Download Ribotyping of Clostridium perfringens from industrially produced

Letters in Applied Microbiology 2002, 34, 238–243
Ribotyping of Clostridium perfringens from industrially
produced ground meat
Ü. Kilic, B. Schalch and A. Stolle
Institute for Hygiene and Technology of Food of Animal Origin, Veterinary Faculty, Ludwig-MaximiliansUniversity Munich., Veterinärstr. 13, D-80539 Munich, Germany
2001/151: received 16 May 2001 and accepted 18 December 2001
Ü . K I L I C , B . S C H A L C H A N D A . S T O L L E . 2002.
Aims: Clostridium (Cl.) perfringens is a common cause of food poisoning outbreaks. Ribosomal
DNA analysis (ribotyping), a method which analyses restriction fragment length polymorphisms in the chromosomal genes that encode rRNA, has been shown to be useful for microbial
species identification and subtyping.
Methods and Results: The current study has used ribotyping to examine 111 Cl. perfringens
isolates from industrially produced ground meat in order to collect a basis for a contamination
survey. Among the 111 isolates 107 distinctly different ribopatterns were detected. In only four
cases two Cl. perfringens isolates showed an identical ribopattern. The isolates gave identical
ribotype patterns in three different runs, carried out 3–4 months apart from each other.
Conclusions, Significance and Impact of the Study: The discriminatory index for EcoRI
ribotyping of the Cl. perfringens isolates was 0Æ99. Results showed that ribotyping is suitable for
subtyping Cl. perfringens isolates from raw meat. Ribotyping appeared to be a useful tool for
profound epidemiologic studies of Cl. perfringens-contamination in food production and
processing.
INTRODUCTION
Clostridium (Cl.) perfringens is a Gram-positive, sporeforming, anaerobic bacillus which is one of the most widely
distributed pathogenic bacteria in the environment. It is
found in the intestinal tract as part of the normal flora.
Cl. perfringens spores are present in small numbers in human
faecal specimens and raw food samples. Human food
poisoning caused by Cl. perfringens is due to an enterotoxin
which is produced during sporulation of the bacteria in the
intestinal tract following ingestion of vegetative cells in large
numbers (Labbe 1989).
In 1986, Grimont and Grimont proposed ribotyping as a
general method for molecular bacterial identification. This
technique can discriminate between isolates of the same
species (Stull et al. 1988). It has proven to be a useful
epidemiological tool in the study of various bacterial
1 pathogens (Altwegg et al. 1989; Blumberg et al. 1991;
Sarafian et al. 1991; Esteban et al. 1993; Sexton et al. 1993).
Correspondence to: A. Stolle, Institute for Hygiene and Technology of Food of
Animal Origin, Veterinary Faculty, Ludwig-Maximilians-University Munich.,
Veterina¨rstr. 13, D-80539 Munich, Germany (e-mail: Sekretariat@lmhyg.
vetmed.uni-muenchen.de).
Ribosomal DNA analysis (ribotyping) is a method for
analysing restriction fragment length polymorphisms in the
chromosomal genes that encode rRNA. Ribotyping is based
on the visualization of segments of these ribosomal genes by
a labelled probe.
Cl. perfringens contains 10 highly conserved operons for
16S and 23S rRNA genes distributed throughout the
2 chromosome (Garnier et al. 1991). Ribotyping was reported
to be an excellent technique for the typing of Cl. perfringens
isolates (Forsblom et al. 1995; Schalch et al. 1997). Typing
of Cl. perfringens is necessary to gain further information
about the origin, the contamination routes of these microorganisms in food producing plants, for investigating and
controlling food poisoning sources (McDonel 1986).
MATERIALS AND METHODS
Strains
In this study, 111 Cl. perfringens isolates from raw meat were
investigated. The isolates examined were taken from the
4 Institute’s collection of sulphate-reducing anaerobes which
originated from industrially produced ground meat. These
isolates had been collected from April 1994 to June 1996
ª 2002 The Society for Applied Microbiology
RIBOTYPING OF CLOSTRIDIUM PERFRINGENS
using the procedure described in the official methods’
5 collection according to § 35 of the German Law on Food and
Commodities. During cultivation and differentiation of the
anaerobic isolates, Reverse CAMP Test and Acid Phosphatase reactions were used for the confirmation of Cl. perfringens. The isolates were stored in a Microbankä storage
system (Mast Diagnostica, Reinfeld, Germany) at ) 18°C.
Prior to ribotyping, the purity of the isolates was re-ensured
by subculturing twice on Columbia sheep blood agar (Oxoid
CM. 331, UK).
DNA preparation
Cl. perfringens isolates were cultured on Columbia sheep
blood agar. The isolates were then incubated under
anaerobic conditions at + 37°C for 16 h in Brain Heart
Infusion Broth (Oxoid CM. 225, UK) and then centrifuged
(13 000 g, 10 min). DNA was isolated from the cell pellet
using the guanidium thiocyanate method according to
Pitcher et al. (1989).
Ribotyping
Ribotyping was carried out according to the method
described by Grimont and Grimont (1986). Following
DNA isolation, the DNA concentrations were measured
with a UV spectrophotometer (UV/VIS spectrometer
Lambda 2; Perkin-Elmer, Norwalk, CT, USA). Five
micrograms of DNA were then digested with EcoRI
at + 37°C for a maximum of 2 h. Digoxigenin (DIG)labelled phage lambda DNA cleaved with HindIII (Boehringer Mannheim GmbH) was used as a molecular weight
marker. The restriction fragments were separated by agarose
gel electrophoresis at 25 V for 16 h, 19 h, 22 h and 24 h.
The fragments were then transferred onto a nylon membrane via Southern Blot. After drying at room temperature,
the DNA was fixed at + 120°C for 0Æ5 h. The membrane
was welded in a plastic bag and prehybridized for 3 h in
a + 58°C water bath. A cDNA probe was prepared from
Escherichia coli 16S and 23S rRNA (Boehringer Mannheim
GmbH) and labelled by incorporation of DIG-dUTP using
Avian myeloblastosis virus reverse transcriptase. The solutions used for hybridization, washes and detection of the
DIG label were prepared according to the DIG DNA
labelling and detection kit (Boehringer Mannheim GmbH).
Finally, the membrane was stained with nitroblue tetrazolium chloride and 5-Bromo-4-chloro-3-indolyl-phosphate,
4-toluidine-salt (Boehringer Mannheim GmbH) overnight
and washed with TE solution (10:1). Ribotyping membranes
were photographed and documented using the Gel Doc
1000 imaging system (Bio-Rad, Hercules, CA, USA) and
Multi-AnalystÒ/PC Software (Bio-Rad, Hercules, CA,
USA).
239
Ribotyping pattern analysis
Cl. perfringens isolates were first examined visually. The
band profiles were then arranged into a dendrogram using
the unweighted pair group method with arithmetic averages
(UPGMA). The ribotype patterns of Cl. perfringens were
analysed via computer cluster analysis with Molecular
AnalystÒ Software (Fingerprinting Version 1Æ12 Applied
Maths 1992–94, Bio-Rad Laboratories). The numerical
index of the discriminatory power of each typing assay
was calculated based on the probability of two unrelated
strains being characterized as the same type (Hunter and
Gaston 1988). In addition, the reproducibility of ribotyping
for the characterization of Cl. perfringens was determined in
this study. 10 of all 111 Cl. perfringens isolates were purely
coincidental selected and ribotyped in three different runs,
carried out 3–4 months apart from each other (Schalch et al.
1997).
RESULTS
Among 111 Cl. perfringens isolates from ground meat 107
distinctly different ribotype patterns were detected. In only
four cases two Cl. perfringens isolates showed an identical
ribopattern. Figure 1 shows an example of the variability of
the ribotype patterns.
The number of DIG labelled bands of Cl. perfringens
varied between 12 and 17. Typical ribotype patterns of
Cl. perfringens were as follows: 7–11 bands between 23 kb
and 4Æ3 kb, 0–4 bands between 4Æ3 kb and 2Æ0 kb and 2–5
bands below 2Æ3 kb. The latter showed typical band patterns
which could be differentiated easily. Based on these patterns
of bands smaller than 2Æ3 kb, all 111 Cl. perfringens isolates
were divided into five groups, named A to E. Seventy-three
percent of all Cl. perfringens isolates examined belonged to
profile type A, 12Æ6% to profile B, 9Æ9% to profile C, 2Æ7%
to profile D and 1Æ8% to profile E (Fig. 1).
6 All Cl. perfringens isolates were typeable with EcoRI
ribotyping and the rDNA patterns were clearly reproducible. In this study, a convincing reproducibility was
observed, as reported before for Cl. perfringens (Schalch
et al. 1997). The discriminatory index for ribotyping of
Cl. perfringens isolates was above 0Æ99.
During the experiments for this study, it was observed
that the following points have to be kept to closely when
ribotyping Cl. perfringens: The DNA from Cl. perfringens
isolates may be stored at + 4°C for a maximum of four
weeks in TE buffer. The incubation time for restriction with
EcoRI at + 37°C must not exceed two hours.
In this study, an electrophoresis running time of 24 h gave
the best results as compared to 16 h, 19 h, and 22 h (Fig. 2).
Figure 3 shows the dendrogram of 74 Cl. perfringens
isolates, which was determined by comparing the complete
ª 2002 The Society for Applied Microbiology, Letters in Applied Microbiology, 34, 238–243
240 Ü . K I L I C ET AL.
kb
M
1
2
3
4
5
6
7
8
9
10
11 12
13
14
15
16
17
18
M
kb
23
23
9.4
9.4
6.5
6.5
4.3
4.3
2.3
2.0
2.3
2.0
(a)
kb
M
1
1
2
2
3
3
4
4
M
kb
23
23
9.4
9.4
6.5
6.5
4.3
4.3
2.3
2.3
2.0
2.0
A
B
C
D
E
(c)
(b)
Fig. 1 Ribotyping of genomic DNA from Cl. perfringens isolates digested with EcoRI and probed for 16S and 23S rRNA genes. Lanes 1 through 18
show the different ribotype patterns of Cl. perfringens isolates. (a). Lanes 1 through 4 show the four identical ribotype patterns within
111 Cl. perfringens isolates (b). Typical patterns of the bands beneath 2,3 kb of Cl. perfringens isolates (c). The digoxigenin-labelled molecular
weight marker II (M) was used a size marker. Band sizes in kb are indicated
band patterns over the whole length of the electrophoretic
run (24 h).
DISCUSSION
Ribotyping has been useful for subtyping diverse bacteria
including species of Listeria monocytogenes (Grimont and
Grimont 1986), Escherichia coli (Lipuma et al. 1989),
Campylobacter spp. (Kiehlbauch et al. 1991), Yersinia
enterocolitica (Lobato et al. 1998), and Cl. perfringens
(Forsblom et al. 1995; Schalch et al. 1997).
Ribotyping has several advantages over other typing
methods. As the ribosomal genes are located on the
chromosome, they are not easily lost like plasmids. Most
bacteria contain multiple copies of the ribosomal operons;
therefore, an acceptable number of fragments (3–15) that
hybridize with the probe are generated. In addition, the
rDNA patterns are not sensitive to antibiotic pressure, and
they are not dependent on susceptibility to phages or
production of bacteriocins.
For molecular subtyping methods, the following
criteria are important in comparative evaluation. First,
ª 2002 The Society for Applied Microbiology, Letters in Applied Microbiology, 34, 238–243
RIBOTYPING OF CLOSTRIDIUM PERFRINGENS
M
241
kb
23
9.4
6.5
M
23
4.3
9.4
6.5
4.3
2.3
2.3
2.0
Fig. 2 Ribopatterns of Cl. perfringens with
electrophoresis running times of 16 h, 19 h,
22 h, and 24 h at 25 V
the reproducibility of a typing method, which is the
percentage of strains with the same subtype on repeated
testing, preferably after a period of a few months. Second,
the discriminatory power of a method is an estimate of its
ability to differentiate between two unrelated strains
(Hunter 1990; Swaminathan and Matar 1993). The
acceptable level of discrimination depends on a number
of factors, but an index of greater than 0Æ90 is considered
necessary if the typing results are to be interpreted with
confidence (Hunter and Gaston 1988). In the current
study, the discriminatory index for ribotyping of the
Cl. perfringens isolates studied was above 0Æ99. This
indicates that ribotyping is very well suited to subtype
Cl. perfringens isolates from raw meat. All isolates included
in this study were typeable with EcoRI ribotyping.
Within 111 Cl. perfringens isolates from ground meat,
ribotyping distinguished 107 distinctly different ribotype
patterns. These results clearly show the great genetic variety
of Cl. perfringens contaminating ground meat.
Several methods are available for the subtyping of
Cl. perfringens isolates; these include bacteriocin typing,
phage typing, antimicrobial susceptibility testing, and
plasmid profile analysis (Traub et al. 1986; Yan 1989;
Mahony et al. 1992). These methods are based on metabolic
features or on the detection of extra-hromosomal genetic
information which are prone to rapid changes. In addition,
plasmid profile and antibiotic susceptibility results are
particularly sensitive to environmental or antibiotic
2.0
16h
19h
22h
24h
pressures. As the genes encoding rRNA are located on
the chromosome and are not readily lost, ribotyping
provides a more reliable discrimination especially for
characterizing strains collected over extended periods of
time (Grattard et al. 1993; Mulligan and Arbeit 1991;
Schmitz et al. 1995).
Manual ribotyping is time and cost intensive and requires
profoundly trained personnel for carrying out the tests and
evaluating the results. Nevertheless, this study underlines
the discriminatory power and reproducibility of ribotyping
of Cl. perfringens isolates from ground meat. Therefore,
ribotyping has to be considered suitable for establishing a
data bank as a basis for comprehensive contamination
studies and seems to be very promising for the epidemiological investigation of contamination studies in the field of
food hygiene and food industry. A recently developed
automated RiboPrinterä system (Qualicon, Wilmington,
Del, USA) allows acquisition and normalization of ribotype
patterns. The system may be used for identification of
bacteria at genus and species levels and to determine
similarity of strain patterns. The RiboPrinterä system was
designed specifically to satisfy the needs of the food
industry. All the process steps required to characterize a
bacterium to the strain level, from cell lysis to image
9 analysis, are performed in 8 h. RiboPrinterä may be an
attractive alternative but, at the present time, the equipment
and supplies are prohibitively expensive for most
10 laboratories.
ª 2002 The Society for Applied Microbiology, Letters in Applied Microbiology, 34, 238–243
242 Ü . K I L I C ET AL.
Fig. 3 Dendrogram obtained from cluster analysis of EcoRI ribotypes
of Cl. perfringens isolates by using the unweighted pair group method
with arithmetic averages (UPGMA)
ACKNOWLEDGEMENTS
We are grateful to Prof. Dr. H. Eisgruber for advice and
helpful comments, and to H. Dietz and A. Fendel for
technical assistance.
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