Bertrand Lombard

Comparison of three chromogenic media and evaluation of two molecular-based identification systems for the detection of Enterobacter sakazakii from environmental samples from infant formulae factories.

Enterobacter sakazakii is an occasional contaminant of powdered infant formula that can cause rare but severe foodborne infections in infants. To determine optimal methods for the detection and identification of E. sakazakii, 38 naturally contaminated samples from infant formulae factories were analyzed by two PCR-based methods and by a method (TS 22964/RM 210) developed by the International Organization for Standardization and the International Dairy Federation (ISO-IDF) using three different commercial chromogenic agars. The ISO-IDF method includes two enrichment steps, plating of the second enrichment broth on E. sakazakii isolation agar (a chromogenic selective agar), picking of five typical colonies for transfer onto tryptone soy agar, and subsequent confirmation of yellow-pigmented colonies by biochemical characterization. Twenty-two of the 38 samples were positive by the culture method. E. sakazakii isolation agar (ESIA; AES Laboratoires), COMPASS agar (Biokar Diagnostics), and Druggan-Forsythe-Iversen agar (Oxoid) compared favorably with violet red bile glucose agar (VRBG, a selective medium for Enterobacteriaceae), with positive predictive values of 86.96, 88, and 74.07%, respectively, in contrast to 47.83% for VRBG. One additional positive sample was detected using the nonpatented real-time PCR method evaluated, and those results were in 97.3% concordance with the ISO-IDF results. Some discrepancies between the results of the DuPont Qualicon BAX system and those of the ISO-IDF method could be explained by heterogeneity of contamination and sampling. Thus, both PCR-based systems were suitable for detecting and specifically identifying E. sakazakii within 1 to 2 days, and COMPASS agar and ESIA could be used interchangeably as a first-step medium to isolate presumptive E. sakazakii colonies.

Impact of pooling powdered infant formula samples on bacterial evolution and Cronobacter detection

Enterobacter sakazakii has been identified as the causative agent of serious neonatal infections, associated with high mortality rates. In most cases, powdered infant formula (PIF) has been identified as the source of infection. Recently, strains commonly referred to as E. sakazakii were proposed for classification in a new genus, Cronobacter. The standardised method for detection of Cronobacter in PIF (ISO/TS 22964; IDF/RM 210) involves pre-enrichment in buffered peptone water (BPW), followed by selective enrichment and plating onto ESIA chromogenic agar. For greater convenience and to reduce analysis cost, the common practice in the food industry is to pool samples at a constant dilution rate, in order to perform a single pre-enrichment and subsequent analysis. The consequences on the sensitivity of Cronobacter detection are not evident. We evaluated the impact of pooling on the growth of Cronobacter and PIF background microflora in samples undergoing pre-enrichment culturing in BPW. Growth of the pathogen was monitored by direct plating onto selective agar or by using a recently developed sensitive enumeration method, based on membrane filtration followed by transfer of the filter onto the selective agar. The evolution of the total bacterial population of the PIF was monitored from a qualitative and quantitative point, using molecular or classical microbiological methods. Results showed that pooling had a negative impact on the maximum population of Cronobacter attained, whereas no clear effect was observed on the onset of growth. This observation suggests strong bacterial interactions with the PIF background microflora, confirmed by a generally higher background microflora growth potential in PIF samples from various origins. These important findings suggest that, in some cases, the practice of pooling samples may affect the performance of the detection method.

Evaluation of an enumeration method for Listeria monocytogenes at low contamination levels in cold-smoked salmon.

For the enumeration of Listeria monocytogenes in cold-smoked salmon, a sensitive enumeration method, based on membrane filtration followed by transfer of the filter on a selective medium has been recently developed (Gnanou Besse et al., 2004, A contribution to the improvement of L. monocytogenes enumeration in cold-smoked salmon. International Journal of Food Microbiology, 91, 119-127). The aim of the study was to assess the performance of this enumeration method through an inter-laboratory study, using cold-smoked salmon artificially contaminated at 2 different levels (approximately 0.6 and 1.6 log10 CFU g(-1)). A reproducibility standard deviation of 0.23 log10 CFU g(-1)and 0.15 log10 CFU g(-1) was obtained for the method respectively at the lower level and the higher level. Under certain conditions, the uncertainty of measurement can be derived from the method reproducibility standard deviation and was calculated to be 0.46 log10 CFU g(-1) for the lower contamination level and 0.30 log10 CFU g(-1) for the higher contamination level. These values can be considered as satisfactory for such low contamination levels.

Reliability of measurement results in food microbiology: the contribution of reference laboratories in the European Union and of international/European standardization

The contribution of reference laboratories in the European Union and of European/international standardization to the reliability of food microbiology measurement results is discussed. A set of European Union reference laboratories has been established. Each of them coordinates a network of national reference laboratories which, in turn, coordinate networks of laboratories in charge of official testing and sometimes own checks in each European Union country. Their contribution to the reliability of food microbiology measurement results is illustrated by three food safety cases: Listeria monocytogenes, coagulase positive staphylococci and milk/milk products. The contribution of European/international standardization focuses on two topics: method validation and measurement uncertainty. The standards covering these topics—EN ISO 16140 and ISO/TS 19036—are briefly discussed, and an update given on their ongoing revision.

The role of standardization bodies in the harmonization of analytical methodology in food microbiology

Publisher Summary The harmonization of analytical methodology is crucial in food microbiology, as it is well known that food microbiological analysis is highly variable. There is therefore a clear need to define common reference methods in food microbiology to minimize as much as possible the variability of microbiological analyses and to define in a less ambiguous way the analytical target: a given microorganism to be detected or a given number of microorganisms to be enumerated. The analytical methods need to be highly reliable since some of them enable detection or quantification of foodborne pathogens, which represent direct hazards to consumer health. Their presence at very low numbers in a given food product may be responsible for the illnesses of consumers of that food, ranging from the most common gastroenteritis to severe renal affections, meningitis, and septicemia, which may if not properly treated lead to death. Standardization has proven to be the preferred way to harmonize reference methods at the international level or regional level and to facilitate trade between or within countries. The set of standards developed is also a sound and common basis for the accreditation of laboratories in food microbiology.

CEN/ISO standards for both culture and molecular methods

Standardized microbial testing and typing methods have become key components for the management of food chain safety. Their harmonization is crucial in the field of microbiology of the food chain. Standardization is an appropriate way to solve this issue, and has been established at European level (European Committee for Standardization: CEN) and international level (International Organization for Standardization: ISO). These organizations promote essentially horizontal standards for microbiological analyses of the sample of the food chain, and mutual recognition of standards. Based on cultural methods for detection, enumeration and characterization of microorganisms, these standards need, if appropriate, to incorporate novel technologies, such as PCR-based methods and genomics. A new challenge for standardization is to adapt its type of standards to the fast development of technologies.