Comparative evaluation of the association among enumeration methods and production of enterotoxins in food-derived Staphylococcus aureus

Staphylococcal food poisoning is one of the most common foodborne diseases worldwide; it results from the ingestion of staphylococcal enterotoxins (SEs) in food, mainly Staphylococcus aureus. This study investigated the statistical relationships among morphological enumerations of food-derived S. aureus and production of SEs using different methodologies. Food samples naturally contaminated with coagulase-positive S. aureus were submitted for enumeration on Baird-Parker (BP) agar, Rabbit Plasma Fibrinogen agar (RPFA), and Petrifilm Staph Express count system (STX), and the morphologically typical colonies were isolated for VIDAS and real-time (RT) PCR tests. RPFA and STX displayed better performance for the enumeration of SE-positive S. aureus when compared with BP, including higher frequencies of SE-positive isolates and better correlation indices between typical and SE-positive counts. Among all the evaluated culture media, no significant difference (P > 0.05) was shown on the frequencies of typical colonies that carried 11 individual se genes. In addition, results for SE identification between VIDAS and RT-PCR assay were unparalleled. These data will be valuable for the selection of methods for inspection of food-derived S. aureus.     

Evaluation of a dry, rehydratable film method for rapid enumeration of Staphylococcus aureus

Results with the new 3M Petrifilm Rapid S. aureus Count (RSA) Plate method were compared with those of the classical Baird-Parker agar (BPA) method for detection and enumeration of Staphylococcus aureus. Studies on 219 bacterial strains demonstrated that the Petrifilm RSA plate is more sensitive than and as specific as the classical BPA method for confirmed identification of S. aureus. Counts of colonies from 71 pure cultures, 61 naturally contaminated food samples, and more than 750 artificially inoculated food samples showed that the Petrifilm RSA method was as effective as the classical BPA method for identification and enumeration of S. aureus. The Petrifilm RSA method gave results in one-third the time required for the classical method.     

Enumeration procedure for monitoring test microbe populations on inoculated carriers in AOAC use-dilution methods

The AOAC Use-Dilution methods do not provide procedures to enumerate the test microbe on stainless steel carriers (penicylinders) or guidance on the expected target populations of the test microbe (i.e., a performance standard). This report describes the procedures used by the U.S. Environmental Protection Agency to enumerate the test microbe (carrier counts) associated with conducting the Use-Dilution method with Staphylococcus aureus (Method 955.15) and Pseudomonas aeruginosa (Method 964.02) and the examination of historical data. The carrier count procedure involves the random selection of carriers, shearing bacterial cells from the carrier surface through sonication, and plating of serially diluted inoculum on trypticase soy agar. For each Use-Dilution test conducted, the official AOAC method was strictly followed for carrier preparation, culture initiation, test culture preparation, and carrier inoculation steps. Carrier count data from 78 Use-Dilution tests conducted over a 6-year period were compiled and analyzed. A mean carrier count of 6.6 logs (approximately 4.0 x 10(6) colony-forming units/carrier) was calculated for both S. aureus and P. aeruginosa. Of the mean values, 95% fell within +/- 2 repeatability standard deviations. The enumeration procedure and target carrier counts are desirable for standardizing the Use-Dilution methods, increasing their reproducibility, and ensuring the quality of the data.     

3M Petrifilm Staph Express Count plate method for the enumeration of Staphylococcus aureus in selected types of meat, seafood, and poultry: collaborative study

The 3M Petrifilm Staph Express Count plate method was compared with AOAC Official Method 975.55 for the enumeration of Staphylococcus aureus in selected foods. Four foods--cooked, diced chicken; cured ham; smoked salmon; and pepperoni--were analyzed for S. aureus by 12 collaborating laboratories. For each food tested, the collaborators received 8 blind test samples consisting of a control sample, a low inoculation level, a medium inoculation level, and a medium inoculation level with background flora, each in duplicate. The mean log10 counts for the methods were comparable for all 4 foods. The repeatability and reproducibility variances of the 24 h Petrifilm Staph Express Count plate method were similar to those of the 72 h standard method.     

3M Petrifilm Staph Express Count plate method for the enumeration of Staphylococcus aureus in selected dairy foods: collaborative study

The 3M Petrifilm Staph Express Count plate method was compared with AOAC Official Method 975.55 for the enumeration of Staphylococcus aureus in selected foods. Five foods--ice cream, raw milk, yogurt, whey powder, and cheese--were analyzed for S. aureus by 12 collaborating laboratories. For each food tested, the collaborators received 8 blind test samples consisting of a control sample, a low inoculation level, a medium inoculation level, and a medium inoculation level with background flora, each in duplicate. The mean log10 counts for the methods were comparable for all 5 foods. The repeatability and reproducibility variances of the 24 h Petrifilm Staph Express Count plate method were similar to those of the 72 h standard method.     

3M Petrifilm Staph Express Count plate method for the enumeration of Staphylococcus aureus in selected types of processed and prepared foods: collaborative study

The 3M Petrifilm Staph Express Count plate method was compared with AOAC Official Method 975.55 for the enumeration of Staphylococcus aureus in selected foods. Five foods--frozen lasagna, custard, frozen mixed vegetables, frozen hashbrowns, and frozen batter-coated mushrooms--were analyzed for S. aureus by 13 collaborating laboratories. For each food tested, the collaborators received 8 blind test samples consisting of a control sample, a low inoculation level, a medium inoculation level, and a medium inoculation level with background flora, each in duplicate. The mean log10 counts for the methods were comparable for all 5 foods. The repeatability and reproducibility variances of the 24 h Petrifilm Staph Express Count plate method were similar to those of the 72 h standard method.     

Comparison of Petrifilm Staph Express Count System with the bacteriological analytical manual direct-plating method for enumeration of Staphylococcus aureus in artificially contaminated hard cheese

The 3M Petrifilm Staph Express Count System was compared with the U.S. Food and Drug Administration's Bacteriological Analytical Manual (BAM) direct-plate count method for the enumeration of Staphylococcus aureus in 6 types of artificially contaminated hard cheese (Asiago, Cheddar, Gruyère, Parmesan, Romano, and Swiss). Five different samples of each cheese type were inoculated with S. aureus (ATCC 25923) to achieve low, medium, and high inoculum levels. S. aureus was enumerated by the Petrifilm and BAM methods, and the results were compared. Multivariate analysis of variance revealed no significant differences (P<0.05) between the 2 methods. The Petrifilm method compared favorably with the BAM procedure. The rapid method was more convenient to use, considerably faster, and less expensive to perform than the BAM method.     

Validation of RAPID E. coli 2 for enumeration and differentiation of Escherichia coli and other coliform bacteria in selected foods--Performance-Tested Method 050601

RAPID'E. coli 2 agar (Bio-Rad Laboratories, Hercules, CA) is a chromogenic medium for differentiation and enumeration of E. coli and non-E. coli coliform bacteria in food. The principle of RAPID'E. coli 2 medium relies on simultaneous detection of 2 enzymatic activities, P-D-glucuronidase (GLUC) and beta-D-galactosidase (GAL). Coliforms, other than E. coli (GAL+/GLUC-), form blue to green colonies, whereas, specifically, E. coli (GAL+/GLUC+) form violet colonies. Eleven foods (raw ground beef, raw boneless pork, fermented sausage, processed ham, processed turkey, frozen turkey breast, raw ground chicken, cottage cheese, ricotta cheese, raw milk, and dry infant formula) were validated, comparing the performance of RAPID'E. coli 2 agar to the reference method AOAC 966.24. Two sample incubation temperatures were evaluated, 37 and 44 degrees C, testing a mixture of naturally and artificially contaminated foods. If naturally contaminated food was not available, the matrix was artificially inoculated with one E. coli strain and one non-E. coli coliform strain. Method comparison studies demonstrated some statistical differences between the 2 methods, which are expected when a plating method is compared to a most probable number method. Inclusivity and exclusivity rates of the medium were 99 and 94%, respectively. The RAPID'E. coli 2 method was shown to be stable when minor variations were introduced.     

Comparison of the compact dry CF with the most probable number method (AOAC official method 966.24) for enumeration of coliform bacteria in raw meats. Performance-Tested Methods" 110401

Compact Dry CF is a ready-to-use test method for the enumeration of coliform bacteria in food. The plates are presterilized and contain culture medium and a cold water-soluble gelling agent. The medium should be rehydrated with 1 mL diluted sample inoculated into the center of the self-diffusible medium, allowing the solution to diffuse by capillary action. The plate can be incubated at 35 degrees C for 20-24 h and the colonies counted without any further working steps. The Compact Dry CF medium plates were validated with 5 different raw meats. The performance tests were conducted at 35 degrees C. In all studies performed, no apparent differences were observed between the Compact Dry CF method and the AOAC Official Method 966.24 results. For the accuracy claim (n = 75), a correlation factor of r2 = 0.91 (coliform) could be assigned, as stated in the application for Performance-Tested Method. No significant variations in coliform bacterial counts were observed with different production lots or plates of diverse storage age by the quality consistency and storage robustness studies.     

Comparison of the compact dry YM with the FDA BAM method for enumeration of yeasts and molds in fruit-based products. Performance-Tested Method 100401

Compact Dry YM is a ready-to-use test method for the enumeration of yeasts and molds in fruit-based products. The plates are presterilized and contain nonwoven incorporated nutrients supplemented with antibiotics and a chromogenic enzyme substrate, 5-bromo-4-chloro-3-indoxyl phosphate, p-toluidine salt (X-phos), to facilitate counting dyes, and a cold water-soluble gelling agent. The medium should be rehydrated with 1 mL of diluted sample inoculated onto the center of the self-diffusible medium, allowing the solution to diffuse immediately by capillary action. The plate can be incubated at 25 degrees C for 7 days. The Compact Dry YM medium plates were validated as an analysis tool determining colony forming units of yeasts and molds from a variety of fruit-based products using 5 different fruit products. The performance tests were conducted at 25 degrees C. In all studies performed, no apparent differences were observed between the Compact Dry YM method and the U.S. Food and Drug Administration's Bacteriological Analytical Manual (FDA BAM) method results (P > 0.05). For the accuracy claim (n = 75), a correlation factor of r2 = 0.99 could be assigned, as stated in the application for Performance-Tested Method.     

Enumeration of probiotic pediococci in animal feed: interlaboratory study

An enumeration method to be used as official control under Council Directive 70/524/EEC for probiotic pediococci used as feed additives was validated for consideration for adoption as Comitée Européen de Normalisation (CEN) and ISO standards. Seventeen laboratories in 11 European countries carried out an interlaboratory study. A spread plate method following BS ISO 15214:1998 using 4 different agars [MRS, acidified MRS, MRS with triphenyl tetrazolium chloride (TTC), and a newly developed pediococci selective medium (PSM)] was validated. Precision data in terms of repeatability (r) and reproducibility (R) of the method for each medium using different feeding stuffs with a high and a low inoculation level were determined. Pediococci were present in the samples in mixtures with other probiotics. The enumeration of pediococci on all agars showed an RSDr value of 0.4-3.1% and an RSDR of 1.3-4.8%. MRS agar was preferred, followed by acidified MRS and MRS + TTC agar. All 4 media gave similar counts. Depending on the presence and concentration of other probiotic, such as enterococci, lactobacilli, and yeast, acidified MRS or MRS + TTC agar are recommended. The PSM was selective for pediococci and can be used if this species is present at a concentration more than 10-fold lower than other species that can grow on the MRS agars. The methodology with all 4 media is not applicable to mineral feed.     

Comparison of the compact dry EC with the most probable number method (AOAC official method 966.24) for enumeration of Escherichia coli and coliform bacteria in raw meats. Performance-Tested Method 110402

Compact Dry E. coli/Coliform Count (EC) is a ready-to-use test method for the enumeration of Escherichia coli and coliform bacteria in food. The plates are presterilized and contain culture medium and a cold water-soluble gelling agent. The medium should be rehydrated with 1 mL diluted sample inoculated onto the center of the self-diffusible medium, allowing the solution to diffuse by capillary action. The plate can be incubated at 35 degrees C for 20-24 h and the colonies counted without any further working steps. The Compact Dry EC medium plates were validated as an analysis tool for determining colony-forming units (CFU) of E. coli and coliform bacteria from a variety of raw meats using 5 different types of raw meats. The performance tests were conducted at 35 degrees C. In all studies performed, no apparent differences were observed between the Compact Dry EC method and the AOAC Official Method 966.24 results. For the accuracy claim (n = 75), a correlation factor of r2 = 0.93 (E. coli) and r2 = 0.93 (coliform bacteria) could be assigned, as stated in the application for "Performance-Tested Method."     

Evaluation of the Sanita-kun coliforms, a dehydrated medium sheet for coliform detection. Performance-Tested Method 100402

The Sanita-kun Coliforms consists of a transparent cover film, an adhesive sheet, a layer of nonwoven fabric, and a water-soluble compound film, including a culture medium formula for the detection of coliforms. The medium sheet was validated with 26 food types belonging to 9 food categories (meat, poultry, fish and seafood, fruits and vegetable, dairy, chocolate or bakery, animal feeds, pasta, and miscellaneous) using violet red bile (VRB) agar method in the U.S. Food and Drug Administration's Bacteriological Analytical Manual as a reference according to the AOAC guideline. The medium sheet showed 100% inclusivity and exclusivity. Ruggedness study suggested allowances in the incubation temperature and time as 33-35 degrees C and 24 +/- 4 h, respectively. The performance of 3 different lots of the medium sheets was equivalent and suggested no change of the performance at least for 3 years. In the comparative recovery study, many samples (84.6%), which were inoculated with a coliform strain, showed no significant difference between the 2 methods. The linear correlation coefficient (r2) to the VRB agar was calculated as 0.94. In the repeatability study, the average relative standard deviation of total foods was 0.10 in the medium sheet. In the independent study, the medium sheet detected significantly more colonies than VRB plates in the frozen raw milk sample, while there was no significant difference between the 2 methods in raw ground beef sample. Comparative recovery study on foods, inoculated and then frozen, showed the medium sheet detected injured cells with better recovery than VRB agar. The analysts in the independent study wrote that the medium sheet was easy to use and read overall. The Sanita-kun sheet provides an alternative method to coliform count agar.     

Modification to the AOAC Sporicidal Activity of Disinfectants Test (Method 966.04): collaborative study

In an effort to improve AOAC Method 966.04, the Sporicidal Activity of Disinfectants Test, selected modifications to the procedure were evaluated in a collaborative study. Method 966.04 is used to generate efficacy data to support the product registration of sporicides and sterilants. The method is a carrier-based test that provides a qualitative measure of product efficacy against spores of Bacillus subtilis and Clostridium sporogenes. The use of garden soil extract and the lack of standard procedures for the enumeration of spores and neutralization of the test chemicals have been considered problematic for many years. The proposed modifications were limited to the B. subtilis and hard surface carrier (porcelain penicylinder) components of the method. The study included the evaluation of a replacement for soil extract nutrient broth and an establishment of a minimum spore titer per carrier, both considered crucial for the improvement and utilization of the method. Additionally, an alternative hard surface material and a neutralization confirmation procedure were evaluated. To determine the equivalence of the proposed alternatives to the standard method, 3 medium/carrier combinations, (1) soil extract nutrient broth/porcelain carrier (current method), (2) nutrient agar amended with 5 microg/mL manganese sulfate/porcelain carrier, and (3) nutrient agar amended with 5 microg/mL manganese sulfate/stainless steel carrier were analyzed for carrier counts, HCI resistance, efficacy, quantitative efficacy, and spore wash-off. The test chemicals used in the study represent 3 chemical classes and are commercially available antimicrobial liquid products: sodium hypochlorite (bleach), glutaraldehyde, and a combination of peracetic acid and hydrogen peroxide. Four laboratories participated in the study. The results of the spore titer per carrier, HCI resistance, efficacy, and wash-off studies demonstrate that amended nutrient agar in conjunction with the porcelain is comparable to the current method, soil extract nutrient broth/porcelain. The nutrient agar method is simple, inexpensive, reproducible, and provides an ample supply of high quality spores. Due to the current use of porcelain carriers for testing C. sporogenes, it is advisable to retain the use of porcelain carriers until stainless steel can be evaluated as a replacement carrier material for Clostridium. The evaluation of stainless steel for Clostridium has been initiated by the Study Director. Study Director recommendations for First Action revisions are provided in a modified method.     

Cellulase Production by Pink Pigmented Facultative Methylotrophic Strains (PPFMs)

Pink pigmented facultative methylotrophs (PPFM) isolated from water samples of Cooum and Adyar rivers in Chennai and soil samples of forests located in various districts of Tamil Nadu, India were screened for cellulase production using carboxymethylcellulose agar (CMC agar) medium. The strains showed wide variations in the production of clearing zones around the colonies on CMC agar medium flooded with Congo red. CMCase and filter paper assays were used to quantitatively measure the cellulase activity of 13 PPFM strains. Among the strains, Methylobacterium gregans, MNW 60, MHW 109, MSF 34, and MSF 40 showed cellulolytic activity ranging from 0.73 to 1.16 U mL⁻¹ with wide temperature (35–65°C) and pH (5 to 8) tolerance. SDS-PAGE analysis of the crude enzyme of PPFM strain MNW 60 exhibited several protein bands, and zymogram analysis revealed two dimeric cellulase bands with molecular mass of ~92 and 42 kDa. Scanning electron microscopic studies revealed significant morphological differences between the cells grown in normal and CMC amended medium. The strain MNW 60 was identified as Methylobacterium sp. based on biochemical, physiological, and morphological analyses, and the methylotrophic nature was authenticated by the presence of mxaF gene, encoding methanol dehydrogenase as a key indicator enzyme of methylotrophs, with 99% similarity to Methylobacterium lusitanum. With the 16S ribosomal RNA sequence showing 97% similarity to M. lusitanum strain MP2, this can be proposed as a novel taxon of the genus Methylobacterium. The study forms the first detailed report on the extracellular cellulase production by pink pigmented Methylobacterium sp., and it is expected that this might be the basis for further studies on cellulase production by PPFMs to explore the molecular mechanism, strain improvement, and large-scale cellulase production for its application.     

磁弹性无线传感器检测不同液体介质中的金黄色葡萄球菌

研制了磁弹性金黄色葡萄球菌无线传感器,用于检测不同液体介质中的金黄色葡萄球菌。取0.2mL一定浓度菌液加到含2mL无菌液体介质的检测玻璃管中,磁弹性传感器共振频率随细菌的生长而改变。通过改变牛肉膏和蛋白胨的浓度,得出传感器在含有2×105cells/mL金黄色葡萄球菌的培养基CM2-2中共振频率响应最大。结果表明,此传感器可以测定的金黄色葡萄球菌浓度范围在CM2-2中是3×103～2×107cells/mL,在牛奶中是1×104～2×107cells/mL,检出限分别是1×103cells/mL和1×104cells/mL。传感器共振频移大小与金黄色葡萄球菌的浓度呈线性相关关系,相关系数在牛奶中是0.98,在培养基中是0.99。     

Effect of monovalent and divalent cations on the photoinactivation of bacteria with meso-substituted cationic porphyrins

It is well established that for successful photoinactivation (PI) of gram-negative bacteria a cationic photosensitizer is required. This requirement suggests a charge-dependent interaction between the photosensitizer and the gram-negative bacterium, which may be influenced by the presence of ions in the suspending medium. The aim of the present study was to investigate the effect of cations Na+ and Ca2+ on the efficacy of the PI of the gram-negative Pseudomonas aeruginosa and the gram-positive Staphylococcus aureus. The bacteria were suspended in buffer containing either meso-tetra(N-methyl-4-pyridyl)-porphyrin or meso-mono-phenyl-tri(N-methyl-4-pyridyl)-porphyrin as photosensitizer and various concentrations of Na+ or Ca2+. The cell suspensions were exposed to a broadband light dose of 9 J/cm2. In buffer without added cations, P. aeruginosa and S. aureus were equally sensitive to PI. Addition of cations strongly decreased the sensitivity of both bacteria to PI, with the PI of P. aeruginosa being much more decreased than that of S. aureus, and Ca2+ being more effective than Na+. The decreased sensitivity was accompanied by a reduced binding of the photosensitizers to the bacteria.     

An immunoassay method for rapid detection of Staphylococcus aureus in cosmetics, pharmaceutical products, and raw materials.

The TECRA Staphylococcus aureus Visual Immunoassay allows a presumptive positive or negative result for the presence of S. aureus to be obtained within 26 h, in contrast to 4-5 days by traditional cultural methods. Presumptive positive immunoassay results are confirmed by streaking the enrichment broth onto conventional agar media. A validation study was undertaken to compare the TECRA assay with a cultural reference method based on the Bacteriological Analytical Manual (8th Ed.), which is also consistent with U.S. Pharmacopoeia requirements. The products tested included a range of cosmetics (toothpaste, shampoos, conditioners, sunscreens, moisturizers, lip and eye creams) and pharmaceuticals (cough mixtures, laxatives, ulcer treatments, infant formulae, antiseptic cream), as well as some pharmaceutical ingredients. Samples were inoculated with S. aureus at 10-20 cfu/g, and then enriched for 24 h at 35 degrees-37 degrees C at a product-to-sample ratio of 1:100. Two different enrichment broths were used for the study: Tryptone Soya Broth with 4% Tween 80 and Modified Letheen Broth. For both enrichment broths, results of the immunoassay and the reference method showed close correlation. The TECRA S. aureus Visual Immunoassay provides a rapid and convenient alternative to cultural methods and provides advantages to industry, such as greater speed of product and ingredient release and faster tracing of contamination problems. Because the immunoassay may be read either visually or with the aid of a plate reader, there is no need for an initial outlay on capital equipment. However, the assay can be automated if required.     

Peppermint oil decreases the production of virulence-associated exoproteins by Staphylococcus aureus

The present study aimed to evaluate the antimicrobial activity of peppermint oil against Staphylococcus aureus, and further investigate the influence of peppermint oil on S. aureus virulence-related exoprotein production. The data show that peppermint oil, which contained high contents of menthone, isomenthone, neomenthol, menthol, and menthyl acetate, was active against S. aureus with minimal inhibitory concentrations (MICs) ranging from 64-256 μg/mL, and the production of S. aureus exotoxins was decreased by subinhibitory concentrations of peppermint oil in a dose-dependent manner. The findings suggest that peppermint oil may potentially be used to aid in the treatment of S. aureus infections.