Dispatch
Molecular Characterization
of Corynebacterium diphtheriae isolates, Russia, 1957–1987
Vegard Skogen,* Valentina V. Cherkasova,† Nina Maksimova,† Chung
K. Marston,‡ Haakon Sjursen,§ Michael W. Reeves,‡ Ørjan Olsvik,*
and Tanja Popovic‡
*University of Tromsø, Norway; †G. N.
Gabrichevsky Institute for Epidemiology and Microbiology, Moscow,
Russia; ‡Centers for Diseases Control and Prevention, Atlanta, Georgia,
USA; and §University of Bergen, Haukeland Hospital, Bergen, Norway
In the 1990s,
the Newly Independent and Baltic States of the former Soviet Union
experienced the largest diphtheria outbreak since the 1960s; it
was caused by Corynebacterium diphtheriae strains of a
unique clonal group. To address its origin, we studied 47 clinical
isolates from Russia and demonstrated that this clonal group was
an integral part of the endemic reservoir that existed in Russia
at least 5 years before the epidemic began.
In the pre-vaccine era, diphtheria was a major cause of childhood
illness and death worldwide. After the diphtheria toxoid vaccine
was introduced, a decline in diphtheria cases was seen where the
vaccine was used. In some areas of the Soviet Union, diphtheria
vaccination started as early as the 1920s, but it was not included
in the general immunization program for children until 1958 (1).
After 1958, reported diphtheria cases declined steadily except for
a small increase in incidence during the 1980s and the epidemic
that started in 1990. In 1991, after the breakup of the Soviet Union,
routine childhood vaccination programs were disrupted due to interruption
of vaccine supplies to countries in Central Asia, the Caucasus,
and the Baltic region. A major diphtheria epidemic began in Russia
in 1990; during the next 4 years, it reached all the Newly Independent
States and Baltic States of the former Soviet Union (FSU) (1,2).
The European Regional Office of the World Health Organization (WHO)
now considers this diphtheria outbreak, which resulted in more than
150,000 cases and 4,000 deaths, to be nearly under control (1).
Several factors, such as an increased proportion of susceptibles
in the population, migration, and a deteriorating health infrastructure,
are suspected to be major catalysts for this outbreak (2).
However, the role of biological factors of the causative organism
is not clear.
To assess the genetic diversity and structure of the bacteria and
its toxin, different molecular typing methods have been used successfully
as a complement to traditional characterization (3–6).
Popovic et al. and de Zoysa et al. identified a particular epidemic
clonal group, characterized by ribotyping, multilocus enzyme electrophoreses
(MEE), and pulsed-field gel electrophoresis (PFGE), associated with
the appearance and spread of this outbreak (7,8).
Our study focuses on the origin of this epidemic clonal group and
is the first to include a limited number of archival isolates collected
more than 30 years before this outbreak began.
The
Study
A convenience sample of 47 Corynebacterium diphtheriae isolates
was available for analysis from a collection of isolates obtained
during 1957–1987, before the onset of the recent diphtheria outbreak.
These isolates were collected from both carriers (n=37) and patients
(n=10) in different regions of Russia. All isolates were kept freeze-dried
at the G. N. Gabrichevsky Institute for Epidemiology and Microbiology,
Moscow, Russia, and were transported on silica gel packages to the
Centers for Disease Control and Prevention, Atlanta, Georgia, for
molecular characterization.
All isolates were biotyped by using the commercial API Coryne kit
(Biomerieux, Lyon, France). Toxigenicity status was determined by
the Elek test, as recommended by WHO (9), and by
the polymerase chain reaction (PCR), which targeted both A and B
subunits of the tox gene (10).
All the strains were characterized by ribotyping as previously
described (11). The hybridization was done by
using five oligonucleotide probes according to Regnault et al. (12).
Ribotyping pattern designations were based on the scheme established
by Popovic et al. (7). A difference in one band
was defined as an individual ribotype (RT).
MEE was carried out as previously described (7,11).
The electromorphs of the same enzyme were visualized in a starch
gel matrix as bands of different migration rates. Each electromorph
was considered to represent a distinct allele of the same enzyme.
By testing 27 different enzymes, a profile of electromorphs, defining
the electrophoretic type (ET) of each strain, was obtained. The
genetic relatedness of ETs was illustrated as a dendrogram, which
was generated by the average-linkage method of clustering ETs described
by Selander et al. (13).
We examined 47 C. diphtheriae isolates collected in the
pre-epidemic period (1957–1987) from 10 patients and 37 carriers
in different areas of Russia. Thirty-nine strains were of the gravis
biotype, 7 were the mitis biotype, and 1 was of the intermedius
biotype. All the mitis biotype strains were toxigenic. Among the
gravis biotype strains, 36 were toxigenic, and 3 were nontoxigenic.
No discrepancies between the results obtained by traditional identification,
the API Coryne test, or toxigenicity testing by the Elek test and
PCR were detected.
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Figure
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Figure 1.Twelve BstEII
ribotypes identified in 47 Corynebacterium diphtheriae
isolates collected in the Russian Federation between 1957
and 1987....
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Figure
2
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Click
to view enlarged image
Figure 2. Dendrogram
showing the genetic relatedness of 85 electrophoretic types
(ETs) of Corynebacterium diphtheriae isolates collected
in different countries around the world.... |
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In the 47 isolates, 12 different RTs were identified (Figure
1). Twenty-two (47%) were of the M11e RT; all were toxigenic
and of the gravis biotype. They were collected from 1957 to 1985.
RT G4, characteristically seen in the recent epidemic clonal group,
was identified in 6 (13%) isolates, all of which were collected
from 1984 through 1987. Four isolates had two new ribotype patterns,
not previously described. They were collected from 1977 through
1981.
Sixteen (6 isolates of RT pattern G4 and 10 isolates of different
RT patterns) of the 47 isolates were analyzed by MEE; 13 different
ETs were identified. Of the six isolates with the G4 patterns, four
also belonged to the ET8 complex (Figure 2).
An additional isolate (strain designation B533 in Table)
collected in 1957 belonged to the ET8 complex but had a different
RT.
Conclusions
In the pre- and early-vaccine era, diphtheria incidence was high
in the Soviet Union. After the diphtheria vaccine was introduced,
a decrease in incidence was seen in the 1950s. During the mid-1970s,
immunization programs resulted in control of diphtheria throughout
the country. However, an increase in incidence was noted at the
end of 1970 and during the 1980s, and a peak was observed in 1983.
This resurgence was associated with a change in the biotype of the
circulating C. diphtheriae strains from gravis, which had
been dominating for several decades, to mitis (14).
To allow better monitoring of the global spread of diphtheria,
the WHO ribotyping database for C. diphtheriae was established
at the Pasteur Institute in Paris, France. The institute demonstrated
that C. diphtheriae RTs are quite diverse worldwide but remain
stable over time (15). Both ribotyping and MEE
have provided a significant level of differentiation and
reliability and subsequently have been accepted as the standard
for molecular subtyping of C. diphtheriae. Thus, we used
these molecular methods to characterize our archival isolates.
Twelve different RTs were found in our 47 isolates. Our data show
that nine C. diphtheriae isolates from the 1950s and 1960s
had an RT pattern (M11e) that was very similar to ribotype M11,
which was only seen occasionally in the FSU in the 1990s. Epidemic
RT G4 was seen in six toxigenic C. diphtheriae isolates collected
from 1984 through 1987 in four distant regions of Russia
(Moscow and Moscow region, Anapa, Smolensk, and Sverdlovsk) from
both diphtheria patients and carriers; four of these isolates were
also members of the ET8 complex.
Our investigation of the origin of the epidemic clonal group determined
that, in our strain collection, the earliest reported strain of
this clonal group was identified in Smolensk in 1985, and that strains
of this clonal group were simultaneously present in several geographically
distant areas in Russia from 1985 through 1987. These findings suggest
that the current epidemic clone was an integral part of the endemic
reservoir that existed in the FSU at least 5 years before the epidemic
began. Further studies that would include a large number of gravis
biotype strains from throughout the Soviet Union isolated from 1980
through 1985 might unveil where and when strains of the epidemic
clone were first associated with disease or carriage.
Dr. Skogen is working at the Department of Microbiology, University
Hospital of North Norway, Tromsø, Norway. His recent research interests
have been in communicable diseases, especially diphtheria, in Russia.
Address for correspondence: Vegard Skogen, Department of Medicine,
Institute of Clinical Medicine, University of Tromsø, N-9037 Tromsø,
Norway; fax: 47 776 44650; e-mail: vegards@fagmed.uit.no
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Table.
Designations and characteristics of 47 Corynebacterium
diphtheriae strains collected in Russia, 1957–1987 |
|
Ribotype
|
Geographic area of isolation
|
No. isolates
|
Year of isolation
|
Biotypea
|
ETb
|
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G4
|
Anapa
|
1
|
1984
|
G
|
286
|
|
Moscow
|
2
|
1985, 1987
|
G
|
291, 8
|
|
Smolensk
|
1
|
1985
|
G
|
8
|
|
Sverdlovsk
|
2
|
1987
|
G
|
8
|
M1b
|
Anapa
|
1
|
1984
|
M
|
287
|
M3
|
Krasnoyarsk
|
2
|
1979
|
M
|
290, ND
|
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Ivanov
|
1
|
1976
|
M
|
ND
|
M6
|
Moscow
|
1
|
1981
|
G
|
ND
|
M7a
|
Moscow
|
2
|
1972, 1973
|
G
|
ND
|
M11e
|
Moscow
|
13
1
|
1964–1977
1964
|
G
G
|
ND
283
|
|
Vladivostok
|
2
1
|
1957
1957
|
G
G
|
280, 281
ND
|
|
Buryatiya
|
1
|
1976
|
G
|
285
|
|
Groznyi
|
1
|
1985
|
G
|
ND
|
|
Vladimir
|
1
|
1977
|
G
|
ND
|
|
Tatarstan
|
1
|
1977
|
G
|
ND
|
|
Omsk
|
1
|
1976
|
G
|
ND
|
M11f
|
Vladivostok
|
1
|
1957
|
G
|
ND
|
|
Omsk
|
2
|
1977
|
Ge
|
ND
|
M11g
|
Kirov
|
1
|
1978
|
G
|
ND
|
M13a
|
Vladivostok
|
1
|
1957
|
Ge
|
282
|
|
Vladimir
|
1
|
1976
|
G
|
284
|
|
Krasnoyarsk
|
1
|
1979
|
G
|
289
|
M13b
|
Moscow
|
1
|
1981
|
Id
|
ND
|
Newc
|
Vladivostok
|
1
|
1981
|
M
|
ND
|
|
Moscow
|
1
1
|
1977
1977
|
M
Ge
|
ND
288
|
|
Krasnoyarsk
|
1
|
1979
|
M
|
ND
|
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aG, biotype gravis; M, biotype
mitis; I, biotype intermedius.
bET, electrophoretic type.
cNew ribotype, pattern has
not been previously observed.
dND, not done.
eNontoxigeic by the Elek
test and polymerase chain reaction.
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