Biology Department, University of Rochester
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Abstract |
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Key Words: antibiotic resistance evolutionary potential phylogenetic analysis aminogylcoside
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Introduction |
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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.
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Materials and Methods |
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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-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-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.
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Results and Discussion |
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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-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-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
-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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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-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 109, and that of having missed a double amino acid substitution molecule is 5 x 105.
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.
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Acknowledgements |
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Footnotes |
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