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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
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