 water samples showed insignificant differences, indicating that the luminescent bacteria retained a full range of AOC measurement capability. This bioluminescence method is amenable to automation with a microplate format with programmable reagent injection.     

不同方法检测鸡蛋表面菌落总数的相关性研究

研究了ATP生物荧光检测法与国标法《GB4789.2-2010食品卫生微生物学检验菌落总数测定》检测鸡蛋壳表面细菌总数的相关性。采用ATP生物荧光检测法和国标法对40个混合样品表面细菌总数进行检测,以log CFU/个蛋壳为横坐标(x),以logRLU/个蛋壳为纵坐标(y),分别进行线性、对数、乘幂、指数拟合。结果表明,ATP荧光检测法与国标法检测结果 Pearson相关系数为0.912,线性模型y=0.7306x-1.0041(R2=0.8322)拟合度较高。该试验结果为ATP荧光检测法在鸡蛋壳表面细菌总数快速检测中应用的可行性提供了依据。     

A robust and fast bacteria counting method using CdSe/ZnS core/shell quantum dots as labels

A rapid, sensitive fluorescence measurement method for detecting the bacterial count using CdSe/ZnS as a fluorescence marker was described. High-quality CdSe/ZnS nanocrystals were synthesized and successfully conjugated with bacteria. The fluorescence intensity was proportional to bacterial count in the range of 10²–10⁸ CFU/mL and the low detection limit was 10² CFU/mL.     

Bacteriophage immobilized graphene electrodes for impedimetric sensing of bacteria (Staphylococcus arlettae)

Bacteriophages are a class of viruses that specifically infect and replicate within a bacterium. They possess inherent affinity and specificity to the particular bacterial cells. This property of bacteriophages makes them an attractive biorecognition element in the field of biosensor development. In this work, we report the use of an immobilized bacteriophage for the development of a highly sensitive electrochemical sensor for Staphylococcus arlettae, bacteria from the pathogenic family of coagulase-negative staphylococci (CNS). The specific bacteriophages were covalently immobilized on the screen-printed graphene electrodes. Thus, the fabricated bacteriophage biosensor displayed quantitative response for the target bacteria (S. arlettae) for a broad detection range (2.0–2.0 × 10⁶ cfu). A fast response time (2 min), low limit of detection (2 cfu), specificity, and stability over a prolonged period (3 months) are some of the important highlights of the proposed sensor. The practical utility of the developed sensor has been demonstrated by the analysis of S. arlettae in spiked water and apple juice samples.     

Antibody–aptamer functionalized fibre‐optic biosensor for specific detection of Listeria monocytogenes from food

Aim: To develop antibody–aptamer functionalized fibre‐optic biosensor for specific detection of Listeria monocytogenes from food products. Methods and Results: Aptamer, a single‐stranded oligonucleotide ligand that displays affinity for the target molecule, was used in the assay to provide sensor specificity. Aptamer‐A8, specific for internalin A, an invasin protein of L. monocytogenes, was used in the fibre‐optic sensor together with antibody in a sandwich format for detection of L. monocytogenes from food. Biotinylated polyclonal anti‐Listeria antibody, P66, was immobilized on streptavidin‐coated optical waveguide surface for capturing bacteria, and Alexa Fluor 647‐conjugated A8 was used as a reporter. The biosensor was able to selectively detect pathogenic Listeria in pure culture and in mixture with other bacteria at a concentration of approx. 10³ CFU ml^?1. This sensor also successfully detected L. monocytogenes cells from artificially contaminated (initial inoculation of 10² CFU 25 g^?1) ready‐to‐eat meat products such as sliced beef, chicken and turkey after 18 h of enrichment. Conclusion: Based on the data presented in this study, the antibody–aptamer functionalized fibre‐optic biosensor could be used as a detection tool for sensitive and specific detection of L. monocytogenes from foods. Significance and Impact of the Study: The study demonstrates feasibility and novel application of aptamer on fibre‐optic biosensor platform for the sensitive detection of L. monocytogenes from food products.     

Characterization of bioluminescent derivatives of assimilable organic carbon test bacteria

The assimilable organic carbon (AOC) test is a standardized measure of the bacterial growth potential of treated water. We describe the design and initial development of an AOC assay that uses bioluminescent derivatives of AOC test bacteria. Our assay is based on the observation that bioluminescence peaks at full cell yield just prior to the onset of the stationary phase during growth in a water sample. Pseudomonas fluorescens P-17 and Spirillum sp. strain NOX bacteria were mutagenized with luxCDABE operon fusion and inducible transposons and were selected on minimal medium. Independent mutants were screened for high luminescence activity and predicted AOC assay sensitivity. All mutants tested were able to grow in tap water under AOC assay conditions. Strains P-17 I5 (with p-aminosalicylate inducer) and NOX I3 were chosen for use in the bioluminescence AOC test. Peak bioluminescence and plate count AOC were linearly related for both test bacteria, though data suggest that the P-17 bioluminescence assay requires more consistent luminescence monitoring. Bioluminescence results were obtained 2 or 3 days postinoculation, compared with 5 days for the ATP luminescence AOC assay and 8 days for the plate count assay. Plate count AOC assay results for nonmutant and bioluminescent bacteria from 36 water samples showed insignificant differences, indicating that the luminescent bacteria retained a full range of AOC measurement capability. This bioluminescence method is amenable to automation with a microplate format with programmable reagent injection.     

THE APPLICATION of ATP BIOLUMINESCENCE FOR the ASSESSMENT of MILK QUALITY and FACTORY HYGIENE

The speed of ATP bioluminescence assays makes them very useful for assessing product quality and efficiency of sanitation at food plants. the suitability of a new method for evaluating the quality of raw milk was compared with plate count. A good correlation was obtained between the ATP method and plate count for milks with counts above 1 × 10⁴ cfu/ml. the combination of ATP bioluminescence with preincubation procedures was investigated for predicting the shelf-life of pasteurized milk. the best correlations were obtained when milks were preincubated at 15C for 25 h or at 21C for 25 h in the presence of an inhibitor system to prevent Gram-positive bacterial growth.
Hygiene monitoring using bioluminescence gave a better indication of surface cleaniness than conventional swab counts. the reasons for the discrepancies in a small percentage of cases are discussed.     

Rapid methods to assess sanitizing efficacy of benzalkonium chloride to Listeria monocytogenes biofilms

Different methods were used to investigate biofilm growth including crystal violet staining, ATP bioluminescence and total viable count. Seven strains of Listeria monocytogenes and 8 of their derivative strains were screened for their capacity to form biofilms. Both adaptation to benzalkonium chloride (BC) and curing of plasmids did not significantly affect biofilm-forming ability. The strains of L. monocytogenes belonging to serotype 1 formed biofilms significantly better as compared to serotype 4 (P = 0.0003). To estimate the efficacy of BC for biofilm elimination the best and the poorest biofilm-formers were used (C719 and LJH 381). It was observed that, L. monocytogenes strain C719 in biofilms is at least 1000 times more resistant to BC than in planktonic form. Cells present in biofilms were shown to recover and grow after BC treatment thus providing a source of recontamination. It was shown that ATP bioluminescence provides good correlation with bacterial counts of L. monocytogenes in biofilms. Staining with crystal violet, on the contrary, did not correlate with bacterial growth in biofilms in the presence of high concentrations of BC but provided information on the concentration of bacterial cells, both live and dead, attached to the surface. ATP bioluminescence was found to be a reliable method for rapid estimation of the efficacy of sanitizers for biofilm disinfection. Crystal violet staining, on the other hand, was shown to be a suitable method to monitor removal of biofilms. Our investigation showed that for Listeria biofilms concentrations of BC higher then 10 mg/ml should be applied for at least 30 min to kill almost all the live cells in biofilms. However, this concentration was still not enough to remove biofilms from the surface of plastic.     

Photodynamic inactivation of recombinant bioluminescent Escherichia coli by cationic porphyrins under artificial and solar irradiation

A faster and simpler method to monitor the photoinactivation process of Escherichia coli involving the use of recombinant bioluminescent bacteria is described here. Escherichia coli cells were transformed with luxCDABE genes from the marine bioluminescent bacterium Vibrio fischeri and the recombinant bioluminescent indicator strain was used to assess, in real time, the effect of three cationic meso-substituted porphyrin derivatives on their metabolic activity, under artificial (40 W m⁻²) and solar irradiation (≈620 W m⁻²). The photoinactivation of bioluminescent E. coli is effective (>4 log bioluminescence decrease) with the three porphyrins used, the tricationic porphyrin Tri-Py⁺-Me-PF being the most efficient compound. The photoinactivation process is efficient both with solar and artificial light, for the three porphyrins tested. The results show that bioluminescence analysis is an efficient and sensitive approach being, in addition, more affordable, faster, cheaper and much less laborious than conventional methods. This approach can be used as a screening method for bacterial photoinactivation studies in vitro and also for the monitoring of the efficiency of novel photosensitizer molecules. As far as we know, this is the first study involving the use of bioluminescent bacteria to monitor the antibacterial activity of porphyrins under environmental conditions.     

Quantitative assessment of total and Gram-positive aerobic bacteria in fresh and ambient-temperature-stored sub-tropical marine fish

The current study was undertaken to enumerate Gram-positive bacteria in fresh sub-tropical marine fish and determine the effect of ambient storage (25°C) on the Gram-positive bacterial count. Total and Gram-positive bacteria were enumerated in the muscles, gills and gut of fresh and stored Pseudocaranx dentex, Pagrus auratus and Mugil cephalus on tryptone soya agar (TSA) and TSA with 0.25% phenylethyl alcohol (PEA), respectively. Initial studies indicated that PEA significantly reduced total aerobic bacterial count (TABC) whereas control Gram-positive bacteria were not affected by 0.25% PEA. TABC significantly increased in all fish body parts, whereas Gram-positive aerobic bacterial count (GABC) significantly increased only in the muscles and gills during ambient storage for 15 h. The TABC of the fish species increased from 4.00, 6.13 and 4.58 log cfu g⁻¹, respectively in the muscles, gills, and gut to 6.31, 7.31 and 7.23 log cfu g⁻¹ by the end of storage. GABC increased from 2.00, 3.52 and 2.20 log cfu g⁻¹ to 4.70, 5.85 and 3.36 log cfu g⁻¹. Within each species, TABC were significantly higher in the gills compared to that of muscles and gut; however, no significant differences were found in GABC between muscles and gills. This study demonstrated the potential importance of Gram-positive bacteria in sub-tropical marine fish and their spoilage.     

基于ATP生物发光法的微生物数量快速检测技术的研究进展

微生物数量的快速检测一直是工业生产与食品行业需要解决的问题,腺嘌呤核苷三磷酸(adenosine triphosphate,ATP)生物发光法具有操作简便、检测周期短等优点,可满足一般微生物检测的需求。然而,ATP生物发光法的准确性也受到不同因素的影响,如微生物的ATP检测限值较高、微生物自身及其他因素(如非微生物ATP、提取剂种类、荧光素酶活性等)均对微生物数量的检测产生影响。本文简述了不同微生物数量检测方法的优缺点,介绍了ATP生物发光法的发展历程及原理,综述了非微生物ATP与游离ATP、微生物量、ATP提取剂、荧光素酶等因素对ATP生物发光法灵敏度与稳定性的影响,归纳总结了ATP生物发光法及检测设备在食品、医疗、污水处理等领域的应用现状,并就ATP生物发光法体系的优化及ATP在线检测的应用等方面进行了展望,以期为ATP生物发光法的高效应用提供新的思路。     

EVALUATION OF ADENOSINE TRIPHOSPHATE (ATP) BIOLUMINESCENCE FOR ESTIMATING BACTERIA ON SURFACES OF BEEF CARCASSES

A rapid (<15 min), inexpensive and simple method has been developed to estimate the concentration of bacteria on surfaces of beef carcasses using adenosine triphosphate (ATP) bioluminescence. Surfaces (5x5 cm²) of beef carcasses (n= 159) were collected by excision. An ATP assay and aerobic plate count were performed on each sample. A significant (p < 0.001) positive linear relationship (r = 0.83) between plate count and ATP assay was obtained for 159 beef carcass samples. When thresholds levels were set at 1 × 10⁴, 1 × 10⁵ and 1 × 10⁶ CFU/cm², there was moderate to good agreement between the ATP bioluminescence assay and the aerobic plate count as determined by the k-statistic. The application of this ATP bioluminescence test to HACCP systems for beef slaughter processes is discussed.     

Construction of a bacterial biosensor for zinc and copper and its application to the development of multifunctional heavy metal adsorption bacteria

In this study, we designed and applied molecular biosensors for heavy metals, zinc and copper, for use in bioremediation strategies. Bacteria utilize two component systems to sense changes in the environment by multiple signal components including heavy metals and control gene expression in response to changes in signal molecules. zraP and cusC promoters were selected from a genetic circuit of the ZraSR and CusSR two-component system and were fused to a dual-labeling reporter protein as an interactive biological component for zinc and copper to generate a signal from the constructed biosensor. The biosensor efficiently senses zinc and copper with a calculated detection limit of 16 μM and 26 μM, respectively, and was shown to be a sensitive and effective heavy metal monitoring bacterial system. To extend the application of the bacterial biosensor, we assembled a bioadsorption system that can trigger bacteria to sense and adsorb 13 ± 0.3 mg/L of zinc and 11.4 ± 0.42 mg/L of copper per gram of dry cell weight with induction at a concentration of 100 mg/L of the respective metal ion.     

Use of ATP bioluminescence to determine the bacterial sensitivity threshold to a bacteriocin

A new ATP bioluminescence‐based method was developed to determine the effectiveness of nisin on a sensitive strain of Lactococcus cremoris. The principle of the method is to quantify the release of adenylic‐nucleotides (AN) by a sensitive strain under the action of the bacteriocin, with the complex luciferin–luciferase. Nisin‐induced leakage of AN included ATP from a sensitive L. cremoris to the external medium immediately after the contact with the bacteria. The growth of L. cremoris was correlated with the extracellular AN content. The extracellular ATP and AN concentration exhibited a linear correlation to the logarithm of the nisin concentration. For the determination of the effectiveness threshold, the concentration of AN was more sensitive and more reliable than the direct quanti?cation of ATP. The effectiveness threshold, corresponding to a 100% inhibition of L. cremoris growth, was obtained for a null concentration of intracellular nucleotides, i.e. for a AN_tot:AN_ext ratio = 1. For an initial concentration of 1.4 × 10⁷ bacteria/mL, the nisin effectiveness threshold is 3.4 ± 0.01 mg nisin/L. It is possible to detect effectiveness threshold concentration by taking into account the physiological state of the cells. Copyright © 2003 John Wiley & Sons, Ltd.     

Analysis of ethanol in fermentation samples by a robust nanocomposite-based microbial biosensor

A robust microbial biosensor was constructed from a bionanocomposite prepared by a direct mixing of bacterial cells of Gluconobacter oxydans and carbon nanotubes with ferricyanide employed as a mediator for enhanced sensitivity of ethanol oxidation. A successful integration of the device into flow injection analysis mode of operation provided a high sensitivity of detection of (74 ± 2.7) μA mM⁻¹ cm⁻², a low detection limit of 5 μM and a linear range from 10 μM up to 1 mM. A short response time of the biosensor allowed a sample throughput of 67 h⁻¹ at 0.3 ml min⁻¹. The biosensor exhibited high operational stability with a decrease in the biosensor response of 1.7% during 43 h of continuous operation. The device was used to analyse ethanol in fermentation samples with a good agreement with a HPLC method.     

Enumeration of phagocytosed Staphylococcus aureus inside cells of the mouse macrophage cell line J774 by bioluminescence,fluorescence and viable counting techniques

In order to quantify intracellular Staphylococcus aureus within a macrophage-like cell line by a bioluminescence technique, the mouse cell line J774 and opsonized Staphylococcus aureus were incubated together to allow phagocytosis to occur. Experiments using UV microscopy and fluorescent stained S. aureus were performed to determine an estimate of the mean intracellular bacterial numbers. For enumeration of intracellular bacteria by a bioluminescence technique, extracellular bacteria were removed by washing, the macrophages lysed mechanically and osmotically and treated with apyrase to remove somatic ATP. Bacterial cells were washed and the intracellular ATP measured by firefly luciferase bioluminescence in a luminometer. This new method of enumerating intracellular bacteria was compared to the conventional method of viable counts and found to correlate (r = 0.78). The bioluminescence assay developed was found to be a relatively rapid alternative method to the techniques currently used to enumerate intracellular bacteria and could prove advantageous in studies of intracellular killing and effects of antimicrobial agents on intracellular pathogens.     

Bacterial bioluminescence as an early indication of marine fish spoilage

Summary The relationships between different microbiological and biochemical parameters and the development of bacterial luminescence associated with the spoilage of marine fish from the Mediterranean-Sea was studied during storage at different temperatures. The bioluminescence level of the bacterial suspensions that were taken from the fish skin increased during the storage; at 20°–25°C the growth and luminescence of the luminuous bacteria correlated well with the total bacterial count while at 5°C the bacterial proliferation was not accompanied by a parallel increase in luminescence.The shift in storage temperature from 25°C to 5°C stabilized the level of the luminescence of bacterial suspension taken from the winter fish which were comprised mainly by Photobacterium phosphoreum, and caused a drop in the luminescence of bacterial suspension taken from the fish caught in the summer which were comprised mainly by Beneckea barveyi. The increase in the bioluminescence level appeared earlier than the increase in trimethylamine level and occured approximately at the same time as the increase in the hypoxanthine concentration. The potential value of the use of bacterial bioluminescence as an early indication for marine fish spoilage is discussed.     

A simple bioluminescence procedure for early warning detection of coliform bacteria in drinking water

Traditional cultivation-dependent tests for coliform bacteria in food and drinking water take 18–24 h to complete. Bioluminescence-based enzyme assays can potentially reduce analysis time for indicator bacteria such as coliforms. In the present study, we developed a simple presence/absence (P/A) bioluminescence procedure for rapid detection of coliform bacteria in groundwater-based drinking water. The bioluminescence procedure targeting β-d-galactosidase activity in coliform bacteria was based on hydrolysis of 6-O-β-galactopyranosyl-luciferin. Bacteria immobilized on membrane filters were enriched for 6–8 h in selective media containing isopropyl-β-d-thiogalactopyranoside (IPTG) to induce β-d-galactosidase activity in coliform bacteria. The equivalent of approximately 300 E. coli cells was required for bioluminescence detection of β-d-galactosidase activity. In comparison, PCR based detection of E. coli in drinking water required approximately 30 target cells. Analysis of contaminated drinking water samples showed comparable results for coliform bacteria using traditional multiple-tube fermentation, Colilert-18, and the bioluminescence procedure. Aeromonas hydrophila or indigenous groundwater bacteria did not interfere with the procedure. The bioluminescence procedure can be combined with commercial substrates such as Fluorocult or Colilert-18, and will allow the detection of one coliform in 100 ml drinking water within one working day. The results suggest the bioluminescence assays targeting β-d-galactosidase activity may be used for or for early warning screening of drinking water and/or rapid identification of contaminated drinking water wells.