Author response: Genetic determinants facilitating the evolution of resistance to carbapenem antibiotics (2021)

Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract In this era of rising antibiotic resistance, in contrast to our increasing understanding of mechanisms that cause resistance, our understanding of mechanisms that influence the propensity to evolve resistance remains limited. Here, we identified genetic factors that facilitate the evolution of resistance to carbapenems, the antibiotic of 'last resort', in Klebsiella pneumoniae, the major carbapenem-resistant species. In clinical isolates, we found that high-level transposon insertional mutagenesis plays an important role in contributing to high-level resistance frequencies in several major and emerging carbapenem-resistant lineages. A broader spectrum of resistance-conferring mutations for select carbapenems such as ertapenem also enables higher resistance frequencies and, importantly, creates stepping-stones to achieve high-level resistance to all carbapenems. These mutational mechanisms can contribute to the evolution of resistance, in conjunction with the loss of systems that restrict horizontal resistance gene uptake, such as the CRISPR-Cas system. Given the need for greater antibiotic stewardship, these findings argue that in addition to considering the current efficacy of an antibiotic for a clinical isolate in antibiotic selection, considerations of future efficacy are also important. The genetic background of a clinical isolate and the exact antibiotic identity can and should also be considered as they are determinants of a strain's propensity to become resistant. Together, these findings thus provide a molecular framework for understanding acquisition of carbapenem resistance in K. pneumoniae with important implications for diagnosing and treating this important class of pathogens. Introduction Antibiotic resistance is one of the most urgent threats to public health. Resistance has emerged to almost all clinically used antibiotics and in nearly all bacterial pathogen species. Numerous studies have focused on identifying and characterizing resistance mechanisms; meanwhile, our understanding of mechanisms that facilitate the evolution of resistance in clinical isolates is less well understood (MacLean and San Millan, 2019). As such, antibiotic efficacy as reflected in minimum inhibitory concentrations (MICs) remains almost the sole criterion to guide clinical antibiotic choice. However, more sophisticated antibiotic stewardship could help to preserve the existing arsenal of antibiotics by better matching individual strains to the antibiotic selected for treatment to minimize the frequency of the evolution of resistance during antibiotic exposure. Such stewardship would need to be informed by an increased understanding of the mechanisms that may affect the evolution of resistance including microbial intrinsic factors such as the genetic background of an isolate and extrinsic factors such as the antibiotic choice. Practically, understanding these mechanisms would result in the identification of genetic markers of a higher likelihood for resistance evolution, which could usher in a new era of more comprehensive diagnostics to guide more sophisticated antibiotic stewardship. Bacteria acquire antimicrobial resistance through horizontal gene transfer (HGT) or mutation, processes that can be influenced by intrinsic microbial genetic factors, such as phage defense systems and error prone polymerases, respectively (Marraffini and Sontheimer, 2008; Rosenberg, 2001). While HGT involves the acquisition of new resistance genes, mutation of existing genes can occur by acquisition of single-nucleotide polymorphisms (SNPs), insertions, deletions, recombination, or transposition events. At the same time, microbe extrinsic factors such as the antibiotic identity can also affect the evolution of resistance, as they vary in their ability to induce mutagenesis (Cirz et al., 2005), have different barriers to resistance (Blázquez et al., 2018), and vary in their spectrum of possible resistance-conferring mutations. The carbapenems, which are the latest generation of β-lactams, are often used to treat infections resistant to almost all antibiotics including extended-spectrum β-lactam antibiotics (Papp-Wallace et al., 2011; Zhanel et al., 2007). Carbapenem resistance thus typically emerges in bacteria that already carry extended-spectrum β-lactamases (ESBLs) and/or other β-lactamases (Cerqueira et al., 2017; Ma et al., 2018; Poirel et al., 2007). Carbapenem resistance is most often mediated by the production of carbapenemases. In the absence of carbapenemases however, resistance can be achieved through the acquisition of a combination of porin mutations to impede drug entry and/or significant increases in β-lactamase expression (Cerqueira et al., 2017; Ma et al., 2018; Poirel et al., 2007; Martínez-Martínez et al., 1999). Therefore, the evolution of carbapenem resistance often involves complex mechanisms of HGT and mutation acquisition. The Gram-negative pathogen K. pneumoniae is one of the most prevalent carbapenem-resistant Gram-negative species (Navon-Venezia et al., 2017; Wyres et al., 2020a). Within this species, carbapenem resistance occurs predominantly in a few clonal groups (CG), such as CG258, CG15, and CG20 (Cerqueira et al., 2017; Wyres et al., 2020a; DeLeo et al., 2014; Bowers et al., 2015; Pitout et al., 2015; Munoz-Price et al., 2013; Wyres et al., 2020b). While clonal spread plays a role in the dissemination of carbapenem resistance (Cerqueira et al., 2017; Bowers et al., 2015), the emergence of new highly resistant lineages (Rada et al., 2020; Marsh et al., 2019; Bonnin et al., 2020; Mathers et al., 2011; Strydom et al., 2020; Peirano et al., 2020) and the independent acquisition of carbapenem resistance by distinct CG258 strains (DeLeo et al., 2014; Marsh et al., 2019; Chen et al., 2014; Eilertson, 2019) suggest that ongoing evolution of carbapenem resistance also plays an important role. These observations suggest that the underlying genetic background of CG258 and other emerging lineages may contribute to a higher propensity for resistance acquisition. Recently, many bioinformatic studies have reported that a major phage defense system, the CRISPR-Cas system, is absent in Sequence Type ST258 and ST11 strains, two major lineages of CG258 (Li et al., 2018; Mackow et al., 2019; Tang et al., 2020). As one of the earliest lineages causing outbreaks of carbapenem resistance, ST258 K. pneumoniae isolates are responsible for the global spread of K. pneumoniae carbapenemases (KPC) (Cerqueira et al., 2017; Bowers et al., 2015). Therefore, it has been suggested that the lack of CRISPR-Cas systems could be one of the genetic factors contributing to the high rates of carbapenem resistance in this group. However, the more recently emerging lineages, such as ST15 and ST307, do contain such systems and so carbapenem resistance more generally cannot be explained so simply. Meanwhile, antibiotic identity may also affect the frequency of evolving resistance. Currently, four different carbapenems are available in an intravenous formulation (Papp-Wallace et al., 2011): imipenem, meropenem, ertapenem, and doripenem. In addition, faropenem, a related oral antibiotic in the penem class, is available but only outside of the USA (Gandra et al., 2016). Although the five drugs share similar structures and mechanisms of action, differences in their pharmacokinetics (ertapenem can be administered once a day while the other carbapenems require administration three to four times per day), stability against β-lactamase hydrolysis, and penicillin-binding protein target preference (Zhanel et al., 2007; Queenan et al., 2010; Kohler et al., 1999; Sutaria et al., 2018) may influence the evolution of resistance differently. For example, previous studies have shown that compared to other carbapenems, ertapenem is more susceptible to hydrolysis by some β-lactamases and its cell entry is more impeded by the loss of porins (Tsai et al., 2017; Jones et al., 2005), raising the possibility that a broader spectrum of mutations on β-lactamase or porin genes may selectively affect ertapenem but not the other carbapenems. In this study, to understand how bacterial genetic background and different carbapenems affect the rates of resistance evolution, we compared mutation frequencies (previously defined as the frequency of independent resistant mutants emerging in a given population [Martinez and Baquero, 2000]) of carbapenem-susceptible K. pneumoniae clinical isolates from 10 lineages and found that isolates from the dominant and emerging carbapenem-resistant lineages had higher mutation frequencies leading to carbapenem resistance than other lineages. We demonstrated that the higher mutation frequencies are caused by high-level transposon insertional mutagenesis, a process leading to resistance gene duplication and reversible porin disruption. We also showed experimentally that one of the major phage defense systems, CRISPR-Cas systems, indeed can play a role in restricting resistance gene acquisition when corresponding spacers sequences are present. Furthermore, we found that a broad spectrum of resistance-conferring mutations for selected carbapenems such as ertapenem contributed to increased resistance rates; importantly, these mutations selected from ertapenem exposure could serve as stepping-stones to high-level resistance to all carbapenems. Taken together, this work identified multiple factors that facilitate the evolution to carbapenem resistance in K. pneumoniae clinical isolates and demonstrated that the evolution of antibiotic resistance can be a complex process with important implications for antibiotic selection tailored to the genetic background of clinical isolates. Results The evolution of carbapenem resistance was affected by genetic background of the isolates We analyzed genomes of 267 previously sequenced K. pneumoniae clinical isolates (Cerqueira et al., 2017) and selected carbapenem-susceptible isolates from 10 lineages (Figure 1A, Table 1, Supplementary files 1 and 2). We chose isolates from the predominant carbapenem-resistant lineage that has caused many outbreaks since 2000 (UCI38 [ST258]), the dominant ESBL-producing lineage that is becoming increasingly carbapenem resistant (MGH222 [ST15]), newly emerging carbapenem-resistant lineages (UCICRE126 [ST147], MGH66 [ST29], BIDMC41 [ST37], MGH74 [ST76], MGH158 [ST152], UCI64 [ST17]), and lineages that have not caused carbapenem-resistant clonal outbreaks (UCI34 [ST34] and MGH21 [ST111]). We measured mutation frequencies of these isolates under ertapenem (Figure 2A) or rifampicin treatment (Figure 2B), using a modified Luria–Delbrück system in which low numbers of bacterial cells were seeded into each well of 384-well plates, thus making the emergence of two independent mutants in the same well extremely unlikely (Gomez et al., 2017; Figure 1B). This format requires that all resistance occurs through mutation acquisition and not HGT. (We define resistance as at least a twofold increase in the MIC for the mutant relative to the MIC against the original susceptible parent strain, and not relative to the clinically defined MIC breakpoints of the antibiotic. Therefore, resistant mutants selected from our experiments do not necessarily have MICs that are greater than the clinical breakpoints.) We found that except for MGH66 (ST29), all isolates showed similar levels of mutation frequencies to rifampicin (Figure 2B), whereas a wide range of mutation frequencies to ertapenem were observed (Figure 2A). In particular, some strains had much higher mutation frequencies to ertapenem than to rifampicin. Since resistance to rifampicin is acquired through point mutation resulting from errors during DNA replication (Goldstein, 2014; Pope et al., 2008), these results suggest that other genetic mechanisms help to determine the mutation frequency to ertapenem. Table 1 Genetic features of 10 selected carbapenem-susceptible clinical isolates. StrainsSTMIC (µg/ml) ofNarrow-spectrum β-lactamaseESBLCarbapenemaseompK36Accession no.CRISPR systemR-M systemETPMEMUCI38ST2580.250.06NoneblaSHV-12NoneIntactGCA_000566805.1NoType II; Type IIIMGH222ST150.030.03NoneblaSHV-28NoneIntactGCA_014902955.1YesNoneUCICRE126ST1470.030.25blaSHV-11NoneNoneIntactGCA_014902315.1YesType IMGH66ST290.060.03blaSHV-187NoneNoneIntactGCA_000694555.1NoType IIBIDMC41ST3720.06blaTEM-1blaSHV-12NoneIntactGCA_000492195.1NoType IIMGH74ST760.030.06blaSHV-1NoneNoneIntactGCA_000694715.1NoType I; Type IIMGH158ST1520.030.06blaSHV-1NoneNoneIntactGCA_002152555.1NoNoneUCI64ST170.030.03blaSHV-11NoneNoneIntactGCA_000688175.1NoType IUCI34ST340.060.03blaSHV-26NoneNoneIntactGCA_000566845.1YesNoneMGH21ST1110.030.03blaSHV-11NoneNoneIntactGCA_000492915.1YesNone Figure 1 Download asset Open asset Ten phylogenetically diverse carbapenem-susceptible K. pneumoniae isolates were selected from a collection of 267 K. pneumoniae clinical isolates. (A) The selected isolates are highlighted in red. In this phylogenetic tree, from inner to outer circles, the content of the CRISPR-Cas systems, restriction–modification systems, susceptibility to carbapenems, and sequence types are indicated. For carbapenem-resistant isolates, the resistance mechanism is also indicated. (B) Scheme of the modified Luria–Delbrück system. Exponential-phase growing cells are diluted and inoculated into 384-well plates, followed by incubation at 37°C for 3 hr. Antibiotics were then added at the concentrations of 1.1× MICs or at specified concentrations, and cultures were incubated at 37°C overnight. OD600 was measured the next day, and positive and negative wells were quantified. Mutants from each plate were sub-cultured in MHB medium supplemented with the same antibiotics at the same concentrations used for the selection, and saved in 25% glycerol stocks for future analysis. Mutants that did not grow up in the sub-culturing were excluded from the calculation of mutation frequencies. Figure 2 with 1 supplement see all Download asset Open asset The evolution of carbapenem resistance is affected by genetic background of the isolates. (A) Mutation frequencies of 10 clinical isolates under treatment with ertapenem. Five isolates, UCI38 (ST258), MGH222 (ST15), UCICRE126 (ST147), MGH66 (ST29), and BIDMC41 (ST37), have relatively greater mutation frequencies to ertapenem (>100 mutants per 108 cells) than the other five isolates. Comparing to UCI38 (ST258) that has the highest mutation frequencies to ertapenem, all isolates have significantly different mutation frequencies. Two-tailed Student's t-test was used for statistical analysis between UCI38 (ST258) and other isolates. (B) Mutation frequencies of 10 clinical isolates under treatment with rifampicin. Isolates with relatively high-level mutation frequencies to ertapenem do not necessarily have high-level mutation frequencies to rifampicin. Two-tailed Student's t-test was used for statistical analysis between UCI38 (ST258) and other isolates. (C) Diagram of pESBL, an ESBL-encoding plasmid isolated from UCI38 (ST258). (D) Diagram of pSHV, a multi-copy laboratory plasmid containing the native promoter and coding region of the ESBL gene blaSHV-12 amplified from pESBL. (E) The ESBL gene, blaSHV-12, was amplified from pESBL and expressed in three isolates lacking an ESBL gene and with relatively low-level mutation frequencies to ertapenem. However, mutation frequencies to ertapenem were not changed compared to the original strains lacking an ESBL gene (red). Two-tailed Student's t-test was used for statistical analysis to compare the original strain with the corresponding strain overexpressing blaSHV-12, with p>0.05 for all three pairs. (F) Transformation efficiencies of pESBL (left) or pSHV (right) in three isolates lacking ESBL genes (red) and with relatively low-level mutation frequencies to ertapenem. As controls, these two plasmids were also transformed into another ST258 strain BWH41 (blue), which does not carry ESBL genes, and a strain of E. coli 10β (black). pESBL could not be transformed into these three isolates but it could be transformed into BWH41 (ST258) and E. coli. In contrast, the laboratory construct pSHV was successfully transformed into all strains tested. For all experiments in (A, B, E, F) two to three independent biological replicates were performed. Data from independent experiments were plotted individually with error bars plotted as the standard deviation. The limit of detection is indicated with a dashed line, and the asterisk (*) under the dashed line indicates frequencies under the limit of detection. *p<0.05; **p<0.005; ***p<0.0005; ns, not significant. Among all strains tested, UCI38 (ST258) had the highest mutation frequency to ertapenem. It carries an ESBL gene blaSHV-12 on the plasmid pESBL (Figure 2C), raising the possibility that ESBL activity could contribute to high-level mutation frequencies. To test this hypothesis, we transformed pSHV (Figure 2D), a multi-copy laboratory plasmid containing blaSHV-12, amplified from pESBL, into three isolates lacking an ESBL gene and with baseline low-level mutation frequencies to ertapenem, including UCI64 (ST17), UCI34 (ST34), and MGH21 (ST111). However, introduction of blaSHV-12 did not change the mutation frequencies of these strains for ertapenem (Figure 2E), even though the expression of blaSHV-12 was higher in strains transformed with pSHV than in UCI38, which naturally carries blaSHV-12 (Figure 2—figure supplement 1). This ruled out the simple presence of the ESBL gene alone as the reason for the differing mutation frequencies. Next, we sought to test the hypothesis that the whole plasmid, pESBL (Figure 2C), might confer high-level mutation frequencies to ertapenem. However, when we attempted to transform pESBL into the same three strains with low-level mutation frequencies to ertapenem, none of them could take up pESBL. In contrast, an ST258 strain BWH41 (the only ST258 isolate lacking an ESBL gene in our collection) and a laboratory strain of E. coli, 10β, could take up pESBL (Figure 2F). Meanwhile, all strains successfully took up pSHV with similar efficiencies, suggesting that pESBL was uniquely restricted in particular strains under regular laboratory conditions. A type I-E CRISPR-Cas system prevented the acquisition of antibiotic resistance genes via HGT, while other genetic factors contribute to high mutation frequencies To understand why pESBL is restricted in these three isolates but not BWH41 (ST258), we analyzed the genomic sequences of the collection of 267 K. pneumoniae isolates for the presence of two major phage defense systems, the CRISPR-Cas systems and restriction–modification (R-M) systems (Supplementary file 3), which function to exclude foreign DNA. We found that of the three strains which could not take up pESBL, MGH21 (ST111), and UCI34 (ST34) have type I CRISPR-Cas systems, while UCI64 (ST17) has no CRISPR-Cas system but carries type I R-M systems. In contrast, among 80 strains of the ST258 lineage, we found no CRISPR-Cas systems and most strains carry type III R-M system (Figure 1A and Supplementary file 3). When we broadened our analysis to include the genomic sequences of 2453 K. pneumoniae strains available in the NCBI database, including 550 ST258 strains, we found that no ST258 strains contain a CRISPR-cas system (Supplementary file 4), confirming that the lack of CRISPR-Cas system is a genetic feature of the ST258 lineage. This finding is consistent with other bioinformatic studies which have tried to link the absence of CRISPR systems in ST258 strains to carbapenem resistance (Li et al., 2018; Mackow et al., 2019; Tang et al., 2020). However, there is no clear association between the absence of CRISPR and the more recently emerging carbapenem-resistant lineages (Figure 1A and Supplementary files 3 and 4). To understand the ability of MGH21 (ST111) to restrict pESBL uptake, a strain that encodes a type I-E CRISPR-Cas system but no R-M systems, we first confirmed by RNA sequencing (RNA-seq) that indeed the CRISPR-Cas system was expressed in MGH21 (Figure 3—figure supplement 1). We then compared the sequence of pESBL with MGH21's CRISPR-Cas system and found that MGH21 has a spacer (spacer 11) (Figure 3A and Supplementary file 5), targeting a gene encoding a DNA-methyltransferase (DNMT) (Bujnicki and Radlinska, 1999) in pESBL (Figure 2C); by searching a curated plasmid database (Brooks et al., 2019), we found that this spacer additionally aligns with sequences found in an additional 94 other plasmids carrying antibiotic resistance genes, including 62 multi-drug resistance plasmids (plasmids carrying resistance genes to more than one class of antibiotics) and 21 plasmids carrying carbapenemase genes (Supplementary file 6 and 7). In addition, spacer24 (Figure 3A and Supplementary file 5) aligned to a conserved hypothetical gene that was also found in UCI38 as well as 66 additional plasmids carrying antibiotic resistance genes, including 44 multi-drug resistant plasmids and 12 plasmids carrying carbapenemase genes (Supplementary file 6 and 8). Collectively, these results pointed to the potential role of the CRISPR-Cas system in excluding the uptake of resistance carrying plasmids such as pESBL. Indeed, after depleting the CRISPR-Cas operon (MGH21∆cas; the CRISPR-Cas system along with two adjacent hypothetical genes was deleted), pESBL could now be successfully transformed, whereas episomal complementation of the CRISPR-Cas system back into MGH21∆cas again restricted pESBL transformation (Figure 3B). Figure 3 with 1 supplement see all Download asset Open asset Type I-E CRISPR-Cas system in MGH21 (ST111) prevents the acquisition of pESBL but the presence of pESBL alone does not account for high mutation frequencies. (A) Type I-E CRISPR-Cas system in MGH21 (ST111). The two CRISPR arrays and the position of two spacers (Spacer11 and Spacer24) that align to plasmids encoding resistance genes are indicated. Spacer11 aligns to the DNMT gene located on pESBL _(Figure 2C). (B, C) Transformation efficiencies of pESBL (B) or the lab construct pSHV (C) in MGH21, MGH21∆cas, MGH21∆cas_R with CRISPR-Cas complementation, and MGH21∆cas_V with control vector complementation. pESBL could only be transformed into MGH21 strains in which the CRISPR-Cas system was deleted (MGH21∆cas and MGH21∆cas_V), whereas pSHV could be transformed into all strains at similar efficiencies. (D) Mutation frequencies of UCI38 (ST258), MGH21, MGH21∆cas, MGH21∆cas(pESBL), and MGH21(pSHV) with ertapenem treatment. The deletion of the CRISPR-Cas system (MGH21∆cas) and the introduction of pSHV (MGH21(pSHV)) did not affect the mutation frequencies. In contrast, the introduction of pESBL (MGH21∆cas(pESBL)) increased mutation frequencies, that some factors on pESBL other than the ESBL gene affect the mutation frequencies. However, mutation frequencies of were significantly than these of UCI38, that more factors in the genetic background of UCI38 contribute to the high-level mutation frequencies. experiments were in and data were plotted bars were plotted as standard deviation. The limit of detection of each is indicated with a dashed line, and the asterisk (*) under the dashed line indicates that the transformation efficiencies are the limit of detection. Two-tailed Student's t-test was used for all statistical an asterisk a a the absence of CRISPR-Cas system increased rates at which resistance by HGT could be acquired but did not change mutation frequencies of MGH21 (Figure In contrast, introduction of pESBL into MGH21∆cas increased the frequency with which resistance to ertapenem emerged in our modified system HGT cannot the frequency for MGH21∆cas was times higher than for the parent MGH21, MGH21∆cas, or MGH21 carrying pSHV (Figure As introduction of the ESBL gene alone in pSHV does not change resistance frequencies, this that factors on pESBL other than the ESBL gene contributed to the high mutation frequencies. Furthermore, while had ertapenem mutation frequencies relative to MGH21, its frequency was times than that of UCI38 from which pESBL was isolated (Figure suggesting that differences between the genetic of MGH21 and UCI38, of pESBL, play additional in high frequency mutation acquisition. insertional mutagenesis caused and reversible of porin genes leading to ertapenem resistance To into other genetic factors that may cause the different levels of mutation frequencies to ertapenem between UCI38 and MGH21, we analyzed whole sequencing data of resistant mutants to the genetic leading to ertapenem resistance. We compared ertapenem resistant mutants from UCI38 (ST258), five mutants from MGH21 (ST111), and five mutants from (Figure We found that the two strains carrying pESBL transposition as a mechanism to resistance while the strain lacking pESBL, MGH21, resistance only through acquisition. resistant mutants from UCI38 were to duplication of the transposon on pESBL in which the blaSHV-12 is (Figure and/or of one of the major porin genes of K. pneumoniae that carbapenem cell by sequences that only carry the the other porin also cell entry for carbapenems, we found no resistant mutants carrying mutations in to the low expression levels of in the used et al., 2018; Figure supplement or mutations already et al., in some for MGH21∆cas(pESBL), four mutants from the same transposon duplication of blaSHV-12 on pESBL, while the mutant from the acquisition of a in In contrast, all resistant mutants from MGH21 from the acquisition of or in porin genes and outer protein pESBL thus increased mutation frequencies relative to pSHV blaSHV-12 on pESBL a transposon that can be to ESBL expression and thus MIC (Figure In contrast, while carrying pSHV a higher baseline MIC of its higher blaSHV-12 expression (Figure 2—figure supplement it could not blaSHV-12 to evolve increased thus its mutation frequencies relative to the parent MGH21 (Figure Figure with see all Download asset Open asset insertional mutagenesis and reversible of porin genes in isolates with high-level mutation frequencies to ertapenem. (A) Mutation types identified via in ertapenem resistant mutants of UCI38 (ST258), MGH21 (ST111), and The of mutants that carry pESBL (UCI38 and have while only or were observed in mutants of (B) of the propensity of 10 selected isolates to transposon acquisition (red) in during ertapenem treatment. For each of these strains, mutants were and the types of mutation in was via at times higher frequencies than the acquisition of or in strains with higher of mutation frequencies to ertapenem. (C) of by four in mutants from UCI38 were selected and the were by (D) of was A ertapenem resistant mutant with an in was in the presence or absence of ertapenem. targeting was on the to the of the population that had the transposon at this Two-tailed Student's t-test was used for statistical analysis at each point to compare the cultures with and F) The relative expression of (E) and (F) of UCI38 and one of experiments were in bars are plotted as the standard deviation. Comparing the two strains that carry pESBL, we that UCI38 was to through transposon while only did so through acquisition. We that the higher likelihood of a transposition than the acquisition of a might the higher mutation frequencies of UCI38 and other strains with relatively high-level mutation frequencies to ertapenem (Figure 2A). Indeed, when we used the modified system to isolate and ertapenem resistant mutants from each of these 10