[Food microbiology: proficiency testing scheme on a naturally contaminated fresh refrigerated food.]

The major objective of the study is to assess the performance of a proficiency testing scheme of a fresh refrigerated dairy product and to compare it with those applied currently in food microbiology. The accuracy is evaluated by using the repeatability and reproducibility standard deviations (respectively referred as s(r) and s(R)). For total mesophilic bacteria, with a mean Log count of around 8 bacteria per g, s(r) was equal to 0.15 and s(R) equal to 0.32. For total and fecal coliforms, these Values were respectively s(r) = 0.19, s(R) = 0.34, and s(r) = 0.20, s(R) = 0,50, at mean Log count of about 3.76 bacteria per g. For Staphylococcus aureus at a contamination level ranging between 1.5 and 4 Log count bacteria per g, these values were s(r) = 0.54 and s(R) = 1.33. Because our samples were originally Salmonella free, spiked samples were used. The sensitivity rate of the method is 95.8 % and the specificity rate 99.1 %. In the course of the present study, a significant progressive improvement of performance among laboratories was observed

Influence of sugar source (lactose, glucose, galactose) on 2,3-butanediol production by Klebsiella oxytoca NRRL-B199.

The ability of Klebsiella oxytoca NRRL-B199 to use either lactose or the mixture of glucose and galactose as substrate for the production of 2,3-butanediol was studied in batch fermentations with different conditions of aeration and pH. 2,3-butanediol was undetected, or present in minute concentration in the fermentation broths with lactose, while it was the main product from glucose + galactose with final concentrations of up to 18.8g/l in media at pH 6.0. Under conditions optimal for 2,3-butanediol synthesis, when aeration limited growth, the rate of biomass growth was more tightly related to the aeration rate in lactose medium than in glucose + galactose medium. These relations suggest that the growth rate is very low on lactose but still considerable on glucose + galactose when aeration rate tends toward zero. Corresponding, the metabolism is more oxidative in the former medium, yielding mainly acetate as product

Gas-chromatographic Analysis of Cellular Fatty-acids in the Identification of Foodborne Bacteria

The cellular fatty acid composition of about 160 strains of Bacillus, lactic acid bacteria, Enterobacteriaceae, and Staphylococcus was analyzed in order to identify foodborne bacteria. In a first step, the reproducibility of the procedure, from the bacterial growth to the extraction and analysis conditions, was evaluated. The stability of fatty acid composition under controlled conditions was confirmed through high similarity levels (So greater than or equal to 94). Secondly, intraspecies similarity was estimated. The existence of low values (So less than or equal to 79) was demonstrated within each of the four groups of strains investigated, which stressed the importance of analyzing a high number of reference strains in each species. The intraspecies similarity was maximal under optimal growth conditions. Finally, interspecies similarity was compared to intraspecies similarity. The best results of discrimination between species was obtained for the heterogeneous genus Bacillus, for which the 12 species studied could be separated into 6 clusters at a similarity level of 79. The association of 27 species of lactic acid bacteria into 8 clusters should be useful to confirm results of other characterization methods. Only 3 clusters were formed for the 16 Enterobacteriaceae species studied. The genus Salmonella could not be discriminated. The 10 Staphylococcus species were separated into 5 clusters. Coagulase-positive and -negative strains were not differentiated. Since the fatty acid composition did not usually appear specific to single species, characterization of foodborne bacteria by the analysis of their cellular fatty acids should be used to complement other taxonomic methods

[Rapid enumeration of bacteria in foods using Direct Epifluorescent Filter Technique.]

The aim of this study was to test the feasibility of a method designed for the rapid counting of bacteria in various kinds of foods i.e. the Direct Epifluorescent Filter Technique (DEFT). With frozen vegetables, total bacteria count found in 103 samples (n = 103) is significantly correlated (r = 0.87) with the standard method (Plate count agar - PCA); with raw meat (n = 14), even if the DEFT seems to overestimate the total bacteria populations, the correlation with PCA is higher (r = 0.99). However, for this last kind of food, an extraction of fat is necessary. When poultry products are examined, the correlation obtained with neck skin samples is unsatisfactory (r = 0.42). As far as the enumeration of more specific bacteria populations is concerned, e.g. the Enterobacteriaceae in frozen spinach (n = 28), a poor correlation is observed with the standard procedure (r = 0.57). However, better results are obtained with the enumeration of Staphylococcus aureus in raw meat (r = 0.8 to 0.95, n = 17). Good agreement is obtained between visual counting (r = 0.95) and semi-automated image analyser counting

RAPD analysis, serotyping, and esterase typing indicate that the population of Listeria monocytogenes strains recovered from cheese and from patients with listeriosis in Belgium are different.

Populations of Listeria monocytogenes strains isolated in Belgium from cheese and from patients with listeriosis were characterised by randomly amplified polymorphic DNA (RAPD) analysis using two 10-mers primers (OPA-04 and OPA-13). High discrimination levels were obtained with each of these primers alone (discrimination indices (DI) of 0.899 and 0.935 for OPA13 and OPA04, respectively) or in combination (DI of 0.960). The clustering of strains obtained by RAPD was compared with a clustering previously made using serotyping and esterase typing. RAPD allowed the subdivision of each serovar cluster and of most of the clusters determined by the polymorphism of the bacterial esterases. Our analysis indicates that the population of strains of L. monocytogenes found in cheese differs from the one isolated from patients with listeriosis during the same period

High Homogeneity of the Yersinia pestis Fatty Acid Composition

The cellular fatty acid compositions of 29 strains of Yersinia pestis representing the global diversity of this species have been analyzed by gas-liquid chromatography to investigate the extent of fatty acid polymorphism in this microorganism. After culture standardization, all Y. pestis strains studied displayed some major fatty acids, namely, the 12:0, 14:0, 3-OH-14:0, 16:0, 16:1ω9cis, 17:0-cyc, and 18:1ω9trans compounds. The fatty acid composition of the various isolates studied was extremely homogeneous (average Bousfield's coefficient, 0.94) and the subtle variations observed did not correlate with epidemiological and genetic characteristics of the strains. Y. pestis major fatty acid compounds were analogous to those found in other Yersinia species. However, when the ratios for the 12:0/16:0 and 14:0/16:0 fatty acids were plotted together, the genus Yersinia could be separated into three clusters corresponding to (i) nonpathogenic strains and species of Yersinia, (ii) pathogenic Yersinia enterocolitica isolates, and (iii) Yersinia pseudotuberculosis and Y. pestis strains. The grouping of the two latter species into the same cluster was also demonstrated by their high Bousfield's coefficients (average, 0.89). Therefore, our results indicate that the fatty acid composition of Y. pestis is highly homogeneous and very close to that of Y. pseudotuberculosis

A Rapid Procedure for the Identification of Lactic-acid Bacteria Based On the Gas-chromatographic Analysis of the Cellular Fatty-acids

This study was conducted to develop a rapid numerical procedure for the analysis of gas chromatograms of lactic acid bacteria fatty acid methylesters (FAMEs), along with an examination of some experimental conditions which affect the fatty acid composition of these bacteria. FAME determination was carried out using high resolution gas chromatography (HRGC). Although the nature and the proportion of fatty acids differed among strains, some major components, n-C14:0, n-C16:1, n-C18:1, n-C18:0, and C19 cycl, were found as a group to represent more than 90% of the whole cellular fatty acids. The differences found in the relative composition of the long-chain fatty acids in paired chromatograms were used to calculate a "distance coefficient", based on the differences found for the most important fatty acids, after their prior ranking. The procedure was initially validated with identified species, used as reference strains; then, unknown lactic acid bacteria isolates were compared to these references. The method proved to be useful for rapid comparisons between strains, provided a strict standardization occurred prior to routine application

Usefulness of cellular fatty acid patterns for identification and pathogenicity of Yersinia species

The cellular fatty acid composition of common non-pathogenic and pathogenic Yersinia strains was determined by gas-liquid chromatography. This study included most of the species of the genus Yersinia, with the exception of Y. pestis. All species possess a rather similar fatty acid pattern characterized by some major compounds, namely C12:0, 14:0, 3-OH-14:0, 16:0, 16,1 omega 9cis, 17:0 cyclic and 18:1 omega 9trans acids, as in other Enterobacteriaceae. By using the overlap coefficient of Bousfield, the similarity value enabled us to identify easily the Yersinia genus of an unknown strain. In the same way, the pathogenicity of strains of Yersinia enterocolitica and related species can be determined by the C12:0/C16:0 and C14:0/C16:0 ratios