Staphylococcus aureus Growth and Enterotoxin Production in Mushrooms

An enterotoxin A (SEA) producing strain of Staphylococcus aureus was inoculated onto mushrooms and incubated under simulated pre-and post-canning conditions. S. aureus grew and produced SEA in mushrooms incubated at 25°C in 10% to 20% NaCl. Growth and SEA production occurred when mushrooms were stored in plastic bags at 37°C, but not at 25°C. Mushrooms heat-processed at 121.1°C supported S. aureus growth and SEA production. No SEA was recovered from mushrooms inoculated with 1 or 10 ng SEA/g and heated at 121.1°C for 4.5 min. At higher inoculated SEA levels (100 and 1,000 ng/g), SEA was detectable after 10 min of heating. Staphylococcal enterotoxin could be present in canned mushrooms as a result of pre-, but more likely post-processing contamination.     

Effects of Frozen Storage on Survival of Staphylococcus aureus and Enterotoxin Production in Precooked Tuna Meat

This study investigated the survival of Staphylococcus aureus in precooked tuna meat for producing canned products during frozen storage (?20 ± 2 °C) as well as its growth and enterotoxin production at 35 to 37 °C after the storage. Samples (50 ± 5 g) of precooked albacore (loin, chunk, and flake) and skipjack (chunk and flake) tuna were inoculated with 5 enterotoxin‐producing strains of S. aureus at a level of approximately 3.5 log CFU/g and individually packed in a vacuum bag after 3 h incubation at 35 to 37 °C. Vacuum‐packed samples were stored in a freezer (?20 ± 2 °C) for 4 wk. The frozen samples were then thawed in 37 °C circulating water for 2 h and incubated at 35 to 37 °C for 22 h. Populations of S. aureus in all precooked tuna samples decreased slightly (<0.7 log CFU/g) after 4 wk of storage at ?20 ± 2 °C, but increased rapidly once the samples were thawed and held at 35 to 37 °C. Total S. aureus counts in albacore and skipjack samples increased by greater than 3 log CFU/g after 6 and 8 h of exposure to 35 to 37 °C, respectively. All samples became spoiled after 10 h of exposure to 35 to 37 °C, while no enterotoxin was detected in any samples. However, enterotoxins were detected in albacore loin and other samples after 12 and 24 h of incubation at 35 to 37 °C, respectively. Frozen precooked tuna meat should be used for producing canned tuna within 6 to 8 h of thawing to avoid product spoilage and potential enterotoxin production by S. aureus in contaminated precooked tuna meat.     

Growth of Staphylococcus aureus in Cooked Potato and Potato Salad—A One‐Step Kinetic Analysis*

Staphylococcus aureus is a Gram‐positive spherically‐shaped bacterium capable of producing heat‐stable enterotoxins that cause acute gastrointestinal diseases. The growth of this pathogen in food is a major threat to public health worldwide. Potato salad is a frequent vehicle for infection and food poisoning caused by S. aureus. Therefore, the objective of this study was to investigate the growth kinetics of S. aureus in cooked potato and potato salad. Samples of potato cubes and potato salad inoculated with S. aureus were incubated at temperatures between 8 and 43 °C to observe its growth for developing growth models. No growth was observed at 8 °C. The experimental results showed that the growth curves did not exhibit lag phases, and can be described by a 3‐parameter logistic model. A one‐step kinetic analysis approach was used to simultaneously analyze all growth curves by direct construction of both the primary and secondary (Ratkowsky square root) models using nonlinear regression to minimize the global residual error. The estimated nominal minimum growth temperature of S. aureus was 6.12 °C in potato cubes and 8.80 °C in potato salad. The estimated maximum growth temperatures of S. aureus in potato cubes and potato salad were very close to each other (46.3 and 46.8 °C, respectively). On the average, the specific growth rates of S. aureus in potato cubes were approximately 70% higher than those in potato salad. This study suggests that cooked potato and potato salad should be stored below 6 °C or above 47 °C to prevent the growth of S. aureus. The mathematical models and kinetic parameters can be used to accurately evaluate the effect of temperature abuse on the growth of S. aureus and conduct risk assessments of S. aureus in cooked potato and potato salad.     

Growth and enterotoxin production of Staphylococcus aureus in Iranian ultra‐filtered white cheese

This study aimed to investigate the growth and enterotoxin production of Staphylococcus aureus during the ripening and storage of Iranian ultra‐filtered white cheese (IUFWC). According to the results, the addition of starter culture had significant inhibitory effects on the growth and enterotoxin production of S. aureus. Moreover, prolong survival and enterotoxin production were observed in the samples with the highest inoculum (5 log CFU/g) of S. aureus. It was concluded that proper activity of the starter culture together with the refrigeration storage of IUFWC was the key element in inhibiting the growth and enterotoxin production of S. aureus.     

Exposure assessment of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> in <Emphasis Type="Italic">Kimbab</Emphasis>, a ready-to-eat Korean food

The exposure assessment was carried out for Staphylococcus aureus in kimbab by predicting growth of S. aureus and the production of enterotoxin using Food MicroModel® program. Environmental parameters selected were pH 5.5, Aw 0.999, and storage temperatures in the range of 10 to 30°C. It was predicted that 6.3 hr could be a critical time for enterotoxin production while kimbab was stored at 30°C. Mild case scenario analysis showed that enterotoxin could not be produced if kimbab was kept at 10°C during preparation and distribution and then left at 25°C for 4 hr before consumption. In the worst case scenario, the keeping time at 25°C was assumed to be 7.0 hr. The level of S. aureus in the worst case was predicted to be 6.8×10⁶ CFU/g which is lower than the critical level (7.8×10⁶ CFU/g) for toxin production.     

Combined Effect of Water Activity, pH and Sub-optimal Temperature on Growth and Enterotoxin Production of Staphylococcus aureus

Staphylococcus aureus growth and enterotoxin production in relation to a, (0.99–0.87), pH (4.0–7.0) and temperature (8–30°C) was determined in Brain-heart-infusion broth with respectively NaCl and sucrose as humectants. Growth of S. aureus was not observed at a_w 0.85, at pH 4.3 or at 8°C. At 12°C no growth occurred at a, 0.90 or at a, 0.93 in combination with pH < 5.5. At a_w 0.96 no growth occurred at pH ≤ 4.9. Production of staphylococcal enterotoxin type A (SEA) occurred under nearly all conditions allowing growth. Production of SEB appears to be determined by a_w; at a_w 0.96 SEB was produced at all temperatures allowing growth, while at a, 0.93 SEB was hardly produced. Production of SEC and SEF was affected both by a_w and temperaure. Production of these toxins was rarely observed at a, 0.93. No SEC and SEF were produced at 12°C. Extrapolation of these findings to food is discussed.     

Antilisterial Properties of Marinades during Refrigerated Storage and Microwave Oven Reheating against Post‐Cooking Inoculated Chicken Breast Meat

This study evaluated growth of Listeria monocytogenes inoculated on cooked chicken meat with different marinades and survival of the pathogen as affected by microwave oven reheating. During aerobic storage at 7 °C, on days 0, 1, 2, 4, and 7, samples were reheated by microwave oven (1100 W) for 45 or 90 s and analyzed microbiologically. L. monocytogenes counts on nonmarinated (control) samples increased (P < 0.05) from 2.7 ± 0.1 (day‐0) to 6.9 ± 0.1 (day‐7) log CFU/g during storage. Initial (day‐0) pathogen counts of marinated samples were <0.5 log CFU/g lower than those of the control, irrespective of marinating treatment. At 7 d of storage, pathogen levels on samples marinated with tomato juice were not different (P ≥ 0.05; 6.9 ± 0.1 log CFU/g) from those of the control, whereas for samples treated with the remaining marinades, pathogen counts were 0.7 (soy sauce) to 2.0 (lemon juice) log CFU/g lower (P < 0.05) than those of the control. Microwave oven reheating reduced L. monocytogenes counts by 1.9 to 4.1 (45 s) and >2.4 to 5.0 (90 s) log CFU/g. With similar trends across different marinates, the high levels of L. monocytogenes survivors found after microwave reheating, especially after storage for more than 2 d, indicate that length of storage and reheating time need to be considered for safe consumption of leftover cooked chicken.     

Production of enterotoxin A by Staphylococcus aureus in food

The relationship between growth of Staphylococcus aureus and enterotoxin A production in various cooked foods at 22°C was determined. Although production of enterotoxin A in artificial media occurs under most conditions that support growth of S. aureus, such a correlation was not found in foods. In meat products and vanilla custard enterotoxin A was detected when ? 7.2 log₁₀ colony forming units/g were present. In vegetables (peas, spinach) however, production of enterotoxin A was markedly delayed despite excellent growth of S. aureus. Production of the toxin was observed when the count of S. aureus had reached ? 8.9 log₁₀ colony forming units/g.     

Application of enterocins A and B, sakacin K and nisin to extend the safe shelf-life of pressurized ready-to-eat meat products

The efficiency of food preservation systems is determined by the technologies that are combined, the intrinsic properties of the food products and the target microorganisms. In the present study, the bacteriocins nisin, enterocins A and B and sakacin K were applied to cooked and dry cured ham spiked with Listeria monocytogenes, Salmonella enterica and Staphylococcus aureus and submitted to a high pressure treatment of 600 MPa. Before pressurization nisin produced significant reductions to the counts of L. monocytogenes and S. aureus, especially in dry cured ham. After the pressurization, Salmonella and L. monocytogenes were not detected in 25 g of both cooked and dry cured ham and remained at this level during the entire storage (57 days at 4 °C + 63 days at 15 °C). S. aureus levels, in contrast, only decreased below the detection limit (1 log CFU/g) in the nisin batches. Afterward, when storage was performed at an abusive temperature, the ability of S. aureus to grow was dependant on the bacteriocin applied and the kind of meat product. Thus, at the end of storage, while S. aureus counts were <1 log CFU/g in all dry cured ham batches, only nisin could inhibit its growth in cooked ham.     

Antimicrobial Effects of Quillaja saponaria Extract Against Escherichia coli O157:H7 and the Emerging Non‐O157 Shiga Toxin‐Producing E. coli

Natural alternate methods to control the spread of Shiga toxin‐producing Escherichia coli (STEC) are important to prevent foodborne outbreaks. Quillaja saponaria aqueous bark extracts (QE), cleared by the U.S. Food and Drug Administration as a natural flavorant, contain bioactive polyphenols, tannins, and tri‐terpenoid saponins with anti‐inflammatory and antimicrobial activity. The objective of this study was to determine the effects of commercial QE against E. coli O157:H7 and non‐O157 strains over 16 h at 37 °C and RT. Overnight cultures of 4 E. coli O157:H7 strains and 6 non‐O157 STECs in Tryptic Soy Broth (TSB) were washed and resuspended in phosphate‐buffered saline (PBS, pH 7.2), and treated with QE and controls including citric acid (pH 3.75), sodium benzoate (0.1% w/w), acidified sodium benzoate (pH 3.75) or PBS for 6 h or 16 h. Recovered bacteria were enumerated after plating on Tryptic Soy Agar, from duplicate treatments, replicated thrice and the data were statistically analyzed. The 4 QE‐treated E. coli O157:H7 strains from initial ～7.5 log CFU had remaining counts between 6.79 and 3.5 log CFU after 16 h at RT. QE‐treated non‐O157 STECs showed lower reductions with remaining counts ranging from 6.81 to 4.55 log CFU after 16 h at RT.  Incubation at 37 °C caused reduction to nondetectable levels within 1 h, without any significant reduction in controls. Scanning electron microscopy studies revealed damaged cell membranes of treated bacteria after 1 h at 37 °C. QE shows potential to control the spread of STECs, and further research in model food systems is needed.     

Growth Potential of Listeria Monocytogenes and Staphylococcus Aureus on Fresh‐Cut Tropical Fruits

The objective of this study was to evaluate the fate of Staphylococcus aureus, Listeria monocytogenes, and natural microbiota on fresh‐cut tropical fruits (pitaya, mango, papaya and pineapple) with commercial PVC film at different storage temperature (5, 13, and 25 °C). The results showed that S. aureus, L. monocytogenes, and natural microbiota increased significantly on fresh‐cut tropical fruits at 25 °C. Both pathogen and natural microbiota were able to grow on fresh‐cut tropical fruits at 13 °C. The maximum population of L. monocytogenes was higher than that of S. aureus on fresh‐cut tropical fruits. L. monocytogenes and S. aureus could survive without growth on fresh‐cut pitaya, mango, and papaya at 5 °C. The population of L. monocytogenes declined significantly on fresh‐cut pineapple at all temperature, indicating composition of fresh‐cut pineapple could inhibit growth of L. monocytogenes. However, S. aureus was still able to grow on fresh‐cut pineapple at storage temperature. Thus, this study suggests that 4 kinds of fresh‐cut tropical fruits (pitaya, mango, papaya, and pineapple) should be stored at low temperature to extend shelf life as well as to ensure the safety of fresh‐cut fruits.     

Development of Staphylococcal Toxin and Sensory Deterioration During Storage of Nitrogen and Vacuum Packaged Nitrite-Free Bacon-Like Product

Toxin production by S. aureus was studied in nitrite-free bacon-like product packaged in air permeable film, vacuum packages, and packages flushed with N₂ during storage at 8°C, 12°C or 26°C. Product wrapped in air permeable film deteriorated rapidly at 26°C and was rejected by sensory evaluation prior to staphylococcal enterotoxin detection. Enterotoxin was not detected in vacuum or N₂-flushed packages stored at 26°C. Samples stored at 12°C supported S. aureus growth although enterotoxin was not detected at 12°C or 8°C in any packaging environment. The potential for staphylococcal food poisoning resulting from the production of a nitrite-free bacon-like product was limited under the conditions studied.     

Determination of Spoilage Microbiota of Pacific White Shrimp During Ambient and Cold Storage Using Next‐Generation Sequencing and Culture‐Dependent Method

This study was conducted to determine the initial and spoilage microbiota of Pacific white shrimp during ambient and cold storage using next‐generation sequencing (NGS) and a culture‐dependent method. The quality changes were also evaluated by sensory analysis and total volatile basic nitrogen (TVB‐N) values. After 1 d of storage, the psychrotrophic bacteria were only 5.97 log CFU/g, accounting for 1.1% of the mesophilic bacteria counts (7.94 log CFU/g). The psychrotrophic bacteria counts exceeded the counts of mesophilic bacteria for shrimp stored at 4 °C after 6 d of storage, indicating that psychrotrophic bacteria became predominant. The NGS was used to identify the bacterial species in samples stored at 25 and 4 °C. The results showed that the dominant microorganisms were Vibrio at 25 °C, and Acinetobacter, Psychrobacter, and Shewanella at 4 °C. By the culture‐dependent method based on 16S rRNA gene and VITEK®2 CompactA system, it showed that the dominant microorganisms were Proteus spp. at 25 °C, and Shewanella putrefaciens, Acinetobacter johnsonii, and Aeromonas sobria at 4 °C. In conclusion, differences in results of microbiota analyzed by culture dependent and independent approaches were observed. The combination of both methodologies may provide more comprehensive information about the dominant spoilage microbiota in Pacific white shrimp during ambient and cold storage.     

Predictive Modeling for Growth of Non‐ and Cold‐adapted Listeria monocytogenes on Fresh‐cut Cantaloupe at Different Storage Temperatures

The aim of this study was to determine the growth kinetics of Listeria monocytogenes, with and without cold‐adaption, on fresh‐cut cantaloupe under different storage temperatures. Fresh‐cut samples, spot inoculated with a 4‐strain cocktail of L. monocytogenes (～3.2 log CFU/g), were exposed to constant storage temperatures held at 10, 15, 20, 25, or 30 °C. All growth curves of L. monocytogenes were fitted to the Baranyi, modified Gompertz, and Huang models. Regardless of conditions under which cells grew, the time needed to reach 5 log CFU/g decreased with the elevated storage temperature. Experimental results showed that there were no significant differences (P > 0.05) in the maximum growth rate k (log CFU/g h^?1) and lag phase duration λ (h) between the cultures of L. monocytogenes with or without previous cold‐adaption treatments. No distinct difference was observed in the growth pattern among 3 primary models at various storage temperatures. The growth curves of secondary modeling were fitted on an Arrhenius‐type model for describing the relationship between k and temperature of the L. monocytogenes on fresh‐cut cantaloupe from 10 to 30 °C. The root mean square error values of secondary models for non‐ and cold‐adapted cells were 0.018, 0.021, and 0.024, and 0.039, 0.026, and 0.017 at the modified Gompertz, Baranyi, and Huang model, respectively, indicating that these 3 models presented the good statistical fit. This study may provide valuable information to predict the growth of L. monocytogenes on fresh‐cut cantaloupes at different storage conditions.     

Behavior and enterotoxin production by coagulase negative Staphylococcus in cooked ham, reconstituted skimmed milk, and confectionery cream

Abstract: In this study, the behavior and enterotoxin production by 10 different coagulase negative Staphylococcus (CNS) strains inoculated in cooked ham, reconstituted skimmed milk, and confectionery cream in the presence or absence of background microbiota have been investigated. After inoculation (10³ CFU/g), foods were incubated at 25, 30, and 37 °C and aerobic mesophilic and CNS counts were carried out at 12, 24, 48, and 72 h. Staphylococcal enterotoxins (SE) detection was performed by SET-RPLA (Oxoid, Basingstoke, U.K.) and mini-Vidas® (bioMérieux, La Balme les Grottes, France). CNS counts increased during incubation and approached 10⁶ to 10⁷ CFU/g after 12 h at 37 °C in the 3 foods studied. At 25 °C, counts reached 10⁶ to 10⁷ CFU/g only after 24 to 48 h. The interference of background microbiota on CNS behavior was only observed when they grew in sliced cooked ham, which presented a high initial total count (10⁵ CFU/g). Significantly higher counts of CNS isolated from raw cow's milk in comparison with food handlers isolates were found in reconstituted milk and confectionery cream. Although CNS strains were able to produce SEA, SEB, and SED in culture media, in foods, in the presence or absence of background microbiota S. chromogenes LE0598 was the only strain able to produce SEs. Despite the scarcity of reports on CNS involvement with foodborne disease outbreaks, the results found here support the CNS growth and SE production in foods even in the presence of background microbiota and may affect food safety.     

Effect of various antimicrobials on the growth kinetics of foodborne pathogens in ready-to-eat, pyeonyuk (cooked and pressed pork)

This study investigated the antimicrobial effects of garlic, potassium lactate and sodium diacetate (PL/SDA) mixture, ?-polylysine, and potassium sorbate (PS) alone or in combinations of them on the growth of foodborne pathogens and production of staphylococcal enterotoxin A (SEA) in ready-to-eat (RTE) pyeonyuk at various temperatures. Lag times (LTs) of Salmonella Typhimurium were longer than those of Staphyloccus aureus at all tested temperatures, regardless of antimicrobial agent. The growth of S. Typhimurium and S. aureus were not observed in pyeonyuk made with 3% PL/SDA mixture or 1.5% ?-polylysine at 10°C. The 0.2% of PS, which is the permitted level for meat products, did not inhibit the growth of S. Typhimurium and S. aureus. Although growth of S. aureus in RTE pyeonyuk with 3% PL/SDA mixture and 1.5% ?-polylysine was observed, SEA was not produced at 17, 24, and 30°C, suggesting the application of PL/SDA mixture or ?-polylysine to reduce the risk related to the consumption of pyeonyuk.     

Prevalence and Characteristics of Enterotoxin B‐Producing Staphylococcus aureus Isolated from Food Sources: A Particular Cluster of ST188 Strains was Identified

The aim of this study was to investigate the prevalence and characteristics of staphylococcal enterotoxin B (SEB) producing Staphylococcus aureus (S. aureus) isolated from food sources. A total of 412 S. aureus isolates were recovered from 1970 milk and dairy samples (n = 236) and 2450 meat samples (n = 176) in China from 2009 to 2014. Of the 412 isolates, 124 isolates were tested positive for 1 or more classical staphylococcal enterotoxin (SE) genes using PCR, and 31 isolates were positive for seb gene and further proved to be SEB‐producing. Four SE profiles were observed among 31 SEB‐producing isolates when investigated using ELISA kit, that is, SEB (16 isolates), SEA+SEB (6 isolates), SEB+SEC (6 isolates), and SEB+SED (3 isolates). Thirteen sequence types (STs) were identified in the 31 SEB‐producing S. aureus isolates using multilocus sequence typing (MLST). The 3 most detected STs were ST1 (7 isolates), ST188 (6 isolates), ST59 (3 isolates). Two distinct clusters were identified by pulsed‐field gel electrophoresis (PFGE), each of which showed excellent consistency with ST188 and ST1 achieved by MLST, respectively. In summary, this study reveals that various SE profiles are observed in SEB‐producing S. aureus isolates and the great part of SEB‐producing S. aureus isolates are showed as clusters. Especially, a particular cluster of ST188 strains was observed in SEB‐producing S. aureus isolates which was associated with outbreaks of SFP and needs further attention.     

Effect of pre-incubation conditions on growth and survival of Staphylococcus aureus in sliced cooked chicken breast

In this work, the effect of pre-incubation conditions (temperature: 10, 15, 37 °C; pH 5.5, 6.5 and water activity, a_w: 0.997, 0.960) was evaluated on the subsequent growth, survival and enterotoxin production (SE) of Staphylococcus aureus in cooked chicken breast incubated at 10 and 20 °C. Results showed the ability of S. aureus to survive at 10 °C when pre-incubated at low a_w (0.960) what could constitute a food risk if osmotic stressed cells of S. aureus which form biofilms survive on dried surfaces, and they are transferred to cooked meat products by cross-contamination. Regarding growth at 20 °C, cells pre-incubated at pH 5.5 and a_w 0.960 had a longer lag phase and a slower maximum growth rate. On the contrary, it was highlighted that pre-incubation at optimal conditions (37 °C/pH 6.5/a_w 0.997) produced a better adaptation and a faster growth in meat products what would lead to a higher SE production. These findings can support the adoption of management strategies and preventive measures in food industries leading to avoid growth and SE production in meat products.     

Inhibitor‐Assisted High‐Pressure Inactivation of Bacteria in Skim Milk

The combined inactivation effects of high hydrostatic pressure (HHP) and antimicrobial compounds (potassium sorbate and ε‐polylysine [ε‐PL]) on 4 different bacterial strains present in skim milk and the effect of these treatments on milk quality were investigated in this study. HHP treatment at 500 MPa for 5 min reduced the populations of Escherichia coli, Salmonella enterica Typhimurium, Listeria monocytogenes, and Staphylococcus aureus from 6.5 log colony‐forming units (CFUs) or higher to less than 1 log CFU/mL. Compared to HHP alone, HHP with potassium or ε‐PL resulted in significantly higher reductions in the bacterial counts. After 5 min of treatment with HHP (500 MPa) and ε‐PL (2 mg/mL), no growth of E. coli, S. enterica Typhimurium, or L. monocytogenes in skim milk was observed during 15 d of refrigerated storage (4 ± 1 °C). Scanning electron microscopy analysis revealed that the synergistic treatments caused more serious damage to the bacterial cell walls. Quality assessments of the treated samples indicated that the combined treatments did not influence the color, the turbidity, the concentrations of –SH group of the proteins, or the in vitro digestion patterns of the milk. This study demonstrates that HHP with potassium or ε‐PL may be useful in the processing of milk or milk‐containing foods.