Growth and survival of Escherichia coli O157:H7 and Listeria monocytogenes in egg products held at different temperatures

Growth and survival of Escherichia coli O157:H7 and Listeria monocytogenes in steamed eggs and scrambled eggs held at different temperatures (5, 18, 22, 37, 55, and 60 degrees C) were investigated in the present study. Among the holding temperatures tested, both pathogens multiplied best at 37 degrees C followed by 22, 18, and 5 degrees C. In general, E. coli O157:H7 grew better in the egg products than L. monocytogenes did at all the storage temperatures tested except at 5 degrees C. E. coli O157:H7 did not grow in steamed eggs and scrambled eggs held at 5 degrees C. L. monocytogenes showed a slight population increase of approximately 0.6 to 0.9 log CFU/g in these egg products at the end of the 36-h storage period at 5 degrees C. The population of both pathogens detected in the egg products was affected by the initial population, holding temperature, and length of the holding period. It was also noted that L. monocytogenes was more susceptible than E. coli O157:H7 in steamed eggs held at 60 degrees C. After holding at 60 degrees C for 1 h, no detectable viable cells of L. monocytogenes with a population reduction of 5.4 log CFU/g was observed in steamed eggs, whereas a lower population reduction of only approximately 0.5 log CFU/ml was noted for E. coli O157:H7.     

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.     

Enterotoxin production by Staphylococcus aureus isolated from mastitic cows

Staphylococcus aureus is an important cause of mastitis in cows. The ability of S. aureus strains to produce one or more enterotoxins in milk and dairy products is linked to staphylococcal food poisoning. To determine whether staphylococci causing bovine mastitis could cause human foodborne intoxication, the production of staphylococcal enterotoxins A through D (SEA, SEB, SEC, and SED) by 160 S. aureus isolates was evaluated with the use of a reverse passive latex agglutination enterotoxin kit. All S. aureus strains were isolated over a 9-month period from 2,343 routine submissions of a composite quarter collection of individual mastitic cows at 18 dairy farms in the San Joaquin Valley in California. Prior to enterotoxin detection, isolates were grown by a method that enhances the in vitro synthesis of enterotoxin. Twenty-two of 160 S. aureus isolates produced enterotoxin. Seven produced SEC, 12 produced SED, and 3 produced both SEC and SED. None of the isolates produced SEA or SEB.     

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.     

Characteristics of enterotoxin H-producing Staphylococcus aureus isolated from clinical cases and properties of the enterotoxin productivity

Staphylococcal enterotoxin H (SEH) is predicted to be involved in staphylococcal food poisoning. To characterize SEH-producing Staphylococcus aureus isolates from staphylococcal food poisoning cases in Japan, we investigated the relationship between SEH production and coagulase serotype, which is an epidemiological marker, and compared the properties of SEH production with those of staphylococcal enterotoxins A (SEA) and B (SEB). SEH production was determined by a newly developed sandwich enzyme-linked immunosorbent assay. Eighty-six (59.7%) of 144 isolates from staphylococcal food poisoning cases produced SEH. Seventy-one of the SEH-producing isolates simultaneously produced SEA, SEB, or both. All SEH-producing isolates belonged to coagulase type VII, which was the predominant type, representing 99 (68.8%) of 144 isolates. The amount of SEH produced in brain heart infusion was almost the same as the amount of SEA and approximately 10-fold lower than that of SEB. SEH and SEA were produced mainly during the late exponential phase of growth, whereas SEB was produced mostly during the stationary phase. The production levels of SEH and SEA were gradually affected by decreases in water activity, but the production of SEB was greatly reduced under conditions of low water activity. These findings indicate that SEH-producing S. aureus isolates are of high prevalence in staphylococcal food poisoning cases. Given the unique epidemiological characteristic of these isolates, SEH and SEA probably are responsible for food poisoning.     

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.     

Staphylococcus aureus growth and enterotoxin A production in an anaerobic environment.

The effects of strict anaerobic conditions on the growth of Staphylococcus aureus and the production of staphylococcal enterotoxin A (SEA) were studied. The growth of S. aureus, a facultative anaerobic bacterium, is slower anaerobically than aerobically. When grown on brain heart infusion broth at 37 degrees C, the anaerobic generation time at mid-log phase was 80 min, compared with 35 min for the aerobic control. In contrast to previous studies demonstrating that staphylococcal cell density was 9- to 17-fold greater in aerobic than in anaerobic cultures, data for a staphylococcal strain implicated in food poisoning showed that the cell density was only two to three times as great in aerobic cultures. Production of SEA was monitored by Western immunoblotting and shown to be growth dependent. With slower anaerobic growth, relatively less toxin was produced than under aerobic conditions. but in both cases SEA was detected after 120 min of incubation. The combined effects of temperature and aeration on S. aureus were also studied. Growth and toxin production of aerobic and anaerobic cultures at temperatures ranging from 14 to 37 degrees C were analyzed. Growth was still observed at low temperatures in both environments. A linear model for S. aureus aerobic or anaerobic growth as a function of incubation temperature was developed from these studies. The model was tested from 17 to 35.5 degrees C, and the results suggest that the model can accurately predict the S. aureus growth rate in this temperature range. The data suggest that anaerobic conditions are not an effective barrier against S. aureus growth.     

利用简并引物检测金黄色葡萄球菌肠毒素A、B基因

通过设计简并引物建立一种PCR技术同步快速检测金黄色葡萄球菌肠毒素A和B基因的方法。根据金黄色葡萄球菌肠毒素A、B基因编码序列,设计一对特异性简并引物SEAB来扩增靶基因片段,长度分别为105bp和135bp,通过对金黄色葡萄球菌肠毒素A、B菌株和4株对照菌株进行PCR检测,评价该引物的特异性;对金黄色葡萄球菌肠毒素B的DNA系列10倍稀释,对其灵敏性进行PCR检测。结果显示,金黄色葡萄球菌肠毒素A和B菌株的DNA检测结果呈阳性,4株对照菌株的检测结果呈阴性,通过基因测序证实了PCR产物的特异性,SEB的DNA最低检测浓度为3.58ng,整个检测过程不超过20h。建立了一个特异、快速灵敏的在同一条件下,金黄色葡萄球菌肠毒素A、B基因的PCR检测方法。     

Development of two multiplex polymerase chain reactions for the detection of enterotoxigenic strains of Staphylococcus aureus isolated from foods

Two multiplex polymerase chain reactions were developed for the detection of enterotoxigenic strains of Staphylococcus aureus: one multiplex reaction for the simultaneous detection of enterotoxigenic strains type A (entA), type B (entB), and type E (entE) and another for the simultaneous detection of enterotoxigenic strains type C (entC) and type D (entD). Both reactions were standardized with the use of the reference enterotoxigenic strains of S. aureus: FRI 722, producer of staphylococcal enterotoxin (SE) type A (SEA); FRI 1007, producer of SEB; FRI 137, producer of SEC1; FRI 472, producer of SED; and FRI 326, producer of SEE. Optimized methods were used to determine the presence of enterotoxigenic types for 51 S. aureus strains isolated from meat (sausage, ham, and chorizo) and dairy (powdered milk and cheese) products by the Baird-Parker technique. The enterotoxigenic capacities of the strains were determined by the indirect enzyme-linked immunosorbent assay (ELISA) with the use of reference staphylococcal toxins and antitoxins. Fifty of the 51 strains isolated were enterotoxigenic and produced one to four enterotoxin types, with the most frequently produced types being SEA and SED. Levels of correlation between the presence of genes that code for the production of SE (as determined by polymerase chain reaction) and the expression of these genes (as determined by the indirect ELISA) were 100% for SEA and SEE, 86% for SEC, 89% for SED, and 47% for SEB.     

Factors influencing inactivation of Salmonella enteritidis in hard-cooked eggs

The inside of a hen's egg, once considered sterile, is now known to occasionally harbor Salmonella Enteritidis. At least two recent outbreaks of salmonellosis in which Salmonella Enteritidis PT34 was involved have been associated with hard-cooked eggs. This study was undertaken to compare D56 degrees C values of Salmonella Senftenberg 775W and six strains of Salmonella Enteritidis isolated from outbreaks associated with eggs. D56 degrees C values for Salmonella Enteritidis in liquid egg yolk ranged from 5.14 to 7.39 min; the D56 degrees C value for Salmonella Senftenberg was 19.96 min. The two PT34 strains from outbreaks associated with hard-cooked eggs did not exhibit significantly higher resistance to heat compared with two PT4 strains and one strain each of PT8 and PT13a. A PT4 strain and a PT34 strain of Salmonella Enteritidis were separately inoculated (10(7) to 10(8) CFU) into the yolk of medium and extra large shell eggs at 10 and 21 degrees C, and survival was monitored using two cooking methods: (i) placing eggs in water at 23 degrees C, heating to 100 degrees C, removing from heat, and holding for 15 min (American Egg Board method) and (ii) placing eggs in water at 100 degrees C, then holding for 15 min at this temperature. Within the 15-min holding periods, inactivation was more rapid using the method recommended by the American Egg Board compared with method 2. Within each cooking method, inactivation was most rapid in medium eggs initially at 21 degrees C. The PT4 strain survived in yolk of extra large eggs initially at 10 degrees C when eggs were held in boiling water 9 min using method 2. The final temperature of the yolk in these eggs was 62.3 +/- 2 degrees C. Of the two methods evaluated for hard cooking eggs, the American Egg Board method is clearly most effective in killing Salmonella Enteritidis in the yolk.     

Coagulase-positive Staphylococci and Staphylococcus aureus in food products marketed in Italy

Staphylococcus aureus is a very common organism capable of producing several enterotoxins (SEs) that cause intoxication symptoms of varying intensity in humans when ingested through contaminated food. This paper reports the results of an investigation on the presence of Coagulase-Positive Staphylococci (CPS) and S. aureus in several food products marketed in Italy and on food contact surface swabs sampled from the food industry. A total of 11,384 samples were examined and 1971 of them (17.3%) were found to contain CPS. The assays performed on 541 CPS strains led to the identification of 537 S. aureus strains on which characterization of type A, B, C and D staphylococcal enterotoxins (SEA, SEB, SEC and SED) was performed. A total of 298 S. aureus strains (55.5%) produced one or more SEs: 33.9% of the strains produced SEC, 26.5% SEA, 20.5% SEA+SED, 13.4% SED, 2.7% SEB, 1.7% SEA+SEB, 0.7% SEC+SED and 0.3% produced SEA+SEC and SEB+SEC. The investigation highlighted that these organisms are very common and constitute a potential risk for consumers' health.     

Growth and production of enterotoxin A by Staphylococcus aureus in cream

Behavior of Staphylococcus aureus strains 100-A, 196-E, 254, 473, 505, and 521 in sweet (18 to 80% milk fat) and neutralized sour cream was studied. Cream was inoculated to contain approximately 10(3) to 10(4) S. aureus/ml, depending on milk fat content, and was incubated at 4, 22, or 37 degrees C. Determinations were made of aerobic plate count, S. aureus count, and pH. When growth in cream exceeded 10(7) S. aureus/ml, enterotoxin analysis was done. Sweet and neutralized sour cream supported growth of all strains of S. aureus tested. Strains 100-A, 196-E, 473, 505, and 521 grew sufficiently to produce enterotoxin in sweet cream of 18 or 32% milk fat held at 37 degrees C for 18 h or at 22 degrees C for 52 h. Populations of strains 100-A, 196-E, 505, and 521 exceeded 10(6) cells/ml in sweet cream of 36% milk fat held for 18 h at 37 degrees C. Strains 100-A and 521 grew to more than 10(6) cells/ml in sweet cream of 40% milk fat held for 18 h at 37 degrees C. No strain of S. aureus grew to levels associated with detectable enterotoxin production at 4 degrees C within 14 d in any cream. Incubation temperature, milk fat content of cream, and variation among strains influenced the ability of S. aureus to grow and produce enterotoxin.     

Optimization of iron supplementation for enhanced detection of Salmonella Enteritidis in eggs

Mixed raw egg contents were inoculated with approximately 10 CFU of Salmonella Enteritidis and supplemented with 0 to 7 mg of FeSO4 per g of egg contents. Egg contents were then incubated at 37 degrees C, and Salmonella Enteritidis colonies were enumerated for up to 106 h. Iron supplementation significantly enhanced the growth of Salmonella Enteritidis. Within the first 24 h of incubation, the optimum iron level for Salmonella Enteritidis growth in egg contents was between 0.2 and 2 mg of FeSO4 per g of egg contents. After 24 h of incubation at 37 degrees C. Salmonella Enteritidis counts in eggs supplemented with 0.5 mg of FeSO4 per g of egg contents consistently reached approximately 1 x 10(9) CFU/ml, whereas Salmonella Enteritidis counts in eggs without iron supplementation varied from less than 5 CFU/ml to 8.4 x 10(6) CFU/ml. A 3 by 3 factorial design was used to study the effect of type of preenrichment and level of iron supplementation on the growth of Salmonella Enteritidis in egg contents. No significant differences in Salmonella Enteritidis counts between preenrichment and nonpreenrichment treatments were observed when egg contents were supplemented with 0.5 mg of FeSO4 per g of egg contents. It was concluded that preenrichment was not necessary for isolation of Salmonella Enteritidis from eggs. The effect of iron supplementation on the sensitivity of detection by the direct plating method was investigated. The direct plating method detected a significantly higher percentage of Salmonella Enteritidis in raw egg contents supplemented with 0.5 mg of FeSO4 per g of egg contents (90%) than in raw egg contents without iron supplementation (63.3%).     

DETECTION OF STAPHYLOCOCCAL ENTEROTOXIN A FROM RICOTTA CHEESE BY CELL CULTURE

Staphylococcal food poisoning (SFP) is a very common foodborne disease. Milk and dairy products are frequently contaminated with enterotoxigenic Staphylococcus aureus, which are often involved in SFP. These foods may become contaminated owing to subclinical staphylococcal mastitis or during handling, storage and trade. For sanitary purposes, an effective approach is to detect the staphylococcal enterotoxins directly in foods, rather than detect, count and type the isolated S. aureus strains, because the toxin is the causative agent of foodborne illness. This paper illustrates a cell‐based bioassay that uses the PEB (bovine embryo lung cells) cell line to detect staphylococcal enterotoxin type A (SEA) directly from artificially contaminated ricotta cheese. The test was able to detect SEA in the contaminated samples after 24 and 48 h of incubation at 37C, while results were uncertain when the samples were incubated for 72 h. The test proved to be easy to use and rapidly provided results.     

Analytical chromatography for recovery of small amounts of staphylococcal enterotoxins from food

Sample preparation is an important element in the detection of toxins in food samples. In this work, a simple analytical sample preparation method for recovery of small amount of staphylococcal enterotoxin B (SEB) and staphylococcal enterotoxin A (SEA) in food samples was developed. Cation exchanger carboxymethylcellulose (CM) was used for small-scale batch chromatography isolation of SEB from infant formula and from mushrooms spiked with SEB. The resulting materials were analyzed for SEB by Western immunoblotting. Nearly all of the extraneous substances in the sample were removed by this procedure with no significant loss of the toxin. Using this method, even small amounts of SE (0.75 ng/g) can be recovered and immunologically analyzed by Western blotting or by ELISA with a very low background. Because this method is effective, rapid, simple and inexpensive, it has the potential to be a general method for the preparation of samples used for analysis of SEs.     

Prevalence of Salmonella spp. in poultry broilers and shell eggs in Korea

This study was conducted to determine the presence of Salmonella spp. in raw broilers and shell eggs in Korea. In total, 135 dozen shell eggs and 27 raw broilers were tested. None of the egg yolks were found to contain Salmonella organisms but Escherichia coli, Escherichia hermanii, and Citrobacter freundii were isolated from egg shells. Salmonella spp. were detected in 25.9% of raw broilers, and Salmonella serotypes isolated from raw broilers were Salmonella Enteritidis, Salmonella Virchow, and Salmonella Virginia. D-values and antibiotic resistance of Salmonella isolates were also investigated. D-values of Salmonella enteritidis, Salmonella Virginia, and Salmonella Virchow in tryptic soy broth at 55 degrees C were 2.36, 2.13, and 0.70 min and 0.53, 0.37, and 0.20 min at 60 degrees C, respectively. All Salmonella isolates showed multiple antibiotic resistance patterns and were resistant to penicillin and vancomycin. One strain of Salmonella Enteritidis showed resistance to 12 antibiotics used in this study.     

多重PCR检测金黄色葡萄球菌六型肠毒素基因的研究

目的:为了建立一种简单、快速检测金黄色葡萄球菌六型肠毒素基因的多重PCR方法。方法:根据相关文献和Genebank报道的编码金黄色葡萄球菌肠毒素A、B、C、D、E、H的基因序列,选择合成了6对特异性引物,建立多重PCR体系,并对反应条件进行了优化。结果:6对引物能同时特异地扩增出120、478、257、319、170、375bp的目的片段,表明6对引物具有良好的特异性。结论:成功地建立了一种同时检测金黄色葡萄球菌六型肠毒素基因的多重PCR方法,在金黄色葡萄球菌肠毒素快速筛查方面具有良好的应用前景。     

Populations of Salmonella enteritidis in artificially inoculated chicken eggs as influenced by the temperatures under which eggs might be held from the day of lay until the day of processing

This study was undertaken to determine the levels of Salmonella Enteritidis in artificially inoculated eggs as affected by the temperatures under which eggs might be held from the day of lay until the day of processing. Unprocessed chicken eggs of different sizes (n=1920, with 480 being laid in each season) were inoculated in the albumen with a five-strain mixture of Salmonella at 102 CFU per egg. The eggs were stored at 4, 10, and 22 degrees C for 3 weeks and sampled twice a week to determine the populations of Salmonella and total aerobic bacteria. The season in which eggs were laid did not significantly impact the growth of the pathogen (P > 0.05). The mean populations of the inoculated Salmonella were not significantly different in eggs stored at 4 versus 10 degrees C (P > 0.05). Eggs stored at 22 degrees C had a mean Salmonella population that was 3.71 or 3.37 log higher than the Salmonella population of eggs stored at 4 or 10 degrees C (P > 0.05). The mean Salmonella population at 22 degrees C increased from the initial 2.12 log CFU/ml to 3.36 log CFU/ml after 2 weeks of storage and to 7.84 log CFU/ml after 3 weeks of storage. A sharp increase in the population of Salmonella occurred after 2 to 2.5 weeks of storage at 22 degree C. This study provided a scientific basis for the current egg handling and transporting temperature requirements and reinforced the importance of maintaining low temperatures in controlling and preventing the growth of Salmonella Enteritidis in eggs from the day of lay until the day of processing.     

Production of staphylococcal enterotoxin D in foods by low-enterotoxin-producing staphylococci.

The goal of this investigation was to determine whether staphylococcal strains producing enterotoxins at nanogram levels per milliliter in laboratory medium, not detectable by gel diffusion methods, could produce sufficient enterotoxin in foods to result in food poisoning. Three low-enterotoxin D (SED)-producing strains were selected for this research because this enterotoxin is produced in smaller amounts than the other enterotoxins. The foods used were cream pie and cooked ham, divided into two portions, sterile and non-sterile. Each portion was inoculated with known concentrations of the staphylococcal strains under study and incubated for 48 h at 25, 30, and 37 degrees C. Samples were taken after 24 and 48 h. Enterotoxin was detectable in both sterilized and unsterilized cream and ham after 24 h at 37 degrees C with an inoculum of 10(3)/g. Some strains produced detectable amounts of enterotoxin in the sterilized foods after 24 h at 30 degrees C and some produced detectable amounts of enterotoxin in the sterilized foods after 24 h at 25 degrees C with inocula of 10(4)/g. It can be concluded that staphylococcal strains producing enterotoxin at ng/ml levels in laboratory medium, not detectable by gel diffusion methods, can produce sufficient enterotoxin (ng/g) in foods to cause food poisoning.