Validation of EN ISO standard methods 6888 Part 1 and Part 2: 1999--enumeration of coagulase-positive staphylococci in foods.

The methods of European and International Organisations for Standardization for the enumeration of coagulase-positive staphylococci (CPS, Staphylococcus aureus and other species) described in EN ISO 6888 Part 1 and Part 2: 1999 were validated by order of the European Commission (Standards, Measurement and Testing Fourth Framework Programme Project SMT4-CT96-2098). EN ISO 6888-1 prescribes the use of Baird-Parker (BP) agar whereas EN ISO 6888-2 prescribes the use of Rabbit Plasma Fibrinogen Agar (RPFA). The objective was to determine the precision of each method in terms of repeatability (r) and reproducibility (R) using three different food types inoculated with various levels of S. aureus and a typical background flora. The results are intended for publication in the associated standards. Cheese, meat and dried egg powder were examined by 24 laboratories from 16 countries in Europe. Each participant received eight test materials per food type: blind duplicates at four inoculum levels (0, 10(3), 10(4) to 10(5), 10(5) to 10(6) cfu/g). In addition, two reference materials (RM) (capsules containing milk powder inoculated with S. aureus) were included in the study. All test materials were subjected to stringent homogeneity and stability testing before being used in the collaborative trial. Two statistical methods were used to calculate the precision parameters. Draft EN ISO 16140: 2000 method appeared more appropriate to the case of microbiological data than ISO 5725-2: 1994 method and was retained to calculate the precision data. Concerning EN ISO 6888-1, overall values for repeatability (r) when used with food test materials was r=log(10) 0.28 (expressed as an absolute difference between log(10)-transformed test results). For the reference materials, r=log(10) 0.19. Overall values for reproducibility (R) when used with food test materials were R=log(10) 0.43. For the reference materials, R=log(10) 0.39. Concerning EN ISO 6888-2, overall values for repeatability (r) when used with food test materials were r=log(10) 0.22. For the reference materials, r=log(10) 0.17. Overall values for reproducibility (R) when used with food test materials were R=log(10) 0.33. For the reference materials, R=log(10) 0.31. These results were presented to the ISO technical committee and to the Comité Européen de Normalisation (CEN). Both committees agreed to incorporate the precision data obtained with food materials as two amendments to EN ISO 6888-1 and -2, and to give an equal status to each part of the standard.

Validation of ISO method 11290 part 1--detection of Listeria monocytogenes in foods.

The European and International Standard method for the detection of Listeria monocytogenes, described in EN ISO 11290 Part 1: 1997 (International Organisation for Standardisation, Geneva) was validated by order of the European Commission (Standards, Measurement and Testing Fourth Framework Programme Project SMT4-CT96-2098). Nineteen laboratories in 14 countries in Europe participated in a collaborative trial to determine the performance characteristics of the method, which are intended for publication in the corresponding standard. An additional objective of this project was to devise a new series of parameters to indicate the 'precision' of microbiological qualitative methods. The method was challenged with three food types, namely fresh cheese, minced beef and dried egg powder and a reference material. Inoculation levels ranged from 5 to 100 cfu/25 g. Each participant examined five replicates of each food type at three inoculum levels and five reference materials. Both PALCAM and Oxford media were assessed. All test materials were subjected to stringent homogeneity and stability testing before being used in the collaborative trial. The results demonstrated that the method prescribed in EN ISO 11290-1 had an overall sensitivity of 85.6% and a specificity of 97.4%. L. monocytogenes was detected in most cases after primary enrichment, although secondary enrichment often yielded further positives. However, a significant number of false-negative results were obtained with all food types when large numbers of L. innocua were present in the test materials. L. innocua tended to dominate L. monocytogenes during the selective enrichment stages and thus masked small numbers of colonies of L. monocytogenes on the isolation media. There was no evidence from this collaborative study to demonstrate a significant difference in performance between Oxford and PALCAM media. Due to the problem of false-negative results with this method as highlighted in this trial, recommendations have been made to ISO to launch a revision of the standard to improve the detection of low numbers of L. monocytogenes in foods. New statistical methods devised to advance the measurement of the performance of qualitative microbiological methods are also described.

Validation of ISO method 11290 part 2. Enumeration of Listeria monocytogenes in foods.

The European and International Standard method for the enumeration of Listeria monocytogenes, described in EN ISO 11290 Part 2: 1998 [EN ISO 11290-2 Microbiology of Food and Animal Feedingstuffs-Horizontal Method for the Detection and Enumeration of L. monocytogenes: Part 2. Enumeration; International Organisation for Standardisation, Geneva.] was validated by order of the European Commission (Standards, Measurement and Testing Fourth Framework Programme Project SMT4-CT96-2098). The objective was to determine the precision of the method in terms of repeatability (r) and reproducibility (R) using three different food types inoculated with various levels of L. monocytogenes and a typical background flora. The results are intended for publication in the associated standards. Cheese, meat, dried egg powder and reference materials were examined by 21 laboratories in 16 countries in Europe. Each participant received eight test materials per food type: blind duplicates at four inoculum levels (0, 10(2), 10(3), 10(4) cfu/g). In addition, two reference materials containing L. monocytogenes were included in the study. All test materials were subjected to stringent homogeneity and stability testing before being used in the collaborative trial. Participants were required to use only PALCAM agar for enumeration of L. monocytogenes, as prescribed by the reference method. Statistical analyses has been performed using a newly introduced approach for food microbiology (draft standard prEN ISO 16140 [prEN ISO 16140 Microbiology of Food and Animal Feedingstuffs-Protocol for the Validation of Alternative Methods, International Organisation for Standardisation, Geneva.], the precision data being calculated using robust estimates. Overall values for repeatability (r) of EN ISO 11290-2 when used with food test materials were r = log10 0.58 (expressed as an absolute difference between log10-transformed test results) or r = 3.8 (expressed as an absolute ratio between test results on the normal scale). For the reference materials (capsules containing approximately 5000 cfu), r = log10 0.34 (expressed as an absolute difference between log10-transformed test results) or r = 2.2 (expressed as an absolute ratio between test results on the normal scale). Overall values for reproducibility (R) of EN ISO 11290-2 when used with food test materials were R = log10 0.81 (expressed as a difference between log10-transformed test results) or R = 6.5 (expressed as an absolute ratio between test results on the normal scale). For the reference materials, R = log10 0.51 (expressed as a difference between log10-transformed test results) or R = 3.2 (expressed as an absolute ratio between test results on the normal scale). Further studies have been initiated by ISO TC34/SC9 to try to enhance the isolation of L. monocytogenes from foods and improve the confirmation procedures.

Development of standard methods with special reference to Europe.

For a long time, many different microbiological methods were used around the world in order to enumerate or detect contaminants in foods. The development of commercial, but also scientific and technical exchanges between countries has stimulated new developments and a desire to harmonize methods. The example of AFNOR (French Association for Standardization) is first presented. Then, the new developments under CEN (European Committee for Standardization) are described, emphasizing a Measurements and Testing project. The MicroVal project, now integrated as a task force within CEN Technical Committee 275/Working Group 6 is described. Finally, the possibility of working towards an international consensus through the Codex Alimentarius is discussed.

A selective medium for the rapid detection by an impedance technique of Pseudomonas spp. associated with poultry meat.

A new medium for detecting and enumerating Pseudomonas spp. associated with poultry meat spoilage by a rapid impedance technique was developed, after testing potential growth promoters for eight Pseudomonas strains and inhibitors against eight competing strains (Enterobacteriaceae) able to grow on the medium of Mead and Adams (1977). Four basal media (brain heart infusion, brucella broth, Shaedler broth and Whitley impedance broth (WIB)) and a synthetic medium were evaluated. Whitley impedance broth was the best basal medium for detecting variations in impedance in relation to Pseudomonas growth. The efficiency of WIB was improved by adding compounds which enhanced the growth of Pseudomonas on the synthetic medium. Among the incubation temperatures tested, 22 degrees C proved to be the best compromise between growth of Pseudomonas associated with poultry meat spoilage and inhibition of competitors. Among the 15 inhibitory substances evaluated against Pseudomonas competitors, five were chosen for inclusion in the final medium: metronidazole, carbenicilline, cetrimide, cycloheximide and diamide (MCCCD medium). Preliminary results obtained from experiments with beef and pork meat showed that this medium could also be used without diamide and at an incubation temperature of 25 degrees C. The impedance technique using MCCCD medium was then compared with an official method which uses the medium of Mead and Adams (1977) on 106 samples of poultry neck skin. The linear regression coefficient between the two techniques was approximately r = 0.85. Impedance was able to detect 10(3) Pseudomonas g-1 within less than 19 h making it a promising technique for predicting poultry meat spoilage.

Predictive microbiology of dairy products: influence of biological factors affecting growth of Listeria monocytogenes.

The growth potential of Listeria monocytogenes was evaluated at low temperature in sterilized milk and raw dairy products. Sterilized and raw milk were inoculated with different strains of L. monocytogenes in 2 physiological states and at various contamination levels. Raw cheese was naturally contaminated with Listeria spp. The results suggest that some biological factors influence the growth capacity of L monocytogenes in dairy products. Significant strain effect was observed at low temperature whatever the growth medium. By contrast, no inoculum effect was observed in the 3 dairy products. In raw matrixes, growth of L. monocytogenes was influenced greatly by bacterial interactions and physiological state of inoculum cells.

[Incidence of Listeria in food]. / Incidence des Listeria dans les denrées alimentaires.

It is only recently that the role of food in the appearance of human listeriosis has been shown up. However, the increasing number of studies made, up to date, don't let us appreciate the influence of Listeria, and more particularly Listeria monocytogenes, in a somehow satisfying way. The problems set by the evaluation of this incidence are called up firstly, and afterwards the data concerning different food-stuffs (meat and meat products, milk and milk products, sea foods, vegetal products) are reviewed, trying to explain the origin for each of it. To conclude, some research axes, susceptible of being developed are called up.

Differential growth of Listeria monocytogenes at 4 and 8°C: Consequences for the Shelf Life of Chilled Products.

Growth rates and lag times of Listeria monocytogenes at 4 and 8°C were compared in dairy products (milk, cream, and cheese), minced beef, and smoked salmon. Results showed that an increase in incubation temperature from 4 to 8°C leads to a significant decrease in time required to reach a given bacterial population density. The decreases were about 50% on cheese surfaces, 60 to 65% in milk and cream, and 75 to 80% in minced beef and smoked salmon. Consequences on the shelf life of chilled products are discussed on the basis of a simple and general linear relationship between the relative decrease in shelf life and generation time. This relationship was experimentally highlighted and theoretically demonstrated.

Use of a competitive exclusion product (Broilact) to prevent Salmonella colonization of newly hatched chicks.

The study described here was carried out at the request of a French company to utilize the authors' experience of 'competitive exclusion' treatment for poultry. The objective of the study was to determinate the safety and efficacy of a particular treatment product (Broilact) in protecting chicks against Salmonella infection.

[Microbial colonisation of the caecal mucosa of specific pathogen-free chicks and adherence of an adult SPF chicken microflora to the caeca of a conventional newly hatched chick: study by scanning electron microscopy]. / La colonisation spontanee de la muqueuse caecale du poulet exempt d'organismes pathogenes specifies et l'implantation provoquee d'une flore de barriere chez le poulet conventionnel: Etude au microscope electronique a balayage.

Two studies were conducted by SEM in order to evaluate the relationship between the caecal microflora morphology and the age of SPF chicks and the colonising of an adult SPF chick caecal microflora to the caecal mucosa of conventional newly hatched chicks. These experiments indicated that the number of 'adhering' bacteria increases with the age of the chick, that the morphological and quantitative changes of the caecal microflora are completed by about 15 days of age and that a complete bacterial colonisation of the caecal wall of a newly hatched chick occurs only 24 h after treatment with an adult caecal microflora.

Rapid identification of Salmonella from poultry meat products by using 'Mucap Test'.

A study was made in order to improve a new Salmonella identification test (Mucap Test) in which umbelliferone is released, giving a blue fluorescent light under a Wood lamp, after contact with Salmonella colonies. The study concerned 354 colonies, previously isolated from 55 poultry meat samples. Two enrichment media [Tetrathionate Bile Broth (TBB) and Rappaport Vassiliadis (RV)] and two isolation media [Brilliant Green Agar (BGA) and Desoxycholate Agar (DA)] were used, and the results of the test obtained respectively with each association were compared. The sensitivity was consistently good, but the specificity of the test was generally poor. The best association seemed to be RV/DA which gave 85% specificity, against 39% for TBB/BGA, 58% for TBB/DA, and 77% for RV/BGA. The predominant genera responsible for false-positive results were Pseudomonas and Proteus Providencia.

Influence of resident Salmonella on contamination of broiler flocks.

An epidemiological survey was made of 5329 samples from 10 poultry operations to determine the relationship between total poultry farm environment and incidences of Salmonella contamination of broiler flocks. Samples were analyzed from walls, drinkers, feeders, litter, insects, water, chicks, broilers, and feed to determine the effect of common sanitary practices on Salmonella contamination of flocks. Results indicated that although similar hygienic practices had been taken on the 10 poultry farms examined, great variation exists in Salmonella contamination among the farms. Among the sources studied, the most important source of contamination was determined to be the resident Salmonella of the flock i.e., the strain isolated on chicks' first day in the poultry house. This source was more important than Salmonella isolated during the rearing period. However, the precise conditions of Salmonella contamination in poultry flocks remain to be elucidated.

Relationship between serotypes of Salmonellae from hatcheries and rearing farms and those from processed poultry carcases.

The influence of the hatchery and the poultry farm on the contamination of poultry carcases by Salmonella species has been studied by examining samples from different stages of production. The incidence of Salmonella serotypes in the hatchery varied considerably in different broiler flocks and decreased from the beginning to the end of the rearing period. Serotypes originating in the hatchery were less important in the final product than those present in the house, or those introduced into the house by vectors during rearing.

Growth Effect of Sorbate and Selected Antioxidants on Toxigenic Strains of Staphylococcus aureus 1, 2.

Effect of potassium sorbate with and without added antioxidants on Staphylococcus aureus 196, S-6, 137 and 326 in a liquid system was evaluated. We found (a) potassium sorbate a 1, 3, and 5% levels in combination with BHA, BHT, PG (50 and 100 ppm) exerted greater bactericidal and bacteriostatic effects on S. aureus strains at pH 5 than at pH 7; at pH 6 the effect was more pronounced at 3 and 5% compared with 1% sorbate, (b) TBHQ was highly inhibitory to S. aureus strains with or without the addition of sorbate, (c) in combination with sorbate, BHA exerted greater bactericidal effects compared with BHT and PG, (d) higher concentration of antioxidants exerted more bactericidal and bacteriostatic effects on test organisms, (e) S. aureus S-6 was more resistant than 196, 326, and 137 in the presence of sorbate and antioxidants and (f) shake cultures of S. aureus grew better than static cultures in the presence of sorbate and BHA.