COMPARISON OF BACARA® AGAR, A NEW CHROMOGENIC MEDIUM AND MYP AGAR FOR THE ENUMERATION OF B. CEREUS IN FOOD SAMPLE

J. THEPAUT, H. SORIANO

AES CHEMUNEX Rue Maryse Bastié - Ker Lann - CS17219 - F-35172 BRUZ Cedex - FRANCE

MYP 30°C BACARA® 37°C • Methods comparison Abstract Material & Methods Strains Number Growth Typical Growth Typical Fig 4: Comparison of the accuracy of BACARA® Method and Iso 7932 reference • Introduction: • Inclusivity and Exclusivity Aerococcus viridans 1 +/- - - / Method on 80 food samples The cereus Group includes species such as B. cereus, B. thuringiensis, B. 100 strains belonging to the group have been tested on both BACARA® Aeromonas hydrophila 1 - / - / mycoides, B. pseudomycoides, B. weihenstephanensis and B.anthracis well known and MYP medium to evaluate the inclusivity and 70 Non Bacillus cereus strains have Bacillus licheniformis 3 +++ - - / ISO 7932 ISO 7932 Bacillus subtilis 6 ++ - - / as human food-borne pathogens. B.cereus is the second pathogen involved in been streaked on these 2 media to study the exclusivity of both methods. All the Positive Results Negative Results Bacillus megaterium 1 +++ - - / BACARA® outbreaks caused by bacterial toxins in Europe. 54 4° strains were cultivated overnight in BHI at 37°C for and in Sabouraud broth Bacillus circulans 1 +++ - - / Positive Results • Purpose: at 25°C for yeasts and moulds and the were all streaked on BACARA® and MYP Agar Bacillus pumilus 2 +++ - - / BACARA® 0 22 The aim of this study was to evaluate the performances of BACARA®, a new chro- and incubated respectively at 37°C +/- 1°C and 30°C +/ 1°C. Bacillus Firmus 1 - / - / Positive Results Bacillus lentus 1 - / - / mogenic medium, versus the MYP agar (mannitol egg yolk polymixin agar - ISO • Method comparison * These strains were confirmed by reference biochemical tests and identified with Bacillus amyloliquefasciens 2 +++ - - / ® Mandatory Medium) for the direct enumeration of B.cereus in Foodstuffs. ® Biolog System as Bacillus cereus A comparison between the BACARA method and the ISO7932 reference method Bacillus coagulans 2 +++ - - / • Methods: has been carried out on more than 80 samples issued from different food catego- Bacillus stearothermophilus 1 +++ - - / 54 samples were confirmed positive withBacillus cereus by both methods. 4 food Inclusivity tests with 100 B. cereus Groups strains and exclusivity tests with 70 ries: Deep frozen vegetables, mushrooms, pastry, cooked meals, catering food… Candida albicans 1 +++ - ++ - samples were only found positive on BACARA® and negative on MYP medium. non B. cereus strains were performed on both BACARA® and MYP. The majority of these samples were naturally contaminated. Citrobacter freundii 1 - / - / Enterococcus hirae 1 - / - / The interfering flora on these 4 samples was very important and the interpretation The accuracy, the sensitivity, the selectivity and the easiness of counting the The assessment of the sensitivity, selectivity and easiness of counting of the Enterococcus faecium 1 + - + - of the MYP medium was impossible. On the contrary, the high selectivity and BACARA® method compared to the ISO7932 method was carried out on a total of typical colonies were compared. Enterococcus faecalis 1 + - - / specificity of the BACARA® allow easy counting of the typical colonies.

80 food samples as a second step. A vast majority of those samples were natu- ® Enterobacter gergoviae 1 - / - / Figure 1: Horizontal Method Figure 2: BACARA method ® rally contaminated. for the enumeration of for the enumeration of Bacillus cereus Enterobacter sakazakii 1 - / - / Fig 5: Difference of interpretation on BACARA (a) and MYP medium (b) with a Escherichia coli 3 - / - / high level of interfering flora Each sample was diluted in a 1/10 Buffered Pepton Water solution and 0.1 ml of presomptive Bacillus cereus Standard Method : NF EN ISO 7932 Escherichia fergusonii 1 - / - / On BACARA®, the enumeration the serial dilutions were inoculated on MYP and BACARA® agar. Both of them, Food Sample preparation and dilution July 2005 according to the ISO 6887- 1 Escherichia vulneris 1 - / - / and differentiation of the typical MYP and BACARA® agar were incubated respectively at 30°C and 37°C. 25 g/225 ml BPW Haemophilus influenzae 1 - / - / colonies (orangey surrounded by Food Sample preparation and dilution Hafnia alvei 1 - / - / • Results: according to the ISO 6887- 1 an opaque halo) from the other 25 g/225 ml BPW Klebsiella oxytoca 1 - / - / Exclusivity data demonstrated a much better selectivity of the BACARA® agar com- Spread 0,1 ml of each chosen dilutions on BACARA® Klebsiella pneumoniae 1 - / - / strains showing a growth was pared to the MYP agar. 27 of non bacillus cereus strains grew on MYP whereas they 1 BACARA® plate/Dilution Lactobacillus acidophilus 1 - / - / a b very easy. The majority of the were all inhibited on BACARA® agar. Although most of these strains didn’t have a Spread 0,1 ml of each chosen dilutions Listeria grayi 1 +++ - - / on MYP Agar interfering flora was completely typical aspect on MYP, they were sources of misinterpretation on high contamina- 2 MYP plates/Dilution Listeria innocua 1 +++ - - / inhibited by the specific mixture of antibiotics (Fig 5 - a). On the other hand, Incubation 24 +/- 2h at 37°C Listeria monocytogenes 1 +++ - - / ted samples due to an interfering flora on the Petri Dishes. in the presence of such a sample, the interpretation of MYP Medium was not On the other hand, the combination of a specific nutrient base, a strong selectivity Listeria seeligeri 1 +++ - - / Listeria welshimeri 1 +++ - - / possible. The lack of selectivity permits the growth of many strains which hide ® Incubation 18-48h at 30°C and a chromogenic mixture utilized in BACARA agar allowed obtaining very large Enumerate the plates Mucor spp 1 ++ - + - the characteristic colonies. In such a case, these standard medium plates were and easy to count colonies. with 10 to 150 typical Enumerate the plates Penicillium commune 1 - - - / not interpretable and are a source of false-negatives results (Fig 5 - b). ® colonies. For both naturally and artificially contaminated samples, BACARA agar performed with 10 to 150 typical Typical colony: Proteus mirabilis 1 - / - / colonies. Large pink/orangey Proteus penneri 1 ++ - - / well for the enumeration of B. cereus compared to the ISO7932 reference method. Typical colony: colonies surrounded Fig 6 : Regression analysis between BACARA® and ISO7932 Methods Bacillus cereus colonies with an opaque halo Providencia spp 1 ++ - - / The comparison of the • Significance: are large flat, rough ® y= 0,9958x - 0,002 Pseudomonas aeruginosa 1 - / - / BACARA vs MYP R2= 0,9786 ® irregular and pink 5 enumeration of Bacillus cereus BACARA method is a reliable alternative method for the enumeration of B. (Mannitol -) surrounded Bacillus cereus Pseudomonas fluorescens 1 - / - / 4 by BACARA® method and cereus in foodstuffs. The high level of selectivity and the specific enzymatic by an opaque halo Saccharomyces cerevisiae 1 + / - / ® detection of typical colonies on this medium can improve interpretation compa- Salmonellla derby 1 - / - / 3 reference method from the 54 red to the classical MYP agar. BACARA® will help the end-user save time and avoid Salmonella dublin 1 - / - / positive samples by a regression Confirmation: Hemolysis positive on sheep blood Salmonella heidelberg 1 - / - / 2 any confirmation protocol. agar after an incubation of 24h +/-2h at 30°C. Log 10 BACARA analysis gave similar results. This confirmation test should be carried out Salmonella infantis 1 - / - / 1 The correlation coefficient R2 = on 5 colonies. Salmonella typhimurium 3 - / - / 0 0,97 with an intercept equal to Introduction Salmonella virchow 1 - / - / 1 2 3 4 5 Salmonella enteritidis 1 ++ - - / - 0,002 and a slope of 0,99 (Fig 6). Log 10 MYP • Bacillus cereus is responsible for food-borne outbreaks. It produces thermoresistant Serratia marcescens 1 ++ - - / spores that make it particularly adapted to foodstuffs submitted to thermal Shigella sonnei 1 - / - / treatment. Some strains of B. cereus can grow at refrigeration temperature, which Results Sphingomonas paucimobilis 1 - / - / is an emerging risk for ready-to-use products. They represent by themselves 5% of Staphylococcus aureus 1 ++ - - / Conclusion Inclusivity and Exclusivity Staphylococcus epidermidis 1 ++ - - / the collective food poisoning in France and are also involved in a lot of opportunistic ® Staphylococcus xylosus 1 ++ - + - This study demonstrated the performances of a novel chromogenic medium, the infections on in-patients. Fig 3: Comparison of the BACARA and MYP AGAR specificity on 100 Bacillus cereus strains and 70 Non Bacillus cereus strains Streptococcus pyogenes 1 ++ - - - BACARA®, for the enumeration of Bacillus cereus in foodstuffs. Vibrio fluvialis 1 - / - / Bacillus cereus belongs to the Bacillus cereus group within can be found The combination of its high selectivity and its specific enzymatic detection sys- MYP 30°C BACARA® 37°C Yersinia enterocolitica 1 - / - / B. thuringiensis, B. Weihenstephanensis, B. mycoides, B. pseudo-mycoides and Strains Number tem improves the sensitivity of the method and the interpretation of the plates B. anthracis. Except the last-mentioned, the phenotypic differentiation of species Growth Typical Growth Typical Bacilllus cereus 53 +++ + +++ + +++ Good growth • ++ Growth • + Growth with small colonies compared to the MYP medium. between Bacillus cereus group is impossible with actual culture methods. ® Bacillus mycoides 16 +++ + +++ + +/- Very weak growth with pinpoint colonies • - Complete inhibition BACARA Method is a reliable alternative method for the enumeration of Bacillus BACARA® agar is a selective chromogenic medium that allows the enumeration Bacillus pseudomycoides 9 +++ + +++ + cereus and will help the end-user save time and avoid any confirmation protocol. of Bacillus of the cereus group without confirmation. On this medium, typical 15 +++ + +++ + The specificity study clearly showed a better selectivity of the BACARA® Agar Bacillus weihenstephanensis 7 +++ + +++ + colonies of B. cereus show a pink/orangey color due to the metabolism of the compare to the MYP Medium. At least 37 non Bacillus cereus strains showed a substrate and are surrounded with an opaque halo due to the phospholipase activity. +++ Good growth • ++ Growth • + Growth with small colonies good growth on MYP (ISO Mandatory Medium) whereas only 4 strains present The selectivity of BACARA® agar has been especially optimized to prevent growth +/- Very weak growth with pinpoint colonies • - Complete inhibition some colonies on BACARA® (Fig 3). Although these strains were not typical on of interfering flora and thus to allow an easy interpretation of plates even when MYP Medium, they were really sources of misinterpretation on high contaminated samples. matrix highly contaminated with competitive flora are analyzed. VA172A The aim of this study was to compare the performances of selectivity, specificity and accuracy of the BACARA® method with the ISO7932 reference method.