oRIGINAl PAPERS

original papers
Adv Clin Exp Med 2011, 20, 6, 717–720
ISSN 1230-025X
© Copyright by Wroclaw Medical University
Aleksandra A. Zasada, Milena Baczewska-Rej, Sebastian Wardak
Differentiation Between Invasive and Non-Invasive
Corynebacterium diphtheriae Isolates
Różnicowanie inwazyjnych i nieinwazyjnych szczepów Corynebacterium
diphtheriae
Department of Bacteriology, National Institute of Public Health – National Institute of Hygiene, Warszawa, Poland
Abstract
Background. In countries with good diphtheria vaccination coverage, diphtheria cases are very rare. But invasive
nontoxigenic C. diphtheriae infections, like bacteriemia, have been emerging during the last decade.
Objectives. The aim of the present investigation was differentiation between C. diphtheriae strains isolated from
invasive and non-invasive infections.
Material and Methods. Toxigenic and nontoxigenic C. diphtheriae strains isolated from invasive and local infections and from carriers in the period of the 1960’s to 2008 were analyzed. A PCR MP technique was used for the
first time for differentiation between C. diphtheriae isolates. The PCR MP results obtained were compared to PFGE
and ERIC-PCR results.
Results. PCR MP differentiates the strains at a similar level as PFGE and ERIC-PCR but, opposite to PFGE and
ERIC-PCR, the technique makes it possible to differentiate strains isolated from invasive infections from strains
isolated from local infections.
Conclusions. This is the first work revealing genetic differences between nontoxigenic C. diphtheriae strains isolated from invasive and local infections. PCR MP is a useful method for the differentiation of C. diphtheriae. The
method gives the possibility for identification of C. diphtheriae strains possessing invasive properties (Adv Clin
Exp Med 2011, 20, 6, 717–720).
Key words: Corynebacterium diphtheriae, invasive infections, PCR MP, genetic differentiation.
Streszczenie
Wprowadzenie. W krajach o dużym stopniu zaszczepienia populacji przeciw błonicy przypadki tej choroby zdarzają
się niezwykle rzadko. W ciągu ostatniej dekady zaobserwowano jednak pojawianie się inwazyjnych zakażeń, takich
jak np. bakteriemia, powodowanych przez nietoksynotwórcze szczepy C. diphtheriae.
Cel pracy. Zróżnicowanie szczepów C. diphtheriae izolowanych z zakażeń inwazyjnych od szczepów izolowanych
z zakażeń miejscowych.
Materiał i metody. Badaniami objęto toksynotwórcze i nietoksynotwórcze szczepy C. diphtheriae izolowane
z zakażeń inwazyjnych, miejscowych oraz od nosicieli od lat 60. XX w. do 2008 r. W badaniach po raz pierwszy
zastosowano technikę PCR MP do różnicowania C. diphtheriae. Uzyskane wyniki typowania techniką PCR MP
porównano z wynikami uzyskanymi technikami PFGE i ERIC-PCR.
Wyniki. Stwierdzono, że technika PCR MP różnicuje badane szczepy na podobnym poziomie jak PFGE i ERIC-PCR, ale w odróżnieniu od PFGE i ERIC-PCR pozwala na odróżnienie izolatów pochodzących z zakażeń inwazyjnych od izolatów z zakażeń miejscowych.
Wnioski. W niniejszej pracy po raz pierwszy wykazano występowanie różnic genetycznych między nietoksynotwórczymi szczepami C. diphtheriae izolowanymi z zakażeń inwazyjnych oraz szczepami izolowanymi z zakażeń
miejscowych. PCR MP jest przydatną metodą różnicowania C. diphtheriae, ponieważ daje możliwość identyfikacji
szczepów mających zdolności inwazyjne (Adv Clin Exp Med 2011, 20, 6, 717–720).
Słowa kluczowe: Corynebacterium diphtheriae, zakażenia inwazyjne, PCR MP, różnicowanie genetyczne.
718
Corynebacterium diphtheriae is a causative
agent of diphtheria – an acute disease – due to
its exotoxin called diphtheria toxin. Strains that
do not produce the diphtheria toxin have been
regarded as nonpathogenic. However, more and
more invasive infections caused by nontoxigenic
C. diphtheriae strains have been recorded in many
developed countries where a diphtheria vaccine is
broadly used. The true number of cases of invasive
nontoxigenic C. diphtheriae infections is unknown
because the reporting obligation only covers cases
of classical diphtheria. Nontoxigenic C. diphtheriae invasive infections affect intravenous drug users, the alcohol addicted and the homeless but also
healthy people [1–6]. There is a question about genome changes which may have given nontoxigenic
C. diphtheriae invasive properties.
The aim of the presented studies was differentiation between C. diphtheriae strains isolated
from invasive and non-invasive infections. In these
studies we demonstrate, for the first time, the use
of a PCR Melting Profile (PCR MP) technique for
differentiation between C. diphtheriae isolates.
PCR MP allows for gradual amplification of the
genomic DNA differing in thermal stability, starting from the less stable DNA fragments amplified
at lower denaturation temperature values to more
stable ones amplified at higher denaturation temperature values. In the present studies, the utility
of the PCR MP method was evaluated with data
obtained using the pulsed-field gel electrophoresis
(PFGE) and enterobacterial repetitive intergenic
consensus PCR (ERIC-PCR) methods.
Material and Methods
The group of isolates tested (Table 1) contained
isolates from Poland, from diphtheria cases in the
1960’s (1-5/A) and 1990’s (6-9/B), nontoxigenic
isolates isolated in 2000 from a diphtheria case and
in 2001 from a carrier (10/C and 11/D, respectively)
and nontoxigenic isolates from blood and wound
infections isolated in 2004–2008 (12-19/E). DNA
was extracted with a DNeasy Tissue Kit (Qiagen)
according to the manufacturer’s procedure. PCR
MP was performed according to Krawczyk et al
[7]. PCR was performed as follows: 7 min at 72°C
followed by initial denaturation at 85.5°C for 90 s
and 22 cycles of denaturation at 85.5°C for 1 min,
and annealing and elongation at 72°C for 2 min.
After the last cycle, samples were incubated for
5 min at 72°C. The denaturation temperature was
calculated during the optimization experiments
using a gradient thermal cycler (Eppendorf) with
a gradient range of 82 to 87°C and 86 to 93°C for
the denaturation step. The PFGE was performed
A.A. Zasada, M. Baczewska-Rej, S. Wardak
as described earlier [8]. The ERIC-PCR was performed according to Steinbruechner et al [9]. Data
analysis was performed with GelCompar II 5.0 for
Windows (AppliedMaths). Dendrograms were
constructed by the unweighted pair group method
with arithmetic averages (UPGMA), using Dice
correlation for PFGE and Pearson correlation for
ERIC-PCR and PCR MP. The computer-assisted
analysis was performed according to the manufacturer’s instructions.
Results and Discussion
The PCR MP results obtained were compared
to PFGE and ERIC-PCR results (Table 1). The similarity level of all tested isolates was 52.3% for PCR
MP and 65% for PFGE and ERIC-PCR. However,
the similarity between strains isolated in the period
2004–2008 was very high. Since all of the nontoxigenic C. diphtheriae strains in this group were collected within a 5-year period from distinct patients
with no epidemiological link (data not shown), we
initially expected diverse genotypes. Surprisingly,
the resulting PFGE, ERIC-PCR and PCR MP patterns were homogeneous for this group of strains.
Only three highly similar PCR MP (genotypes a, a1
and b) and PFGE patterns (genotypes A, A1 and
A2) and two ERIC-PCR patterns (V and VI) could
be distinguished in the nontoxigenic isolates isolated during last 5 years. This remarkable genetic
homogeneity may suggest that Polish nontoxigenic C. diphtheriae isolates are clonal currently.
But this raises the question whether it is a more
virulent genetic clone causing infections or it is the
only clone circulating currently in the Polish population. There are no C. diphtheriae isolates from
carriers in this time frame.
It is worth underlining that all of the compared
methods differentiated the studied group of strains
at a similar level, but, as opposed to PFGE and
ERIC-PCR, PCR MP differentiated strains isolated
from invasive infections (from blood samples) from
strains isolated from local infections (Fig. 1). The
additional DNA band in PCR MP profiles of invasive C. diphtheriae isolates may suggest some kind
of mutations that give the strains invasive potential,
for example the acquisition of new virulence genes
located within a pathogenicity island or a profage
genome. Only one strain isolated from blood did
not reveal the additional band (12/E). Possibly, the
strain did not possess all the same virulence genes
as the other invasive strains tested, but the pathogenicity level of the strain was enough to cause invasive infection in the patient. The patient could
be more sensitive to the infection due to additional
diseases or the genetic specificity of the patient’s
719
Invasive and Non-Invasive C. diphtheriae Isolates
Table 1. Characteristics of tested isolates of C. diphtheriae
Tabela 1. Charakterystyka badanych izolatów C. diphtheriae
Strain
(Szczep)
Toxigenicity
(Toksynotwórczość)
Site of isolation
(Miejsce izolacji)
PCR MP type
PFGE type
ERIC-PCR type
1/A
+
nosopharynx
c1
C
X
2/A
+
nosopharynx
c
C1
X
3/A
+
nosopharynx
c
C
X
4/A
+
nosopharynx
f
H
XI
5/A
+
nosopharynx
d
G
IV
6/B
–
nosopharynx
i
F
VII
7/B
+
nosopharynx
e
B1
I
8/B
+
nosopharynx
e
B
III
9/B
+
nosopharynx
e
B
II
10/C
–
nosopharynx
g
E
VIII
11/D
–
nosopharynx
h
D
IX
12/E
–
blood
a
A2
V
13/E
–
blood
a1
A
V
14/E
–
blood
a1
A
V
15/E
–
wound
a
A
V
16/E
–
fistula
b
A
V
17/E
–
wound
a
A1
VI
18/E
–
blood
a1
A
V
19/E
–
blood
a1
A
V
Fig. 1. The dendrogram illustrating the
genetic similarity of PCR MP profiles obtained
for nontoxigenic C. diphtheriae strains isolated in Poland in 2004–2008 from infections.
Information about the site of the isolation in
brackets
Ryc. 1. Dendrogram przedstawiający
podobieństwo genetyczne profili PCR MP
uzyskanych dla nietoksynotwórczych szczepów
C. diphtheriae izolowanych w Polsce w latach
2004–2008. W nawiasie podano miejsce izolacji
body. Unfortunately, the authors were not able to
verify this hypothesis because they did not have the
necessary information about the patient.
On the basis of the results, the authors cannot say what kind of mutation it is and this needs
to be studied further. The additional DNA band
detected in PCR MP profiles will be sequenced
in the near future in order to clarify the molecular mechanisms of the acquisition of additional
genes involved in the invasive properties of some
nontoxigenic C. diphtheriae strains. To the best of
authors knowledge, this is the first report of the
detection of small genotype differences between
nontoxigenic C. diphtheriae strains isolated from
invasive and non-invasive infections, although the
domination of specific clones of toxigenic C. diphtheriae, regarded as more virulent, was reported
during diphtheria outbreaks [10–12], as well as the
720
A.A. Zasada, M. Baczewska-Rej, S. Wardak
domination of a single clone of nontoxigenic C. diphtheriae isolated from infections [1, 3, 13, 14].
To summarize, the PCR MP technique differentiates C. diphtheriae isolates slightly better than
ERIC-PCR and PFGE and might be useful for the
differentiation of strains possessing invasive properties from non-invasive strains. Moreover, the presented study showed that nontoxigenic C. diphtheriae strains isolated from invasive infections could
possess additional genes or other mutations that
result in higher invasiveness and virulence regardless of toxin production. But both these findings
must be confirmed using a larger group of strains.
The results of the study also point at the necessity
of carrying out screening studies of nontoxigenic
C. diphtheriae carriage in the Polish population to
answer the questions about the clonality of strains
circulating in Poland.
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Address for correspondence:
Aleksandra A. Zasada
National Institute of Public Health
National Institute of Hygiene
Department of Bacteriology
Chocimska 24
00-791 Warszawa
Poland
Tel.: +48 22 542 12 69
E-mail: [email protected]
Conflict of interest: None declared
Received: 21.02.2011
Revised: 21.03.2011
Accepted: 5.10.2011