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Previous Article | Next Article 
Journal of Clinical Microbiology, January 2001, p. 175-182, Vol. 39, No. 1
Service de Bactériologie-Virologie,
Hôpital de Bicêtre, Assistance Publique-Hôpitaux de
Paris, Faculté de Médecine Paris-Sud, 94275 Le
Kremlin-Bicêtre, France1;
Department of Microbiology, Faculty of Medicine Siriraj
Hospital, Mahidol University, Bangkok 10700, Thailand2; and Department of
Microbiology and Immunology, Finch University of Health Sciences,
The Chicago Medical School, North Chicago, Illinois
600643
Received 20 July 2000/Returned for modification 4 September
2000/Accepted 31 October 2000
Over a 21/2-month period in 1999, 37 ceftazidime-resistant
nonrepetitive enterobacterial isolates were collected from 37 patients in a Bangkok hospital, Thailand. Eighty-one percent of these strains expressed a clavulanic acid-inhibited extended-cephalosporin resistance profile. An identical extended-spectrum Plasmid-mediated
extended-spectrum Strains expressing the ESBL VEB-1 (also named CEF-1) have rarely been
identified, i.e., one Escherichia coli and one K. pneumoniae isolate from the same Vietnamese patient and two
Pseudomonas aeruginosa isolates from two patients
hospitalized in Thailand (23, 30, 38). Genetic analysis of
blaVEB-1 revealed either its
chromosome or its plasmid location and always its integration within
class 1 integrons of variable structure. Integrons contain a
site-specific recombination system able to capture and express genes as
gene cassettes (4, 9). The essential components of class 1 integrons are the 5' conserved segment (5'-CS) that includes an
integrase gene, intI, which encodes a site-specific
recombinase, an adjacent site, attI, that is recognized by
the integrase and acts as a receptor for gene cassettes, and a common
promoter region(s), Pant (P1) and/or
P2, from which integrated gene cassettes are expressed
(5, 32). The 3' conserved segment (3'-CS), located downstream of the integrated gene cassettes, usually contains a
combination of the three genes qacE1 (antiseptic
resistance), sulI (resistance to sulfonamides), and an open
reading frame (orf5) of unknown function (27).
Each gene cassette is associated with a site-specific recombination
site designated the 59-base element (59-be) and located downstream of
the gene. Among the cassette-integrated The aim of the present study was to evaluate the prevalence of
nosocomial Enterobacteriaceae isolates that
produced VEB-1 among nonrepetitive ceftazidime-resistant
Enterobacteriaceae isolates over a
21/2-month period in 1999 from patients hospitalized in a hospital in Bangkok, Thailand. To determine whether the enterobacterial isolates were epidemiologically related, they were compared for their
Bacterial isolates.
Thirty-seven nonrepetitive
ceftazidime-resistant entero-bacterial isolates were consecutively
collected in the bacteriology laboratory in the Department for
Microbiology at Siriraj Hospital, Bangkok (the biggest hospital
facility in Thailand) from June to August 1999. The total number of
enterobacterial isolates in the studied period was as follows: E.
coli (n = 266), K. pneumoniae (n = 62), and
Enterobacter sp. (n = 46).
Isolates were identified by using the API20E system (bioMérieux
SA, Marcy-l'Etoile, France). Electrocompetent E. coli DH10B
(GIBCO BRL, Life Technologies, Cergy Pontoise, France) was used as a
recipient strain in transformation experiments. Nalidixic
acid-resistant E. coli JM109 was used as host in conjugation
experiments (30). E. coli NCTC 50192, harboring 154-, 66-, 38-, and 7-kb plasmids, was used as a
plasmid-containing reference strain (30). E. coli DH10B harboring recombinant plasmid pRLT1 that carries the
blaVEB-1 gene was used as a VEB-1-producing reference strain, as described previously (30).
Susceptibility testing and screening for ESBL-producing
strains.
The antibiotic susceptibility of
Enterobacteriaceae clinical isolates and
their E. coli transformants was first determined by the disk
diffusion method on Mueller-Hinton (MH) agar plates with
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.1.175-182.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Molecular Epidemiology of the Integron-Located VEB-1
Extended-Spectrum
-Lactamase in Nosocomial
Enterobacterial Isolates in Bangkok, Thailand
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
-lactamase (ESBL), VEB-1, was found in 16 unrelated enterobacterial isolates (Escherichia coli, n = 10; Enterobacter
cloacae, n = 2;
Enterobacter sakazakii, n = 1; and Klebsiella pneumoniae, n = 3) and
in two clonally related E. cloacae isolates. The
blaVEB-1 gene was located on mostly
self-conjugative plasmids (ca. 24 to 200 kb) that conferred additional
non--lactam antibiotic resistance patterns. Additionally, the
blaVEB-1 gene cassette was part of class 1 integrons varying in size and structure. The
blaVEB-1-containing integrons were mostly
associated with blaOXA-10-like and
arr-2-like gene cassettes, the latter conferring resistance
to rifampin. These data indicated the spread of
blaVEB-1 in Bangkok due to frequent transfer of
different plasmids and class 1 integrons and rarely to clonally related
strains. Plasmid- and integron-mediated resistance to rifampin was also
found in enterobacterial isolates.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
-lactamases (ESBLs) were first identified in
a Klebsiella pneumoniae isolate in Germany in 1983 (15). Since then, the infections caused by ESBL-producing members of the family Enterobacteriaceae
have rapidly increased (20). These enzymes confer variable
degrees of protection against expanded-spectrum cephalosporins such as
cefotaxime, ceftazidime, and the monobactam aztreonam.
Their activity is inhibited by clavulanic acid in vitro (13,
15). Most of the ESBLs that are disseminated worldwide are
derivatives of narrow-spectrum TEM- and SHV-type -lactamases,
with one or more amino acid substitutions surrounding their active
site, thus explaining the extension of their hydrolytic profile
(20). In addition to these ESBLs, non-TEM, non-SHV
derivatives with weak structural relationships have been detected with
specific geographical distributions, such as CTX-M derivatives in
Europe and South America, TOHO-1 and TOHO-2 in Japan, PER-1 in Turkey, and PER-2 in South America (2, 3, 7, 8, 11, 19, 25, 40,
41).
-lactamase genes, most of
them encode -lactamases of Ambler class D (oxacillin-hydrolyzing
-lactamases) and only rarely of class B (such as IMP-1, VIM-1, and
VIM-2 carbapenem-hydrolyzing -lactamases) or of class A
(carbenicillin-hydrolyzing -lactamases) (10, 22, 31,
32). The blaVEB-1 and
blaGES-1 gene cassettes are the only
class A ESBL gene cassettes so far known as part of class 1 integrons (29, 30).
-lactamase content, their plasmid profile, and their genotype using
arbitrary primer PCR analyses. Conjugation experiments (and/or electrotransformation) were performed to analyze the self-conjugative property of the plasmids. Finally, class 1 integrons were searched and
analyzed in the VEB-1-positive isolates. This is, to our knowledge, the
first work designed to study the distribution of an Ambler class A
ESBL in this part of the world and the first study on the molecular
epidemiology of an integron-located class A ESBL gene.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
-lactam and
non--lactam antibiotic-containing disks (Sanofi Diagnostics Pasteur,
Marnes-La-Coquette, France), according to the guidelines of the
antibiogram committee of the French Society for Microbiology
(30). Since in some cases transconjugants were obtained,
plasmid DNAs of the transconjugants were electroporated into E. coli DH10B in order to make valid comparisons of the MICs for all
transformants in an identical E. coli genetic background. The double-disk synergy test was performed with cefotaxime,
ceftazidime, aztreonam, and amoxicillin-clavulanic acid disks on MH
agar plates, and the results were interpreted as described previously
(13).
-lactams by an agar dilution
technique on MH agar with an inoculum of 104 CFU per spot,
as described previously (28). MICs of some -lactams were determined alone or in combination with a fixed concentration of
either clavulanic acid (2 µg/ml) or tazobactam (4 µg/ml). MIC results were interpreted according to the guidelines of the National Committee for Clinical Laboratory Standards (24).
Conjugation, electroporation, and plasmid DNA content analysis. Conjugation experiments were performed between clinical isolates and E. coli JM109 in solid and liquid media at 37°C as reported previously (28). Transconjugants were selected on Trypticase soy (TS) agar plates containing 100 µg of nalidixic acid per ml and 2 µg of ceftazidime per ml. Non-self-conjugative plasmid DNAs of the enterobacterial isolates and plasmid DNAs of the transconjugants were extracted as described previously (28). They were electroporated into E. coli DH10B, and recombinant strains were selected on ceftazidime-containing (2 µg/ml) TS agar plates. Plasmid DNAs of these transformants were analyzed as described previously (28).
IEF analysis. Cultures of the E. coli electroporants were grown overnight at 37°C in 10 ml of TS broth containing 2 µg of ceftazidime per ml. Then, 1 ml of each overnight culture was further grown for 3 h at 37°C in 10 ml of TS broth with ceftazidime. Analytical isoelectric focusing (IEF) was performed using an ampholine polyacrylamide gel as described previously (28).
PCR amplification for detection of ESBL genes, analysis of
class 1 integrons, and sequencing.
Under standard PCR conditions
(33), a series of primers was designed for detection of
Ambler class A -lactamase genes. Detection was performed for genes
encoding TEM (TEM-A and TEM-B [29, 35]), SHV (SHV-F and
SHV-B [21]), PER-1/PER-2 (PER-A and PER-B [25,
29]), VEB-1 (VEB-1A and VEB-1B [23]), CTX-M (CTXM-2A and CTXM2B [8]), TOHO-1/TOHO-2 (TOHO-A and
TOHO-B [11, 29]), and GES-1 (GES-1A and GES-1B
[29]) (Table 1). For each
reaction, 0.5 µg of whole-cell DNA of the ESBL-possessing Enterobacteriaceae isolates or 0.5 µg of
plasmid DNA from E. coli DH10B electroporants was used.
Primers for the detection of class 1 integrons were located in the
5'-CS and in the 3'-CS regions (primers 5'-CS and 3'-CS
[16]). A combination of 5'-CS or 3'-CS and
blaVEB-1 primers was also used for the
determination of the genetic content of the class 1 integrons (primers
5'-CS and VEB-B, or 3'-CS and VEB-F [16, 23]).
Additionally, since oxa-10 and arr-2 genes had
been found to be associated with the
blaVEB-1 gene (23, 38), their
detections by PCR amplification were performed (primers OXA-10casF and
-casB [Table 1] and ARR-2-F and ARR-2-B [37]). Their
position relative to the blaVEB-1 gene was
determined by PCR using primers for blaVEB-1
(primers VEB-1A and VEB-1B [23] and VEB-INV4F and
VEB-INFV3B [Table 1]), for blaOXA-10
(primers OXA-10promB [Table 1] and OPR-1 and OPR-2 [42]), and for arr-2 (primers ARR-2-F and
ARR-2-B).
|
RAPD fingerprinting. Random amplified polymorphism DNA (RAPD) analysis was performed and interpreted as described by Williams et al. (43) with some modifications as reported previously (28). The RAPD primers were ERIC-2 and 628 (28) and AP1 and AP4 (1).
Hybridizations. DNA-DNA hybridizations were performed as described by Sambrook et al. with a Southern transfer of an agarose gel containing plasmid DNA from E. coli DH10B electroporants as a template (33). The probe consisted of a 650-bp PCR fragment generated from recombinant plasmid pRLT-1 and internal to blaVEB-1 (30). Labeling of the probe and signal detection were carried out using a nonradioactive labeling and detection kit according to the manufacturer's instructions (Amersham Pharmacia Biotech).
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
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Epidemiology and preliminary PCR detection of -lactamase
genes.
A total of 37 nonrepetitive ceftazidime-resistant
enterobacterial isolates were collected from hospitalized patients in a 2,000-bed hospital in Bangkok from June to August 1999. Ceftazidime resistance accounted for 4.5% (12 of 266) of E. coli, 41%
(19 of 46) of Enterobacter sp., and 9.6% (6 of 62) of K. pneumoniae isolates. Preliminary antibiotic
susceptibility testing by disk diffusion showed synergy between
ceftazidime- and clavulanate-containing disks for 30 of these 37 isolates. The seven isolates that did not show synergy image were
Enterobacter cloacae. They likely had a
derepressed expression of their cephalosporinase since the diameter
around the ceftazidime disk increased when antibiograms were performed
on oxacillin-containing MH plates (200 µg/ml) (data not shown).
|
MICs of -lactam antibiotics and IEF analysis.
In all cases,
the MICs of ceftazidime and aztreonam were higher than those of
cefotaxime, which is consistent with the presence of VEB-1
-lactamase (Table 3)
(30). The addition of clavulanic acid consistently
decreased the MICs of ceftazidime, cefotaxime, and aztreonam (Table 3).
The MICs of -lactams for transformants were very similar to those
found for clinical isolates (Table 3). IEF analysis showed that the
transformants expressed a -lactamase pI value of 7.4 that
corresponded to VEB-1 in all cases (Table 4). An additional -lactamase with a pI
value of 6.3 was found in all cases except for three transformants
(E. cloacae strains 11, 12, and 14) (Table 4). In one case
(transformant E. cloacae 14) a -lactamase with a pI value
of 6.5 instead of 6.3 was found. Additionally, a -lactamase with a
pI value of 5.4 that may correspond to a TEM derivative was found
in four transformants (E. coli 1, E. cloacae
13, E. cloacae 15, and K. pneumoniae
18), all of them being blaTEM PCR positive
(Table 4).
|
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Transfer of resistance and plasmid analysis.
The
transmissibility of the ceftazidime resistance marker was tested by
conjugation. The transfer of resistance was obtained for 8 of 18 strains at a high frequency (10
3 to 104).
For the remaining 10 strains, transfer of the ceftazidime resistance marker was obtained by electroporation using plasmid extracts from the
clinical isolates. Plasmid DNAs of the transconjugants were extracted
and retransformed into E. coli DH10B in order to make a
valid comparison between transconjugants and transformants. All
transformants were checked to be blaVEB-1
positive by hybridization (data not shown). The plasmid sizes of the
transformants ranged from ca. 24 to 210 kb (Table 4). A correlation was
established between plasmids of similar size and non--lactam
antibiotic resistance markers of the transformants (Table 4). This was
the case for transformants of E. coli strains 2, 3, 4, 6, 7, and 9 on the one hand and transformants of E. coli 1 and K. pneumoniae 18 on the other hand. It is likely that,
in some cases, plasmid transfer may explain interspecies transfer of
blaVEB-1. Additionally, E. cloacae 11 and 12 were clonally related and harbored identically sized plasmid conferring identical non--lactam antibiotic resistance phenotypes in their transformants (Table 4).
Identification of blaVEB-1 and integron
structure determination.
From each of the 18 blaVEB-1-positive strains, cassette-located
external primers for the blaVEB-1 gene were
used to PCR amplify and sequence the entire
blaVEB-1 genes. An identical
blaVEB-1 gene was identified in all cases,
showing the widespread distribution of this ESBL gene and
underlining the genetic stability of this coding sequence and its
cassette (data not shown). Since the
blaVEB-1 gene cassette had been reported as
integron located, PCR amplifications were performed to identify its
precise location. Using 5'-CS, 3'-CS, and
blaVEB-1 primers, PCR fragments of various
sizes were obtained (Table 4), indicating that
blaVEB-1 was part of class 1 integrons.
Taking into account the size of the amplified fragments, it could be
deduced that the surrounding sequences upstream of blaVEB-1 varied from 0.1 to 4.2 kb. Since
the MICs of -lactams were similar for each of the transformants
(except E. cloacae 14 [Table 4]), the expression of VEB-1
was not related to the position of the
blaVEB-1 cassette relative to that of the
5'-CS where the integron promoter sequences are located.
-lactamase with a pI value of 6.3 and
blaOXA-10-like positive PCR results (Table
3). These blaOXA-10-like genes likely coded for the narrow-spectrum clavulanic acid-resistant -lactamase OXA-10
because (i) the MICs of ceftazidime, cefotaxime, and aztreonam for the
transformants were decreased by the addition of clavulanic acid (almost
at the level for wild-type E. coli DH10B) and (ii) the pI value of 6.3 corresponded to OXA-10. In the case of transformant E. cloacae 14, the -lactamase with a pI value of 6.5 did
not correspond to any of the class A ESBL genes tested nor to
oxa-1, oxa-2, and pse-1 derivatives
(data not shown).
PCR detection of the arr-2 gene that conferred resistance to
rifampin was found for all clinical isolates and transformants, thus
indicating a plasmid location for this gene. Expression of rifampin
resistance was found for all transformants except E. coli 8 and E. cloacae 11, 12 and 14. These
transformants and transformant E. cloacae 13 gave a weakened
PCR signal compared to the other transformants (data not shown).
By performing PCR experiments with primers for
blaVEB-1,
blaOXA-10, and arr-2 genes and
comparing the results with those obtained with primers for
blaVEB-1, 5'-CS, and 3'-CS, the structures
of integrons were deduced. The most common structure of the integrons was the downstream location of the arr-2 gene from
blaVEB-1 followed by a
blaOXA-10-like gene (transformants E. coli 1, 2, 3, 4, 5, 6, 7, 9, and 10, E. cloacae 15, and
K. pneumoniae 16, 17, and 18) (Table 4 and Fig.
1). In one case (transformant E. cloacae 13), a blaOXA-10-like gene was
located outside the 3'-CS sequence (Table 4). For transformants
E. coli 8 and E. cloacae 11, 12, and
13, which gave a weak PCR signal for the arr-2 gene, no
blaVEB-1-arr-2 fragment was
amplified by PCR. In these cases, an arr2-like gene may be
involved in conferring either no or a low level of resistance to
rifampin (Table 4). Although the sul1 gene is associated
with class 1 integrons, expression of resistance to sulfonamides was not found in several transformants (E. coli 10, E. cloacae 11 and 12, and E. sakazakii 15 [Table 4]). In
these cases, the sul1 gene is either lacking or not
expressed, as reported previously (4, 5, 32).
|
-lactam antibiotic-resistance markers in the
transformants and similar integrons was found for E. coli 1 and K. pneumoniae 18 on the one hand and for
E. coli 6, 7, and 9 on the other hand (Table 4).
Thus, the spread of the blaVEB-1 gene among
these nosocomial isolates may be explained rarely by the spread of
clonally related strains and often by similar plasmids and integrons.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
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Taking into account the total number of enterobacterial isolates in the studied period, the prevalence of ESBL-producing organisms was 4.5% for E. coli, 9.6% for K. pneumoniae, and 26% for Enterobacter sp. These values ranged within those reported for isolates of North American and European hospitals (12, 18, 20, 36, 46). In a multicenter study performed in Thailand, the prevalence of ceftazidime-resistant E. coli strains ranged from 0 to 62.5% (37). In this same study, the prevalence of ceftazidime-resistant K. pneumoniae was 45% on average, and that is higher than the value reported here. In Korea, the prevalence of ESBL-producing E. coli was 4.8% with mostly TEM- and SHV-type ESBLs and was 22% in K. pneumoniae isolates (14). In Taiwan, the prevalence of ESBL-producing K. pneumoniae was quite high (30%), mostly involving TEM-type ESBLs (17, 26, 45). On the contrary, in Japan ESBL-producing organisms are rarely encountered (<0.001%) and the ESBLs are mostly of the TOHO-2 type and rarely of the TEM and SHV type (44). In China, ESBLs have been reported but their prevalence is unknown (34).
In Bangkok, as reported previously in other countries (39), Enterobacter sp. but not K. pneumoniae may represent the main reservoir of ESBL-producing enteric isolates. This may result from either an outbreak or a situation in which such organisms are endemic. The blaVEB-1 gene seemed to be highly prevalent in these ceftazidime-resistant Thai isolates since it accounted for 60% of the ESBL-possessing isolates. However, frequent identifications of blaTEM and blaSHV derivatives may correspond also to additional ESBL genes in the same clinical isolates. The epidemiological analysis at the strain and plasmid levels indicated that blaVEB-1 had spread among various enterobacterial species. Its dissemination was not due to a single strain or a single plasmid type. Most of the blaVEB-1-positive isolates harbored a self-conjugative plasmid of large size, as found for E. coli MG-1 from Vietnam (30).
The present work established the dissemination of an Ambler class A
ESBL gene via various structures of class 1 integrons. A
dissemination of an unusual Ambler class A ESBL gene has been reported for blaPER-1 in Turkey (40,
41). However, in this case the plasmid location but not the
integron location of blaPER-1 is
known. In the case of the Ambler class B IMP-1 -lactamase, its
integron-located gene has been reported extensively in Japan (10). However, as opposed to
blaIMP-1 (10), the
blaVEB-1 location on a class 1 integron is
always followed by its expression.
Structure analysis of the
blaVEB-1-containing integrons showed their
variability compared to the three other known
blaVEB-1-containing integrons, which are
from E. coli MG-1 (from Vietnam) and P. aeruginosa JES-1 and Thl-1 (from Thailand) (23, 30,
38). Various structures of class 1 integrons have been detected
in Vibrio cholerae isolates from Thailand carrying a
carbenicillinase gene not related to blaVEB-1 (6). Spread of
another class A -lactamase (blaPSE-1) has
also been reported to be related to integron location in
Salmonella enterica serotype Typhimurium DT104
(28).
In the present study, most of the integrons containing the blaVEB-1 gene cassette possessed arr-2 and blaOXA-10 gene cassettes. This result raises the question of how these genes have evolved and if they have been transferred among soil organisms such as mycobacteria, Enterobacteriaceae, and Pseudomonas species. Indeed, arr-2 shares a structural relationship with arr-1 from Mycobacterium smegmatis (38) and blaOXA-10, and their extended-spectrum derivative genes are mostly found in P. aeruginosa isolates (22). Once located on integrons, these resistance genes may have been transferred in block through a transposition-related mechanism. It would be interesting to test if blaVEB-1-carrying integrons are located on transposons such as Tn21 derivatives, as described for some class 1 integrons (4).
Our results also raise the question of whether antibiotic selective
pressure in hospitals in Thailand may have led to dissemination of this
integron-located gene. -Lactam as well as aminoglycoside, sulfonamide, and disinfectant resistance genes are associated with
class 1 integrons that may enhance the in vivo dissemination of these
integrons. Restricted clinical use of broad-spectrum antibiotics and
rigorous hygiene measures are the most important means to prevent the
spread of this ESBL gene as much as possible. Since
blaVEB-1 has been detected also in two
P. aeruginosa isolates from Thailand, a further study would
evaluate its spread among other gram-negative rods.
The important prevalence of blaVEB-1 among enterobacterial isolates may lead to heavy use of carbapenems in this Bangkok hospital, which in turn may favor the selection of, at least, carbapenem-resistant P. aeruginosa isolates.
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ACKNOWLEDGMENTS |
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We thank Venus Tolun for help in preliminary experiments.
This work was financed by a grant from the Ministères de l'Education Nationale et de la Recherche (grant UPRES, JE-2227), Université Paris XI, France.
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FOOTNOTES |
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* Corresponding author. Mailing address: Service de Bactériologie-Virologie, Hôpital de Bicêtre, 78 rue du Général Leclerc, 94275 Le Kremlin-Bicêtre Cedex, France. Phone: 33-1-45-21-36-32. Fax: 33-1-45-21-63-40. E-mail: nordmann.patrice{at}bct.ap-hop-paris.fr.
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Abstract
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Discussion
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