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A Simple and Rapid Biochemical Screening Test for the Differentiation of Campylobacter spp. from other Contaminating Micro-organisms Smith, C.M., Colborne, N.R., Stephens, P.J. and Druggan, P. Oxoid Ltd., Wade Road, Basingstoke, RG24 8PW, UK INTRODUCTION negative. A strong purple colour KOH/NaOH –ve L-ALA +ve (=Gram +ve) Campylobacter is universally Bacillus Clostridium Lactobacillus Staphylococcus Listeria Micrococci acknowledged as a significant bacterial cause of enteritis Enterococci Moraxella Pseudomonas Vibrionaceae Flavobacterium worldwide. Despite its widespread incidence, detection of Campylobacter spp. is difficult for Campylobacter Arcobacter Helicobacter3 many reasons including its ability to in the O.B.I.S. campy test (Fig. 2) indicated the organism to be not Escherichia Acinetobacter Bordatella Klebsiella Citrobacter Burkholderia Proteus Salmonella Yersinia Edwardsiella Shigella Plesiomonas Hafnia Aeromonas Serratia Stenotrophomonas Morganella Campylobacter. Only organisms that appeared Gram negative and L-ala negative were considered to be probable Campylobacter spp. L-ala –ve KOH/NaOH +ve present polymorphic characteristics with regards to its physical appearance on solid media. RESULTS AND DISCUSSION Fig. 1 Specificity of the two tests in detecting Campylobacteraceae All the Campylobacter, Arcobacter spp. and Step 1: Inoculate test area and add 1 drop O.B.I.S. Buffer and wait 30 seconds Additionally, despite the selective nature of Campylobacter isolation methods, there are still micro-organism species that present determined as Gram negative using the KOH/NaOH test and L-ala negative using campylobacter-like morphologies on Step 2: Add 1 drop O.B.I.S. DMAC Developer and read after 20 seconds Campylobacter isolation media. With a view to simplifying the identification of Campylobacter the O.B.I.S. test. Of the competing bacterial species screened only 1 strain (Bacillus pumilis) produced a similar profile as the Positive we developed a simple two-test procedure (Gram test according to KOH/NaOH reaction Bacteroides ureolyticus strains were Negative Campylobacteraceae that was due to an 1 incorrect Gram string reaction. The and detection of L-alanyl aminopeptidase activity using the Oxoid Biochemical sensitivity and specificity for these = Not Campylobacteraceae Identification System (O.B.I.S.) campy test) to = Presumptive Campylobacteraceae (in conjunction with a Gram negative (KOH/NaOH positive) reaction) differentiate presumptive Campylobacter combined tests was therefore determined as 100% and 99.6% respectively. Fig. 2 O.B.I.S. campy procedure colonies on agar media from those of other micro-organism species. Campylobacter spp. are unique in the fact that they are both Gram negative and L-ala negative (Fig. 1). CONCLUSIONS Combining the Gram-lysis and O.B.I.S. campy tests gives the microbiologist a powerful and convenient method for differentiating MATERIALS AND METHODS A wide range of strains comprising: Campylobacter spp. (n=46), Arcobacter spp. (n=6) Bacteroides spp. (n=2) and competing microorganisms (n=250) were tested for their KOH/NaOH and L-ala all Campylobacteraceae from competing bacterial species on agar media. To perform both tests takes a maximum of two minutes and the strong colour of the product of the O.B.I.S. biochemical reaction means that it is easy to differentiate positive from negative results. reactions. Strains were grown on Anaerobe Basal Agar (Oxoid) ACKNOWLEDGEMENTS containing 5% (v/v) laked horse blood incubated at 37°C for 48hrs in 2 an atmosphere of 0.45% O2, 11% CO2 and 7.5% H2. Arcobacter spp. were incubated at 25°C. The Gram-lysis test was performed by emulsifying a colony in a drop of KOH / NaOH (0.5M) on a glass slide. A string of DNA (between the emulsifying loop and the slide) This work was part funded by the EC FP5 CAMPYCHECK project (QLK CT 2002 02201). REFERENCES 1. Carlone, G.M. et al. (1982) Methods for distinguishing gram-positive from gram-nagative bacteria. Journal of Clinical Microbiology 16 (6), 1157-1159. 2. Smith, C.M., Colborne, N.R. and Stephens, P.J. (2006) Creation of an Optimal Gas Atmosphere for Growth of all Campylobacter spp. using Conventional Gas Jars and Atmosphere Generation Systems. In: th Abstracts of Emerging Campylobacter spp. in the food chain, CAMPYCHECK. Feb 8 2006, Croke Park Conference Centre, Dublin, Ireland. 3. Hoosain, N., A.J. Lastovica 2005. Evaluation of the Oxoid Biochemical Identification System (O.B.I.S.) for the differentiation of Campylobacter and Arcobacter from other Gram-negative organisms. In: Abstracts of CHRO 2005. 13th International Workshop on Campylobacter, Helicobacter and related organisms. Sept 48, 2005, Gold Coast, Queensland, Australia. Griffith University. produced within 1 minute indicated the organism to be Gram LT1084A www.oxoid.com