Experimental Prediction of the Natural Evolution of Antibiotic Resistance

Copyright 2003 by the Genetics Society of America Experimental Prediction of the Natural Evolution of Antibiotic Resistance Miriam Barlow and Barry G. Hall 1 Biology Department, University of Rochester, Rochester, New York 14627-0211 Manuscript received July 22, 2002 Accepted for publication December 20, 2002 ABSTRACT The TEM family of -lactamases has evolved to confer resistance to most of the -lactam antibiotics, but not to cefepime. To determine whether the TEM -lactamases have the potential to evolve cefepime resistance, we evolved the ancestral TEM allele, TEM-1, in vitro and selected for cefepime resistance. After four rounds of mutagenesis and selection for increased cefepime resistance each of eight independent populations reached a level equivalent to clinical resistance. All eight evolved alleles increased the level of cefepime resistance by a factor of at least 32, and the best allele improved by a factor of 512. Sequencing showed that alleles contained from two to six amino acid substitutions, many of which were shared among alleles, and that the best allele contained only three substitutions. 2001). Developing effective strategies requires under- standing how antibiotic resistance genes are most likely to evolve in response to the introduction of new antibi- otics. Because nature has already shown that one resistance gene can give rise to many phenotypically diverse de- scendant alleles, protein engineers have used in vitro evolution of resistance genes as models for exploring better strategies for protein engineering (Stemmer 1994). The resistance gene most commonly used as a model for protein engineering is the TEM-1 -lacta- mase. TEM-1 is one of the best-studied antibiotic resis- tance genes because it exists at high frequencies in anti- biotic-resistant bacteria across the globe (Medeiros 1997; Chanal et al. 2000; Yan et al. 2000). TEM-1 is clinically important because it confers resistance to peni- cillin and other -lactam antibiotics. -Lactams are often the preferred antibiotics because of their low toxicity and the broad spectrum of bacteria that they affect (Livermore 1996). While TEM-1 has a spectrum that is limited to penicillins and early cephalosporins, it has given rise to 90 descendent alleles that confer resistance to most modern -lactam antibiotics (http://www. rochester.edu/college/bio/labs/halllab/tem_phylo. html and http://www.lahey.org/studies/webt.htm). At this point, there are only a handful of -lactam antibiotics to which the TEM -lactamases do not confer resistance. The TEMs are still evolving, and it will be valuable to know what their potential for new resistance is before that potential is realized. While others have used the TEM enzyme to demon- strate the power of their in vitro evolution methods for protein engineering, we have developed an in vitro evolution method for the specific purpose of predicting how resistance genes will evolve in nature. We recently showed that we could reproduce the natural evolution of the TEM -lactamases that has occurred during the WHEN penicillin was first shown to be an effective antibiotic in the 1940s, the expectation that infectious diseases could be controlled or even eradicated appeared to be realistic. Dreaded infections such as pneumonia and tuberculosis became curable, but just when we started to feel secure about our victory over microbes, antibiotic-resistant strains of bacteria started appearing at high frequencies (Cohen 2000). While it is true that antibiotics continue to cure most infections, it is increasingly difficult to identify antibiotics that can be used as effective treatments because of the diversity of resistant strains that exist. Resistance to every antibiotic in clinical use has been observed throughout the world (Cohen 2000), and in many cases microbial strains exhibit resistance to multiple antibiotics. In most cases, resistance to a new antibiotic arises within 3 years of the antibiotic s FDA approval date (Medeiros 1997). Resistance genes produce enzymes that either modify or eliminate an antibiotic. Resistance genes can be spread by vertical transmission from parent to offspring or by horizontal transfer between different strains and species of bacteria. Sensitive microbes become resistant to antibiotics either through the acquisition of plasmid- borne resistance genes or through mutations that either upregulate the expression of a resistance gene or alter the binding or substrate specificity of an enzyme en- coded by a resistance gene. As resistance arises, the pharmaceutical industry races to create new antibiotics more quickly than the current antibiotics become obsolete (McGowan 2001). Doctors and hospitals continuously revise their strategies for treating infections to limit the occurrence and spread of antibiotic resistance (Courvalin and Trieu-Cuot 1 Corresponding author: Biology Department, Hutchison Hall, River Campus, University of Rochester, Rochester, NY 14627-0211. E-mail: drbh@mail.rochester.edu Genetics 163: 1237 1241 (April 2003)

1238 M. Barlow and B. G. Hall past 20 years. Phylogenetic analysis showed that nine Our in vitro evolution method differs from other methods amino acid substitutions have arisen multiple times in in that (a) it permits precise control over the average number of mutations introduced into each molecule; (b) it utilizes nature in response to the use of a class of -lactams an enzyme whose mutagenic spectrum is very similar to the known as the extended spectrum cephalosporins. Be- spontaneous mutagenic spectrum of E. coli; (c) it introduces cause those nine substitutions were selected multiple mutations at random positions throughout each molecule; times in nature, it is clear that they are important for (d) it selects at clinically realistic drug concentrations; and resistance to extended spectrum cephalosporins (Barfor maintenance of important wild-type resistance phenotypes, (e) it uses more realistic selection regimes, including selection low and Hall 2002). Structural and biochemical analy- e.g., continued resistane to ampicillin. While other approaches sis of those substitutions has confirmed their importance have incorporated various elements of our method (Stemmer for increasing cephalosporin resistance (Knox 1995). 1994; Vakulenko et al. 1998; Blazquez et al. 2000), none Among 10 alleles that were independently evolved in have incorporated all. vitro by our method, we repeatedly recovered seven of Following selection, plasmid from each library was prepared from the highest concentration of cefepime at which growth the nine substitutions that have arisen multiple times occurred. Those plasmid preparations were then used as startin nature (Barlow and Hall 2002). This means that ing material for the next round of mutagenesis. our in vitro evolution method creates and selects the same mutations that are found in natural TEM alleles. Cefepime is a relatively new -lactam antibiotic that RESULTS AND DISCUSSION received FDA approval in 1996. Because our in vitro Resistance to an antibiotic can be quantified by deevolution method accurately reproduces the natural termining the MIC of the antibiotic on a bacterial strain. evolution of the TEM -lactamases, we can use the same The MIC is the lowest concentration of the antibiotic method to predict how the TEM -lactamases will evolve that can completely block microbial growth. An MIC of in the future. In this study we have used our in vitro 32 g/ml is the breakpoint for clinical resistance to evolution method to determine whether a new pheno- cefepime (National Committee for Clinical Labotype, resistance to cefepime, will arise through natural ratory Standards 2001), but the highest reported evolution of the TEM -lactamases. level of TEM-conferred cefepime resistance is an MIC of 8 g/ml (Perilli et al. 2000; Rebuck et al. 2000). While it is possible that the TEM -lactamases lack the MATERIALS AND METHODS potential to evolve high levels of activity toward cefepime, Esherichia coli strain DH5 E [F φ80dlacz M15 (laczyaduction it is also possible that because of the recent intro- argf)u169 enda1 reca1 hsdr17(r m ) deor thi-1 phoa supe44 and relatively low use of cefepime the TEM gyra96 rela1 gal ] (GIBCO, Gaithersburg, MD) was used -lactamases have not had time to evolve resistance to as the host for all plasmids. Plasmid pacse3 (Barlow and this antibiotic. Hall 2002) was used as the vector for cloning and expressing To determine which amino acid substitutions, if any, TEM alleles. Site-directed mutagenesis was performed acincrease the specificity of the TEM -lactamases for cefecording to the manufacturer s instructions using the Quickpime, we created, in E. coli K12 strain DH5- E, eight Change kit from Stratagene (La Jolla, CA). In vitro mutagenesis, cloning, sequencing, and determina- independent libraries of mutant TEM alleles and setion of minimum inhibitory concentrations (MICs) of antibiot- lected for increased cefepime resistance using an apics were as previously described (Barlow and Hall 2002). proach similar to the one previously described (Barlow Selection of evolved mutants was as previously described (Barand Hall 2002). Four cycles of mutagenesis and seleclow and Hall 2002) except that only one drug, cefepime, was used as the primary selective agent, and only ampicillin tion were required to obtain cells that could grow in was used as a selective agent to ensure that a preexisting cefepime at a concentration of at least 64 g/ml. Plasresistance phenotype had been retained. Briefly, the TEM-1 mid from those cells was harvested and retransformed gene was mutagenized using the error-prone polymerase Muinto naïve DH5- E to eliminate any host selection that tazyme (Stratagene) in a PCR reaction under conditions that generated an average of two mutations per molecule. Muta- had occurred during growth in cefepime or ampicillin. genized genes were cloned into pacse3, a low-copy-number Ten individual colonies from each transformation were vector based on plasmid pacyc184 (Barlow and Hall 2002) screened for resistance to cefepime by disk diffusion and transformed into E. coli strain DH5- E, and the resulting test as previously described (Barlow and Hall 2002) libraries were grown in increasing concentrations of cefepime. and a single cefepime-resistant colony from each trans- Each of the eight mutant libraries was passaged twice through twofold serial dilutions of cefepime (64 0.5 g/ml), once formation was chosen. In each set among the 10 colo- through ampicillin (64 g/ml), and then once again through nies, only one resistant phenotype was present. This a dilution series of cefepime (64 0.5 g/ml). For each library, pattern is consistent with our earlier observation (Bar- cells taken from the highest concentration of cefepime at low and Hall 2002) that during selection a single clone which growth occurred were used to inoculate the next cul- comes to dominate the population. The final result was ture. Multiple passages through cefepime ensured that cefetherefore eight independent alleles, each of which was pime-resistant alleles dominated the culture. The single pasderived from an independent library of mutated TEM-1 sage through ampicillin required that the mutant alleles also maintain the ability to confer resistance to ampicillin, a commonly alleles, and each of which was the result of four rounds used -lactam. of mutation and selection.

Predicting Evolution 1239 TABLE 1 MICs (in micrograms per milliliter) Drug Cefepime Ampicillin Pipericillin Cefuroxime Cefotaxime Ceftazidime Aztreonam Clinical resistance MIC a 32 32 128 32 64 32 32 pacse3 b 0.03125 8 2 16 0.5 1 1 TEM1 0.5 4096 2048 0.25 0.125 1 0.5 Clone 1 128 4096 4096 16 16 1024 32 Clone 2 32 4096 4096 16 4 256 16 Clone 3 32 4096 4096 16 8 256 32 Clone 4 32 4096 4096 16 2 256 8 Clone 5 32 4096 4096 16 4 512 32 Clone 6 16 2048 2048 16 2 256 16 Clone 7 64 4096 4096 16 8 512 64 Clone 8 256 4096 4096 32 32 2048 256 R164H 2 4096 2048 8 1 32 4 I173V 0.5 4096 4096 16 1 2 1 R178S 0.25 4096 256 16 0.25 2 0.5 R164H I173V 32 4096 4096 16 4 256 16 R164H R178S 16 4096 2048 16 2 128 8 R164H I173V 256 4096 4096 32 32 2048 256 R178S a MIC necessary to be considered clinically resistant as defined by the National Committee for Clinical Laboratory Standards (1999). b The vector into which all alleles are cloned. The MICs of several -lactam antibiotics for each cho- simultaneously introduces multiple substitutions, it is sen allele are shown in Table 1. All alleles increased possible to recover phenotypes from in vitro mutagenesis cefepime resistance relative to TEM-1. When TEM-1 is that would never arise in nature (Hall 2002). For example, expressed, cefepime has an MIC of 0.5 g/ml whereas, if two substitutions are individually deleterious, but when the evolved alleles are expressed, cefepime has advantageous when they are together, they would probably an MIC of 16 256 g/ml. Expression of allele 8 confers not go to fixation in nature, but they might well be the highest level of resistance. Although the libraries recovered through in vitro evolution procedures that were never exposed to ceftazadime or aztreonam, all introduce mutations at a high frequency. To verify that alleles confer significantly higher resistance than TEM-1 the mutations we recovered in the best allele, allele to those antibiotics. This demonstrates that resistance to 8, can also be recovered from natural evolution, we cefepime indirectly selects resistance to both aztreonam determined whether a pathway exists between TEM-1 and ceftazadime. and allele 8 in which the three substitutions found in The sequences of the evolved TEM alleles were deter- clone 8 can be introduced one at a time such that each mined and the differences between those sequences additional substitution confers an increase in cefepime and the sequence of TEM-1 are shown in Table 2. All resistance. eight alleles have an amino acid substitution at position To do that, we introduced each of the amino acid 164 and six of the eight alleles contain the substitution substitutions into the TEM-1 gene by site-directed mutagenesis. R164H. Six alleles also contain the substitution I173V. Of the three substitutions, we found that the The high frequency of those amino acid substitutions substitution R164H conferred the largest increase in suggests that both are important for resistance to cefepime. cefepime resistance (Table 1) with an MIC of 2 g/ml. The importance of those two substitutions is fur- To the R164H allele we added the substitution I173V ther suggested by the presence of both substitutions or R178S to produce two distinct double-mutant alleles, within the most resistant allele, allele 8. However, nei- R164H/I173V and R164H/R178S. Of those, we found ther of those substitutions exist in allele 1. Because allele that the R164H/I173V allele conferred the greatest in- 1 confers the second highest level of cefepime resis- crease in resistance, giving a cefepime MIC of 32 g/ tance, there are clearly multiple potential pathways for ml. To that allele we added the substitution R178S and TEM-1 to evolve cefepime resistance. found that cefepime resistance increased to a final MIC Because natural mutations generally occur one at a of 256 g/ml. Because sequential addition of those time and because our in vitro mutagenesis technique three single substitutions confers an increase in cefe-

1240 M. Barlow and B. G. Hall TABLE 2 Amino acid substitutions in evolved TEM alleles Amino acid substitutions DNA site Mutation Allele 1 Allele 2 Allele 3 Allele 4 Allele 5 Allele 6 Allele 7 Allele 8 22 G A V10I V10I 73 C T P27S 106 G T D38Y 123 A G S a 138 A G S 189 T C S 232 G A V80I 243 T C S 280 C G H96D 399 C T S 420 C A S 433 G A E147K 444 T C S 459 G T M155I 484 C G R164G 484 C A RI64S 485 G A R164H R164H R164H R164H R164H R164H 507 A G S 511 A G I173V I173V I173V I173V I173V I173V 514 C T P174S 517 A G N175D 526 C A R178S 539 T C M182T M182T 579 T C S S 639 A G S 665 C T A224V 672 G A S 705 C A S 791 G A S268N 847 A G I287V 853 C T H289Y a Silent mutation. pime resistance with each addition, there is a pathway through which natural selection can create a TEM allele that confers high levels of cefepime resistance. Other enzymes such as the OXA and metallo -lactamases confer resistance to cefepime; however, neither of those families of resistance genes are as widely dispersed or as common as the TEM genes. The substitution R164H has already been observed in numerous TEM alleles that are found in clinical isolates (http:// www.lahey.org/studies/webt.htm). To our knowledge, the substitutions I173V and R178S have never been observed in natural TEM alleles. Because only three amino acid substitutions are required to greatly increase the activity of the TEM enzymes toward cefepime, and because one of those mutations already exists in nature, it seems likely that the naturally occurring TEM -lactamases will evolve the ability to confer cefepime resistance. While we have used in vitro evolution to make specific predictions about how cefepime resistance will arise through modification of the TEM enzymes, the in vitro evolution method we have developed can also be used to make predictions about how other resistance genes may evolve in nature. The resources available to pharmaceutical companies would enable a more thorough analysis of the evolutionary pathways through which antibi- otic resistance may arise. In addition to predicting the ways in which specific enzymes can evolve the ability to confer resistance to different antibiotics, pharmaceutical companies could also use this method to test the effectiveness of using different antibiotic combinations to inhibit the evolution of resistance. For example, while it is clear that selection for resistance to cefepime indirectly selects TEM alleles that confer high levels of resis- tance to aztreonam and ceftazadime, it is possible that the TEM enzymes are not capable of conferring high levels of resistance to cefepime and cefotaxime at the same time. Combination of those two antibiotics may increase the time required for resistance to those antibiotics to evolve. Again, the resources available to industry

Predicting Evolution 1241 would permit screening a sufficient number of libraries Hall, B. G., 2002 Predicting evolution by in vitro evolution requires determining evolutionary pathways. Antimicrob. Agents Chemo- (at least 100) to determine whether combining cefotax- ther. 46: 3035 3038. ime and cefepime would effectively preclude resistance Knox, J. R., 1995 Extended-spectrum and inhibitor-resistant TEMto either drug from arising. The in vitro evolution type beta-lactamases: mutations, specificity, and three-dimen- sional structure. Antimicrob. Agents Chemother. 39: 2593 2601. method we have developed will allow pharmaceutical Livermore, D. M., 1996 Are all beta-lactams created equal? Scand. companies to assess the relative ease or difficulty with J. Infect. Dis. Suppl. 101: 33 43. which resistance to a new antibiotic or combination of McGowan, Jr., J. E., 2001 Economic impact of antimicrobial resis- tance. Emerg. Infect. Dis. 7: 286 292. antibiotic will arise. This information will help physi- Medeiros, A. A., 1997 Evolution and dissemination of beta-lactamases accelerated by generations of beta-lactam antibiotics. Clin. cians and hospitals to develop intelligent strategies for prescribing antibiotics before they ever see resistance Infect. Dis. 24: S19 S45. National Committee for Clinical Laboratory Standards, 1999 to them arise. The method we developed will also give Performance Standards for Antimicrobial Susceptibility Testing: Ninth pharmaceutical companies the ability to implement Informational Supplement. NCCLS Document M100 S9, National structural information about how resistance evolves into Committee for Clinical Laboratory Standards, Wayne, PA. National Committee for Clinical Laboratory Standards, 2001 designs for future antibiotics that they will design and Performance Standards for Antimicrobial Susceptibility Testing: Supplemental Tables. NCCLS Document M100 S11, National Committee produce. for Clinical Laboratory Standards, Wayne, PA. This study was supported by grant GM-60761 from the National Perilli, M., B. Segatore, M. R. de Massis, M. L. Riccio, C. Bianchi Institutes of Health. et al., 2000 TEM-72, a new extended-spectrum beta-lactamase detected in Proteus mirabilis and Morganella morganii in Italy. Antimicrob. Agents Chemother. 44: 2537 2539. Rebuck, J. A., K. M. Olsen, P. D. Fey, K. L. Bergman and M. E. Rupp, LITERATURE CITED 2000 In vitro activities of parenteral beta-lactam antimicrobials against TEM-10-, TEM-26- and SHV-5-derived extended-spectrum Barlow, M., and B. G. Hall, 2002 Predicting evolutionary potential: beta-lactamases expressed in an isogenic Escherichia coli host. in vitro evolution accurately reproduces natural evolution of the J. Antimicrob. Chemother. 46: 461 464. TEM -lactamase. Genetics 160: 823 832. Stemmer, W. P. C., 1994 Rapid evolution of a protein in vitro by Blazquez, J., M. I. Morosini, M. C. Negri and F. Baquero, 2000 DNA shuffling. Nature 370: 389 390. Selection of naturally occurring extended-spectrum TEM beta- Vakulenko, S. B., B. Geryk, L. P. Kotra, S. Mobashery and S. A. lactamase variants by fluctuating beta-lactam pressure. Anti- Lerner, 1998 Selection and characterization of beta-lactammicrob. Agents Chemother. 44: 2182 2184. beta-lactamase inactivator-resistant mutants following PCR muta- Chanal, C., R. Bonnet, C. De Champs, D. Sirot, R. Labia et al., genesis of the TEM-1 beta-lactamase gene. Antimicrob. Agents 2000 Prevalence of beta-lactamases among 1,072 clinical strains Chemother. 42: 1542 1548. of Proteus mirabilis: a 2-year survey in a French hospital. Antimi- Yan, J. J., S. M. Wu, S. H. Tsai, J. J. Wu and I. J. Su, 2000 Prevalence crob. Agents Chemother. 44: 1930 1935. of SHV-12 among clinical isolates of Klebsiella pneumoniae pro- Cohen, M. L., 2000 Changing patterns of infectious disease. Nature ducing extended-spectrum beta-lactamases and identification of 406: 762 767. a novel AmpC enzyme (CMY-8) in Southern Taiwan. Antimicrob. Courvalin, P., and P. Trieu-Cuot, 2001 Minimizing potential resis- Agents Chemother. 44: 1438 1442. tance: the molecular view. Clin. Infect. Dis. 33 (Suppl. 3): S138 S146. Communicating editor: H. Ochman