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Previous Article | Next Article 
Antimicrobial Agents and Chemotherapy, July 2000, p. 1860-1864, Vol. 44, No. 7
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Rapid Discriminatory Detection of Genes Coding for
SHV 
-Lactamases by Ligase Chain Reaction
Jungmin
Kim1,* and
Hoan-Jong
Lee2
Department of Microbiology, College of
Medicine, Dankook University, Cheonan,1 and
Department of Pediatrics, Seoul National University College
of Medicine, Seoul,2 Korea
Received 11 October 1999/Returned for modification 15 February
2000/Accepted 21 April 2000
 |
ABSTRACT |
Ligase chain reaction (LCR) is a recently developed technique that
employs a thermostable ligase and allows for the discrimination of DNA
sequences differing in only a single base pair. The method has been
adapted and applied to differentiation of
blaSHV genes. We have developed an LCR typing
method to characterize point mutations in genes for SHV-derived
extended-spectrum 
-lactamases with four different sets of
biotinylated LCR primers. To evaluate the applicability of the current
technique, we tested seven Escherichia coli strains producing SHV-1, SHV-2, SHV-2a, SHV-3, SHV-4, SHV-5, and SHV-12. With
the LCR typing, seven SHV genes can be distinguished according to their
incorporating point mutations. In an attempt to characterize SHV
-lactamases by LCR typing in clinical isolates, 46 strains carrying
blaSHV genes (32 Klebsiella
pneumoniae, 10 Enterobacter cloacae, and 4 E. coli) were subjected to antibiotic susceptibility testing,
isoelectric focusing, and LCR typing. LCR typing allowed the
characterization of -lactamases, and genotypes obtained by LCR
typing were in accordance with phenotypes such as antibiotic resistance
profile and pI value of -lactamase. Therefore, we concluded that LCR
typing may permit defining the SHV families with simplicity and
reliability and can be applied to the detailed characterization and
molecular epidemiology of SHV-type -lactamases.
| |
INTRODUCTION |
The SHV-type -lactamases
represent one of the most clinically significant families of
plasmid-encoded -lactamases. Point mutations in the nucleotide
sequences of the structural genes for the SHV-type -lactamases can
broaden their substrate spectrum towards all -lactams except
cephamycins and carbapenems (12, 28). Detection of such
mutations usually requires sequencing of the genes, which is
time-consuming and technically demanding. Other approaches used to
study the -lactamases of SHV-group have limitations: isoelectric
focusing (IEF) is inadequate since the same pI can correspond to
different -lactamases and characterization of enzymatic substrate
profiles does not allow one to differentiate between closely related enzymes.
Here, we report a new strategy for differentiation of
blaSHV genes based on a nonradioactive ligase
chain reaction (LCR) method. LCR employs a thermostable ligase and
allows for the discrimination of DNA sequences differing in only a
single base pair (4). In the LCR, a target DNA sequence is
denatured at 94°C and the four primers anneal to their complementary
strands at 60°C. Then, thermostable ligase only ligates primers that
are perfectly complementary to their target sequence and hybridize
directly adjacent to each other. Because the oligonucleotide products
from one round may serve as substrates during the next round, the
signal is amplified exponentially, analogous to PCR amplification. A
single-base mismatch at the oligonucleotide junction will not be
amplified and is, therefore, distinguished. Thus, LCR allows for the
detection and discrimination of parental and mutated nucleotide
sequences of SHV enzymes. We have developed an LCR typing method by
using four different sets of biotinylated LCR primers to characterize
point mutations in genes for SHV-derived extended-spectrum
-lactamases (ESBLs). With LCR typing, we distinguished seven SHV
genes encoding SHV-1, SHV-2, SHV-2a, SHV-3, SHV-4, SHV-5, or SHV-12
-lactamase. (This work was presented at the 39th Interscience
Conference on Antimicrobial Agents and Chemotherapy, September 1999.)
| |
MATERIALS AND METHODS |
Bacterial strains.
Seven strains, each producing one of
seven recognized SHV -lactamases, were used: Escherichia
coli C600(R1010), encoding SHV-1; E. coli C600(pMG229),
encoding SHV-2; E. coli J53-2(pUD18), encoding SHV-3;
E. coli J53-2(pUD21), encoding SHV-4; E. coli HB101(pAFF2), encoding SHV-5; E. coli J53(pKS39), encoding
SHV-2a; and E. coli J53(pKS12), encoding SHV-12 (13,
14). Forty-six clinical isolates harboring
blaSHV genes were also included in this study: 4 strains of E. coli, 32 strains of Klebsiella
pneumoniae, and 10 strains of Enterobacter cloacae.
These isolates were selected by SHV-specific PCR from 82 clinical
isolates with reduced susceptibility or resistance to
oxyiminocephalosporins that were obtained from blood specimens of
pediatric patients at Seoul National University Hospital during 1995 and 1999.
SHV-specific PCR.
An 870-bp fragment of the SHV gene was
amplified with the primers S1 (5'-TGGTTATGCGTTATATTCGCC-3')
and S2 (5'-GGTTAGCGTTGCCAGTGCT-3'), corresponding to
nucleotides 120 to 140 and 990 to 972, respectively, of the SHV-1
bla gene (18). PCR amplification was performed in
100-µl reaction mixtures containing 1 µl of crude cellular lysate,
50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.0 mM MgCl2, 0.1 µM
oligonucleotide primers, 200 µM deoxynucleoside triphosphate mix, and
2.5 U of Taq DNA polymerase (Promega). PCR assay was performed in a Gene Cycler thermal cycler (Bio-Rad, Hercules, Calif.)
with the following cycling parameters: denaturation at 94°C for 5 min; 35 cycles of 94°C for 30 s, 58°C for 1 min, and 72°C
for 1 min; and a final extension period of 72°C for 10 min. Two
microliters of each PCR product was diluted with 38 µl of distilled
water, and then 2 µl of diluted PCR product was used as a DNA
template for LCR. Before addition to the LCR mixture, the DNA template
was boiled for 8 min.
Oligonucleotide primers.
For discriminatory detection of
genes coding for SHV variants, we synthesized four different
biotinylated primer sets that were designed to detect the following
amino acid substitutions: Gln for Leu at position 35, Leu for Arg at
205, Ser for Gly at 238, and Lys for Glu at 240 (Table
1). Each primer set included four
oligonucleotides (i.e., two pairs of oligonucleotides) to amplify the
target sequence. One pair of oligonucleotides was complementary to one
strand of the target DNA sequence, and the second pair was
complementary to the first pair. Two oligonucleotides of each pair were
hybridized to denatured target DNA so that the 3' end of one primer is
next to the 5' end of the other primer. Consensus primers contained a
phosphate at the 5' end that is required for the ligation reaction.
Mutant-specific primers of each oligonucleotide set allowed
discrimination of parental and mutated nucleotide sequences of SHV
enzymes. For colorimetric detection, one of the mutant-specific primers
contained a biotin at the 5' end for capture on a streptavidin-coated
microwell, and the other mutant-specific primer contained an additional
21-base sequence (5'-TGGCACTGGCCGTCGTTTTAC-3') at its 5'
end, complementary to the universal primer sequence. This sequence
hybridizes to the detection oligonucleotide provided by the AmpLiTek
LCR detection kit (Bio-Rad).
LCR and colorimetric detection.
LCRs were performed with an
AmpLiTek LCR kit (Bio-Rad) according to directions provided by the
manufacturer. The reactions took place in 25-µl reaction mixtures
with 2 µl of target DNA containing 16 fmol of oligonucleotide mix per
µl, 50 ng of salmon sperm DNA per µl, 1 U of Taq ligase,
and 10× ligase buffer. After samples were covered with 30 µl of
mineral oil, they were run in a Gene Cycler thermal cycler (Bio-Rad)
with the following cycling parameters: 1 cycle of 94°C for 4 min and
60°C for 4 min and 10 cycles of 91°C for 30 s and 60°C for 5 min. Amplified products from the reaction were identified by
colorimetric detection in a microtiter plate with the AmpLiTek LCR
detection kit (Bio-Rad) according to the directions provided by the
manufacturer. After thermal cycling, 5 µl of each amplified product
was diluted with 45 µl of 1× SSC (0.15 M NaCl plus 0.015 M sodium
citrate) and was placed in the streptavidin-coated wells. After
incubation for 1 h at 37°C, each well was washed five times with
300 µl of 1× well wash solution. After washing, the detection
oligonucleotide supplied with the kit was added to each well. This
alkaline-phosphatase-conjugated oligonucleotide contained the universal
primer sequence that would hybridize to its complementary sequence on
the LCR-amplified products bound in the well. After incubation for
1 h at 37°C, each well was washed again five times with 300 µl
of 1× well wash solution. The substrate (NADPH) for alkaline
phosphatase was added to all wells, and a second reagent, which uses
the product of the NADPH-alkaline-phosphatase reaction to generate a
redox cycle that produces a red formazan dye, was added. The color
change reaction was assayed through endpoint determination at 490 nm in
a precision microplate reader (Molecular Device). If the optical
density value of the color change was above 0.1 at 10 min after
addition of amplifier, we interpreted it as a positive reaction.
Antibiotics and susceptibility testing.
Antimicrobial agents
tested were cefotaxime, ceftazidime, aztreonam, cefotetan,
piperacillin, and piperacillin-tazobactam. MICs were determined by the
agar dilution method according to the guidelines of the NCCLS
(20). In the piperacillin-tazobactam combination, the
concentration of tazobactam was 4 mg/liter.
Analytical IEF.
Crude preparations of -lactamases from
clinical isolates were obtained by two sonications for 30 s each
time in 0.1 M phosphate buffer (pH 7.0). IEF was performed by the
method of Matthew et al. (17) with a Mini-IEF cell system
(Bio-Rad). Enzyme activities were detected by overlaying the gel with
0.5 mM nitrocefin in 0.1 M phosphate buffer, pH 7.0. -Lactamases
were identified by comparison to reference enzymes run in tracks
adjacent to the test samples. Inhibition assay was performed by
overlaying the gels with 0.5 mM nitrocefin with and without 0.3 mM
cloxacillin or 0.3 mM clavulanic acid in 0.1 M phosphate buffer, pH 7.0 (15).
| |
RESULTS AND DISCUSSION |
The evolutionary relationship of seven members of the SHV family
is summarized in Fig. 1. Many SHV-derived
extended-spectrum enzymes have the same change of amino acid at
position 238 and thus must all be derived from SHV-2. This first
mutation from SHV-1, changing glycine to serine, was associated with a
large increase in the MIC of cefotaxime but only a moderate increase in
the MIC of ceftazidime. SHV-4 was derived from SHV-2 by substitution of
two amino acids through either SHV-3 or SHV-5. A change in the amino
acid at position 240 from glutamic acid (SHV-2) to lysine (SHV-5)
considerably increased the MIC of ceftazidime, but had a lesser effect
on the MIC of cefotaxime (8). On the other hand, although
SHV-2a and SHV-12 were indistinguishable from SHV-2 and SHV-5,
respectively, by isoelectric point and substrate profiles, SHV-2a and
SHV-12 share the same substitution of glutamine for leucine at position
35 as SHV-2 and SHV-5, respectively. SHV-12 conferred a relatively low
level of resistance to cefotaxime but higher resistance to ceftazidime
and aztreonam (14). The substrate profile and the sites of
amino acid variation suggest that SHV-12 may be derived from SHV-2a by
replacement of glutamic acid with lysine at position 240. Continued
challenge of an SHV-2a-producing strain with 7-oxyiminocephalosporins,
particularly ceftazidime, is likely to select for the E240K
substitution leading to SHV-12 (14).
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|
FIG. 1.
A diagram of the evolutionary relationship of six
SHV-derived ESBLs. Location of selected amino acids is according to
the consensus numbering of Ambler et al. (1). Amino acids in
boldface type represent changes from SHV-1 at these positions.
|
|
For discriminatory detection of genes coding for SHV variants, we
performed an LCR typing with the four different primer sets that are
designed to detect the following amino acid substitutions: Gln for Leu
at position 35, Leu for Arg at 205, Ser for Gly at 238, and Lys for Glu
at 240.
Discrimination of SHV enzymes by LCR typing.
The seven
reference strains, each producing one of the seven recognized SHV
-lactamases, were subjected to LCR typing. Each point mutation of
blaSHV genes targeted by each primer set was precisely detected (Fig. 2). LCRs with
the primer set of Ser-238 gave a positive signal with all
blaSHV-ESBL. Similar results were obtained with
the primer sets of Gln-35, Leu-205, and Lys-240 on
blaSHV-2a and blaSHV-12;
blaSHV-3 and blaSHV-4;
and blaSHV-4, blaSHV-5,
and blaSHV-12 target DNAs, respectively.
Target-independent ligation or false-negative reactions were not
observed on any occasion. Therefore, we could distinguish seven SHV
bla genes rapidly and precisely with LCR typing according to
their incorporating point mutations (Table
2).
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|
FIG. 2.
Identification and discrimination of
blaSHV genes by LCR typing. The seven reference
strains (lanes 1 to 7), each producing one of seven recognized SHV
-lactamases, were subjected to LCR typing with four different
biotinylated primer sets (A to D).
|
|
The phenotypic characterization of -lactamases that relies on the
determination of their substrate profile, biochemical data, and IEF
properties is poorly reproducible from one laboratory to another and
can be technically demanding. Recently, the genotypic identification of
-lactamases such as oligotyping (16, 29), PCR-single-strand conformational polymorphism (SSCP) (14),
PCR-restriction fragment length polymorphism (2, 6, 22),
immunoassay (7), and direct sequencing of PCR product has
been developed, and in the future these approaches may be more
reliable. Oligotyping using biotinylated probes has been used to
characterize TEM variant enzymes (29), and PCR-SSCP has been
used to distinguish SHV-1 to -5 (19). We had also tried
differentiation of SHV variants by PCR-SSCP according to the method
described by M'Zali et al. (19) with some modifications. By
that method, SHV-1 to SHV-5 were distinguished consistently, but SHV-2a
and SHV-12 were not distinguished from SHV-2 and SHV-5, respectively
(data not shown). In contrast, LCR typing enabled us to discriminate
all tested SHV enzymes, including SHV-2a and SHV-12. Moreover, LCR
typing of this study offers many advantages. First, LCR with the primer set of Ser-238 allows detection of serine-for-glycine substitution at
amino acid position 238 incorporated in many
blaSHV-ESBL genes, so this can be used to screen
clinical isolates producing a majority of SHV ESBLs. Second, LCR with
the primer set of Gln-35 allows differentiation of SHV-2a and SHV-12
from SHV-2 and SHV-5, respectively, without sequencing. Third,
combining LCR and simple colorimetric detection using the AmpLiTek LCR
kit provides an acquisition of the results in 1 day. And finally, the
use of stripwell and microplate reader affords the opportunity to
screen a large number of strains. Therefore, we believe that the LCR
typing may also become a very useful genotypic method to detect and
distinguish -lactamases.
Identification and discrimination of SHV ESBLs in clinical isolates
with LCR typing.
To test the applicability of the LCR technique
for identification of unknown SHV -lactamases, clinical isolates
were also included in this study. Of 82 strains with reduced
susceptibility to extended-spectrum cephalosporins which had been
collected, 46 strains (32 K. pneumoniae, 10 E. cloacae, and 4 E. coli) were positively reacted with
the PCR of blaSHV genes. By LCR with the primer
set of Ser-238, 16 of 32 K. pneumoniae strains were
considered to carry chromosomal blaSHV-1 gene
giving no color change, and they were excluded from further studies.
The remaining 30 strains were subjected to antibiotic susceptibility
testing, IEF, and LCR typing. The results obtained are shown in Table
3.
Upon IEF, 21 strains revealed the SHV-type -lactamase of pI 7.6, and
nine isolates revealed the SHV-type -lactamase of pI 8.2. Additional
TEM-type enzymes with pI of 5.4, 5.7, or 5.9 appeared in 14 strains.
One isolate of E. coli E15 also produced a class C
-lactamase with a pI of 8.0 which was sensitive to inhibition by
cloxacillin but not clavulanic acid, and most strains of E. cloacae also produced a class C chromosomal -lactamase with a pI of 8.1 or 8.5.
For 21 strains showing the -lactamase of pI 7.6, LCR with the primer
set of Gln-35 enabled us to distinguish
blaSHV-2a from blaSHV-2
(10), blaSHV-7 (5), and
blaSHV-8 (27). Similarly, for nine
strains showing the -lactamase of pI 8.2, LCR with the primer set of
Gln-35 enabled us to distinguish blaSHV-12 from blaSHV-5, blaSHV-9, and
blaSHV-10 (25, 26). LCR with the
primer set of Lys-240 allowed us to discriminate
blaSHV-2a and blaSHV-12. The results revealed that 21 strains carried the presumptive
blaSHV-2a gene and nine strains carried the
presumptive blaSHV-12 gene. Unfortunately, the
present method does not detect SHV ESBLs lacking the four covered
substitutions, e.g., SHV-6 (3), SHV-7 (5), and
SHV-8 (27). But this can be overcome by implementing two additional primer sets for the amino acid substitutions R43S and D179.
It is also noted that there are two different codons that give rise to
the Lys-240 substitution: AAG, which our primer set of Lys-240 is based
on, and AAA, found in one of the SHV-5 genes deposited in GenBank and
in SHV-7. If one of the genes that have AAA would be present, it would
be a mismatch for our primer set of Lys-240. (Information for the
various ESBL sequences for SHV -lactamases was obtained from the
website http://www.lahey.org/studies/webt.htm#SHV.)
Antimicrobial resistance patterns of these isolates were in accordance
with LCR data. MICs of ceftazidime and aztreonam for the strains
harboring the blaSHV-2a gene were frequently
low, while the strains harboring the blaSHV-12
gene usually showed high resistance to those antibiotics
(14). For the strain of E. coli E15, the presence
of an enzyme with a pI of 8.0, resembling CMY-1, raised the MICs of
cefotaxime, ceftazidime, aztreonam, and cefotetan up to 64, 32, 4, and
256 µg/ml, respectively. Two strains of E. cloacae, En18
and En19, were highly resistant to all antibiotics tested, including
cefotetan, indicating that they produced the Bush group 1 -lactamase
constitutively at high levels due to the derepressed mutation of a
chromosomal gene, ampD. Therefore, they were considered to
be derepressed mutants and SHV-2a -lactamase producers.
The results obtained indicated that LCR typing was also applied
successfully to the identification of SHV -lactamases from clinical
isolates. It is important to note that IEF and biochemical data can be
used in conjunction with the LCR typing data to confirm observations.
In addition, as LCR typing detects a limited number of amino acid
changes but other changes in the sequence may also exist, care is
required in the interpretation of the data.
So far, SHV ESBLs have been found predominantly in
Klebsiella spp. and E. coli. ESBLs of the TEM
and, particularly, the SHV type are very rarely found in other
Enterobacteriaceae genera, such as Enterobacter,
Serratia, Citrobacter, etc., in which chromosomal AmpC cepahlosporinases predominate (11, 15). Nevertheless, Serratia marcescens isolated in Greece has been reported to
produce an SHV-like enzyme (identified by IEF) (9), and
S. marcescens as well as E. cloacae have been
found to carry SHV-4 (23). In addition, Pitout et al.
(24) presented evidence for the production of SHV-3 and
SHV-4 by E. cloacae and Enterobacter aerogenes in the United States. A single isolate of E. cloacae from
Switzerland was found to carry SHV-2 by DNA sequencing (21).
In this context, it is important to note that this is the first report
describing a number of strains of E. cloacae producing
SHV-2a or SHV-12.
Although the prevalence of SHV-2a and SHV-12 in
Enterobacteriaceae in Western Europe or in the United
States is not known, they are widespread among K. pneumoniae
strains in Korea (14). This study also revealed the
prevalence of SHV-2a and SHV-12 even among E. cloacae
strains. Although the reason for the widespread distribution of SHV-2a
and SHV-12 is not clear, the spread of resistant organisms or mobile
elements like transposons or insertion sequences may have played a role
in the spread of common ESBLs. Moreover, intrahospital spread of common
organisms or similar selective pressure among the institutions could be
another possible explanation.
In summary, we have developed a new genotypic method to characterize
point mutations in genes for SHV-type -lactamases based on a
nonradioactive LCR technique. This technique permits detailed characterization and molecular epidemiology of SHV-type -lactamases more easily and rapidly than sequencing. The SHV family of ESBLs is
well defined and has proved to be a good model for the development of
LCR technology as applied to the characterization of antibiotic resistance genes in bacteria. This technique could also be extended to
characterize the mutations that have given rise to the much larger
family of TEM-derived ESBLs and any other resistance genes that differ
by only point mutations.
| |
ACKNOWLEDGMENTS |
We are very grateful to G. A. Jacoby, who provided the five
strains carrying plasmids encoding SHV-1, SHV-2, SHV-3, SHV-4, and
SHV-5 -lactamases.
This work was supported by grant KOSEF 971-0712-102-2 from the Korean
Science and Engineering Foundation.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology, College of Medicine, Dankook University, San 29, Anseo-dong, Cheonan, Choongnam, 330-180, Republic of Korea. Phone:
82-417-550-3853. Fax: 82-417-550-3905. E-mail:
minkim{at}anseo.dankook.ac.kr.
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Antimicrobial Agents and Chemotherapy, July 2000, p. 1860-1864, Vol. 44, No. 7
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
