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Macedonian Journal of Medical Sciences. 2011 Dec
15;
4(4):372-375.
http://dx.doi.org/10.3889/MJMS.1957-5773.2011.0200
Basic Science
Serogrouping and Randomly Amplified Polymorphic DNA Fingerprinting of
Campylobacter Jejuni
Elena Trajkovska-Dokic1, Snezana Stojkovska2, Kostadin
Icev1, Aleksandra Grozdanova3
1Institute of Microbiology and Parasitology, Medical Faculty,
“Ss Cyril and Methodius” University, Skopje, Republic of Macedonia; 2University
Clinic for Infective Diseases, Skopje, Republic of Macedonia; 3Faculty
of Pharmacy, “Ss Cyril and Methodius” University, Skopje, Republic of
Macedonia
Background: Thermophilic campylobacters are of worldwide significance
in human and animal diseases. Sources of human infection remain mainly
undetermined, but contaminated food, like poultry and row milk, are widely
regarded as important vehicles of infection. Accurate methods of strain
identification, differentiation and typing are essential for diagnosing and
epidemiological purposes.
Aim: The aim of our study was to determine the serologic and genetic
diversity among the strains and to assess the discriminatory power of both
typing methods in epidemiological studies.
Material and Methods: Within a period of six months were isolated 26
strains of C. jejuni from faecal samples of children with acute
gastroenteritis. Heat-stable specific antigen of C. jejuni was used
for serogrouping of the strains by the reaction of passive hemagglutination.
Purified genomic DNA was obtained and used in RAPD-PCR reaction.
Results: Twenty one C. jejuni strains belonged to the
following Penner’s serogroups: 8 strains were group A, 13 strains were group
O. The remaining 5 strains were non-typable. RAPD-PCR analysis of 26 strains
of Campylobacter jejuni yielded multiple amplification products in
all of them. None of the strains processed by this method was non-typable.
According to the number and sizes of amplification bands, 4 different
genotypes (a, b, c, d) of C. jejuni strains were distinguished within
26 investigated strains.
Conclusion: In this study we found that RAPD-PCR analysis provide
better discrimination of C. jejuni strains than serogrouping by
Penner’s method. Each of the three Penner’s antigenic groups comprised
different genotypes. RAPD-PCR analysis of C. jejuni resulted in the
generation of highly specific and reproducible DNA fingerprints that enable
discrimination even between isolates of a single bacterial serogroup.
...................
Citation: Trajkovska-Dokic E, Stojkovska S, Icev K, Grozdanova A.
Serogrouping and Randomly Amplified Polymorphic DNA Fingerprinting of
Campylobacter Jejuni. Maced J Med Sci. 2011 Dec 15; 4(4):372-375.
http://dx.doi.org/10.3889/MJMS.1957-5773.2011.0200.
Key words: Campylobacter jejuni; serogrouping; RAPD-PCR
fingerprinting.
Correspondence: Elena Trajkovska-Dokic, MD, PhD. Institute of
Microbiology and Parasitology, Medical Faculty, Skopje, Republic of
Macedonia. E-mail: elenatdokic@gmail.com
Received: 21-Oct-2011; Accepted: 13-Nov-2011; Online first: 25-Nov-2011
Copyright: © 2011 Trajkovska-Dokic E. This is an open access article
distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are credited.
Competing Interests: The authors have declared that no competing
interests exist.
Thermophilic campylobacters are of worldwide significance in human and
animal diseases. Campylobacter jejuni in particular, is recognized as
a major cause of acute bacterial enteritis in man in most developed
countries [1, 2]. Sources of human infection remain mainly undetermined, but
contaminated food, like poultry and row milk, are widely regarded as
important vehicles of infection [3].
Accurate methods of strain identification, differentiation and typing are
essential for diagnosing enteritis caused by campylobacters as well as for
detecting the source or the origin of the infection. Conventional phenotypic
methods based on biotyping [4], serotyping [5] or phage typing [6] have been
applied to campylobacters for over a decade. New molecular genotyping
methods based on chromosomal DNA analysis are more stable and avoid
dependence on expressed and possibly variable phenotypic features. Plasmid
profiling has not been developed as a typing method, because less than 50%
of campylobacters carry plasmids [7]. Two molecular methods that provide
precise and stable strain markers applicable to pure culture of
campylobacters are ribosomal DNA (rDNA) gene profiles from Southern blot
hybridization (ribotyping) and polymerase chain reaction (PCR) generated
fingerprints from random primer sequences [8].
Ribotyping is of value in typing most bacterial pathogens including
Campylobacter species [9], but is time-consuming and not well suited to
routine use. The PCR has revolutionized molecular biology through the
introduction of new genetic assays based on selective DNA amplification
[10]. In 1990, a PCR method based on the amplification of random DNA
fragments using a single primer of arbitrary sequence was described as a
method for typing of different pathogenic microorganisms including isolates
of the genus Campylobacter.
In the present study, we evaluated the application of C. jejuni
serogrouping by Penner and RAPD fingerprinting with a 10-mer primer of
arbitrary sequence, for detecting DNA polymorphisms in C. jejuni
strains.
The aim of this study was to determine the serologic and genetic diversity
among the strains and to assess the discriminatory power of both typing
methods in epidemiological studies.
Microorganisms and growth media
The primary isolation was done on blood agar base no. 2 (Oxoid CM271)
supplemented with 5% defibrinated sheep blood (Oxoid SR51) and Butzler
selective supplement (Oxoid SR85). Inoculated media were cultivated at 420C,
under microaerophilic conditions for 42 hours. Isolates were confirmed as
Campylobacter by colonial (Fig. 1) and microscopic morphology, catalase and
oxidase reactions. Differentiation of C. jejuni was done by positive
reaction of hippurate hydrolysis (purple color) (Fig. 2).
Figure 1: Colonial morphology of Campylobacter.
Using this conventional method for isolation and differentiation of
campylobacters, during a period of six months were isolated 26 strains of C.
jejuni from faecal samples of children with acute gastroenteritis.
Figure 2: Hippurate hydrolysis reaction for differentiation of C. jejuni.
Serogrouping of C. jejuni by Penner
Heat-stable specific antigen of C. jejuni extracted by nitric acid was
sensitized to the blood cells (DENKA SEIKEN, Japan). When the sensitized
cells were mixed with the antiserum, specific reaction of passive
hemagglutination was observed. All 26 isolates of Campylobacter were
biotyped by the reactions for hippurat hydrolysis, H2S production and DNA
hydrolysis.
RAPD-PCR analysis
Purified genomic DNA was obtained in 200 ml suspension of bacterial cells by
boiling for 10 min and centrifuging for 5 min. 10 ml of each dilution was
used in a 40 ml PCR reaction volume by addition of 10 mM Tris-HCl pH 8.8;
2.5 mM MgCl2, 200 mM of each deoxynucleotide triphosphate, 0.2 mM of the
primer OPA-11 with sequence 5’-CAATCGCCGT-3’, 1U Taq DNA Polymerase and
sterile destiled water. The solutions were overlaid with 100 ml of paraffin
oil and cycled through the following temperature profile: an initial
denaturation step at 940C for 1 min, 45 cycles of 940C for 1 min (denaturation),
360C for 1 min (annealing), and 720C for 2 min (DNA chain extension), and
final elongation step at 720C for 5 min. Incubation was performed in a
thermocycler (JR Instrumentation, UK). The amplified DNA products were
electrophoresed on 1.8 % agarose gels and stained in ethidium bromide
solution. A 100 bp ladder was used as a marker for the PCR products.
Twenty one C. jejuni strains belonged to the following Penner’s
serogroups: 8 strains were group A, 13 strains were group O. The remaining 5
strains were non-typable.
Table 1: Biotyping of Campylobacter jejuni.
Biotyping of 26 Campylobacter isolates revealed four different
biotypes ( I, II, III and IV) (Table 1). 19, 4, 2 and 1 strains belonged to
biotype I, II, III and IV respectively. All the strains from the Penner
serogroup A and 11 strains from the Penner serogroup O were biotype I.
Figure 3: RAPD fingerprints of Campylobacter jejuni. M - Marker; Lines:1
and 2 - genotype a; Lines: 3 and 4 - genotype b; Line: 5 - genotype c; Line:
6 - genotype d; Line: 7 - negative control (n.c.).
RAPD analysis of 26 isolates of Campylobacter jejuni yielded multiple
amplification products in all of them. None of the strains processed by this
method was non-typable. The resultant fingerprints comprised between 1 and 7
bands with sizes between 0.3 and 1.5 kb. The majority of strains had
characteristic amplification bands of 1.5, 1.4, 1.3, 0.8, 0.7, 0.6 and 0.3
kb. According to the number and sizes of amplification bands, 4 different
genotypes (a, b, c, d) of C. jejuni strains were distinguished within 26
investigated strains. Control assays in which cell suspensions were replaced
by plain distilled water yielded no detectable amplified product.
Representative example is shown in Figure 3.
In this study we found that RAPD-PCR analysis provide better discrimination
than biotyping and serogrouping by Penner’s method. Each of the three
Penner’s antigenic groups comprised different genotypes. RAPD fingerprinting
proved to have an excellent discrimination ability, confirming the higher
degree of variation among C. jejuni strains. A simple procedure in
which bacteria were boiled and the lysate was directly introduced in the PCR
vessel enabled reproducible typing of serologically non-typeable strains.
RAPD fingerprinting of Campylobacter jejuni is well suited for
epidemiological studies.
RAPD-PCR process resulted in the generation of highly specific and
reproducible DNA fingerprints that enable discrimination even between
isolates of a single bacterial serotype. Apparently, minimal genetic
differences can be determined through this relatively simple technique. For
differentiation of Campylobacter jejuni strains, RAPD-PCR analysis
has been applied successfully and may replace biotyping and serotyping
tests. Such data are needed in the epidemiological surveillance for
investigating the origin of the infection, and the distribution of
particular bacterial types in different environments. This procedure may be
especially time and cost effective, when in a single specimen are required
the detection and typing of multiple infectious agents.
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Med Microbiol. 1992;3:159-167.
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5. Penner JL, Hennessy JN. Passive haemagglutination technique for
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8. Owen RJ, Hernandez J. Ribotyping and arbitrary-primer PCR fingerprinting
of campylobacters. Blackwell Scientific Publ. : Oxford, 1993.
9. Fayos A, Owen RJ, Hernandez J. Ribosomal RNA gene restriction fragment
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FEMS Microbiol Letter. 1992;95:87-94.
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