Characterization of Strains of Staphylococcus aureus by Their Lipolytic Activity on Various Agar Media

Staphylococcus aureus strains 100-A, 196-E, 254, 473, 505 and 521 were plated on Spirit Blue Agar, Spirit Blue Milk Fat Agar, Nutrient Milk Fat Agar, and Double-Layer Agar plates containing milk fat to determine lipolytic activity. All strains produced dark blue colonies surrounded by blue zones and clearing on Spirit Blue Milk Fat Agar. Results varied for Spirit Blue Agar, Nutrient Milk Fat Agar, and double-layer agar plates, although all strains were at least weakly lipolytic on all agars. S. aureus strains 100-A, 196-E, 505 and 521 demonstrated greater lipolytic acitivity on milk fat than did strains 254 and 473. Degree of lipolytic activity on agars containing milk fat is a means for characterizing different strains of S. aureus.     

Fate of Staphylococcus aureus in whey, whey cream, and whey cream butter

Fresh Cheddar cheese whey was inoculated with ca. 10(6) Staphylococcus aureus/ml and held at 4, 25, and 37 degrees C for 48 h. Numbers of staphylococci decreased in whey at 25 and 37 degrees C and decreased or remained constant in whey at 4 degrees C. When Cheddar cheese whey was neutralized with sodium hydroxide before inoculation with ca. 10(2) or 10(6) S. aureus/ml, numbers of the bacterium increased at all incubation temperatures. Viability of S. aureus strains in whey butter made from inoculated whey cream (from Cheddar cheese whey) was determined. Whey cream was either neutralized to a titratable acidity of .15% or untreated before inoculation with ca. 10(4) S. aureus/ml. Butter churned from the whey cream was held at 4, 25, and 30 degrees C for up to 4 wk. Viability of S. aureus was enhanced in lightly salted (1%) whey cream butter and in butter made from neutralized whey cream. Strains of S. aureus did not survive in unsalted or in salted (1.5%) butter made from untreated whey cream.     

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.     

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.     

Influence of holding temperature on the growth and survival of Salmonella spp. and Staphylococcus aureus and the production of staphylococcal enterotoxin in egg products

In this study, growth and survival of Salmonella spp. and Staphylococcus aureus in steamed egg and scrambled egg held at 5, 18, 22, 37, 55 and 60 degrees C are investigated. The production of staphylococcal enterotoxin in steamed egg is also examined. Results reveal that Salmonella spp. and Staph. aureus in the egg products multiply best at 37 degrees C, followed closely by 22 and 18 degrees C. Neither pathogen showed growth in the egg products held at 5 degrees C. Initial inoculation dose, holding temperature and holding time affected the population of both organisms found in the egg products. Staphylococcal enterotoxin A (SEA) and B (SEB) are detected only in the egg products held at 37 or 22 degrees C. After holding at 37 degrees C for 36 h, scrambled egg inoculated with ca. 5.0 log cfu/g Staph. aureus contains the highest levels of SEA (> 64 ng/g) and SEB (> 64 ng/g). Although Salmonella spp. and Staph. aureus grow better in steamed eggs than in scrambled eggs, production of staphylococcal enterotoxin, in general, was higher in scrambled eggs than in steamed eggs. On the other hand, a repaid destruction of the test organisms in steamed eggs held at 60 degrees C was observed. Holding the steamed eggs at 60 degrees C, Salmonella spp. and Staph. aureus with an initial population of ca. 5.9 and 5.6 log cfu/g, respectively, reduced to a non-detectable level in 1 h.     

Growth and toxin profiles of Bacillus cereus isolated from different food sources

Eleven strains of Bacillus cereus isolated from milk and meat products have been used to study growth and sporulation profiles in detail. Polymerase chain reaction (PCR) using primers detecting cold shock protein A gene signatures (cspA), showed that none of the strains were the newly suggested species in the B. cereus group, B. weihenstephanensis, comprising psychrotolerant cereus strains, although one of the strains grew at 4 degrees C, two at 6 degrees C and seven grew at 7 degrees C. One of the two strains that grew at 6 degrees C had a maximum growth temperature of 42 degrees C, while the remaining 10 strains all grew at temperature of 43 degrees C or higher. Only three strains grew at 48 degrees C. At 42 degrees C, the generation time varied between 11 and 34 min. Spore germination was much faster for the two strains that grew at 6 degrees C than for the other nine strains in milk at 7 degrees C and 10 degrees C. All strains were cytotoxic and contained the non-haemolytic enterotoxin gene (nhe), 10 strains contained the enterotoxin T gene (bceT), and only six had the gene (hbl) encoding haemolytic enterotoxin. Two strains showed some microheterogeneity in the nhe operon. but contained all three genes. We can conclude that true B. cereus strains can have growth profiles as expected for B. weihenstephanensis, and that nhe and bceT were not correlated with growth profiles. However, the two psychrotolerant strains with minimal growth temperature of 4 degrees C and 6 degrees C did not contain hbl, as judged from our PCR results.     

Growth of Staphylococcus aureus and synthesis of enterotoxins in home-made yoghurt

Staphylococcus aureus strains FRI-100, S6, FRI-137 and FRI 472 were inoculated into milk to study growth and enterotoxin production in home-made yogurts. The yogurt used as starter was progressively weakened by successive inoculations (up to four) in milk to prepare other yogurts in order to study the ability of yogurt microflora to inhibit staphylococci. After elaboration, yogurts were stored at 4 degrees C, 22 degrees C, and 37 degrees C for a maximum of 21 days. Periodically, staphylococcal counts, pH and the production of enterotoxins A, B, C, and D were determined. Enterotoxins were only detected in the last batch. It was concluded that the inhibitory effect of the starter culture is not only due to the decrease of pH, but also to other factors.     

Development of a multiplex polymerase chain reaction assay for detection and differentiation of Staphylococcus aureus in dairy products

A multiplex polymerase chain reaction (PCR) assay was developed for the detection and differentiation of enterotoxigenic Staphylococcus aureus in dairy products. A solvent extraction procedure was successfully modified for extraction of S. aureus DNA from 10 ml of artificially contaminated skim milk or 20 g cheddar cheese. Primers targeting the enterotoxin C gene (entC) and thermostable nuclease gene (nuc) were used in the multiplex PCR. PCR products were confirmed using restriction fragment length polymorphism analysis. DNA was consistently quantified and amplified by uniplex PCR from 10 CFU/ml of S. aureus in skim milk or 10 CFU/20 g cheddar cheese. The sensitivity of the multiplex PCR was 100 CFU/ml of skim milk or 100 CFU/20 g cheddar cheese. The developed methodology allows presumptive identification and differentiation of enterotoxigenic S. aureus in less than 6 h.     

Influence of temperature shifts on survival, growth, and toxin production by psychrotrophic and mesophilic strains of Bacillus cereus in potatoes and chicken gravy.

A study was done to determine the influence of temperature on growth and toxin production characteristics of psychrotrophic and mesophilic strains of Bacillus cereus when inoculated into mashed potatoes and chicken gravy containing various concentrations of sodium chloride and held at temperatures different from those at which cells had been cultured. Logarithmic growth phase cells (10 h, 30 degrees C) of psychrotrophic (F3802A/84) and mesophilic (B4ac-1) strains of Bacillus cereus were inoculated into rehydrated commercially processed instant mashed potatoes and chicken gravy supplemented with 0, 2, or 4% sodium chloride. Growth, survival, and diarrheal toxin production in potatoes and gravy held at 30, 37, and 10 degrees C (strain F3802A/84) or 30, 40, and 10 degrees C (strain B4ac-1) were monitored. Both strains grew in both foods containing no added sodium chloride or 2% sodium chloride when held at 30, 37, or 40 degrees C for 2 days. Strain B4ac-1 grew better than strain F3802A/84 in foods containing 4% sodium chloride. Maximum amounts of enterotoxin (1024 ng/g) were produced by strain B4ac-1 in chicken gravy held at 30 and 40 degrees C. Strain F3802A/84 grew to populations of 7 log10 CFU/g in foods containing no added sodium chloride or 2% sodium chloride at 10 degrees C. Strain F3802A/84 produced the highest amount of enterotoxin (1024 ng/g) at 30 degrees C in chicken gravy containing 0.7 or 2% sodium chloride; however, little or low amounts of toxin (4-16 ng/g) were produced in chicken gravy at 10 degrees C. Compared to strain B4ac-1, cells of strain F3802A/84 subjected to a downward shift in incubation temperature (10 degrees C) grew more rapidly in chicken gravy. Strain B4ac-1 produced the highest amount of toxin (1024 ng/g) at 30 degrees C in gravy containing 4% sodium chloride and at 40 degrees C in gravy containing 0.7% sodium chloride. Toxin was not detected in inoculated mashed potatoes. Results of this study indicate that shifts in incubation temperature influence growth and toxin production by psychrotrophic and mesophilic strains of B. cereux differently. It is important to store pasteurized, ready-to-eat foods at a temperature low enough to prevent the growth of B. cereus.     

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.     

High pressure inactivation of microorganisms inoculated into ovine milk of different fat contents

High hydrostatic pressure inactivation of Escherichia coli, Pseudomonas fluorescens, Listeria innocua, Staphylococcus aureus, and Lactobacillus helveticus were studied. These microorganisms were inoculated at a concentration between 10(7) and 10(8) cfu/ml in Ringer solution and in ovine milk adjusted to 0, 6, and 50% fat content to evaluate the baroprotective effect of fat content on inactivation of microorganisms. Treatments of pressurization consisted of combinations of pressure (100 to 500 MPa) and temperature (4, 25, and 50 degrees C) for 15 min. Gram-negative microorganisms were more sensitive than were Gram-positive ones (more destruction P. fluorescens > E. coli > or = List. innocua > Lb. helveticus > S. aureus). Pressurizations at low temperature (4 degrees C) produced greater inactivation on P. fluorescens, List. innocua, and Lb. helveticus than at room temperature (25 degrees C), whereas for E. coli and S. aureus the results were opposite. Ovine milk per se (0% fat) showed a baroprotective effect on all microorganisms, but percentage of fat (6 and 50%) did not show a progressive baroprotective effect in all pressurization conditions or for all microorganisms.     

一起由金黄色葡萄球菌引起食物中毒的实验室诊断

目的对一起食物中毒进行相关样品的实验室检测,针对分离病原菌进行分子分型溯源和耐药分析。方法对可疑食品(冰激凌、牛奶)样品、患者呕吐物和粪便标本使用国家标准或其他方法进行沙门菌、志贺菌、致泻大肠埃希菌、金黄色葡萄球菌、单核细胞增生李斯特菌、蜡样芽胞杆菌和诺如病毒检测。对检出的金黄色葡萄球菌进行肠毒素检测、脉冲场凝胶电泳(PFGE)分型和抗生素敏感性检测。结果从生产冰激凌剩余牛奶及冰激凌样品中检出金黄色葡萄球菌12株,患者呕吐物和粪便标本中检出4株,菌株均产生A+C+E混合型肠毒素,PFGE显示患者呕吐物、粪便、中毒同批冰激凌样品、生产冰激凌用牛奶中检出的金黄色葡萄球菌菌株同源性为100.0%;药敏结果显示,这16株菌株均对青霉素和红霉素耐药。结论本起事件是由冰激凌加工点使用了被金黄色葡萄球菌污染且产生大量肠毒素的牛奶生产冰激凌所致。建议监管部门加强对农村食品小作坊、牛奶供应站等生产经营场所的监管,预防类似事件的发生。     

Effect of six organic acids on staphylococcal growth and enterotoxin production.

Four Staphylococcus aureus strains were incubated at 37 degrees C for 24 h in broth progressively acidified with lactic, citric, ascorbic, acetic, pyruvic and propionic acids, and their survival rate and enterotoxin producing ability was studied. Acids were chosen based on their frequent use by the food industry. Periodically, samples were withdrawn to determine counts, pH and the presence of enterotoxins A, B, C, and D. For a given acid, the effect on growth and enterotoxin synthesis was different. The most inhibitory acid for the growth of strains FRI-100 and FRI-472 was pyruvic acid, for strain FRI-137 was lactic acid, all six acids were equally effective on strain S6. Lactic acid was very inhibitory to enterotoxin synthesis, but the effect on this parameter of acetic and citric acids was almost nil. Enterotoxins were seen to be inactivated at acid pH values; enterotoxin B was the most resistant to inactivation.     

Antimicrobial activity of reuterin in combination with nisin against food-borne pathogens

Antimicrobial activity of reuterin individually or in combination with nisin against different food-borne Gram-positive and Gram-negative pathogens in milk was investigated. Reuterin (8 AU/ml) exhibited bacteriostatic activity against Listeria monocytogenes, whereas its activity was slightly bactericidal against Staphylococcus aureus at 37 degrees C. Higher bactericidal activity was detected against Escherichia coli O157:H7, Salmonella choleraesuis subsp. choleraesuis, Yersinia enterocolitica, Aeromonas hydrophila subsp. hydrophila and Campylobacter jejuni. A significant synergistic effect on L. monocytogenes and a slight additive effect on S. aureus after 24 h at 37 degrees C were observed when reuterin was combined with nisin (100 IU/ml). The combination of reuterin with nisin did not enhance the antimicrobial effect of reuterin against Gram-negative pathogens.     

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.     

Inhibition of growth of Enterobacter sakazakii in reconstituted infant formula by the lactoperoxidase system

Neonatal bacteremia and meningitis caused by the opportunistic pathogen Enterobacter sakazakii have been associated with the consumption of reconstituted powdered infant formula. Lactoperoxidase (LPO), present in mammalian milk, is known to inhibit the growth of enteric pathogens. We undertook a study to determine if the lactoperoxidase system (LPOS) will inhibit the growth of E. sakazakii in a milk-based powdered infant formula reconstituted with water. Initially at 0.04 CFU/ml, E. sakazakii grew to 2.40 to 2.74 log CFU/ml in reconstituted infant formula held at 30 or 37 degrees C for 8 h and to 0.6 log CFU/ ml in formula held for 12 h at 21 degrees C. The pathogen was not detected (less than 1 CFU/227 ml) by enrichment of formula treated with 10 to 30 microg/ml LPO and stored for 24 h at 37 degrees C or 30 microg/ml LPO and stored for 24 h at 30 degrees C. Populations of E. sakazakii, initially at 4.40 log CFU/ml of reconstituted infant formula containing 5 microg/ml LPO, did not increase significantly (P > 0.05) for up to 12 h at 21 and 30 degrees C. Populations either decreased significantly or were unchanged in formula supplemented with 10 microg/ml LPO and stored at 21, 30, or 37 degrees C for up to 24, 8, and 8 h, respectively. Results indicate that LPOS can be used to control the growth of E. sakazakii in reconstituted infant formula, thereby potentially reducing the risk of neonatal infections resulting from consumption of formula that may be contaminated with the pathogen.     

Effect of water activity on growth and enterotoxin D production of Staphylococcus aureus

Four enterotoxin D-producing strains of Staphylococcus aureus, were cultured at 37 degrees C in brain heart infusion broth adjusted to various water activity (aw) levels by means of NaCl. For high cell inocula growth of all strains and enterotoxin D production were observed within 6 days at aw levels down to 0.86.     

Effect of temperature of CO2 injection on the pH and freezing point of milks and creams

The objectives of this study were to measure the impact of CO2 injection temperature (0 degree C and 40 degrees C) on the pH and freezing point (FP) of (a) milks with different fat contents (i.e., 0, 15, 30%) and (b) creams with 15% fat but different fat characteristics. Skim milk and unhomogenized creams containing 15 and 30% fat were prepared from the same batch of whole milk and were carbonated at 0 and 40 degrees C in a continuous flow CO2 injection unit (230 ml/min). At 0 degree C, milk fat was mostly solid; at 40 degrees C, milk fat was liquid. At the same total CO2 concentration with CO2 injection at 0 degree C, milk with a higher fat content had a lower pH and FP, while with CO2 injection at 40 degrees C, milks with 0%, 15%, and 30% fat had the same pH. This indicated that less CO2 was dissolved in the fat portion of the milk when the CO2 was injected at 0 degree C than when it was injected at 40 degrees C. Three creams, 15% unhomogenized cream, 15% butter oil emulsion in skim milk, and 15% vegetable oil emulsion in skim milk were also carbonated and analyzed as described above. Vegetable oil was liquid at both 0 and 40 degrees C. At a CO2 injection temperature of 0 degree C, the 15% vegetable oil emulsion had a slightly higher pH than the 15% butter oil emulsion and the 15% unhomogenized cream, indicating that the liquid vegetable oil dissolved more CO2 than the mostly solid milk fat and butter oil. No difference in the pH or FP of the 15% unhomogenized cream and 15% butter oil emulsion was observed when CO2 was injected at 0 degree C, suggesting that homogenization or physical dispersion of milk fat globules did not influence the amount of CO2 dissolved in milk fat at a CO2 injection temperature of 0 degree C. At a CO2 injection temperature of 40 degrees C and at the same total CO2 concentration, the 15% unhomogenized cream, 15% vegetable oil emulsion, and 15% butter oil emulsion had similar pH. At the same total concentration of CO2 in cream, injection of CO2 at low temperature (i.e., < 4 degrees C) may produce a better antimicrobial effect during refrigerated shelf life due to the higher concentration of CO2 in the skim portion of the cream.     

Characterization of Staphylococcus aureus strains isolated from raw milk utilized in small-scale artisan cheese production

Staphylococcus aureus is an important agent of bacterial mastitis in milking animals and of foodborne intoxication in humans. The purpose of this study was to examine the genetic and phenotypic diversity, enterotoxigenicity, and antimicrobial resistance of S. aureus strains isolated from raw milk used for the production of artisan cheese in Vermont. Cross-tabulations revealed that the 16 ribotypes identified among the 90 milk isolates examined were typically associated with a specific animal species and that more than half of these ribotypes were unique to individual farms. In general, specific EcoRI ribotypes were commonly associated with specific phenotypical characteristics, including staphylococcal enterotoxin production or the lack thereof. Limited antimicrobial resistance was observed among the isolates, with resistance to ampicillin (12.51%) or penicillin (17.04%) most common. Two isolates of the same ribotype obtained from the same farm were resistant to oxacillin with 2% NaCl. More than half (52.22%) of isolates produced toxin, and 31 of the 32 isolates solely produced staphylococcal enterotoxin type C. Although these data demonstrate that S. aureus strains found in raw milk intended for artisan cheese manufacture are capable of enterotoxin production, staphylococcal enterotoxin C is not typically linked to foodborne illness. Because S. aureus is a common contaminant of cheese, an understanding of the ecology of this pathogen and of the antimicrobial susceptibility and toxigenicity of various strains will ultimately contribute to the development of control practices needed to enhance the safety of artisan and farmstead cheese production.