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Antimicrobial Agents and Chemotherapy, October 2000, p. 2759-2763, Vol. 44, No. 10
Institute of Medical Microbiology, University
of Zürich, CH-8028 Zürich,
Switzerland,1 and Department of
Bacteriology, National Institute of Infectious Diseases, Shinjuku-ku,
Tokyo 162-8640, Japan2
Received 29 November 1999/Returned for modification 28 March
2000/Accepted 16 July 2000
SHV extended-spectrum Since 1983 (15, 16),
clinical isolates resistant to expanded-spectrum cephalosporins have
increasingly been reported. They were derived through single amino acid
substitutions from one of three parental enzymes, TEM-1, TEM-2, or
SHV-1. The resulting structures were designated extended-spectrum
Phenotypic differences due to the variably mutated In order to examine the influence of amino acid substitutions in known
SHV Bacterial strains and plasmids.
Escherichia coli
DH5
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Contribution of Natural Amino Acid Substitutions in
SHV Extended-Spectrum
-Lactamases to Resistance against
Various -Lactams
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
-lactamases (ESBLs) arise through single
amino acid substitutions in the parental enzyme, SHV-1. In order to
evaluate the effect of genetic dissimilarities around the structural
gene on MICs, we had previously devised an isogenic system of strains.
Here, we present an extended version of the system that now allows
assessment of all major types of SHV -lactamases as well as of two
types of promoters of various strengths. Moreover, we devised a novel
vector, pCCR9, to eliminate interference of the selection marker. A
substitution within the signal sequence, I8F found in SHV-7, slightly
increased MICs, suggesting more efficient transfer of enzyme precursor
into the periplasmic space. We also noted that combination of G238S and
E240K yielded higher resistance than G238S alone. However, the
influence of the additional E240K change was more pronounced with
ceftazidime and aztreonam than with cefotaxime and ceftriaxone. The SHV
enzymes characterized by the single change, D179N, such as SHV-8,
turned out to be the weakest SHV ESBLs. Only resistance to ceftazidime
was moderately increased compared to SHV-1.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
-lactamases (ESBLs) (13, 29), and they were classified in
a new subgroup, 2be (4). Since the responsible genes are
easily transferable due to frequent localization on plasmids
(34) the situation has recently been called a "plague of
plasmids" (6).
-lactamases were
noted early in vitro, and, although ESBL production appears to
frequently lead to treatment failure, MICs for ESBL producers may be
barely significantly increased compared to those for fully susceptible
variants (18). Therefore, it is crucial to be able (i) to
detect ESBLs or blaESBL genes easily and
reliably (19) and (ii) to judge the clinical significance of
given ESBLs by studying the structure-function relationships of the
various ESBLs. The present study is a contribution to the second aim.
-lactamases as well as of various promoter strengths on the
level of resistance, we exploited a previously developed system of
strains (26) which allows direct phenotypic comparison of
such derivatives under isogenic conditions. To some extent, the effect
of the plasmid copy number could also be estimated through introduction
of a novel low-copy-number vector system.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
(10) was used as a recipient for transformation with
cloned blaSHV genes based on a novel plasmid vector, pCCR9 (this study). Relevant information on recombinant strains
and plasmids are given in Tables 1 and
2.
TABLE 1.
Susceptibilities of isogenic strains carrying recombinant
plasmids based on the low-copy-number vector pCCR9
TABLE 2.
Susceptibilities of isogenic strains carrying amino acid
substitutions within the signal sequence
Antibiotics. Ampicillin was obtained from SmithKline Beecham Pharmaceuticals (Surrey, England), and tetracycline was from Pfizer (Groton, Conn.).
Susceptibility tests. Inhibition zone diameters were ascertained by disk testing according to the guidelines of the NCCLS (23). E tests (AB Biodisk, Solna, Sweden) were performed on Mueller-Hinton agar plates (Difco, Detroit, Mich.), according to the manufacturer's instructions.
DNA preparation. Plasmid DNA was prepared using the Qiagen (Hilden, Germany) plasmid kit according to the manufacturer's instructions. Total DNA was extracted following standard protocols (32).
Cloning and sequencing of -lactamase genes.
Total DNA or
recombinant plasmid as well as vector DNA was digested with appropriate
restriction enzymes following the supplier's protocols (Hoffmann La
Roche, Basel, Switzerland). Size and orientation of inserts were
determined by visualizing the restriction fragments under UV light
after agarose gel electrophoresis (0.7% agarose, 1 mg of ethidium
bromide per liter, 4 V/cm). The gels were photographed using a Polaroid
camera (type 667 Professional). Calf intestinal phosphatase, T4 DNA
ligase and ligation buffer were obtained from Hoffmann La Roche, and
cloning of purified restriction fragments into vector pCCR9 followed by
transformation of competent E. coli DH5 cells was
performed according to the method of Sambrook et al. (32).
Clones were selected and purified on Mueller-Hinton agar plates (Difco)
with 10 µg of tetracycline per ml. DNA sequencing was performed by
the dideoxy chain termination method (33) using the ABI
Prism Big Dye terminator cycle sequencing ready reaction kit
(Perkin-Elmer, Foster City, Calif.) and an ABI Prism 310 genetic analyzer.
SDM and oligonucleotides.
Single nucleotide mutations were
introduced using the QuikChange site-directed mutagenesis (SDM) kit
(Stratagene, La Jolla, Calif.). Introduction of each point mutation was
confirmed, and the entire gene and promoter region were checked, all by
sequencing. Correctly mutated genes were recloned as 3.6-kb fragments
into fresh pCCR9 vector using EcoRI and
HindIII restriction sites for genes with promoter A and
Asp718 and SphI sites for those governed by
promoter B. The recombinant plasmids were used to transform E. coli DH5. Oligonucleotides for sequencing (20-mers) and SDM (20- and 23-mers) were custom synthesized (Microsynth, Balgach, Switzerland). Oligonucleotides for SDM are listed in Table
3.
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Nucleotide sequence accession number. The complete nucleotide sequences of vectors pAW9 and pCCR9 have been deposited in the EMBL database under the accession numbers AJ289102 and AJ277764, respectively.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Construction of a low-copy-number plasmid vector, pCCR9.
The
original vector of the isogenic system, pTZ18R, is a high-copy-number
system and expresses TEM-1 as a selection marker, which can interfere
with part of the phenotype of the product of cloned
blaSHV genes. In order to eliminate such adverse
effects, an alternative vector, pAW9, was constructed, a 2,553-bp
low-copy-number vector with a tetracycline selection marker. The
low-copy-number replicon originated from pACYC184 (EMBL accession no.
X06403), and the tetracycline resistance determinant originated from
the shuttle vectors pHY300PLK (12) and pHY325PLK (EMBL
accession no. D00054). The multiple cloning site (MCS) from pHY300PLK (12) is also contained in pAW9. Since, however, this MCS did not contain a KpnI/Asp718 site, we replaced it by
the MCS from pTZ18R. The resulting new 2,568-bp vector was designated
pCCR9, and a physical map is given in Fig.
1. The complete nucleotide sequences of
both pAW9 and pCCR9 have been determined.
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Derivation of isogenic blaSHV mutants. The two described isogenic systems are based on a stronger promoter (PA) and a weaker promoter (PB) (26). PA originates from blaSHV-2a, and PB originates from blaSHV-2. PA leads to four to eight times more transcript (30). Introduction of point mutations into the bla genes of plasmids pMPA-1, pMPB-1, pMPA-5, and pMPB-5 resulted in further recombinants according to the genealogy shown in Table 3. Thus, the naturally occurring genes for SHV-1, SHV-2, SHV-2a, SHV-3, SHV-4, SHV-5, SHV-7, and SHV-8 were included in both systems, PA and PB. Moreover, two additional genes were constructed, coding for SHV-5F8 and SHV-7I8, which are not found in nature. Together with the producers of SHV-5 and SHV-7, these constructions provided a system for the study of a particularly interesting amino acid variation within the signal sequence. All mutations were verified by sequencing the entire blaSHV genes and flanking regions, 400 nucleotides upstream and 300 nucleotides downstream of the open reading frame, and no mistakes were found.
Since, after mutagenesis, the derived genes were all associated with the pTZ18R background (MPA-MPB system), they were recloned into the pCCR9 vector, using EcoRI/HindIII in the PA background and Asp718/SphI in the PB background to recover the desired 3.6-kb fragments for ligation. In order to indicate the promoters PA and PB, the final plasmids were designated pCTA or pCTB, respectively, followed by the number of the encoded SHV enzyme, e.g., pCTA-1 or pCTB-5F8. Correct recloning and orientation of inserts was confirmed by restriction analysis (not shown).Resistance phenotypes of naturally occurring SHV
-lactamases.
The MICs for carriers of gene constructions cloned
on vector pCCR9 are listed in Table 1. As expected, pCCR9 had barely
any measurable effect on MICs as is shown by rows of data for
ECDH5 and ECDH5(pCCR9), which represent
host alone and host with unmodified vector, respectively. In contrast
to the ESBLs, except SHV-8, SHV-1 caused MICs of cephalothin (CF) of
only 8 or 96 µg/ml depending on the promoter. The cephamycins,
represented by cefoxitin (FOX), were completely unaffected by any SHV
derivative, regardless of the promoter or the plasmid copy number.
-lactamase against CF
and amoxicillin-clavulanic acid (AMC). These MICs were even markedly
below those of the non-ESBL SHV-1 and in the range of those reached by
the non--lactamase control strains (Table 1). Interestingly, the
only drug to which SHV-8 conferred a level of resistance, 16 µg/ml,
that was intermediate according to NCCLS breakpoints (24)
was ceftazidime (CAZ), even though the MICs of other oxyimino
cephalosporins rose by 
10-fold to levels still 
1 µg/ml.
The enzymes SHV-3 and SHV-4, carrying an additional mutation, R205L
(25, 27), compared to SHV-2 and SHV-5, respectively, conferred 1.5- to 2-fold less resistance than the latter. However, some
exceptions to this trend were found (Table 1): looking at resistance to
expanded spectrum cephalosporins, 7 of 20 producers of an enzyme with
R205 gave rise to MICs higher than those reached by the respective L205
counterpart, while the opposite was true in only 4 of the 20 comparisons. The remaining nine pairs showed identical MICs (Table 1).
The second non-active-site substitution, R43S, which turned SHV-5 into
SHV-7 (3), also led to a general but slight decrease of
resistance. Again, the results were slightly ambiguous: in a total of
20 comparisons, 11 producers of a -lactamase with R43 were more
resistant to expanded spectrum cephalosporins than those with S43,
while only 2 were less resistant and 7 were equally resistant (Tables 1
and 2).
As expected, the promoter PA gave rise to higher MICs, and the effect
varied from 1.5- to 16-fold. Finally, the results obtained by the disk
method (data not shown) were in good agreement with the E tests.
Influence on resistance of amino acid substitution within the
signal sequence.
Since one difference between SHV-7 and all other
SHV ESBLs involved amino acid position 8 within the signal sequence,
the influence of this change on the level of resistance was
investigated. The MICs of the respective strain constructions carrying
either isoleucine or phenylalanine at position 8 are listed in Table 1.
With the introduction of phenylalanine at amino acid position 8 in
SHV-5 we obtained SHV-5F8, which mediated increased resistance against
all -lactam antibiotics tested. Conversely, replacement of the
phenylanine at position 8 within SHV-7 by isoleucine resulted in
SHV-7I8, which conferred decreased resistance. This effect was very
small in SHV-5 derivatives but more pronounced in those of SHV-7. In
summary, judging from our two examples the substitution I8F within the
SHV signal sequence was always likely to increase resistance.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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A previously conceived isogenic system for accurate phenotypic
comparison of SHV -lactamases carrying single amino acid
substitutions (26) has been exploited to analyze the
contribution of all natural SHV sequence changes that are important to
the level of resistance conferred. The system has now further been
refined by recloning all inserts carrying blaSHV
genes into an especially constructed low-copy-number vector, pCCR9.
This allowed (i) elimination of any interference by the
blaTEM-1 gene present on the previously used
vector, pTZ18R, and (ii) greater sensitivity for small phenotypic changes thanks to the low gene dosage.
Substitution at position 238 (15, 37) is a hallmark of the
SHV ESBLs, since, except in six of them, it is found in all 26 derivatives, as well as in some TEM ESBLs conferring high ceftazidime resistance (36). S238 causes displacement of the B3
-strand in ESBLs, thus giving rise to a slight opening of the active
site, allowing the entrance of bulkier oxyimino cephalosporins (7, 17, 21). The second-most important modification is the
neighboring E240K, found in at least 8 SHV ESBLs, including
uncharacterized ones such as SHV-15 (EMBL accession no. AJ011428). The
effect in SHV ESBLs of E240K alone is not known because it is always associated with G238S. Together with S238, it does, however, improve interaction of the enzyme with the carboxylic acid group on oxyimino substituents of CAZ and aztreonam (ATM) (11). The effects of the two substitutions, G238S and E240K, are well documented by this
study: G238S alone had a rather low to moderate impact on resistance,
but caused a 50- to 100-fold increase in MICs towards cefotaxime (CTX)
and ceftriaxone (CRO) while altering MICs of CAZ and ATM only two- to
fivefold (Table 1). Conversely, addition of E240K to G238S boosted
resistance to CAZ and ATM by another 20- to 250-fold, while increasing
the MICs of CTX and CRO further by only 1.5- to 3-fold.
Concerning the three non-active-site substitutions, the effects are generally much smaller. Earlier results, e.g., the 1.5- to 3-fold increase of resistance by L35Q when governed by PA on the multicopy replicon (26), are confirmed by the present study. However, the effects are minimized under the control of the weaker promoter PB and by the low-copy-number effect. SHV-3 and SHV-4 differ from SHV-2 and SHV-5, respectively, by the substitution R205L, which is thought to cause cephalosporins with large C-3 substituents to fit better into the active-site pocket (17). Our results do not seem to support this view. Despite ambiguities in some of the tested strain pairs, the MICs for derivatives expressing arginine rather than leucine at position 205 were slightly elevated.
SHV-6 (1), SHV-8 (31), and four uncharacterized
enzymes
SHV-16 (EMBL accession no. AF072684), SHV-24 (EMBL accession no. AB023477), SHV-25 (EMBL accession no. AF208796), and SHV-26 (EMBL
accession no. AF227204)are the only SHV ESBLs that do not contain the
typical G238S substitution. SHV-6, SHV-8, and SHV-24 carry D179A, -N,
and -G, respectively. D179 and R164 form a salt bridge and are the
anchor points of the omega loop that contains the catalytically
important E166 (20). Disruption of this salt bridge leads to
extension of the substrate profile (2, 28). For unknown
reasons, many natural TEM ESBLs are altered in position 164 (22), but none are altered in position 179, while in SHV
ESBLs the opposite is true. The effects of D179A, -N, and -G had been
poorly understood either for a lack of investigation, or, in the case
of SHV-8, because the enzyme was masked by additional mechanisms
(31). Examination of SHV-8 in our isogenic system revealed
that the D179N change resulted (i) in a moderate increase in CAZ
resistance, (ii) in a small but insignificant increase of resistance to
CTX, CRO, cefepime (FEP), and ATM, and (iii) in a significant decrease
of resistance to narrow-spectrum cephalosporins and AMC. In fact, SHV-8
conferred the lowest level of CF resistance of all derivatives tested,
even with the strong promoter (Table 1). Thus, the future will show
whether SHV derivatives, lacking the G238S substitution, will emerge as
important ESBLs and disseminate successfully.
The precursor of SHV-7 carries a substitution, I8F (3), that
can have no influence on the native -lactamase because it is trimmed
off with the signal sequence after secretion into the periplasmic
space. Since testing of the I8F substitution in the SHV-7 and SHV-5
background revealed slightly higher MICs for derivatives with
phenylalanine in position 8 (Table 2), we propose that the signal
sequence of the original SHV-7 precursor may lead to more efficient
transfer of -lactamase into the periplasm. The effect is stronger in
SHV-7 than in SHV-5. These results suggest that, apart from primary
structure and expression, the level of resistance conferred by a
particular -lactamase can be modulated by the rate of transfer into
the periplasm.
Increased resistance has been ascribed to hyperproduction of
-lactamases (9), and hyperproduction may be due to
promoter variations (8). This effect is clearly documented
by the two presented isogenic systems. In some cases, the contribution
of the different promoters to the level of resistance appeared to be
even greater than that of certain amino acid substitutions, as
exemplified by the MICs of CTX for strains CTA-2, CTB-2, CTA-2a, and
CTB-2a (Table 1). Moreover, the low-copy-number system, pCCR9, yielded
generally greater promoter effects than the high-copy-number system
based on vector pTZ18R (data not shown). This may be due to saturation
effects in the latter system.
The detection of ESBLs is still not satisfactory (19). The data in Table 1 demonstrate that cefpodoxime (CPX) is the expanded-spectrum cephalosporin most efficiently destroyed by these ESBLs. As suggested earlier (5, 35), it is therefore the most sensitive single screening agent for SHV ESBLs, clearly superior to ceftazidime (14). This is another example for the versatility and also for the practical use of the isogenic system for the investigation of the resistance phenotype of the clinically important SHV-type ESBLs.
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ACKNOWLEDGMENT |
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This work was supported by the Swiss National Foundation (grants 3200-39466.93/3 and 3200-52532.97).
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FOOTNOTES |
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* Corresponding author. Mailing address: Institute of Medical Microbiology, University of Zürich, P.O. Box, CH-8028 Zürich, Switzerland. Phone: 41-1-634-26 48. Fax: 41-1-634-4906. E-mail: haechler{at}immv.unizh.ch.
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Abstract
Introduction
Materials and Methods
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Discussion
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