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Journal of Clinical Microbiology, March 2000, p. 1269-1271, Vol. 38, No. 3
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Multiple Clones within Multidrug-Resistant
Salmonella enterica Serotype Typhimurium Phage Type
DT104
Antonis
Markogiannakis,1
Panayotis T.
Tassios,1
Maria
Lambiri,2
Linda R.
Ward,3
Jenny
Kourea-Kremastinou,2
Nicholas J.
Legakis,1
The Greek Nontyphoidal
Salmonella Study Group,
and
Alkiviadis C.
Vatopoulos4,*
Department of
Microbiology1 and Department of Hygiene
and Epidemiology,4 Medical School, University of
Athens, and National Reference Centre for Salmonella and
Shigella, National School of Public Health,2
Athens, Greece, and Laboratory of Enteric Pathogens, CPHL,
PHLS, Colindale, London, United Kingdom3
Received 2 July 1999/Returned for modification 24 August
1999/Accepted 27 November 1999
 |
ABSTRACT |
Six distinct clones were present among Greek multidrug-resistant
Salmonella enterica serotype Typhimurium phage type DT104, since isolates belonging to resistance phenotypes including the ACSSuT
(ampicillin, chloramphenicol, streptomycin, sulfonamides, and
tetracycline) core could be distinguished with respect to their
pulsed-field gel electrophoresis patterns, int1 integron structures, and presence or absence of antibiotic resistance genes ant(3")-Ia, pse-1, and tem-1.
| |
TEXT |
In recent years, a marked increase
in the number of multidrug-resistant Salmonella enterica
serotype Typhimurium isolates belonging to definitive phage type 104 (DT104) and having a core pattern of resistance to ampicillin,
chloramphenicol, streptomycin, sulfonamides, and tetracycline (ACSSuT)
has been reported (3, 4, 13, 14). Molecular studies have
demonstrated that in DT104 some of the resistance genes responsible for
the ACSSuT phenotype are integron associated (8, 12).
To date, all reports have suggested that multidrug-resistant DT104
isolates are clonally related (R. Prager, A. Liesegang, W. Streckel, B. Gerike, G. Seltmann, R. Helmuth, W. Rabsch, and H. Tschape, Fourth Int.
Meet. Bacterial Epidemiol. Markers, abstr. P40, 1997; 7, 8). In this
study, we aimed to analyze the genetic relationships of
multidrug-resistant serotype Typhimurium isolates from Greece, with
respect to their chromosomal fingerprints and mechanisms of resistance.
During 1989 to 1997, a total of 1,005 Salmonella isolates of
human, animal feed, animal, and food origins from various parts of
Greece were referred to the National Reference Center for
Salmonella and Shigella. They were identified as
serotype Typhimurium by the API 20E system (BioMerieux S.A., Marcy
l'Etoile, France) and the Kauffman serotyping scheme (5),
using commercially obtained antisera (BioMerieux). Serotype Typhimurium
represented the second most frequent serotype isolated from humans, as
for 1987 to 1993 (10), standing at 17% in 1997. The
organism was also an important serotype in isolates of animal and food sources.
Of the 1,005 serotype Typhimurium isolates, 328 (33%) were randomly
selected for antimicrobial susceptibility testing by a disk diffusion
method on Mueller-Hinton agar (Oxoid Ltd., Basingstoke, United
Kingdom), evaluated according to the standards of the National Committee for Clinical Laboratory Standards (6). Disks
containing ampicillin, amoxicillin and clavulanic acid, cefalothin,
cefamandole, ceftriaxone, ceftazidime, cefotaxime, streptomycin,
kanamycin, gentamicin, chloramphenicol, doxycycline, nalidixic acid,
ciprofloxacin, sulfonamides, sulfomethoxazole and trimethoprim, and
nitrofurantoin were purchased from Oxoid. The isolates were grouped
into 26 resistance phenotypes, the major ones being shown in Table
1. Eighty-four (26%) isolates were
resistant to the ACSSuT core alone or to additional drugs as well.
Thus, 11 distinct resistance phenotypes were compatible with the ACSSuT
core resistance phenotype of multidrug-resistant serotype Typhimurium
DT104. Fourteen isolates (17%) representing seven of these resistance
phenotypes were therefore randomly selected for further analysis.
Phage typing was performed by the methods described by Callow
(2). The scheme extended by Anderson et al. (1)
uses 34 typing phages and differentiates in excess of 250 types. Of the 14 isolates, 11 belonged to phage type DT104 and 2 belonged to the
related type DT104b, differing by a single phage reaction from DT104,
while the remaining isolate belonged to the unrelated phage type DT193.
In conjugation experiments for the transfer of antibiotic resistance,
carried out as previously described (16), only two DT104
isolates (195 and 1041) were able to transfer resistance to ampicillin,
tetracycline, chloramphenicol, streptomycin, and cotrimoxazole.
Typing by pulsed-field gel electrophoresis (PFGE) of genomic DNA
digested with XbaI (New England Biolabs, Beverly, Mass.) was
performed as previously described (10). DNA fragment
patterns were assessed visually and compared by the GelCompar software package (Applied Maths, Kortrijk, Belgium), using the Dice coefficient, UPGMA (unweighted pair group method using arithmetic averages) clustering, and 1% tolerance in band position differences. Isolates were considered as belonging to different types if differing by four or
more DNA fragments (11). Given the genetic homogeneity of
Salmonella populations (10), this criterion can
be considered stringent. Five distinct types, A to D and F, were
observed among DT104 isolates (Fig. 1).
Four isolates belonged to type A, with the remaining seven distributed
among types B (two isolates), C (two), D (two), and F (one).
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FIG. 1.
(a) PFGE patterns of 14 multidrug-resistant serotype
Typhimurium isolates. Isol. no., isolate number. The sizes, in
kilobases, of lambda phage DNA concatemers ( ) are shown to the left
of the gel. All lanes are from the same gel. (b) Dendrogram of isolate
similarities based on the chromosomal fingerprints shown in panel a.
Isolate number, phage type, resistance phenotype code, and PFGE type
are shown to the right of the dendrogram. A percent scale of similarity
is shown above the dendrogram.
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|
For PCR amplification of integron-related and resistance gene
sequences, DNA was prepared as previously described (17). PCR was carried out in 50-µl-total-volume reactions containing 1.5 mM
MgCl2, 100 µM (each) deoxynucleoside triphosphate, 0.1 U
of Taq polymerase (Promega, Madison, Wis.), 1 µl of
bacterial lysate, and 0.4 µM primers for int1 integron
(8), ant(3")-Ia (17), pse-1
(8), and tem-1 sequences (9). Under
conditions used, three distinct amplicons were obtained from the DT104
isolates with the int1 primers (Fig.
2). Two PCR products of approximately 0.9 and 1.1 kb were generated from five isolates belonging to PFGE types A
and D, while a product of approximately 1.4 kb was generated from the
two DT104 isolates belonging to PFGE type C. No int1 PCR
product could be obtained from the remaining four DT104 isolates
belonging to PFGE types B, D, and F. All DT104 isolates that were
positive for int1-type integron sequences also yielded an
ant(3")-I product of the expected size, 0.5 kb (Table 2). Of the four isolates (1308, 461, 385, and 1511) from which no int1 or ant(3")-I
products could be generated, all were intermediately resistant to
streptomycin. Amplicons of the expected size, 0.3 kb, were obtained
with the pse-1 primers from five DT104 isolates, while
amplicons of the expected size for tem-1, 0.7 kb, were
detected in four DT104 isolates (Table 2). In contrast to
ant(3")-Ia and pse-1 sequences, which were shown
by PCR to be internal to int1 amplicons, the
tem-1 gene was not contained in an integron gene cassette
(data not shown). All strains positive for int1 sequences also contained qacE-1 and sul1 sequences (not
shown).
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FIG. 2.
PCR amplification products of class 1 integrons from
representative multidrug-resistant serotype Typhimurium isolates. Isol.
no., isolate number. The sizes, in kilobases, of the indicated
amplification products are shown to the left of the gel. All lanes are
from the same gel.
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|
Therefore, of the five PFGE types within phage type DT104, type A was
prevalent. Type A isolates contained two distinct int1 integrons, detected by PCR products of 0.9 and 1.1 kb, as well as
ant(3")-Ia and pse-1 sequences. Similar studies
have found that the small integron harbors the aminoglycoside-resistant
gene cassette ant(3")-Ia, conferring resistance to
streptomycin and spectinomycin, while the large amplicon harbors the
pse-1 
-lactamase gene (7, 8). The variety in
integron size and presence or absence of aminoglycoside-modifying
enzymes and -lactamases, set against a background of different
chromosomal types, resulting in six distinct types among Greek
multidrug resistant DT104 isolates, is reminiscent of other published
data on multidrug resistant serotype Typhimurium (15). In
their study, Tosini et al. (15) found three distinct class 1 integrons located on two conjugative plasmids. In our study, transfer
of resistance by conjugation was possible only with isolates in which
the tem-1 gene was not inserted in a 1.4-kb integron. In
contrast, resistance of isolates harboring the ant(3")-Ia
and pse-1 gene cassettes inserted in two integrons was not
transferable by conjugation, presumably due to the chromosomal location
of these integrons (12).
It therefore appears that common phenotypic characteristics of
antibiotic resistance and phage type may have been acquired by
genotypically distinct multidrug-resistant serotype Typhimurium DT104
strains in Greece. This differentiates them from those isolated in
other countries (Prager et al., Fourth Int. Meet. Bacterial Epidemiol.
Markers; 7, 8), where clonality of multidrug-resistant serotype
Typhimurium DT104 has been suggested. While we cannot rule out that the
presence of various clones within multidrug-resistant DT104 isolates
may be characteristic of the Greek situation, additional molecular
studies from regions with a high rate of multidrug resistance in
serotype Typhimurium, among both human and nonhuman isolates, would
help to elucidate the origin and extent of this problem.
| |
ACKNOWLEDGMENTS |
We gratefully acknowledge the expert technical assistance of
Vasiliki Kontogianni, Zannina Sarandopoulou, and Yanna Pournou. We also
thank Leonidas S. Tzouvelekis for critical reading of the manuscript.
This work was funded in part by the Ministry of Health.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Hygiene and Epidemiology, Medical School, University of Athens, M. Asias 75, 115 27 Athens, Greece. Phone: 301 771 165. Fax: 301 770 4225. E-mail: avatopou{at}cc.uoa.gr.
Members are as follows: V. Danielides and A. Kansouzidou
(Salmonella Reference Centre for Macedonia and Thrace, Hospital of Communicable Diseases, Thessaloniki), I. Tselentis (Laboratory of
Clinical Bacteriology, Parasitology, Zoonoses and Geographical Medicine, University of Crete, Herakleion), A. Pangali and E. Chryssaki
(Microbiological Laboratory, Ag. Sophia Children's Hospital, Athens),
J. Leonardopoulos (Department of Microbiology, University of Athens),
and E. Kouskouni (Microbiological Laboratory, Aretaieion Hospital, Athens).
| |
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Journal of Clinical Microbiology, March 2000, p. 1269-1271, Vol. 38, No. 3
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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