The development and evaluation of reference materials for food microbiology

Since 1986 the National Institute of Public Health and the Environment (RIVM) has worked on the development and evaluation of microbiological reference materials (RMs) with support from the European Communities Bureau of Reference (BCR), now called Standards Measurement and Testing (SM&T). The RMs are the result of efforts, which was started many years ago, to develop standardised samples that could be used in collaborative studies and in particular for the evaluation of methods for the detection of Salmonella . This work ultimately led to the development of capsules filled with artificially contaminated milk powder. The objectives of this thesis were to evaluate the possibility of producing RMs that fulfil the general requirements for such materials i.e. stability, homogeneity and representativity, to produce certified RMs (CRMs) and to set up (commercial) production of RMs.

The basic process for preparation of the RMs is the spray drying of bacteria suspended in milk with subsequent mixing of the contaminated milk powder (called highly contaminated milk powder; HCMP) with sterile milk powder until the desired level of contamination is achieved. The mixed powder is then filled into gelatin capsules. Various RMs were developed using this process, for example, the RMs for Bacillus cereus and Listeria monocytogenes as described in chapter 1. The B. cereus RM is an RM containing ca 10 ⁴colony forming particles (cfp) per capsule and is intended for the evaluation of enumeration (quantitative) methods (they are also referred to as quantitative RMs). This RM is first reconstituted in 10 ml peptone saline solution after which 0.1 ml is plated onto an agar plate for enumeration of the organism. The L. monocytogenes RM is an RM containing only ca 5 cfp per capsule and is intended for the evaluation of detection (qualitative) methods (also called qualitative RMs). This RM is added, whole, to a (pre-warmed) enrichment broth.

Both RMs were used to characterise the behaviour of the material in relation to the general requirements for RMs. They were stable at storage temperature (-20 °C) over a period of more than 96 weeks. The B. cereus RM was stable for at least four weeks at higher temperatures (tested up to 37 °C). The L. monocytogenes RM showed a significant decrease in the level of contamination when stored for four weeks at 5 °C or higher, indicating the need for cooling during transport of this material. Both RMs could be produced homogeneously, meaning that the variation between the number of cfp was less than twice the value expected from a Poisson distribution.

The L. monocytogenes HCMP 2-2 could not be diluted homogeneously to a level of 5 cfp per capsule due to the high level of contamination of this HCMP. In general, mixing using a mortar and pestle improved homogeneity of the final mixed powders. The representativity of the B. cereus RM was tested by examining the effects of osmotic shock and heat injury and of heat shock, storage time and lysozyme on spore germination. The parameters examined for the L. monocytogenes RM included the effects of osmotic shock, heat injury, pre-warming of enrichment broth and incubation time on recovery of the organism. For the B. cereus RM no effect of the various parameters tested could be observed, but with the L. monocytogenes RM there was an effect due to both heat injury and osmotic shock. As a result of heat shock (tested by comparing direct selective enrichment and non selective pre-enrichment) 40 - 50 % less positive isolations were observed with the direct selective enrichment. The effect of osmotic shock (tested by comparing free milk powder and the powder filled in gelatin capsules) led to ca 10 % less positive isolations using the free milk powder. The growth rate of Listeria using RMs was faster than with a heat treated culture of the same strain ( ca 2 log ₁₀ -units difference after 31 h incubation at 30 °C).

As a part of the evaluation of the RMs a number of collaborative studies were organised. Chapter 2 describes three international collaborative studies using the L. monocytogenes RM. This RM was tested both with and without competitive micro-organisms. The competitors were added either as a capsule containing a mixture of strains or as a food sample. The food samples were tested in combination with RMs at two contamination levels ( ca 5 cfp per capsule and ca 100 cfp per capsule). Based on the known level of contamination of the RMs the number of positive isolations expected was calculated and compared to the results obtained by the laboratories. Most found the expected number of L. monocytogenes isolations when no competitors were present. However, the addition of a capsule containing competitive micro-organisms ( ca 3 x 10 ⁴cfp per capsule) reduced the rate of positive isolations from 97 % to 80 %.

In the presence of food samples the positive rate decreased even further. No relationship between the effect of the competitive micro-organisms and the type of food product and/or the detection method used was observed. The RMs proved to be useful in evaluating the performance of a detection method in the presence of competitive micro-organisms.

Once the stability, homogeneity and representativity of an RM are proven it can be used to produce an CRM. The procedure necessary obtaining a qualitative CRM for Salmonella typhimurium is described in chapter 3 together with the results of the collaborative study organised to establish the certified values. The batch was certified based on the results from 9 or 10 laboratories for the number of Salmonella cfp in a single capsule (mean value 5.9) and the fraction of capsules in which no Salmonella could be detected (2.7 % using the ISO detection method). The certification of the quantitative B. cereus RM is described in chapter 4. For certification, the number of cfp on two media, MEYP (incubated at 30 °C) and on PEMBA (incubated at 37 °C) was determined. The certified geometric mean value on MEYP after 24 h incubation was 53.4 cfp per 0.1 ml reconstituted capsule solution (95 % confidence interval 51.7 - 55.2) and on PEMBA 55.0 cfp per 0.1 ml (95 % confidence interval 52.8 - 57.4), both sets of figures are based on the results from 11 laboratories. The certificate for both CRMs gives the mean expected value and the 95 % confidence limits. From these results user tables are constructed presenting the 95 % confidence limits for the number of capsules (and replicates per capsule in the case of quantitative CRMs) likely to be examined in practice.

Chapter 5 describes the evaluation of an alternative method for the preparation of HCMPs by means of fluid bed spray granulation. This method was chosen as this drying procedure (spraying of concentrated milk containing the micro-organism onto sterile milk powder held fluidised by means of air) does not require drying temperatures as high as those necessary for spray drying. A number of different micro-organisms ( B. cereus, Campylobacter jejuni, Escherichia coli, L. monocytogenes, Pseudomonas aeruginosa, S. enteritidis and Staphylococcus aureus )were dried using this procedure and, the resulting materials tested for homogeneity and stability when stored at -20 °C and 22 °C. The strain of C. jejuni did not survive the drying procedure, no organisms could be recovered from the milk powder the day after the drying. The homogeneity of the various HCMPs produced varied widely. The time used for spraying the milk onto the powder was a critical factor in obtaining homogeneous HCMPs. An initial decrease in numbers of organisms was found comparable to that obtained with spray dried material. This indicates that the drying temperature is not the only factor determining survival of organisms through the drying process. The stability at 22 °C showed, for most strains, a non linear decrease and the results obtained were not as good as those for silica gel or spray dried material. Satisfactory results were obtained with stability tests at -20 °C, but more information is needed on long term stability. Production of HCMP by spray granulation is less laborious than by spray drying and the spray granulation process is better suited for testing the influence of culturing and drying conditions on the survival of micro-organisms.

Chapter 6 describes the use of RMs and CRMs. A distinction is made between the use of both quantitative and qualitative RMs for quality assurance of routine and occasional examinations. Control charts such as the Shewhart chart can be constructed for the routine use of quantitative RMs. The control limits for the Shewhart chart are calculated using log-transformed counts and can be back-transformed to produce a chart for counts on the original scale. Each data point on the chart is the result from the examination of a single capsule enumerated in singluar. It is possible to adapt the method of calculating the control limits for RMs that are not fully stable by using the principle of Kalman's filtering. For routine use of qualitative RMs it is recommended that a single capsule is examined on each test occasion. The results from a series of tests can be compared to the expected number of positive isolations as calculated for the occasional use of RMs. Certified RMs are used only occasionally and are mainly intended for the determination of trueness in a laboratory. Results of power analyses are presented to demonstrate the minimum difference that can be detected between the certified value and the true laboratory mean (for quantitative CRMs) or the fraction of negatives (for qualitative CRMs). For the qualitative RM and CRM a fraction of negatives of 0 % is needed in order to minimise the number of capsules necessary for evaluation of laboratory performance. Additional information is required on the variation between laboratories for occasional use of non-certified RMs in order to interpret results. The laboratory variance component of a CRM can be used to adjust the confidence limits of an RM determined by a single laboratory.

During the development and evaluation of the RMs market research was undertaken to obtain the opinions of potential customers on the characteristics of the product. In this market research, reported in 1993, both inspection and production laboratories (all with some knowledge of the materials) were interviewed. The results of this market research are presented in chapter 7. A number of requirements for the materials became apparent and these were compared with the product developed. Most of the requirements could be or already were included in the product, but some would be difficult or even impossible to incorporate. The specific requirement for a simple short pre-treatment for the quantitative RMs was evident and has not been resolved. On the basis of the results of the market research production of the materials was set up at the SVM with assistance of RIVM. The actual sale of RMs is gradually increasing but the increase is much slower than was predicted by the market research. In order to obtain information on the organisation of quality assurance in laboratories and the role played by RMs, a second series of interviews was organised in 1998. It became apparent from these interviews that the use of RMs in quality assurance of routine microbiological examination is important; this was not as apparent at the time of the original market research. Some laboratories used a type of control chart for evaluation of results from the use of quantitative RMs over a period of time. It was also agreed that the level of contamination of the qualitative RMs need to be increased to eliminate the chance of finding capsules that do not contain the target organism. To increase the sale of the RMs more information on their use in QA should be made available to laboratories throughout Europe, especially to those that are accredited and also for routine examinations. A comparison between the RMs developed and alternatives from other sources indicates that they are competitive with respect to homogeneity, stability and price. However, as far as the quantitative RMs are concerned, the alternative RMs are much easier to reconstitute than the gelatine capsules.

Based on the studies undertaken it can be concluded that RMs and CRMs can be produced that fulfil the accepted requirements for such materials. Market research indicated that there is a need for this kind of materials and, therefore, the production of RM's was set up at SVM. Although sale of the RMs is less than expected they are increasing. The RMs developed are competitive with existing alternative RMs and are useful for quality assurance purposes.

List of available reference materials (non-certified and certified).

reference materials (non-certified) (available from SVM, Bilthoven, The Netherlands)

Bacillus cereus RM (5.000 cfp per capsule)Clostridium perfringens RM (5.000 cfp per capsule)Enterobacter cloacae RM (500 cfp per capsule)Enterococcus faecium RM (500 cfp per capsule)Escherichia coli RM (500 cfp per capsule)Listeria monocytogenes RM (5 cfp per capsule)Listeria monocytogenes RM (5.000 cfp per capsule)Salmonella panama RM (5 cfp per capsule)Staphylococcus warneri RM (500 cfp per capsule)

certified reference materials (available from EC, IRMM, Geel, Belgium)

Enterococcus faecium CRM 506Salmonella typhimurium CRM 507Enterobacter cloacae CRM 527Bacillus cereus CRM 528Escherichia coli CRM 594Listeria monocytogenes CRM 595