Department of Veterinary Medicine, Faculty of
Agriculture, Tokyo University of Agriculture and Technology, Fuchu,
Tokyo 183-8509, Japan
Received 29 May 2001/Returned for modification 16 July
2001/Accepted 3 September 2001
 |
INTRODUCTION |
Erysipelothrix
rhusiopathiae is a gram-positive, slender, and straight or
slightly curved rod that causes a wide spectrum of diseases in animals,
birds, and humans (14, 49). This bacterium has been
isolated in most parts of the world, not only from sick and healthy
animals but even from pork, seafood, retail game meat, and chicken meat
(11, 19, 26, 27, 38, 40, 44). Human infections with this
bacterium are usually related to occupational exposure
(33). However, infection after consumption of undercooked pork and infections of patients with no history of contact with animals
or skin lesions have been reported, and in many cases, the source of
infection has not been identified (6, 13, 20, 28, 37).
Moreover, potential errors in the recognition of this organism isolated
from human infections due to unusual clinical presentations and the
possibility of underdiagnosed infections have been reported (3,
10, 34). PCR-based assays for the rapid diagnosis of
Erysipelothrix species have been described (22, 39,
42). However, to proceed with an epidemiological study and
identify the source of infection, it is necessary to be able to
identify each strain isolated from a case or outbreak, as well as the
relatedness among the strains isolated from the possible source.
During the last few years, molecular biological methods such as
randomly amplified polymorphic DNA (RAPD), ribotyping, and pulsed-field
gel electrophoresis (PFGE) have been demonstrated to be reliable tools
for the differentiation of species and strains of one genus and for use
in epidemiological studies of several pathogenic bacteria (9, 15,
16, 24, 32, 46-48). Although PFGE has been considered to be the
"gold standard" among these methods (30), studies have
shown that this method can be less sensitive than ribotyping and
PCR-based methods with regard to the ability to differentiate between
bacterial strains of some species (5, 36, 45). Moreover,
there is no standard and universal PFGE protocol for all species of
bacteria and it is necessary to adapt the procedures and choose a
suitable enzyme for each genus or species. However, the use of this
method for strains of the genus Erysipelothrix has not been
reported. Therefore, we describe herein the first analysis of a large
collection of Erysipelothrix species strains by PFGE.
| |
MATERIALS AND METHODS |
Bacterial strains.
Seventy strains, including 55 of E. rhusiopathiae and 12 of E. tonsillarum, as well as
2 strains of serovar 13 and 1 strain of serovar 18 that have been
considered to be members of a possible new species (41),
were chosen from our previous study (29). The sources for
and details regarding each strain are shown in Table
1.
DNA preparation.
Chromosomal DNAs from the strains were
prepared by using the CHEF Bacterial Genomic DNA Plug Kit (Bio-Rad
Laboratories, Richmond, Calif.) with some modifications of the
manufacturer's instructions. All of the buffers and solutions, except
those for which the manufacturer are identified, were supplied with the
DNA Plug Kit. The strains were inoculated in 50 ml of tryptose
phosphate broth (Difco Laboratories) and cultured overnight with
shaking at 37°C. Bacterial cells were harvested by centrifugation,
suspended in 1 ml of phosphate-buffered saline (8.45 mM
NaH2PO4, 5.12 mM
KH2PO4, 0.12 M NaCl [pH
7.2]), transferred to 2-ml Eppendorf tubes, washed once, resuspended, and then diluted with the same buffer to an optical density at 600 nm
of 0.8 to 1.0. Chloramphenicol was added to a final concentration of
180 µg/ml, and the suspension was incubated at 37°C for 1 h. One milliliter of the suspension was centrifuged, and the harvested cells were resuspended in 50 µl of cell suspension buffer. The suspension was heated to 50°C in warm water and combined with the
same amount of melted 2% CleanCut agarose, also equilibrated to
50°C. The mixture was transferred to disposable plug molds and
allowed to solidify at 4°C for 10 min. Each plug was removed from the
molds and placed in 250 µl of lysozyme solution, prepared by adding
200 µl of the lysozyme stock (25 mg/ml) and 100 µl of N-acetylmuramidase (1 mg/ml; Seikagaku Corp., Tokyo, Japan)
to 2.5 ml of lysozyme buffer, and then incubated for 48 h at
37°C in a water bath. The lysozyme solution was removed, and the
plugs were rinsed once with sterile deionized water, after which they were replaced in 250 µl of proteinase K reaction buffer containing 10 µl of proteinase K stock, yielding a final proteinase K concentration of 100 µg/ml. After further incubation for 24 h at 50°C, the
plugs were washed four times in 1× wash buffer for 1 h each time
at room temperature. Phenylmethylsulfonyl fluoride (Sigma) was added to
a final concentration of 1 mM at the second wash. Plugs were stored in
1 ml of 1× wash buffer until enzyme treatment.
Restriction enzymes and PFGE.
DNA plugs were sliced, and
DNAs were digested with SmaI (Takara Co. Ltd., Tokyo,
Japan), AscI (New England BioLabs), and NotI (Takara). A slice of each plug was placed twice in 0.1× wash buffer and incubated for 1 h each time at room temperature. After removal of the wash buffer, the DNAs were digested with 10 U of each enzyme in
the respective reaction mixture in accordance with the manufacturer's instructions. The DNA fragments were separated in 1% agarose NA gel
(Amersham Pharmacia) that was prepared in 0.5× Tris-borate-EDTA buffer
(50 mM Tris base, 50 mM boric acid, 2 mM EDTA) on a Gene Navigator
(Pharmacia Biotech). Electrophoresis was carried out for 24 h at
12°C and 200 V with pulse times of 1 to 35s. The CHEF DNA Size
Standard Lambda Ladder (Bio-Rad) was used as a DNA size marker.
Thereafter, the gels were stained with ethidium bromide for 1 h, destained in distilled water, and photographed under UV light. PFGE patterns were inspected visually, each PFGE
pattern that differed by one or more DNA fragment bands was identified, and the relatedness among the patterns was analyzed based on the guidelines described by Tenover et al. (43) and Maslow et
al. (24).
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RESULTS |
Of the three restriction enzymes tested, only SmaI
produced several bands in repeated screening tests and was able to
clearly differentiate between strains. Thus, SmaI was used
for further analysis of all 70 strains. The PFGE patterns produced from
four strains with the three enzymes are shown in Fig.
1. To determine the optimal
electrophoresis protocol, a series of trials changing the pulse and
electrophoresis times was carried out (data not shown). Of the
protocols tested, the one described above was best able to discern the
high- and even low-molecular-weight DNA fragment bands produced by
SmaI.
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FIG. 1.
PFGE patterns produced from four strains of
Erysipelothrix spp. by SmaI,
AscI, and NotI. Lanes: 1, E.
rhusiopathiae strain ME-7, serovar 1a; 2, E.
rhusiopathiae strain ATCC 19414, serovar 2; 3, E.
tonsillarum strain ATCC 43339, serovar 7; 4, Erysipelothrix species strain 715, serovar 18; M,
CHEF DNA Size Standard Lambda Ladder (Bio-Rad).
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From the 70 strains analyzed, 63 distinct PFGE patterns with 8 to 27 DNA fragment bands were produced (Fig. 2;
Table 1). Twelve strains showed five distinct PFGE patterns with
no DNA fragment band difference observed among strains
sharing the same PFGE pattern. These were strains 47, N008,
r4.1a, and r6.1a, which shared pattern P28 and strains 17.2a and 10.2a,
212 and 213, 136 and 20.4a, and 88 and 97, which shared, respectively,
patterns P12, P17, P25, and P38 (Table 1). Single and distinct
PFGE patterns were produced for 58 strains. Among them, patterns
P50 and P51 of strains ATCC 43339 and ATCC 43338 differed by two bands,
patterns P4 and P36 of strains 422/1E1 and K021 differed by three
bands, patterns P32 and P39 of strains 280 and E146 differed by four bands, and patterns P19 and P20 of strains Pécs 67 and AKO
differed by five bands. Patterns P24 of strain 36.4a and P25 of strains 136 and 20.4a differed by six bands from pattern P26 of strain K002, as
well as patterns P34 and P45 of strains K024 and E051. The remaining 45 patterns differed by at least seven bands (Fig. 2). Although some
similar bands were observed among many strains, no characteristic PFGE
patterns related to the species or even serovar could be
differentiated (Fig. 2). The PFGE analysis was repeated
twice, and the same results were obtained.
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FIG. 2.
Schematic representation of the 63 PFGE patterns
produced from the 70 Erysipelothrix spp. strains studied
by using SmaI. E. spp.,
Erysipelothrix species. The serovar and PFGE pattern
designation of each strain are given, respectively, in parentheses.
Lanes: 1, ME-7 (1a, P1); 2, E176 (1a, P2); 3, E157 (1a, P3); 4, 422/1E1
(1b, P4); 5, E019 (1b, P5); 6, K040 (1b, P6); 7, K075 (1b, P7); 8, ATCC
19414 (2, P8); 9, R32E11 (2, P9); 10, NF4E1 (2, P10); 11, 115 (2, P11);
12, 17.2a (2, P12); 13, E037 (2, P13); 14, K003 (2, P14); 15, N026 (2, P15); 16, Doggerscharbe (4, P16); 17, 212 (4, P17); 18, E127 (4, P18);
19, Pécs 67 (5, P19); 20, AKO (5, P20); 21, 2.2a (5, P21); 22, K059 (5, P22); 23, Tuzok (6, P23); 24, 36.4a (6, P24); 25, 136 (6, P25); 26, K002 (6, P26); 27, Goda (8, P27); 28, 47 (8, P28); 29, E024
(8, P29); 30, Kaparek (9, P30), 31, E112 (9, P31); 32, 280 (9, P32);
33, K052 (9, P33); 34, K024 (10, P34); 35, IV 12/8 (11, P35); 36, K021
(11, P36); 37, Pécs 9 (12, P37); 38, 88 (12, P38); 39, E146 (12, P39); 40, Pécs 3597 (15, P40); 41, Tanzania (16, P41); 42, 545 (17, P42); 43, 2017 (19, P43); 44, E053 (19, P44); 45, E051 (19, P45);
46, K031 (19, P46); 47, Bãno 36 (21, P47); 48, MEW22 (N, P48);
49, Wittling (3, P49); 50, ATCC 43339 (7, P50); 51, ATCC 43338 (7, P51); 52, P-43 (7, P52); 53, K015 (7, P53); 54, Lengyel-P (10, P54); 55, Iszap-4 (14, P55); 56, E073 (15, P56); 57, K037 (16, P57);
58, 2553 (20, P58); 59, Bãno 107 (22, P59); 60, KS20A (23, P60);
61, Pécs 56 (13, P61); 62, Shiribeshi 17 (13, P62); 63, 715 (18, P63); M, CHEF DNA Size Standard Lambda Ladder (Bio-Rad).
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| |
DISCUSSION |
Distinct and reproducible PFGE patterns were produced by using
SmaI, and this allowed us to differentiate 63 PFGE patterns among the 70 strains analyzed. Studies have shown that for some bacterial species, the PFGE method is less sensitive than ribotyping and PCR-based methods. In addition, a high incidence of DNA
degradation, which leads to a decrease in typeability by this method,
has been cited (17, 23). Although DNA degradation should
be avoided by addition of thiourea to the gel buffer (8,
35), no DNA degradation was observed with the strains used in
this study and PFGE patterns were obtained for all of the strains
analyzed. In a previous study using the same strains, we determined
that the RAPD method is able to differentiate between species and to
distinguish strains of this genus (29). By that method, 14 distinct amplification profiles were obtained (Table 1). Arhné et
al. (1) have analyzed 39 strains by ribotyping and
identified nine different patterns. Of those 39 strains, 23 producing
six different patterns by ribotyping were also analyzed in our study
and unique PFGE patterns were produced for each strain (Table 1).
Although analysis including more detailed epidemiological data and
comparing the PFGE patterns of more strains of one species and one
serovar and the inclusion of human isolates may be desirable, based on
those previous results and on the results of this study, in which all
of the strains studied were typed by PFGE and distinct PFGE patterns
were obtained for 90% of the strains analyzed, it might be concluded
that this method is better than RAPD analysis and ribotyping for
epidemiological studies of strains of this genus.
Of the 70 strains, 12 were clustered in five PFGE patterns and no band
differences were observed among strains classified into the same
cluster. Since this study did not include an epidemiological study,
only the sources from which the strains were isolated are shown in
Table 1. However, although those strains that shared the same PFGE
patterns were isolated in our laboratory at different times and from
different samples, all of the strains were isolated from chickens or
chicken meat from the same abattoir or processing plant, and each group
was composed of strains sharing the same serovar. Moreover, by the
guidelines for the interpretation of PFGE patterns of bacterial
isolates in an epidemiological study proposed by Tenover et al.
(43), isolates might be considered genetically
indistinguishable when their PFGE patterns have the same numbers of
bands and the corresponding bands are the same apparent size.
Therefore, it might be considered that these strains are possibly
members of the same clonal line. According to the same guidelines,
isolates might be considered closely related when PFGE patterns differ
by one to three bands, which is consistent with a single genetic event
(e.g., a point mutation resulting in the loss or gain of a restriction
site, an insertion, a deletion, or a chromosomal inversion); possibly
related when PFGE patterns differ by four to six bands; and different
when they differ by seven or more bands. Among the PFGE patterns
obtained in this study, two pairs of strains showed PFGE patterns
differing by three or fewer DNA fragment bands. The PFGE analysis of
strains ATCC 43339 and ATCC 43338 showed, respectively, patterns
P50 and P51, which differed by two bands, and that of strains
422/1E1 and K021 showed, respectively, patterns P4 and P36, which
differed by three bands. The difference between the PFGE patterns of
the first pair would be explained by a single genetic event, as
described by Tenover et al. (43). Therefore, it might be
that one strain is a subtype of the other and corroborate the results
obtained by RAPD analysis and ribotyping since both of the strains
showed, respectively, the same RAPD pattern (j) and ribopattern (E)
(Table 1). However, the differences between the patterns of the
second pair do not match any pattern differences proposed in
those guidelines. Maslow et al. (24) reported that
when there are a very limited number of band differences that are not
explained by a single genetic event, the isolates may be closely
related but distinct. Thus, we believe that although these two strains
are closely related, they should be classified as distinct
strains. Similarly, and as described elsewhere (12),
the other strains that differed by fewer than seven bands but by four
or more were classified as distinct strains. In an epidemiological
study using PFGE, a common pattern must be identified and the
comparisons among PFGE patterns must be done based on that pattern
(43). Some common bands could be identified among the
strains analyzed in this study. However, few strains showed
nearly identical patterns. Based on results of
previous studies, in which many distinct PFGE patterns were
produced from strains of only one species (2, 21,
23), the high variability of PFGE patterns observed in
this study is not surprising since we included not only the two species
of the genus Erysipelothrix but also numerous strains from
many sources, strains representing the 23 serovars and type N, as well
as serovars 13 and 18, which are considered possible members of new and
separate species (41).
SmaI, AscI, and NotI have been
described as effective enzymes in producing a clear and reliable number
of DNA fragments producing distinct PFGE patterns for several bacterial
species (4, 7, 18, 25, 31). However, with DNAs of strains
of this genus, few fragments, such as two or three, were produced with
AscI and NotI (Fig. 1). In the same set of
guidelines for the interpretation of DNA restriction patterns generated
by PFGE described above, Tenover et al. (43) emphasized
the need for at least 10 distinct DNA fragment bands for reliable
analysis of the relationship among strains. Thus, AscI and
NotI were considered to be inappropriate for analysis of
strains of this genus.
To avoid the labor necessary to prepare the buffers and solutions
required to obtain DNA from the strains studied, a commercial kit was
used. Although DNA could not be obtained by using the protocol
described by the manufacturer, reliable results were obtained with
small modifications such as an increase in the lysozyme stock amount
supplied with the kit and the addition of
N-acetylmuramidase, which has been used for DNA extraction
from strains of this genus (22, 29), to the lysis
solution, as well as an increase in the incubation time.
In this report, we have described a protocol for the performance of
PFGE with strains of the genus Erysipelothrix, demonstrating that PFGE performed with SmaI might be more sensitive than
RAPD and ribotyping and also that this method might be a useful and reliable tool for epidemiological studies of strains of this genus. Moreover, as this was the first study to apply this method to strains
of this genus, without disregarding the need to screen other enzymes,
we expect that the procedure will be of value as a basis for new
approaches using this method with strains of Erysipelothrix species.
We thank Toshio Takahashi (National Veterinary Assay Laboratory,
Tokyo, Japan) for kindly providing us with
Erysipelothrix strains.
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Abstract
Introduction
