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Antimicrobial Agents and Chemotherapy, November 2002, p. 3568-3573, Vol. 46, No. 11
0066-4804/02/$04.00+0     DOI: 10.1128/AAC.46.11.3568-3573.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Resistance to ß-Lactamase Inhibitor Protein Does Not Parallel Resistance to Clavulanic Acid in TEM ß-Lactamase Mutants

William A. Schroeder, Troy R. Locke, and Susan E. Jensen^*

Department of Biological Sciences, University of Alberta, Edmonton, Alberta T6G 2E9, Canada

Received 20 February 2002/ Returned for modification 22 May 2002/ Accepted 19 July 2002

Top Abstract Introduction Materials and Methods Results Discussion References

 
In order to compare patterns of resistance to inhibition by clavulanic acid with patterns of resistance to inhibition by a ß-lactamase inhibitor protein (BLIP), R164S, R244S, and R164S/R244S mutant forms of TEM ß-lactamase were prepared by site-directed mutagenesis. When kinetic parameters were determined for these mutant and wild-type forms of TEM, the single mutants showed properties that were similar to those in the literature but the double mutant showed properties that were very different. The R164S/R244S double mutant form of TEM retained its resistance to inhibition by clavulanic acid (characteristic of the R244S mutation) but lost all its ability to hydrolyze ceftazidime (characteristic of the R164S mutation). While these characteristics are contrary to those previously reported for an R164S/R244S double mutant, this discrepancy resulted from the use of a defective mutant in the earlier study. Both the single and double mutant forms of TEM remained highly sensitive when tested for inhibition by BLIP, showing only slightly increased resistance compared to that of the wild type; this pattern of resistance is quite different from the pattern of clavulanic acid resistance. The slight increases in resistance to inhibition by BLIP seen in the mutants may have been related to the fact that all of the mutations effected changes in the net charge on the TEM protein that could impede interactions with BLIP.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacterial resistance to antibiotics is an important and growing concern in the treatment of infectious diseases. Although ß-lactam compounds still compose more than 50% of all prescribed antibiotics, the development of resistant strains represents a serious threat to the continued usefulness of these agents. In particular, the emergence of mutant forms of the ß-lactamase TEM, the single most prevalent ß-lactamase found in gram-negative bacteria, provides a striking example of the evolution of antibiotic resistance. In response to the appearance of microbial resistance mediated by TEM and other ß-lactamases, aminothiazole oxime-containing, ß-lactamase-resistant antibiotics such as ceftazidime, cefotaxime, and aztreonam were developed. As an alternative approach, ß-lactamase inhibitors used in combination with conventional penicillins and cephalosporins were also introduced. However, the advent of each new control strategy was met with the emergence of mutant forms of TEM that are resistant to the new agent. The number of naturally occurring mutant forms of TEM now exceeds 100 (http://www.lahey.org/studies/temtable.htm). Although there are many examples of TEM mutants showing activity against extended-spectrum cephalosporins, and others that are insensitive to inhibition by clavulanic acid (3-6, 10, 12, 14; D. Sirot, C. Chanal, R. Bonnet, C. DeChamps, and J. Sirot, Letter, Antimicrob. Agents Chemother. 45:2179-2180, 2001), the coexistence of mutations associated with these resistance characteristics within a single enzyme is less common and typically results in the loss of one phenotype or the other (9, 13, 18). This has led to the suggestion that both the structural constraints associated with the ability to hydrolyze broad-spectrum compounds and those associated with high-level resistance to ß-lactamase inhibitors may be incompatible.

Given the health care challenges posed by the continuing spread of resistance to traditional and extended-spectrum ß-lactam antibiotics, coupled with the emergence of ß-lactamase inhibitor-resistant mutants, the need for new agents that are effective against resistant strains is pressing. In previous studies, a proteinaceous ß-lactamase inhibitor (BLIP) was shown to bind to and inhibit TEM with very high affinity (K_i of less than 1 nM) (21). Crystallographic analyses of BLIP and TEM alone and in complex gave insights into the nature of the inhibitory interaction and defined a peptide loop exposed on the surface of BLIP which interacts with the active site of the ß-lactamase (20). While the proteinaceous nature of BLIP limits its value as a therapeutic agent, the fact that BLIP is structurally unrelated to existing small-molecule ß-lactamase inhibitors, such as clavulanic acid and tazobactam, suggests that new classes of ß-lactamase inhibitors based on small-molecule derivatives of BLIP might hold clinical potential (15-17). However, this theory is predicated upon the assumption that clavulanic acid-resistant forms of ß-lactamases will not show a parallel resistance to inhibition by BLIP. The present study was undertaken to investigate the sensitivity to inhibition by BLIP demonstrated by mutant forms of TEM that show resistance to small-molecule inhibitors and/or activity against broad-spectrum compounds. Single and double mutant forms of TEM were prepared by site-directed mutagenesis, and the kinetic properties of the mutants, in the presence of either clavulanic acid or BLIP, were compared.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Plasmids and ß-lactam compounds. The expression plasmid pT7-4 (22) was generously provided by S. Tabor (Harvard University) and modified for the expression of wild-type and mutant forms of the bla gene. pT7-4 was digested with BspHI to release the resident ß-lactamase-encoding bla gene, and an alternate selectable marker was provided by the insertion of a cassette carrying an apramycin resistance gene. The apr cassette was released from the plasmid pUC120Apr (23) by digestion with EcoRI and PstI and ligated to the digested pT7-4 plasmid after both had been made blunt by treatment with the Klenow fragment of DNA polymerase I. Analysis of the resulting 2.75-kb plasmid, called pT7-apr, showed that the apr gene was inserted in the orientation opposite to that of the T710 promoter. Wild-type and mutant forms of the bla gene were then inserted into the multiple-cloning site of pT7-apr in the orientation necessary to place them under the control of the T710 promoter.

Ampicillin and 2-{[p-(dimethylamino)phenyl]azol-pyridino} cephalosporin (PADAC) were purchased from Sigma and Calbiochem, respectively. Ceftazidime and clavulanic acid were generously provided by SmithKline Beecham.

Mutagenesis of the TEM ß-lactamase. The amino acid residues in TEM were numbered according to the convention described by Ambler et al. (2). All mutations were introduced into the bla gene with the altered-site mutagenesis kit from Promega. To form the R164S mutation, an oligonucleotide primer with the sequence 5'-CTCGCCTTGATAGTTGGGAACCGG was used. To form the R244S mutation, an oligonucleotide with the sequence 5'-GCGTGGGTCTAGCGGTATCATTGC was used. The R164S/R244S double mutant was generated by conducting a second round of mutation on the R164S mutant by using the R244S mutational oligonucleotide. Each mutant form of bla was sequenced in its entirety by using a combination of universal and sequence-specific oligonucleotide primers. The Molecular Biology Service Unit (Biological Sciences Department, University of Alberta) used the DYEnamic ET terminator cycle sequencing procedure (Amersham Pharmacia Biotech) to conduct sequence analyses. The mutant forms of bla were then transferred to pT7-apr for expression.

Purification of the ß-lactamases and BLIP. Wild-type and mutant forms of TEM were produced in the Escherichia coli host strain BL21DE3 (Novagen) and purified from the osmotic shock fluid by a combination of anion-exchange and gel filtration chromatographies as described previously (8). Purified TEM preparations were stored in aliquots in 0.1 M NaPO₄, pH 7.0, at -20°C. The concentration of the preparations was determined by using a molar extinction coefficient for TEM of 28,960 M^-1 cm^-1 (1).

BLIP was purified from the culture supernatant of Streptomyces clavuligerus grown in Trypticase soy broth plus 1% soluble starch by using a combination of anion-exchange and gel filtration chromatographies as described previously (8). Purified BLIP was dialyzed against distilled H₂O, lyophilized, and stored at -20°C. The concentration of the BLIP preparations was determined by using a molar extinction coefficient of 28,460 M^-1 cm^-1 (1).

Enyme kinetics. Kinetic measurements were made with a Unicam UV3 spectrophotometer. All assays were carried out in 0.1 M NaPO₄ buffer, pH 7.0, at 25°C in a final reaction volume of 1.0 ml. Initial rates were determined for five or more substrate concentrations, and all rate measurements were conducted in duplicate on two or more occasions. The values for each substrate were as follows: ampicillin (240 nm), 538 M^-1 cm^-1; PADAC (466 nm), 9,590 M^-1 cm^-1; and ceftazidime (260 nm), 10,500 M^-1 cm^-1.

An iterative robust nonlinear regression analysis was used to determine the values for the kinetic parameters k_cat and K_m, with initial estimates computed by the Eadie-Hofstee method; Dixon plots and the Enzyme Kinetics software package (Trinity Software) were used to determine the dissociation constant K_i for clavulanate. For K_i determinations, three concentrations of ampicillin, 100, 200, and 500 µM, and three concentrations of PADAC, 50, 100, and 200 µM, were used. Series of assay mixtures containing each substrate, together with various concentrations of clavulanate, were prepared and prewarmed to 25°C before the reactions were started by the addition of enzyme.

BLIP inhibition assay. Various concentrations of BLIP were preincubated with ß-lactamase for 2 h at 25°C. The enzyme-inhibitor incubations were carried out in 0.1 M NaPO₄ buffer, pH 7.0, containing bovine serum albumin (New England Biolabs) at a concentration of 100 µg/ml. Following the preincubation, PADAC was added at a concentration at least 10-fold lower than the K_m for the enzyme under study. The hydrolysis of PADAC was monitored at 570 nm (to gain greater sensitivity), and the change in molar extinction coefficient () used for PADAC was 52, 700 M^-1 cm^-1. Plots of the concentration of free enzyme versus the concentration of BLIP were fit by nonlinear regression analysis as described by Petrosino et al. (15).

Top Abstract Introduction Materials and Methods Results Discussion References

 
In one laboratory study, the combination of a mutation conferring inhibitor resistance (R244S) to TEM ß-lactamase and a mutation conferring activity against broad-spectrum cephalosporins (R164S) resulted in a double mutant form that lost inhibitor resistance while retaining activity against ceftazidime (11). In order to see if this same pattern of resistance and activity would extend to BLIP, mutant forms of TEM that carried each of the individual mutations as well as the double mutation were prepared. The pALTER mutation system was used to introduce mutations by site-directed mutagenesis. Mutations were verified by sequencing the resulting bla genes, and the genes were then transferred into pT7-apr for expression. Expression of each of the mutant genes yielded large amounts of ß-lactamase protein that were purified to apparent homogeneity in accordance with established procedures.

Kinetic properties of mutant and wild-type forms of TEM. The purified enzymes were tested for their activity against several ß-lactam substrates (Table 1). In most respects, the wild-type and mutant forms of TEM behaved in accordance with published reports (11). The R164S mutant form of TEM showed greatly reduced k_cat values against ampicillin and PADAC compared to those of the wild-type enzyme. The K_m values for these substrates were also somewhat reduced for the R164S mutant compared to those of the wild type; thus, catalytic efficiencies remained quite high. In keeping with its description as an extended-spectrum ß-lactamase, the R164S mutant TEM showed activity against ceftazidime, whereas no activity was detected in preparations of the wild-type enzyme, even at an enzyme concentration of 170 nM.

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TABLE 1. Kinetic parameters for the wild type and mutant forms (R164S, R244S, and R164S/R244S) of TEM ß-lactamase

The R244S mutant form of TEM showed good activity against ampicillin but with an increased K_m compared to that of the wild type. With PADAC as the substrate, the k_cat was greatly reduced, the K_m was increased, and no activity against ceftazidime was observed. Again, these values were in agreement with those previously published (11).

However, when similar kinetic assays were conducted with the R164S/R244S double mutant form of TEM, unexpected values were obtained. Compared to the wild type, the double mutant showed greatly reduced k_cat values with ampicillin and PADAC as the substrates; the K_m for ampicillin of the double mutant was relatively unchanged but that for PADAC was greatly increased relative to that of the wild type. The most striking observation, however, was that the double mutant form had no detectable ability to hydrolyze ceftazidime, even when assayed at an enzyme concentration of 170 nM.

Inhibition by clavulanic acid. When these same mutant forms of TEM were assayed for their sensitivity to inhibition by clavulanic acid, the results were again mixed (Table 2). In comparison to the wild-type enzyme, the R164S mutant showed an increased sensitivity to inhibition by clavulanic acid with more than a threefold drop in the K_i with PADAC as the substrate. Compared to the wild type, the R244S mutant showed decreased sensitivity to inhibition by clavulanic acid and nearly a 50- to 100-fold increase in K_i with ampicillin and PADAC as the substrates, in keeping with its classification as an inhibitor-resistant form of TEM. All of these results agreed with those of previously reported studies concerning these mutants. However, the R164S/R244S TEM double mutant again gave unexpected results, showing an increase in resistance to inhibition by clavulanic acid compared to that of the wild type. The level of resistance of the R164S/R244S TEM double mutant was of the same order of magnitude as that seen in the R244S single mutant. While differences in assay procedures may explain minor discrepancies among the results of separate groups, the explanation for these major differences was not readily apparent. In order to clarify the situation, we obtained a sample of the expression construct used to produce the R164S/R244S mutant form of TEM described previously (11). R164S/R244S mutant TEM protein was prepared with this construct and purified by the same procedures described for the other TEM preparations in this study. When assayed for its kinetic properties, the enzyme was found to behave much as had been reported in the earlier study, demonstrating both an ability to hydrolyze ceftazidime and a sensitivity to inhibition by clavulanic acid. Thus, the two preparations of R164S/R244S TEM protein showed kinetic properties that were very different from one another.

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TABLE 2. Inhibition of the wild type and mutant forms (R164S, R244S, and R164S/R244S) of TEM ß-lactamase by clavulanic acid

In considering other factors of possible relevance, we were able to discount any effects due to the form of bla gene used. In the present study, we employed a form of the bla gene that was originally derived from the plasmid pUC19. This bla gene (designated bla_TEM-Bs) has been modified for use as a tool in molecular genetic procedures and carries mutations that result in amino acid substitutions of V84I and A184V with respect to the sequence of TEM-1 (7). The effects of these mutations on TEM activity have been investigated and found to result in somewhat altered kinetic properties, but this particular bla_TEM-Bs form of the gene was also used in the earlier study. Therefore, any effects on kinetic properties should be consistent between the two studies.

The methodological description of the creation of the mutant forms of bla genes used in the earlier study indicated that only the 3' end of the mutated R164S/R244S-encoding gene had been verified by DNA sequence analysis. While the sequenced region encompassed the R164S and R244S mutations, it seemed possible that another unrecognized mutation in the 5' end of the gene which could alter kinetic properties might have occurred. With this in mind, the entire bla gene encoding the R164S/R244S TEM used in the earlier study was sequenced. No unanticipated mutations were identified in the 5' end of the gene, but the DNA sequence data indicated that the gene was wild type at the position corresponding to R244. Therefore, the intended R164S/R244S double mutant from the earlier study was, inadvertently, a second example of an R164S single mutant.

Inhibition by BLIP. The wild-type and single and double mutant forms of TEM prepared in this study were investigated with respect to the relationship between their sensitivities to inhibition by clavulanic acid and their sensitivities to inhibition by BLIP. Samples of each TEM preparation were incubated with various BLIP concentrations for 2 h at 25°C before the addition of PADAC as a substrate. Residual TEM activity was plotted as a function of BLIP concentration by using a nonlinear regression analysis to fit the data as described by Petrosino et al. (15).

Wild-type TEM was found to be sensitive to inhibition by BLIP, demonstrating an apparent K_i of 2.76 nM (Table 3). In comparison, the R244S mutant form of TEM, which showed increased resistance to inhibition by clavulanic acid, showed only slightly increased resistance to inhibition by BLIP, with an apparent K_i of 9.48 nM. When inhibition of the R164S form of TEM by BLIP was investigated, another slight increase in resistance, with an apparent K_i of 14.6 nM, was observed. The R164S/R244S double mutant form of TEM also showed a slightly increased resistance to inhibition by BLIP. An apparent K_i value of 6.86 nM was obtained in assays using the R164S/R244S mutant enzyme at a 100 nM concentration. However, the apparent K_i value was observed to be sensitive to changes in the enzyme concentration used in the assay. A higher value, 13.3 nM, for the apparent K_i was obtained when a 200 nM concentration of enzyme was used. The change in the shape of the inhibition curve as the enzyme concentration was doubled is shown in Fig. 1.

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TABLE 3. Inhibition of the wild type and mutant forms (R164S, R244S, and R164S/R244S) of TEM ß-lactamase by BLIP

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FIG. 1. Effect of enzyme concentration on the inhibition of the R164S/R244S mutant form of TEM by BLIP. Inhibition of ß-lactamase was determined by measuring the amount of free ß-lactamase in assays of BLIP and the R164S/R244S mutant enzyme at 100 (A) and 200 (B) nM concentrations.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Site-directed mutagenesis was used to prepare TEM ß-lactamases carrying mutations previously shown to be associated with enhanced activity against ceftazidime (R164S), enhanced resistance to inhibition by clavulanic acid (R244S), and a complex mutant carrying both mutations. When the kinetic properties of the various mutant forms of TEM were determined and compared to those of the wild type, the R164S and R244S TEM mutants each performed according to expectation, demonstrating the ability to hydrolyze ceftazidime and the ability to resist inhibition by clavulanic acid, respectively. Values for k_cat, K_m, and k_cat/K_m for each mutant accorded with those previously published. However, the same kinetic parameters for the R164S/R244S mutant were at variance with those in the literature. The double mutant showed a loss of ability to hydrolyze ceftazidime while it retained its resistance to inhibition by clavulanic acid, whereas the same mutant was previously reported to hydrolyze ceftazidime and demonstrate sensitivity to inhibition by clavulanic acid. Upon further analysis, it became clear that the reason for this disagreement was that the bla gene believed to encode the R164S/R244S mutant form of TEM used in the previous study had actually carried only the single R164S mutation. Whether this discrepancy arose as a result of the reversion of the double mutant, or through the outgrowth of a contaminating R164S single mutant, it seems clear that the kinetic properties reported earlier were, inadvertently, those of a single R164S mutant.

While the kinetic properties of the R164S/R244S TEM enzyme prepared in this study were at odds with those described by Imtiaz et al. (11), they did agree with the findings of Stapleton et al. (19), who studied a closely related R164S/R244C mutant form of TEM. Although kinetic studies on purified enzyme preparations were not undertaken, Stapleton et al. observed that the R164S/R244C double mutant form of TEM had lost the extended-spectrum ß-lactamase activity associated with the R164S single mutant. In contrast, the resistance to inhibition by clavulanic acid associated with the R244C mutation was still markedly increased compared to that of wild-type TEM. Stapleton et al. remarked that these characteristics seemed to disagree with the kinetic properties noted previously for the R164S/R244S mutant TEM but suggested that some unanticipated structural effect of replacing arginine with cysteine rather than with serine in position 244 might explain the differences. In view of our present findings, it is now apparent that the results of mutation in TEM of the arginine residue at position 164 to serine, together with mutation of the arginine residue at position 244 to either serine or cysteine, are to abolish activity against ceftazidime while retaining resistance to inhibition by clavulanic acid.

The wild-type form of TEM gave an apparent K_i for BLIP of 2.76 nM. This value is higher than most published values for K_i but does correspond quite well with the value of 2.3 nM reported by Albeck and Schreiber for the equilibrium dissociation constant of BLIP interacting with wild-type TEM (1).

When the properties of the various mutant forms of TEM were examined with respect to their sensitivities to inhibition by BLIP, similar values for apparent K_i were found for all of the mutant enzymes. In comparison to wild-type TEM, the R244S mutant form of TEM showed approximately 50- to 100-fold-greater resistance to inhibition by clavulanic acid but showed only 3.4-fold-greater resistance to inhibition by BLIP. Similarly, the R164S mutant TEM showed a very slight increase in resistance to BLIP (about 5.2-fold) whereas it showed decreased resistance to clavulanic acid. Finally, the R164S/R244S double mutant form of TEM showed very slightly increased resistance to inhibition by BLIP (2.5-fold with a 100 nM concentration of enzyme) while it showed high-level resistance to inhibition by clavulanic acid, similar to the R244S single mutant. Despite fluctuations of a few fold in the apparent K_i values, all of the enzymes examined, including the inhibitor-resistant TEM mutants, remained susceptible to inhibition by BLIP.

The high activity of wild-type TEM against the substrate PADAC means that a low concentration of TEM (1 nM) is sufficient for the assay of BLIP inhibition of enzyme activity. The R164S and R244S mutations so greatly decreased the activity of the resulting enzymes against the substrate PADAC that larger concentrations (10 nM) of the mutant enzyme were required in the assay. Combining the two mutations in one molecule further reduced the activity against PADAC, so that even greater concentrations of enzyme (100 nM) were required for assay. When BLIP inhibition studies were repeated for the R164S/R244S mutant form of TEM with enzyme concentrations of 200 nM rather than 100 nM, the calculated value of apparent K_i nearly doubled. Therefore, the apparent K_i values obtained for inhibition of the mutant form of TEM by BLIP changed with the enzyme concentration used in the assay, at least in the case of the double mutant, and cannot be considered constants at these high enzyme concentrations.

While the reasons for this unexpected variation in apparent K_i with enzyme concentration are not clear, they may be related to both the nature of the kinetic analyses carried out and the mutations themselves. Although the present study was not designed to consider the effects of change in net charge, the R164S and R244S mutations that were selected for their properties of enhancing activity against ceftazidime and resistance to inhibition by clavulanic acid effected increases in the net negative charge of TEM. Since both the TEM and BLIP molecules already bear a net negative charge, mutations which increase the negative charge of the TEM protein might affect its interaction with BLIP.

The majority of the interactions between BLIP and TEM are concentrated in the region of a ß-hairpin loop, amino acid residues 46 to 51 of BLIP, which makes critical contacts with the active site of TEM. R244 is one of the active-site residues that lies at the interface of the two interacting proteins and is believed to be involved in strong hydrogen bonding interactions that stabilize the enzyme inhibitor complex. In addition to specific hydrogen bonding interactions, other protein-protein contacts contributing to the binding and ionic interactions are also certainly important. As a result, mutations that change the net charge on either TEM or BLIP may affect the interaction between the two proteins.

Albeck and Schreiber have studied mutant forms of BLIP and their interactions with wild-type and mutant forms of TEM (1). They observed that changes which increase the net negative charge on BLIP can reduce its inhibitory effectiveness by decreasing the association rate between the two proteins and increasing the dissociation rate of the enzyme inhibitor complex. The BLIP inhibition assay of Petrosino et al. (15) that was used in this study features a 2-h preincubation step, which is described as sufficient to achieve binding equilibrium in a small volume. However, when mutant forms of TEM with altered net charge are used, the mutations may affect the rate at which equilibrium is reached, making this assay less suitable for the analysis of such mutants. Therefore, any attempt to relate the resistance of a mutant form of TEM to inhibition by clavulanic acid with its resistance to inhibition by BLIP must also entail consideration of all aspects of the particular mutation being imposed and the effects that it may have on the kinetic analyses being performed.

The present study suggests that the resistance or sensitivity of the TEM mutants to inhibition by BLIP is unrelated to the resistance or sensitivity of those same mutants to inhibition by clavulanic acid; rather, the effects of the mutations on the net charge of the proteins may be of greater significance in predicting their effects on BLIP inhibition.

 
This work was supported by both a grant from the Natural Sciences and Engineering Research Council of Canada and a fellowship (to W.A.S.) from the Alberta Heritage Foundation for Medical Research.

 
* Corresponding author. Mailing address: Department of Biological Sciences, University of Alberta, CW-405 Biological Sciences Building, Edmonton, Alberta T6G 2E9, Canada. Phone: (780) 492-0830. Fax: (780) 492-9234. E-mail: susan.jensen{at}ualberta.ca.

Present address: Cargill Central Research, Minneapolis, MN 55440-5702.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Antimicrobial Agents and Chemotherapy, November 2002, p. 3568-3573, Vol. 46, No. 11
0066-4804/02/$04.00+0     DOI: 10.1128/AAC.46.11.3568-3573.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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