Determining the Limits of the Evolutionary Potential of an Antibiotic Resistance Gene

Stephen J. Salipante and Barry G. Hall

Biology Department, University of Rochester


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 Literature Cited
 
The AAC(6') enzymes inactivate aminoglycoside antibiotics by acetylating their substrates at the 6' position. Based on functional similarity and size similarity, the AAC(6') enzymes have been considered to be members of a single family. Our phylogenetic analysis shows that the AAC(6') enzymes instead belong to three unrelated families that we now designate as [A], [B], and [C] and that aminoglycoside acetylation at the 6' position has evolved independently at least three times. AAC(6')-Iaa is a typical member of the [A] family in that it acetylates tobramycin, kanamycin, and amikacin effectively but acetylates gentamicin ineffectively. The potential of the aac(6')-Iaa gene to increase resistance to tobramycin, kanamycin, or amikacin or to acquire resistance to gentamicin was assessed by in vitro evolution. Libraries of PCR mutagenized alleles were screened for increased resistance to tobramycin, kanamycin, and amikacin, but no isolates that conferred more resistance than the wild-type gene were recovered. The library sizes were sufficient to conclude with 99.9% confidence that no single amino acid substitution or combination of two amino acid substitutions in aac(6')-Iaa is capable of increasing resistance to the antibiotics used. It is therefore very unlikely that aac(6')-Iaa of S. typhimurium LT2 has the potential to evolve increased aminoglycoside resistance in nature. The practical implications of being able to determine the evolutionary limits for other antibiotic resistance genes are discussed.

Key Words: antibiotic resistance • evolutionary potential • phylogenetic analysis • aminogylcoside


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 Literature Cited
 
Some antibiotic resistance genes, notably the TEM family of ß-lactamases, have evolved rapidly in response to the clinical use of ß-lactam antibiotics. Descendants of the TEM-1 ß-lactamase, for instance, have evolved the ability to hydrolyze an extended range of substrates, including the cephalosporins and oxyiminocephalosporins, the so-called "extended spectrum" phenotype (Medeiros 1997). Other TEM-1 descendants have evolved resistance to the ß-lactamases inhibitors clavulanic acid, tazobactam, and sulbactam (Nordmann 1998), the "inhibitor-resistant" phenotype. Thus, while TEM-1 has a substrate spectrum that is limited to penicillins and early cephalosporins, it has given rise to more than 90 descendent alleles that confer resistance to most modern ß-lactam antibiotics (http://www.rochester.edu/College/BIO/labs/HallLab/TEM_Phylo.html and http://www.lahey.org/studies/webt.htm). With a few exceptions, within 3 years of the introduction of a new ß-lactam antibiotic, a TEM ß-lactamase has evolved to confer resistance to the new drug (Medeiros 1997).

In vitro evolution mediated by error-prone PCR using the Mutazyme (Stratagene) polymerase has been shown to accurately mimic natural evolution (Barlow and Hall 2002b). Specifically, we showed that we could reproduce the natural evolution of the TEM ß-lactamases that has occurred during the past 20 years (Barlow and Hall 2002b). Through phylogenetic analysis, we determined that nine amino acid substitutions have arisen multiple times in nature in response to the use of a class of ß-lactams known as the "extended spectrum cephalosporins." Because those nine substitutions were selected multiple times in nature, it is clear that they are important for resistance to extended spectrum cephalosporins. Among 10 alleles that were independently evolved in vitro by our method, we repeatedly recovered seven of the nine substitutions that have arisen multiple times in nature (Barlow and Hall 2002b). We have recently used that system to evolve the TEM ß-lactamase to confer resistance to cefepime, a phenotype that has not yet arisen in nature (Barlow and Hall 2003b). We were therefore able to predict that the TEM ß-lactamase is likely to evolve resistance to cefepime in the near future. Similar results for the Citrobacter freundii AmpC ß-lactamase (Barlow and Hall, 2003a) allow us to conclude that our in vitro evolution method is generally applicable to predicting the natural evolution of antibiotic resistance genes.

Resistance to aminoglycoside antibiotics such as kanamycin, tobramycin, amikacin, and gentamicin is often mediated by enzymes that modify those drugs by acetylation, adenylation, or phosphorylation. The AAC(6') enzymes inactivate their substrates by acetylating the aminoglycosides at the 6' position. Two families of AAC(6') enzymes have been distinguished on the basis of their substrate specificities. AAC(6')-I enzymes confer resistance to amikacin but not to gentamicin. AAC(6')-II enzymes confer resistance to gentamicin but not to amikacin (Rather et al. 1992; Shaw et al. 1993)

In contrast to the situation exemplified by the TEM ß-lactamases, the AAC(6')-I enzymes are remarkably homogeneous (Shaw et al. 1993; Casin et al. 1998) and show no evidence of having increased either their substrate range or the efficiency with which they acetylate their substrates in response to the selection pressure imposed by the clinical use of aminoglycoside antibiotics. The failure to evolve increased efficiency or an increased substrate range might reflect insufficient exposure to selective pressure from aminoglycoside antibiotics, or it might reflect the fact that the AAC(6')-I enzymes are not nearly as widespread or found as frequently on plasmids as are the TEM ß-lactamases. There simply may not have been sufficient time for the evolutionary potential for improvement yet to be realized. Alternatively, it might be the case that the potential for improvement simply does not exist and that the AAC(6')-I enzymes have reached the limits of their evolutionary potential.

The AAC(6')-Ib enzyme is sufficiently similar to the AAC(6')-II enzymes that a Leu119Ser substitution in AAC(6')-Ib shifts its substrate spectrum to that of an AAC(6')-II enzyme (Rather et al. 1992), and naturally occurring variants of AAC(6')-Ib that possess a serine at position 119 also acetylate gentamicin but not amikacin (Casin et al. 1998). Here we use in vitro evolution of a typical AAC(6')-I enzyme in order to determine whether those enzymes possess the evolutionary potential to (1) increase the efficiency with which they acetylate tobramycin, kanamycin, or amikacin or (2) acquire the ability to acetylate gentamicin effectively. Changing Leu119Ser in AAC(6')-Ib allows the enzyme to acetylate gentamicin but results in the loss of the ability to acetylate amikacin (Rather et al. 1992; Casin et al. 1998). There is a trade-off in substrate specificities. In contrast, in vitro evolution of TEM ß-lactamases does not result in such trade-offs when new substrate specificities are evolved (Barlow and Hall 2002b). If typical AAC(6')-I enzymes possess the potential to acetylate gentamicin, it is of interest to determine whether or not that potential involves substrate trade-offs.

Magnet, Courvalin, and Lambert (1999) isolated a tobramycin-resistant strain of Salmonella enterica from the clinical environment and determined that the resistance was conferred by a 6'-N-acetyltransferase designated aac(6')-Iy. Genetic analysis showed that the only difference between that isolate and an aminoglycoside sensitive strain of the same organism was a 60 kilobase deletion present in the chromosome of the tobramycin-resistant strain that fused the cryptic aac(6')-Iy to a strong promoter, thus the aac(6')-Iy gene was silent, but fully functional, in the tobramycin-sensitive strain. Both when expressed in the original host, Salmonella enterica, and when cloned onto a plasmid and expressed in E. coli, AAC(6')-Iy exhibited the resistance profile typical of an AAC(6')-I enzyme. A Blast search of the Salmonella enterica serovar typhimurium strain LT2 complete genome (GenBank accession number NC_003197) showed that the LT2 genome includes a gene whose product differs from AAC(6')-Iy by only two amino acids. We have designated that gene aac(6')-Iaa. For reasons of convenience we have cloned aac(6')-Iaa and used it as the target of our in vitro evolution studies.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 Literature Cited
 
Phylogenetic Analysis
Phylogenetic trees were constructed by the Bayesian method as previously described (Barlow and Hall 2002b). Briefly, protein sequences were aligned using ClustalX (Thompson et al. 1997) and gaps were introduced into the corresponding DNA coding sequences using CodonAlign (Hall 2001). MrBayes (Huelsenbeck and Ronquist 2001) was used to construct a Bayesian tree from the DNA alignment. Table 1 shows the GenBank accession numbers of the coding sequences used to construct the trees.


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Table 1 Sequences Used to Construct Phylogeny Shown in Figure 1.

 
Isolation of the aac(6')-Iaa Gene
The aac(6')-Iaa gene was amplified from genomic DNA of Salmonella enterica serovar typhimurium strain LT2 using the Failsafe PCR System with Premix B Buffer (Epicentre Technologies) and primers 1 and 2 according to manufacturer's instructions and purified with QIAquick PCR Purification Kit (Qiagen). Primer 1 (5'-GGCCATGGACGGGCATCACCAACGCTAC-3') included a NcoI site upstream of the start codon, and primer 2 (5'-GGGAGCTCGTTGGGTGGCGCCAGAAATA-3') included a SacI site downstream of the stop codon. The aac(6')-Iaa amplicon was digested with restriction endonucleases SacI and NcoI (New England Biolabs) and ligated into similarly digested plasmid pACSE2 (Barlow and Hall 2002b) to create plasmid pAAC(6')-Iaa. The aac(6')-Iaa gene in plasmid pAAC(6')-Iaa is expressed from the strong pTAC promoter under the control of the plasmid-encoded lacIq gene by induction with 1 mM IPTG (isopropyl-ß-D-thiogalactopyranoside). The authenticity of the sequence of the cloned aac(6')I-Iaa gene was confirmed by sequencing using the ABI PrismTM BigDyeTM Terminator Cycle Sequencing Ready Reaction Kit (PE Applied Biosystems).

Antibiotics and Determination of Resistance by the Minimum Inhibitory Concentration (MIC) and Disk Diffusion Methods
Amikacin (Bedford Laboratories), kanamycin (Sigma), gentamicin (Abbott Laboratories), and tobramycin (Eli Lilly and Company) were used in this project. Stock solutions of antibiotics were prepared in 0.1 M NaPO4 buffer, pH 7.0, filter sterilized, and stored at –80°C in single-use aliquots. All MICs were determined in Mueller Hinton broth containing 1 mM IPTG according to Barlow and Hall (2002a). To evaluate resistance by the disk diffusion methods, cultures were grown in L-broth containing tetracycline overnight, spread onto Mueller-Hinton medium agar plates containing 1 mM IPTG, and incubated for 1 h to allow gene expression. BBL antibiotic disks containing 30 µg amikacin, 10 µg tobramycin, 10 µg gentamicin, or 30 µg kanamycin or 10 µg streptomycin were applied to all plates. After 16 h of incubation, the zones of inhibition observed for the wild-type strain were compared with those of each mutant strain.

Artificial Evolution of the aac(6')-Iaa Gene
The aac(6')-Iaa gene was PCR amplified from plasmid pAAC(6')-Iaa with an annealing temperature of 65°C using the GeneMorphTM PCR Mutagenesis Kit (Stratagene) with primers 3 (5'-ACTCTCTTCCGGGCGCTATCAT-3') and 4 (5'-TCATCCGGCTCGTATAATGTGTGGA-3') according to the manufacturer's instructions. Amplification was carried out using the number of cycles expected to result in a mutation frequency of two mutations per gene copy. To obtain sufficient DNA for cloning, the mutagenized amplicon was reamplified using the Failsafe PCR System, the product was cloned as described above and transformed into competent E. coli DH5{alpha}-E. After expression, the cells were transferred to L-broth containing 1mM IPTG, 10-fold serial dilutions of the culture were plated onto L-tetracycline medium to estimate the library size, and tetracycline was added to the culture. To estimate the fraction of cells containing insert-bearing plasmid, nine colonies were randomly selected from the plates and analytical PCR was performed with primers 3 and 4 using Taq PCR Master Mix (Gibco). To estimate the true mutation frequency, plasmid purified from 10 random isolates of the library was sequenced with primer 3.

Selection for Enhanced Resistance
The library was distributed to aliquots of Mueller-Hinton broth containing 1 mM IPTG so that each aliquot contained a number of cells equal to five times the original library size. In twofold increments, antibiotics were added at concentrations ranging from the MIC of the wild-type clone to 32 times that value, and after 48 h, the populations growing at the highest concentration of each antibiotic were harvested. Plasmid was purified, electroporated into DH5{alpha}-E, and transformants were selected on L-tetracycline medium. The resistance conferred by the original wild-type clone and an average of five isolates from each transformed population was determined by the disk diffusion method.


    Results and Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 Literature Cited
 
Phylogenetic Analysis
The aminoglycoside acetyl transferases are classified on the basis of the substrate atom that is acetylated (Shaw et al. 1993), and the AAC(6') enzymes have, on the basis of functional similarity and similar protein sizes (145 to 193 amino acids, mean 161 ± 3.7 amino acids), been considered to form a single family that is subdivided into two subfamilies, AAC(6')-I and AAC(6')-II, on the basis of substrate range. It has been pointed out, however, that on the basis of sequence similarity, the AAC(6')-I subfamily can be further divided into two distinct groups, one being AAC(6')-Ia, -Ii, and -Il, and the other being all of the other AAC(6')-I group except AAC(6')-Ib, which belongs to the AAC(6')-II subfamily (Shaw et al. 1993; Hannecart-Pokorni et al. 1997). Nevertheless, the several published phylogenetic trees of the AAC(6') enzymes all place the entire AAC(6') family onto a single tree, and the enzymes are always discussed as though they are descended from a common ancestor (Shaw et al. 1992, 1993; Hannecart-Pokorni et al. 1997; Wu et al. 1997). A Bayesian phylogeny of all of the currently known AAC(6') genes shows extremely long branches connecting the three clades (fig. 1). Those branches suggest that the three clades may not be homologous. There is no single, well-accepted criterion for determining whether two sequences are or are not homologous, but a reasonable criterion for nonhomology is that a pairwise Blast alignment of the two sequences fails to find a significant match between the two sequences. Three representatives of each the major clades shown in figure 1—AAC(6')-Ig, AAC(6')-Ih, and AAC(6')-Iy from clade A; AAC(6')-Ia, AAC(6')-Ii, and AAC(6')-Iq from clade B; and AAC(6')-IIb, AAC(6')-IIc, and AAC(6')-Im from clade C—were subjected to pairwise Blast alignments of the protein sequences in all possible pairwise combination. In no case did members of different clades yield significant alignments, whereas members of the same clade always produced significant alignments with the E-scores of those alignments ranging from 3 x 10–67 to 1 x 10–10. Furthermore, Blast searches of the nonredundant protein sequence database using each of the representative sequences showed that each produced significant alignments with all of the other members of its group but not with any members of the other groups. Thus, despite their functional and protein size similarities, the three "subfamilies" exhibit no sequence homology with each other and should no longer be referred to as members of the same family. We suggest the following families of AAC(6') aminoglycoside acetyltransferases: AAC(6')[A] consisting of AAC(6')-Ic, -Ig, -Ih, -Ij, -Ik, -Il, -Ir, -Is, -It, -Iu, -Iv, -Iw, -Ix, -Iy, -Iz, and -Iaa; AAC(6')[B], consisting of AAC(6')-Ia, -Ii, and -Iq; and AAC(6')[C] consisting of AAC(6')-Ib, -Ie, -Im, -IIa, -IIb, -IIc, and -IId. Proteins of each of the three families were aligned separately, and those alignments were used to construct phylogenetic trees of the families (fig. 2). Acetylation of aminoglycosides at the 6' position may well have evolved independently on at least three occasions.



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FIG. 1. False Bayesian phylogenetic tree of the AAC(6') coding sequences. MrBayes was run for 3,000,000 generations and the first 1,000 of the total of 30,000 trees were excluded from the consensus tree. See table 1 for organisms and GenBank accession numbers corresponding to abbreviations shown

 


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FIG. 2. Phylogenetic trees of the AAC(6')[A], AAC(6')[B], and AAC(6')[C] families. Trees of the [A] and [C] families were constructed by the Bayesian method during which MrBayes was run for 1,000,000 generations and the first 2,000 of the total of 10,000 trees were excluded from the consensus tree. MrBayes does not permit constructing trees of less than four taxa, therefore the [B] tree was constructed by the Neighbor-Joining method using PAUP* (Swofford 2000). Probabilities of clades that are less than 90% are shown for families [A] and [C]

 
This situation is completely analogous to that of the serine ß-lactamases in which there are three families, A, C, and D, that exhibit no sequence homology within classes. Those families do, however, exhibit clear homology at the structural level (Ambler 1980; Jaurin and Grundstrom 1981; Ouellette, Bissonnette, and Roy 1987) and are clearly descended from a common ancestor. Only three aminoglycoside-N-acetyl transferases have been crystallized and characterized at the structural level: AAC(6')-Ii (Wybenga-Groot et al. 1999), AAC(2')-Ic (Vetting et al. 2002), and AAC(3')-I (Wolf et al. 1998). All are members of the GCN5-related acetyltransferases (Wolf et al. 1998; Vetting et al. 2002). Although it seems likely that the three families of AAC(6') enzymes are also members of the GCN5-related acetyltransferases, in the absence of structural information on members of the A and C families of AAC(6') enzymes, it is not possible to be sure of that. In the absence of that information we do not know whether the three families of AAC(6') enzymes are more closely related to each other than they are to the AAC(2') or AAC(3') enzymes or, indeed, whether they are more closely related to other aminoglycoside acetyl transferases than they are to other members of the GCN5-related acetyl transferase superfamily.

Because enzymes with typical type I and typical type II substrate specificities are present in the AAC(6')[C] family, it is clear that the potential for effective acetylation of both gentamicin and amikacin can exist within a single family. To determine whether a typical member of the AAC(6')[A] family has the potential either for gentamicin acetylation or for improved acetylation of tobramycin, kanamycin, or amikacin, we subjected the aac(6')[A]-Iaa gene to in vitro evolution.

In Vitro Evolution of the aac(6')-Iaa Gene
Gene aac(6')-Iaa of Salmonella typhimurium LT2 was cloned into E. coli strain DH5{alpha}-E, and sequencing confirmed that plasmid pAAC(6')-Iaa contained a copy of the gene identical to the reported nucleotide sequence. All further experiments were carried out using pAAC(6')-Iaa. Synthesis of AAC(6')-Iaa from the plasmid is under control of the plasmid-encoded Lac repressor and is induced by IPTG (isopropyl-ß-D-thiogalactopyranoside). The addition of IPTG at a concentration of 1 mM resulted in an MIC of 16 µg/mL tobramycin, identical to the tobramycin resistance conferred by the tobramycin-resistant S. enterica strain identified by Magnet, Courvalin, and Lambert (1999). That concentration of IPTG was used in the remainder of the experiments.

MIC analysis showed that both E. coli strain DH5{alpha}-E containing the vector pACSE2 and Salmonella typhimurium LT2 were sensitive to all of the aminoglycoside antibiotics used: tobramycin, amikacin, gentamicin, and kanamycin (table 2). DH5{alpha}-E containing pAAC(6')-Iaa exhibited greater than a clinical level of resistance (National Committee for Clinical Laboratory Standards 1999) to tobramycin and kanamycin, as well as a significantly improved resistance to amikacin. The characterization of aac(6')-Iaa as a 6'-N-aminoglycoside acetyltransferase enzyme of the type I classification was supported by the enzyme's substrate specificities (Shaw et al. 1993). The aac(6')-Iaa gene appeared to confer similar resistance properties to gene aac(6')-Iy (Magnet, Courvalin, and Lambert 1999), although the difference in host strains used in the two experiments makes a direct comparison impossible.


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Table 2 Minimum Inhibitory Concentrations.

 
It has recently been shown that mutagenic PCR can be used to accurately mimic the natural evolution of antibiotic resistance genes (Barlow and Hall 2002b). That same process has also been employed to make strong predictions about how current resistance genes are likely to continue evolving under conditions of antibiotic-mediated selection (Barlow and Hall 2003b). The GeneMorphTM PCR Mutagenesis system utilizes an error-prone DNA polymerase that is capable of introducing mutations with the same mutagenic spectrum as E. coli (Barlow and Hall 2002c). Using that system, the rate of mutation can be controlled by varying the number of cycles in the PCR reaction. Mutagenic PCR was performed so that an average of two mutations would be introduced per copy of aac(6')-Iaa. Sequencing random isolates from the libraries later revealed that an average mutation rate of 1.7 mutations per gene copy had been achieved. The amplicons were cloned into E. coli, producing seven libraries with 3.3 ± 0.79 x 106 (mean ± standard error) mutants per library.

In order to mimic the conditions of antibiotic mediated selection in the clinical setting, the four most commonly prescribed aminoglycoside antibiotics were used as selective agents; tobramycin, amikacin, gentamicin, and kanamycin (Top 200 Most Prescribed Drugs, 2002, http://www.rxlist.com/top200.htm). Cells were harvested from cultures containing the highest concentration of each antibiotic that permitted growth, and plasmid was purified from those populations and transformed into E. coli strain DH5{alpha}-E. Because mutants exhibiting the highest level of antibiotic resistance come to dominate a population during growth in the presence of those antibiotics, it was necessary to sample only a small number of isolates from each retransformed population to ensure that the most fit alleles had been recovered. A total of 140 isolates from the seven libraries were screened by the disk diffusion method, but none exhibited a greater level of aminoglycoside resistance than the wild-type clone. MICs were determined for a subset of 20 of those isolates, and it was confirmed that none exhibited higher MICs for any of the drugs than did the wild-type clone.

These results lead us to predict that the aac(6')-Iaa gene does not have the potential to evolve either increased activity toward any of its present substrates, amikacin, kanamycin, and tobramycin, or resistance toward gentamicin.

Our confidence in that evolutionary prediction is based on a simulation of the in vitro evolution process using the program In vitro Evolution Simulator (Hall 2002). That program first calculates the number of possible single and double amino acid substitutions that can result from single and double mutations for the input coding sequence. Then, given the average number of mutations per molecules, in each cycle the program simulates the introduction of random mutations poisson distributed about the average number of mutations per molecule and determines the resulting amino acid substitutions. As the program is run for a number of cycles equivalent to the library size, it calculates the fraction of possible single-substitution and double-substitution sequences that are present in a library of that size. For the AAC(6')-Iaa sequence, there are 2,165 possible single amino acid substitutions and 1,699,110 possible double amino acid substitutions that can result from one or two mutations. Each library of 3.3x106 molecules is expected to include 93.3% of the possible single amino acid substitutions and 75.6% of the possible double amino acid substitutions. (Note that some single amino acid substitutions require two base substitutions in the same codon.) With a total of seven such libraries, the probability of having missed any particular single amino acid substitution molecule by chance alone is 6 x 10–9, and that of having missed a double amino acid substitution molecule is 5 x 10–5.

In nature, mutations almost always arise one at a time, and each mutation must be fixed into microbial populations by selection. For populations as large as microbial and plasmid populations, fixation by random drift of any particular neutral mutation that might potentially be advantageous in the presence of another otherwise neutral mutation can largely be ignored. With respect to increased antibiotic resistance, this means that during selection for antibiotic resistance, each mutation must confer an increase in effectiveness in order to be fixed into the population. Therefore, it is only important to consider the effects of one or two independent base substitution mutations when attempting to predict the natural evolutionary potential of a given gene.

The relatively short coding sequence of aac(6')-Iaa has allowed us the rare opportunity of being able to fully evaluate the evolutionary potential of a gene. It possible to conclude with a 99.99% confidence that no single amino acid substitution or combination of two independent amino acid substitutions in aac(6')-Iaa is capable of increasing resistance to any of the antibiotics used. In light of its inability to evolve either an increase in activity or an extended substrate specificity, aac(6')-Iaa, even if mobilized to a plasmid, is unlikely to become a problem of clinical significance.

The finding that aac(6')-Iaa has reached the limit of its evolutionary potential with respect to the selected substrates is consistent with the observation that there has been little phenotypic evolution of the AAC(6') proteins in nature. More importantly, the attempt to evolve aac(6')-Iaa serves as a model for exploring the limits of evolutionary potential for other antibiotic resistance genes. Our expectations for the evolution of antibiotic resistance genes has been shaped to a great extent by the enormous phenotypic plasticity of the TEM ß-lactamases. Those expectations have led us to assume that all resistance genes will evolve new substrate specificities in response to the selection imposed by the clinical use of new antibiotics, and those expectations, in turn, shape public policy with respect to administration of those drugs. For instance, it is the expectation that the metallo-ß-lactamases, which already exhibit a low level of activity toward the carbapenem ß-lactams, will evolve sufficient activity to confer clinical resistance that leads to strong limitations on the use of carbapenems in hospitals. Experiments similar to those described above could be used to determine whether potential actually exists with the possible result of relaxing restrictions on carbapenem use if it can be shown that metallo-ß-lactamases do not have the potential for increased carbapenem hydrolysis.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 Literature Cited
 
We thank S. Mookerjee for her preliminary work on this project. This project was supported by grant GM60761 from the National Institutes of Health.


    Footnotes
 
E-mail: drbh{at}mail.rochester.edu. Back


    Literature Cited
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 Literature Cited
 

    Ambler, R. P. 1980. The structure of beta-lactamases. Philos. Trans. R. Soc. Lond. B Biol. Sci. 289:321-331.[ISI][Medline]

    Barlow, M., and B. G. Hall. 2002a. Origin and evolution of the AmpC ß-lactamases of Citrobacter freundii. Antimicrob. Agents Chemother. 46:1190-1198.[Abstract/Free Full Text]

    Barlow, M., and B. G. Hall. 2002b. Predicting evolutionary potential: in vitro evolution accurately reproduces natural evolution of the TEM ß-Lactamase. Genetics 160:823-832.[Abstract/Free Full Text]

    Barlow, M., and B. G. Hall. 2003a. Experimental prediction of the evolution of cefepime resistance from the CMY-2 AmpC ß-lactamase. Genetics (in press).

    Barlow, M., and B. G. Hall. 2003b. Experimental prediction of the natural evolution of antibiotic resistance. Genetics (in press).

    Casin, I., F. Bordon, P. Bertin, A. Coutrot, I. Podglajen, R. Brasseur, and E. Collatz. 1998. Aminoglycoside 6'-N-acetyltransferase variants of the Ib type with altered substrate profile in clinical isolates of Enterobacter cloacae and Citrobacter freundii. Antimicrob. Agents Chemother. 42:209-215.[Abstract/Free Full Text]

    Hall, B. G. 2001.. CodonAlign, Rochester, N.Y.

    Hall, B. G. 2002.. In vitro Evolution Simulator, Rochester, N.Y.

    Hannecart-Pokorni, E., F. Depuydt, L. de wit, E. van Bossuyt, J. Content, and R. Vanhoof. 1997. Characterization of the 6'-N-aminoglycoside acetyltransferase gene aac(6')-Im [corrected] associated with a sulI-type integron. Antimicrob. Agents Chemother. 41:314-318.[Abstract]

    Huelsenbeck, J. P., and F. Ronquist. 2001. MrBayes: Bayesian inference of phylogeny. Bioinformatics 17:754-755.[Abstract/Free Full Text]

    Jaurin, B., and T. Grundstrom. 1981. ampC cephalosporinase of Escherichia coli K-12 has a different evolutionary origin from that of beta-lactamases of the penicillinase type. Proc. Natl. Acad. Sci. USA 78:4897-4901.[Abstract]

    Magnet, S., P. Courvalin, and T. Lambert. 1999. Activation of the cryptic aac(6')-Iy aminoglycoside resistance gene of Salmonella by a chromosomal deletion generating a transcriptional fusion. J. Bacteriol. 181:6650-6655.[Abstract/Free Full Text]

    Medeiros, A. A. 1997. Evolution and dissemination of beta-lactamases accelerated by generations of beta-lactam antibiotics. Clin. Infect. Dis. 24:S19-45.[ISI][Medline]

    National Committee for Clinical Laboratory Standards. 1999. Performance standards for antimicrobial susceptibility testing; ninth informational supplement. NCCLS Document M100-S9. National Committee for Clinical Laboratory Standards, Wayne, Penn.

    Nordmann, P. 1998. Trends in beta-lactam resistance among Enterobacteriaceae. Clin. Infect. Dis. 27:S100-106.[ISI][Medline]

    Ouellette, M., L. Bissonnette, and P. H. Roy. 1987. Precise insertion of antibiotic resistance determinants into Tn21-like transposons: nucleotide sequence of the OXA-1 beta-lactamase gene. Proc. Natl. Acad. Sci. USA 84:7378-7382.[Abstract]

    Rather, P. N., H. Munayyer, P. A. Mann, R. S. Hare, G. H. Miller, and K. J. Shaw. 1992. Genetic analysis of bacterial acetyltransferases: identification of amino acids determining the specificities of the aminoglycoside 6'-N- acetyltransferase Ib and IIa proteins. J. Bacteriol. 174:3196-3203.[Abstract]

    Shaw, K. J., P. N. Rather, R. S. Hare, and G. H. Miller. 1993. Molecular genetics of aminoglycoside resistance genes and familial relationships of the aminoglycoside-modifying enzymes. Microbiol. Rev. 57:138-163.[ISI][Medline]

    Shaw, K. J., P. N. Rather, and F. J. Sabatelli, et al. (11 co-authors). 1992. Characterization of the chromosomal aac(6')-Ic gene from Serratia marcescens. Antimicrob. Agents Chemother. 36:1447-1455.[Abstract]

    Swofford, D. L. 2000. PAUP*: phylogenetic analysis using parsimony (*and other methods). Sinauer Associates, Sunderland, Mass.

    Thompson, J. D., T. J. Gibson, F. Plewniak, F. Jeanmougin, and D. G. Higgins. 1997. The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25:4876-4882.[Abstract/Free Full Text]

    Vetting, M. W., S. S. Hegde, F. Javid-Majd, J. S. Blanchard, and S. L. Roderick. 2002. Aminoglycoside 2'-N-acetyltransferase from Mycobacterium tuberculosis in complex with coenzyme A and aminoglycoside substrates. Nat. Struct. Biol. 9:653-658.[CrossRef][ISI][Medline]

    Wolf, E., A. Vassilev, Y. Makino, A. Sali, Y. Nakatani, and S. K. Burley. 1998. Crystal structure of a GCN5-related N-acetyltransferase: Serratia marcescens aminoglycoside 3-N-acetyltransferase. Cell 94:439-449.[ISI][Medline]

    Wu, H. Y., G. H. Miller, M. G. Blanco, R. S. Hare, and K. J. Shaw. 1997. Cloning and characterization of an aminoglycoside 6'-N- acetyltransferase gene from Citrobacter freundii which confers an altered resistance profile. Antimicrob. Agents Chemother. 41:2439-2447.[Abstract]

    Wybenga-Groot, L. E., K. Draker, G. D. Wright, and A. M. Berghuis. 1999. Crystal structure of an aminoglycoside 6'-N-acetyltransferase: defining the GCN5-related N-acetyltransferase superfamily fold. Struct. Fold Des. 7:497-507.[Medline]

Accepted for publication December 9, 2002.