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Journal of Clinical Microbiology, November 1999, p. 3774-3777, Vol. 37, No. 11
Department of Microbiology,
Received 7 June 1999/Returned for modification 23 July
1999/Accepted 6 August 1999
Twelve Salmonella typhimurium strains resistant to
broad-spectrum cephalosporins were isolated from cases of
gastroenteritis during 1996 to 1998 in Russia, Hungary, and Greece.
Resistance was due to the production of CTX-M-type extended-spectrum
Over the years, an increasing
proportion of Salmonella isolates have been acquiring
resistance to various "older" antimicrobial drugs (4, 10,
19). Lately, sporadic appearance of nontyphoid salmonella
isolates that are resistant to broad-spectrum cephalosporins due to
production of various plasmid-mediated Twelve cefotaxime-resistant S. typhimurium strains were
included in the study. They were identified by the API 20E system and
were serotyped with respect to cell wall (O) and flagellar (H) antigens
(17). Six strains were isolated from an equal number of
patients during an outbreak of gastroenteritis in a psychiatric institution in St. Petersburg, Russia, in 1997 (R-strains). Strain S-661 was representative of an outbreak of gastroenteritis that had
occurred in St. Petersburg in 1996 (7). Three strains were isolated from sporadic cases of gastroenteritis in Budapest, Hungary, during 1998 (H-strains). The two Greek strains, AS30 and AS31, were
also epidemiologically unrelated (20).
Genomic DNA was extracted and pulsed-field gel electrophoresis (PFGE)
was performed as described previously (17). Cell lysis with
lysozyme was followed by a proteinase K treatment and DNA digestion
with XbaI. Electrophoresis through 1% agarose-0.5×
(wt/vol) Tris-borate-EDTA was performed by using a CHEF DRIII apparatus (Bio-Rad). Isolates with electrophoretic patterns differing by four or
more DNA fragments were assigned to distinct types (16, 18).
Following visual inspection, PFGE banding patterns were also analyzed
with GelCompar software (Applied Maths).
Conjugal transfer was carried out in broth with E. coli
26R793 (Rifr Lac Susceptibility to Isolation of plasmids was performed by an alkaline lysis procedure
(11). Agarose-purified plasmid DNA was digested with SacII or HaeIII restriction endonucleases and
subjected to agarose gel electrophoresis.
The DNA sequences of blaCTX-M genes were
determined directly from the wild-type plasmids by the dideoxy chain
termination method with the Sequenase 2.0 kit (United States
Biochemicals) and a set of blaCTX-M-specific
oligonucleotide primers based on the blaCTX-M-4
sequence (8).
All cefotaxime-resistant S. typhimurium isolates exhibited
similar resistance phenotypes to Conjugal transfer of cefotaxime resistance was attempted by using R-893
and H-140 as representative donors. Cefotaxime-resistant E. coli transconjugants were readily obtained from both isolates at a
frequency of 10 Nucleotide sequencing with purified plasmid preparations from R-893 and
H-140 confirmed the presence of blaCTX-M genes.
The coding and promoter regions of the blaCTX-M
gene from R-893 were identical to those of the previously described
blaCTX-M-4 gene found in isolate S-661
(8). Sequencing of the coding region of the
blaCTX-M gene from H-140 showed that its deduced
amino acid sequence differed from that of CTX-M-4 only at position 211, where a leucine had been replaced by an isoleucine.
PFGE can successfully identify epidemiological and clonal relationships
among S. typhimurium isolates, either in concordance with or
with higher discrimination than phage typing (1, 5, 14).
This method showed that all cefotaxime-resistant S. typhimurium isolates were also highly related at the chromosomal
level. Their PFGE patterns differed by three bands at most, thus
classifying them in the same type, D, clearly distinguishable from PFGE
types A, B and C, obtained with cefotaxime-susceptible control isolates (Fig. 2). Types A and B are the dominant
types in Greek S. typhimurium (unpublished data). Type D, on
the other hand, was observed for the first time with AS-30 and AS-31.
The macrorestriction pattern of the Greek strain AS-30 was 94%
identical to those of the Russian or Hungarian cefotaxime-resistant
isolates, while that of AS-31 was 84% identical to the rest of the
cluster.
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Spread of a Salmonella typhimurium Clone
Resistant to Expanded-Spectrum Cephalosporins in Three European
Countries
ABSTRACT
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Abstract
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References
-lactamases encoded by similar 12-kb plasmids. By pulsed-field gel
electrophoresis, all strains shared the same chromosomal type. These
data suggest that an S. typhimurium clone resistant to
broad-spectrum cephalosporins is present in at least three European countries.
TEXT
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Abstract
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References
-lactamases, including the
CTX-M-type extended-spectrum (ES) enzymes, has been noted (summarized
in reference 15). We have reported previously the emergence of resistance to cephalosporins in Salmonella
typhimurium. Strains from Russia and Greece displayed an unusual
phenotype, being resistant to cefotaxime, ceftriaxone, and aztreonam,
but susceptible to ceftazidime (7, 20). These strains
possessed plasmids encoding CTX-M-type -lactamases (8,
9). In the meantime, additional S. typhimurium strains
with similar resistance phenotypes were isolated in Russia and Hungary.
In the present study, we examined the possibility of the older and
newer strains being clonally related and analyzed the resistance
mechanisms to -lactams of the recent isolates.
) as the recipient
(21). Transconjugant clones were selected on nutrient agar
containing cefotaxime (10 µg/ml) and rifampin (200 µg/ml).
-lactam antibiotics was assessed by an agar
dilution method (12). Susceptibility to other antibiotics was assessed by a disk diffusion assay (13).
-Lactamase extracts were obtained after ultrasonic treatment of
mid-log-phase cultures in tryptone-soy broth. The lysates were
clarified by ultracentrifugation and dialyzed against phosphate buffer
(50 mM, pH 7.0). Isoelectric focusing (IEF) of -lactamases was
performed in polyacrylamide gels containing ampholytes (pH range 3.5 to
9.5) (Pharmacia-LKB). -Lactamase bands were visualized with
nitrocefin (Oxoid).
-lactams, displaying at most 1-dilution differences in MIC; hence, results for only two
representative strains are shown in Table
1. They were resistant to penicillins, cefotaxime, ceftriaxone, and aztreonam, but susceptible to ceftazidime. Piperacillin-tazobactam was highly active, while the combinations of
clavulanate with amoxicillin or ticarcillin were less active. In IEF
experiments, the isolates produced a single -lactamase species that
focused at 8.4 (Fig. 1). The resistance
phenotype and pI of the enzymes were indicative of a CTX-M-type
-lactamase. Resistance phenotypes to various non--lactam
antibiotics were also similar (Table 1).
TABLE 1.
Antibiotic susceptibilities of representative S. typhimurium isolates and E. coli transconjugant clones
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FIG. 1.
IEF of -lactamase preparations from the indicated
cefotaxime-resistant isolates (Isol.). -Lactamases of the indicated
pIs are in lane 1.
4. Based on their resistance phenotypes,
they were divided into two types. In type 1, which included the
majority of transconjugants, all resistance characters of the donor
strain had been transferred. The remaining transconjugants (type 2)
were resistant only to -lactams (Table 1). The latter clones had
acquired relatively small plasmids (12 kb) and produced a -lactamase
of pI 8.4 (data not shown). In type 1 clones, the other resistance
determinants were most likely transferred by larger plasmids (60 to 80 kb) which could be detected along with the small CTX-M-encoding
plasmids. Therefore, the genes encoding the cefotaxime-hydrolyzing
-lactamase resided in the small plasmids observed in both
transconjugant types. In subsequent conjugation experiments, however,
type 2 clones were unable to transfer resistance to -lactams to
another E. coli recipient strain. When the 12-kb plasmid DNA
from different isolates was digested with restriction endonuclease
HaeIII or SacII, the patterns obtained were
indistinguishable, with the exception of plasmids derived from the
Hungarian isolates, which differed in one band of approximately 0.6 kb
(data not shown).
View larger version (46K):
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FIG. 2.
(A) PFGE of cefotaxime-resistant and -susceptible
S. typhimurium isolates. The sizes (in kilobase pairs) of
bacteriophage 
concatamers are indicated on the right. All lanes are
from the same gel. (B) Dendrogram based on the similarity of the PFGE
patterns shown in panel A. Isolates are indicated on the right, and a
percentage similarity scale is shown at the top.
S. typhimurium strains producing CTX-M-type -lactamases
have also been isolated in Argentina (2) and Latvia
(3). These plasmid-mediated class A enzymes are related to
the species-specific -lactamases of Klebsiella oxytoca
(6) and constitute a small but rapidly expanding group of ES
-lactamases. CTX-M -lactamases preferentially hydrolyze
cefotaxime and ceftriaxone, but, unlike most ES TEM and SHV enzymes,
they spare ceftazidime. They are inhibited by low concentrations of
tazobactam, while clavulanic acid exerts a less potent inhibitory
activity (3, 8).
Based on all assays performed, the cefotaxime-resistant isolates should
be considered as clonally related. However, it was not possible to
demonstrate a clear epidemiological relationship among them. The
R-strain isolates might be connected with the outbreak isolates
represented by S-661 (7), both outbreaks having occurred in
the region of St. Petersburg within 1 year. Interestingly, an ongoing
epidemic of cefotaxime-resistant S. typhimurium producing
plasmid-mediated CTX-M-type -lactamases that may be related to those
described here, has been reported in nearby Latvia (3). As
reported previously, strains AS-30 and AS-31 may have been acquired in
Eastern Europe (20). The available patients' data did not
reveal any epidemiological association of the Hungarian isolates with
the rest of the cluster.
In addition to the nearly identical PGFE patterns, the similarity of
plasmids encoding the CTX-M--lactamase variants further supported
the clonal origin of the cefotaxime-resistant isolates. These plasmids
were probably not self-transmissible but were mobilized by coexisting
conjugative plasmids. Under such circumstances, further spread of the
blaCTX-M genes is likely to occur. The
CTX-M-5-encoding plasmids found in S. typhimurium isolates
from Latvia were also small (10 kb) and non-self-transferable
(3).
In this study, we showed that an oximino-cephalosporin-resistant clone
of S. typhimurium is present in geographically distinct areas across Eastern and Southeastern Europe. By its resistance phenotype, its mechanism of resistance to -lactams, and its phage type, DT193 (22), this clone is not related to the
widespread multidrug-resistant clone of S. typhimurium DT104.
To date, it has not been possible to obtain information as to the
presence of S. typhimurium isolates with similar phenotypes of resistance from other parts of Eastern Europe. The scarcity of
relevant reports may indicate that such strains have not spread widely
yet. The unusual resistance to -lactams of this CTX-M-producing clone, together with its multidrug resistance, are traits that can
hardly pass unnoticed during susceptibility testing. Nevertheless, the
establishment and spread of an S. typhimurium clone that is resistant to therapeutically important broad-spectrum -lactams are
causes for concern.
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ACKNOWLEDGMENTS |
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We thank Linda R. Ward (Laboratory of Enteric Pathogens, Central Public Health Laboratory, London, United Kingdom) for kindly phage typing isolate AS-31 and Zannina Sarandopoulou for excellent technical assistance with PFGE. We are grateful to Jenny Kourea-Kremastinou and Maria Lambiri (National Reference Centre for Salmonella and Shigella, National School of Public Health, Athens, Greece) for providing the Greek control strains and Antonios Markogiannakis for their initial characterization.
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ADDENDUM IN PROOF |
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Phage typing performed in the laboratory of W. Rabsch (National Reference Center for Salmonella and other enterobacteria, Robert Koch-Institute, Wernigerode, Germany) revealed that all cefotaxime-resistant isolates from the three countries belonged to DT193 (14a).
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Microbiology, Medical School, University of Athens, M. Asias 75, 115 27 Athens, Greece. Phone: 301 7785 638 or 301 7714 432. Fax: 301 7709 180. E-mail: Ltzouvel{at}cc.uoa.gr.
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