Significance of the detection of staphylococcal enterotoxin A gene in low fat milk which caused a serious outbreak of food poisoning

In June 2000, there was a large-scale outbreak of food poisoning after consumption of Snow Brand low fat milk. In the evening of a day the incident made public, some cartons of low fat milk were brought to our laboratory for examination. Next day, we detected only staphylococcal enterotoxin (SE) A gene among SE (A-E) genes by PCR in left-over milk samples or samples from the same lots that patients had consumed. We presumed that the outbreak was caused by the intake of SEA. We subsequently confirmed the presence of SEA in these samples. To investigate the existence of SE (A-E) genes in milk, we examined 100 samples of commercial low fat milk and milk by PCR, but none of the genes was detected. We estimated the detection limit of SEA gene in low fat milk by PCR. Four strains of SEA-producing Staphylococcus aureus cultures were serially diluted in low fat milk. The SEA gene was detected at levels of 5.5 x 10(2) to 1.6 x 10(4) cfu/mL of S. aureus. These amounts of S. aureus are higher than the values in raw milk reported previously. Therefore we consider that SE genes in low fat milk should usually be undetectable by our PCR. This study shows that quick detection of SE genes by PCR is very helpful to analyze outbreaks, especially if no significant bacterium can be cultured.     

Feasibility of a reference material for staphylococcal enterotoxin A.

A reference material for staphylococcal enterotoxin A (SEA), was produced by spray-drying the toxin in milk. With this procedure the SEA was distributed homogeneously in the material. For ease of handling the reference material was encased in gelatin capsules, each containing 405 ng of SEA. Simply dissolving the milk powder in distilled water resulted in a 100% recovery of the SEA present. The reference material would appear suitable for testing laboratory performance, comparison of detection methods and to validation of extraction procedures.     

Risk evaluation for staphylococcal food poisoning in processed milk produced with skim milk powder

The growth of S. aureus and the production of staphylococcal enterotoxin A (SEA) in skim milk concentrates stored at inappropriate temperatures in a recovery milk tank (tank for excess concentrated skim milk) used in the manufacture of skimmed milk powder were investigated. Also, it was estimated if a possible outbreak of food poisoning would occur if the contaminated skimmed milk powder was used in the manufacture of processed milk. Skim milk concentrates with milk solid content of 15, 25, and 35% were inoculated with S. aureus at 1-2 log CFU/ml and incubated at 15, 25, or 35 degrees C for 0 to 24 h with or without shaking. Bacterial growth and the level of SEA production were measured. At 35 degrees C with shaking, there was a significant difference (p<0.05) in one way layout analysis of variance, and it was demonstrated that the growth of S. aureus and SEA production could be milk solid content-dependent. Shaking accelerated the growth of S. aureus and SEA production at 35 degrees C. Generally, skim milk powder is produced by mixing a set percentage of skim milk concentrates (recovery milk) from the recovery milk tank into raw milk. If recovery milk contaminated with S. aureus at levels of 1-2 log CFU/ml is kept at 15 to 35 degrees C due to a power failure, it was estimated that processed milk consumption of 670-1200 ml, 420-1500 ml and 18-83 ml would trigger the onset of food poisoning symptoms when skim milk concentrates (recovery milk) are stored at 25 degrees C for 24 h, 35 degrees C for 10 h, and 35 degrees C for 24 h, respectively, during the production of the skim milk powder. Based on these consumption levels, it was concluded that, if recovery milk cannot be refrigerated and is stored at room temperature (25 to 35 degrees C), it must be used within 8 h and preferably within 6 h.     

Quantitative risk assessment for type A staphylococcal enterotoxin poisoning due to consumption of Minas Frescal cheese in Brazil

This study aimed to estimate the risk of staphylococcal toxin type A (SEA) poisoning from consuming Minas Frescal cheese (MFC) in Brazil. A Quantitative Microbiological Risk Assessment model was developed, focussing on the production of SEA while still in the raw material. The baseline scenario yielded a simulated mean concentration of SEA in the MFC portion of 16.20 ng. The concentration of Staphylococcus aureus in raw milk is proved to be the most influential parameter for the risk, followed by the serving size and the prevalence of toxin genes.     

Comparison of antibiogram, staphylococcal enterotoxin productivity, and coagulase genotypes among Staphylococcus aureus isolated from animal and vegetable sources in Korea

Staphylococcal food poisoning is caused by enterotoxin-producing Staphylococcus aureus. We investigated the prevalence of such organisms in samples of bovine mastitic milk (n = 714), raw meat (n = 139), and vegetables (n = 616). We determined the degrees of relatedness of isolates as indicated by antibiogram, staphylococcal enterotoxin (SE) productivity, and coagulase gene restriction fragment length polymorphism analysis. We examined 297 S. aureus isolates and found SE production in 57 (31.8%), 4 (7.8%), and 49 (73.1%) isolates from raw milk, raw meat, and vegetables, respectively. A high proportion of the isolates obtained from milk produced more than two types of toxins (mainly SEA, SEB, and/or SEC), whereas isolates from raw meat and vegetables primarily produced SEA alone. Most isolates were sensitive to cephalothin (97.6%), gentamicin (80.8%), erythromycin (79.5%), and tetracycline (72.7%), but were resistant to penicillin (90.2%) and ampicillin (88.9%). The proportion of antibiotic-resistant isolates differed according the source of the bacteria; the milk and vegetable isolates were more resistant to penicillin and ampicillin than were the meat isolates (P < 0.05), whereas tetracycline resistance was limited to the milk and vegetables isolates. The coagulase genotypes (I to XII) varied with the source of the organism, and only a few genotypes prevailed in each source: II (42.4%) and IV (24%) types in isolates from milk, IX (35.3%) and XI (45%) from raw meat, and III (40.3%) and XII (32.8%) from vegetables. These findings suggest that remarkable differences exist in antibiogram, SE productivity, and coagulase genotypes, resulting in limited clonal transmission of S. aureus into various food sources. As enterotoxin production only occurs when S. aureus grows to high numbers, staphylococcal food poisoning can be prevented by proper refrigeration.     

Real time biosensor analysis of staphylococcal enterotoxin A in food.

Currently there is no 'real-time' detection system to identify food borne toxins. In order to develop such a system, we have used a evanescent wave biosensor for real time detection of staphylococcal enterotoxin A (SEA) in foods. The approach used here is sandwich biosensor, a method utilizing two antibodies. The toxin binds initially to a capturing antibody which is bound covalently on the surface of the biosensor detector. The second antibody binds to the captured toxin. We were able to measure SEA in foods with little or no background interference, demonstrating that biosensor-based measurement of SEA was possible not only with purified SEA but also in complex food matrices such as hot dogs, potato salad, milk and mushrooms. Autoclaved samples of SEA did not evoke a positive response. With both purified SEA and SEA-spiked foods, the assay sensitivity is 10-100 ng/g depending on the material tested and the assay is rapid ( <4 min) when a single antibody is used.     

食品基质中不同金黄色葡萄球菌肠毒素基因型表达差异研究

目的 比较研究食品基质中不同金黄色葡萄球菌肠毒素(Staphylococcal enterotoxins ,SEs)基因型的蛋白表达差异,为预防金黄色葡萄球菌食物中毒提供参考依据。方法 采用特异聚合酶链反应方法(polymerase chain reaction,PCR)对食品中分离的金黄色葡萄球菌菌株进行肠毒素基因型检测;选择sea、seb、sec、sed等基因型阳性菌株,分别接种于胰酪大豆胨液体培养基(trypticase soy broth,TSB)、牛奶、鸡肉中,按照国家标准GB 4789.10—2016酶联免疫吸附试验法(enzyme linked immunosorbent assay,ELISA)定量检测TSB培养基、牛奶和鲜鸡肉中不同金黄色葡萄球菌肠毒素基因型的蛋白表达量。结果 30株金黄色葡萄球菌中检测到14株肠毒素基因型阳性菌株,所占比例为46.67%,其中sea基因携带率最高(16.67%),而seb、sec、sed、seh则各占6.67%。SEA 在TSB、牛奶、鸡肉3种基质中的平均表达量为7.37 ng/mL,高于SEB、SEC、SED;不同基质环境对肠毒素的表达具有一定影响,如SEA、SEB、SEC、SED在TSB中的表达水平最高,平均表达量为9.04 ng/mL,牛奶次之,鸡肉最低。结论 肠毒素基因型的表达与菌株自身的调控及环境作用密切相关,本研究对肠毒素的产生机制进行初步了解,有助于进一步降低食物中毒风险。     

Statistical tools for net quantity control in the dairy industry

A pilot study was carried out to assist management in the development of an improved strategy for net quantity control in a medium volume, liquid milk, carton line operation. A low cost statistical quality control system was used to demonstrate that computerization for net quantity data handling and interpretation must be part of an improved strategy to prove compliance with weights and measures legislation. A process capability study identified differential behaviour between filling valve sets which required an improved maintenance plan in order to prevent process instability when filling one litre cartons. Preliminary and ongoing process capability indices for skimmed, semi-skimmed and whole milk in half pint and one pint cartons were two to four times the minimum required to prove capability. Overfill was typically of the order 0.2 to 0.4% of the nominal quantity for half pint and one pint fills, but it tended to be higher for whole milk due to an overestimation of the milk density. Overfill on the one litre carton was shown to be excessive at 0.8% of the nominal quantity.     

Effect of heat treatment on activity of staphylococcal enterotoxins of type A,B, and C in milk

Intoxication by staphylococcal enterotoxins (SE) is among the most common causes of food-poisoning outbreaks resulting from the consumption of raw milk or products made thereof. The aim of our study was to analyze the thermal stability of SE and evaluate the inactivation of SE types A, B, and C (SEA, SEB, SEC) by autoclaving at 100°C, 110°C, and 121°C. Milk samples were inoculated with 38 Staphylococcus aureus strains that possessed the ability to produce SEA, SEB, or SEC and incubated at 37°C for 24 h. This incubation was followed by heat treatment at 100°C, 110°C, or 121°C for 3 min. Samples were analyzed by Staph. aureus plate count method on Baird-Parker agar and specifically for the presence of SE. An enzyme-linked immunofluorescent assay (ELFA) on a MiniVIDAS analyzer (bioMérieux, Marcy l'Étoile, France) was used to detect SE, which were determined semi-quantitatively based on test values. The obtained results were analyzed by means of nonparametric statistical methods. All samples (100%; 38/38) were SE-positive before heat treatment, and the positivity rates decreased after heat treatment at 100°C, 110°C, and 121°C to 36.8% (14/38), 34.2% (13/38), and 31.6% (12/38), respectively. The rates of positive samples differed between SEA, SEB, and SEC producers: SEA was detected in the highest amounts both before and after heat treatment. The amount of SE (expressed as test values) decreased significantly after heat treatment. Comparing amounts of SE in positive and negative samples before and after heat treatment, we can conclude that the success of SE inactivation depends on the amount present before heat treatment. The highest amount of SE and the highest rate of SE-positive samples after all heat treatments were found in samples with strains producing SEA. For SEB and SEC, lower amounts of enterotoxin were present and were inactivated at 100°C. Although temperatures of 100°C, 110°C, and 121°C may inactivate SE in milk, the key measures in prevention of staphylococcal enterotoxicosis are avoiding initial contamination of milk by Staph. aureus, promoting consumption of heat-treated milk, and preventing disruption of the cold chain during milk production and processing.     

LABORATORY EVALUATION OF PAPERBOARD CARTONS FOR THE NONREFRIGERATED STORAGE OF STERILIZED MILK1,2

The suitability of various carton materials for the nonrefrigerated storage of sterilized milk was investigated. One quart paperboard cartons were fabricated from the same base sheet of stock but varied in the type of sizing used to make them resistant to penetration by liquids and whether or not they were aluminum foil-lined. They were preformed and sterilized with ethylene oxide. The four types of paperboard were: (a) rosin (sizing) paperboard (R); (b) rosin paperboard with foil lining (RF); (c) cyanasize (sizing) juice paperboard (CJ); and (d) cyanasizejuice paperboard with foil lining (CJF). Each carton was aseptically filled and sealed, in a glovebox. Incubation was carried out at 20°C for up to nine weeks. Every week five cartons of each type were randomly selected and the milk tested for microbial stability and flavor. The candidate cartons were also tested for degradation of the physical characteristics of static bulge, wicking, tensile strength, and stiffness. Of these, it appears as if selection of carton type will be determined mostly by wicking resistance. The most acceptable carton type is CJF, which had minimal wicking, acceptable bulge, acceptable stiffness, and acceptable tensile strength during the testing period.     

双通道超高速条烟提升机的设计与应用

当条烟输送系统中的单道S型提升机的输送能力>50条/min时,容易导致链板磨损加快、条烟折角、表面BOPP薄膜划痕、条烟破损率高等问题,无法满足超高速包装机组的生产输送需求,为此对条烟提升机进行了改进。在单道S型提升机条烟平条进烟、平条出烟方式的基础上,设计了双通道超高速条烟提升机,实现了条烟与输送链板运动方向的一致,减少了条烟在滚轮和链板上表面的滑动摩擦。应用效果表明,条烟输送稳定可靠,提升机最大输送能力由50条/min提高到100条/min,链板使用寿命由2～3年提高到5～6年,条烟折角、表面BOPP薄膜划痕、破损等缺陷由5%～7%降低到0.5%以下,提高了条烟输送能力和产品质量。     

Quantum dot bead-based competitive immunochromatographic assay for enterotoxin aureus A detection in pasteurized milk

Staphylococcal enterotoxin A (SEA) is an important biotoxin, produced by Staphylococcus aureus under appropriate conditions, and often contaminates milk and dairy products. Herein, an anti-SEA monoclonal antibody (anti-SEA mAb) was prepared by injecting the SEA protein in BALB/c mice, and a novel immunochromatographic assay (ICA) was developed for the rapid and sensitive determination of SEA in pasteurized milk by using highly luminescent quantum dot beads (QB) as signal amplification probe. Given the 1020-fold enhancement in the photoluminescence intensity of QB to the original quantum dot, the proposed QB-ICA exhibits high sensitivity for SEA determination in real milk samples with a limit of detection of 1.89 ng/mL, and shows good dynamic linearity for SEA quantitative detection from 2 to 150 ng/mL within 15 min of test time. The proposed QB-ICA also shows good selectivity to SEA detection with a negligible cross-reaction to common analogs, including staphylococcal enterotoxins B, C, D, and E. In addition, the accuracy and precision of QB-ICA were assessed by analyzing SEA-fortified milk samples. The average recoveries of intra- and interassays range from 85.5 to 128.1%, and the coefficients of variation range from 4.6 to 14.2%, indicating an acceptable accuracy for the quantitative detection of SEA in real milk samples. In summary, this work provides a powerful and rapid analysis tool for the sensitive monitoring of SEA contamination in pasteurized milk samples.     

Inactivation of Bacillus subtilis spores on aluminum and polyethylene preformed cartons by UV-excimer laser irradiation

The efficacy of UV KrF-excimer laser light (at 248 nm) to inactivate Bacillus subtilis spores loaded onto preformed cartons was found to be dependent on the interior carton coating and scheme by which the irradiation was applied. When the carton was held static during UV laser treatment, the majority of the dose was delivered to the base of the carton and to a lesser extent to the upper part of the pack. In this arrangement no irradiation of the interior sides of the carton was observed. A more even distribution of dose was achieved, however, by moving the carton within the laser beam during irradiation treatment. The distribution of UV was also found to be dependent on the type of carton interior coating. With aluminum cartons the dose measured was found to be significantly greater (P < 0.01) and more evenly distributed across the interior compared to when polyethylene packs were tested. Under optimized conditions no spore survivors were detected on aluminum cartons preloaded with 9.5 x l0 B. subtilis spores by applying a UV laser output dose of 160 J. In comparison, the same conditions only achieved a significantly lower (P < 0.01) reduction in spore numbers (log count reduction 4.2) when polyethylene cartons were used. This difference in lethality and UV distribution of laser light was associated with the higher internal reflection of photons with aluminum cartons. The suitability of UV-excimer lasers for sterilizing preformed cartons over traditional germicidal lamp-based methods is discussed.     

An outbreak of food poisoning due to egg yolk reaction-negative Staphylococcus aureus

An outbreak of staphylococcal food poisoning due to an egg yolk (EY) reaction-negative strain occurred in Japan. Twenty-one of 53 dam construction workers who ate boxed lunches prepared at their company cafeteria became ill, and eight required hospital treatment. The outbreak showed a typical incubation time (1.5-4 h with a median time of 2.7 h) and symptoms (vomiting and diarrhea) of staphylococcal food poisoning. Staphylococcus aureus, which produces staphylococcal enterotoxin (SE) A, was isolated from four fecal specimens of eight patients tested. Scrambled egg in the boxed lunches contained 20-40 ng/g of SEA, and 3.0 x 10(9)/g of viable S. aureus cells that produced this toxin. All isolates from patients and the food were EY reaction-negative, coagulase type II, and showed the same restriction fragment length polymorphism (RFLP) pattern. We concluded that the outbreak was caused by scrambled egg contaminated with EY reaction-negative S. aureus. In Japan, outbreaks of staphylococcal food poisoning are mainly caused by EY reaction-positive S. aureus, and EY reaction-negative colonies grown on agar plates containing EY are usually not analyzed further for detection of S. aureus. The present outbreak suggested that EY reaction-negative isolates should be subjected to further analysis to detect the causative agents of staphylococcal food poisoning.     

A MODEL FOR PREDICTING THE GROWTH OF LISTERIA MONOCYTOGENES IN PACKAGED WHOLE MILK

Experiments were conducted to determine growth characteristics of Listeria monocytogenes in sterilized whole milk at nine temperatures in the range of 277.15 to 308.15K (4 to 35C). Based on these data, the parameter values of the Baranyi dynamic growth model were statistically determined. Finite element software, ANSYS, was used to determine temperature distributions in milk cartons subject to a time‐varying ambient temperature profile. The space‐time‐temperature data were input to the Baranyi dynamic growth model, to predict the microbial population density distribution and the average population density in the milk carton. The Baranyi dynamic growth model and the finite element model were integrated and validated using experimental results from inoculated sterilized whole milk in half‐gallon laminated paper cartons. In all experiments, the milk cartons were subjected to the same temperature profile as the Baranyi dynamic growth model. Experimental microbial counts were within predicted upper and lower bounds obtained using the integrated Baranyi dynamic growth and finite element models. In addition, the growth curve at the mean value of initial physiological state parameter for L. monocytogenes underpredicted the microbial growth (standard error = 0.54 log (cfu/mL) and maximum relative difference = 15.49%).     

Production of enterotoxins and thermonuclease by Staphylococcus aureus in cooked egg-noodles

Production of enterotoxin A (SEA), enterotoxin C (SEC), and thermonuclease (TNase) by Staphylococcus aureus was determined during growth in cooked egg-noodles at different temperatures (15-37 degrees C). Both SEA and SEC and TNase were detected when greater than or equal to 4.0 x 10(7) colony forming units (cfu)/g were present. The contents of SEA, SEC, and TNase in egg-noodles mainly increased at the end of the exponential growth phase. In contrast with SEA and SEC the production of TNase always continued till the end of each experiment. Recovery rates of SEA and TNase in cooked noodles were dependent on their amounts. High amounts (64 ng SEA/g; 1 unit TNase/g) were recovered at a rate of 93% (SEA) and 54% (TNase) respectively, whereas low concentrations (1 ng SEA/g; 0.004 units TNase/g) were recovered at a rate of only 45% (SEA) and 1.1% (TNase). TNase usually is produced at all conditions which allow growth of S. aureus. Evidence of TNase was proposed for screening for staphylococcal enterotoxins (SE) in foods. But sometimes foods contain no TNase but SE. For this reason the ELISA-test which is simple and sensitive should be used for determination of SE-production in foods.     

Rapid immunofluorescence assay for staphylococcal enterotoxin A using magnetic nanoparticles

A competitive immunoassay for staphylococcal enterotoxin A (SEA) detection in milk was developed, using immobilised antibody onto magnetic nanoparticles (MNPs). MNPs were prepared and then modified to introduce amino groups on them. The morphology and size of the obtained both unmodified and modified MNPs were characterized using TEM analyses. Monoclonal anti-SEA antibody was immobilised onto the modified MNPs (MNP-Ab). Staphylococcal enterotoxin A was conjugated with fluorescent dye ATTO620NHS. The characteristics of fluorescence conjugate were examined. The amount of MNP-Ab and concentration of the fluorescent conjugate used for competitive immunoassay were optimized: 0.25 mg and 53 μg mL⁻¹, respectively. The detection limit of developed immunoassay was determined – 0.23 ng mL⁻¹ SEA in spiked milk samples. The immunoassay takes only 30 min, the magnetic separation is fast (<10 s) and the volume of the sample for analysis is very small (200 μL).     

Detection of Enterotoxigenic Staphylococcus aureus in Raw Milk and Dairy Products by Multiplex PCR

Abstract: The aim of this study was to investigate the prevalence of enterotoxigenic Staphylococcus aureus in 122 samples, including 60 raw milk, 32 white cheese, 10 kashar cheese, 10 butter, and 10 ice cream samples obtained from Samsun province, Turkey. In this study, S. aureus was detected in 64 samples, including raw milk (45/60; 75%), white cheese (12/32; 37.5%), kashar cheese (3/10; 30%), butter (3/10; 30%), and ice cream (1/10; 10%) samples. A total of 81 isolates were identified as S. aureus by PCR with the presence of 16S rRNA and nuc genes. The presence of genes encoding the staphylococcal enterotoxins (SEs) SEA, SEB, SEC, and SED was detected by multiplex PCR. According to the analysis, seven isolates from the raw milk samples (7/51; 13.7%) were enterotoxigenic; five of them produced SEA (5/7; 71.4%), one produced SEB (1/7; 14.2%), and one produced SEA+SEB (1/7; 14.2%). Four isolates from the white cheese samples (4/21; 19%) produced the SEA (1/4; 25%), SEC (1/4; 25%), SED (1/4; 25%), and SEA+SED (1/4; 25%) toxins. Two isolates from the kashar cheese samples (2/4; 50%) were found to be enterotoxigenic; one produced SEA (1/2; 50%) and the other produced SED (1/2; 50%). One isolate from the butter samples (1/4; 25%) showed enterotoxigenic character (SEB, 1/1; 100%). The products were found to be potentially hazardous to public health because of the fact that levels of contamination were higher than 105–106 cfu/g ml in 39% (25/64, 17 raw milk, 7 white cheese, and 1 butter) of the analyzed samples.     

Development of an analytical method for the determination of photoinitiators used for food packaging materials with potential to migrate into milk

Photoinitiators are used in the curing process during UV printing of food carton labels. The alarm concerning the detection of a photoinitiator, 2-isopropyl thioxanthone (ITX), in food samples packed with cartons printed with UV-cured inks has focused the attention of legislative authorities on the potential migrants from packaging inks into foods. For this reason it is very important to carry out analytical methods for the detection of those compounds in food as potential migrants from packaging. The aim of the present work was to develop a multimethod for the analysis of 6 photoinitiators in milk. The selected photoinitiators were Irgacure 184, benzophenone, Irgacure 651, Irgacure 907, Quantacure ITX, and Quantacure EHA (2-ethylhexyl-4-dimethylaminobenzoate). Milk (10 mL) extraction was carried out by using ammoniac and hexane. The supernatant was evaporated and the residue was redissolved with acetonitrile. Then, the extract was analyzed by HPLC-UV. Calibration lines were carried out over the concentration range of 0.1 to 10 mg/L. The calibration data presented high correlation coefficients (>0.9999). Mean recoveries (n = 6) of the 6 photoinitiators were 83.4% (residual standard deviation = 2.3%) at 0.5 mg/kg and 81.0% (residual standard deviation = 4.6%) at 1 mg/kg. Several milk samples and their respective packaging cartons were analyzed. Results were confirmed by HPLC-mass spectrometry.