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.

[Verocytotoxin-producing Escherichia coli O157 in Dutch veal calves and beef cattle]. / Verocytotoxine-producerende Escherichia coli O157 bij Nederlandse vleeskalveren en slachtrunderen.

In 1994 (August-September) and in 1995 (July-October) faeces of Dutch veal calves and adult cattle was examined for the presence of verocytotoxin-producing Escherichia coli O157 (O157 VTEC). The samples were collected at slaughterhouses. In 1994, O157 VTEC were isolated from three (0.9%) of 365 fecal samples of veal calves. Faeces of adult cattle was not collected. In 1995, O157 VTEC were isolated from one (0.5%) of 18.3 fecal samples of veal calves and from 30 (11.1%) of 270 fecal samples of adult cattle. In 1994, the organisms were isolated by selective plating onto both sorbitol MacConkey agar (SMAC) and SMAC containing tellurite and cefixime (TC-SMAC) following selective enrichment in modified trypton soya broth with acriflavin (mTSB + a). In 1995, the samples were enriched in modified E. coli broth with novobiocin (mEC + n), and in addition to directly plating onto TC-SMAC the enriched cultures were plated after incorporation of an immunomagnetic separation step of E. coli O157. All 30 strains isolated from adult cattle were isolated with the immunomagnetic separation procedure. Four of the 30 samples were determined positive also by directly plating onto TC-SMAC. The sample from the veal calve was determined positive only by directly plating onto TC-SMAC. Plating onto SMAC resulted only in negative results. Characterization of the O157 VTEC isolates showed similarities between isolates of humans and cattle. Additional experiments need to be done concerning this aspect. From this study it appeared that also Dutch cattle are a reservoir of O157 VTEC.