Progression ofampCamplification during de novo amoxicillin resistance development inE. coli

Progression of ampC amplification during de novo amoxicillin resistance development in E. coli | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Progression of ampC amplification during de novo amoxicillin resistance development in E. coli Luyuan Nong , Martijs Jonker , Wim de Leeuw , View ORCID Profile Meike T. Wortel , View ORCID Profile Benno ter Kuile doi: https://doi.org/10.1101/2024.05.24.595737 Luyuan Nong 1 Biology and Microbial Food Safety, Swammerdam Institute for Life Sciences, University of Amsterdam , Amsterdam, The Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site Martijs Jonker 2 RNA Biology & Applied Bioinformatics, Swammerdam Institute for Life Sciences, University of Amsterdam , Amsterdam, The Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site Wim de Leeuw 2 RNA Biology & Applied Bioinformatics, Swammerdam Institute for Life Sciences, University of Amsterdam , Amsterdam, The Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site Meike T. Wortel 1 Biology and Microbial Food Safety, Swammerdam Institute for Life Sciences, University of Amsterdam , Amsterdam, The Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Meike T. Wortel Benno ter Kuile 1 Biology and Microbial Food Safety, Swammerdam Institute for Life Sciences, University of Amsterdam , Amsterdam, The Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Benno ter Kuile For correspondence: b.h.terkuile{at}uva.nl Abstract Full Text Info/History Metrics Preview PDF ABSTRACT Beta-lactam antibiotics are the most applied antimicrobials in human and veterinarian health care. Hence, beta-lactam resistance is a major health problem. Gene amplification of AmpC beta-lactamase is a main contributor to de novo β-lactam resistance in E. coli . However, the time course of amplification and the accompanying DNA mutations are unclear. Here, we study the progression of ampC amplification and ampC promoter mutations in the evolution of resistance by stepwise increasing amoxicillin concentration. AmpC promoter mutations occur by day two, while the amplification by a factor of approximately eight occurs after more than six days of amoxicillin exposure. The combination of amplification and promoter mutations increase ampC mRNA level by an average factor of 200 after 22 days. An IS1 insertion was identified in the amplification junction, suggesting the amplification is facilitated by mobile genetic elements transposition. In order to identify the essential genes for ampC amplification, the chromosomal mutations of strains with induced amoxicillin resistance were compared a similarly evolved resistant Δ ampC knockout. The evolved Δ ampC contained several resistance mutations that were absent in the WT, which accumulated more mutations in stress response genes. The amoxicillin evolved Δ ampC does not show amplification of the fragment around the original ampC position but exhibits a large duplication or triplication at another position, suggesting selection of genes to amplify is essential for resistance adaption. IMPORTANCE Amoxicillin is one of the most used antimicrobial against bacterial infections. DNA fragments containing ampC are amplified upon prolonged and stepwise increasing exposure to amoxicillin. These ampC amplification fragments have been identified in extended-spectrum beta-lactamases (ESBLs) plasmids, which are considered the main cause of beta- lactam resistance. Understanding the progression of ampC amplification enables amoxicillin resistance prevention. In this study, we show the time course of two important factors for ampC transcription enhancement, ampC amplification and ampC promoter mutations, during de novo amoxicillin resistance evolution. We propose that the transposon IS1 contributes to the amplification and that the sigma factor 70 regulates ampC overexpression. INTRODUCTION Although alternative antibiotics are available ( 1 )( 2 ), beta-lactam antimicrobials remain the most commonly used antibiotics for human infection therapy ( 3 ). Exposure to sublethal levels of antibiotics is the main reason for resistance emergence ( 4 ). When the effectiveness of antibiotics is reduced, exposure to sublethal levels during antibiotic treatment is unavoidable ( 5 ). The risk of non-lethal concentrations occurs especially in the veterinary sector when livestock is treated with antibiotics added to water or feed. Under long-term exposure, antimicrobial resistance may accumulate in the microbiome and this resistance can be transmitted to human pathogens through horizontal gene transfer. AmpC beta-lactamases (AmpC) are widely distributed cephalosporinases causing beta-lactam degradation ( 6 ). The ampC gene can be encoded by both chromosomal and plasmid DNA. Unlike extended-spectrum beta-lactamases (ESBLs) which are considered the main cause of the prevalence of beta-lactam resistance ( 7 ), the overexpression of ampC is underestimated. In Escherichia coli , the chromosomal ampC gene is poorly expressed in the wildtype, as it lacks the transcription activator AmpR ( 8 ). However, after laboratory evolution of amoxicillin resistance, the transcription of ampC is enhanced more than 100-fold compared to the naive strain, mainly because of two reasons: ampC promoter mutations and gene multiplication ( 9 ). The AmpC gene is controlled by a weak promoter (P ampC ) which has three main elements, a - 10 box, a -35 box and an attenuator ( 10 ). The mutations C > T in the -10 box at position -11 and T > A in the -35 box at -32 are conservative, increasing AmpC production by 21- and 7- fold, respectively ( 10 ). In addition, ampC gene amplification plays a considerable role in its transcriptional enhancement ( 11 ). Chromosomal ampC amplification was first reported increasing resistance ( 12 ). This amplification is a RecA-independent event occurring in tandem ( 13 )( 14 ). Duplicated antibiotic resistance genes could undergo horizontal gene transfer in microbial communities, with mobile genetic elements serving as a vehicle ( 15 ). A chromosomal DNA fragment containing the ampC gene was amplified from strains made resistant to amoxicillin by exposure to step-wise increasing concentrations and isolated with plasmid isolation techniques ( 16 ). This fragment can be exchanged between E. coli strains by horizontal gene transfer. A similar E. coli chromosomal fragment harboring ampC and two nearby genes can be identified in several ESBL plasmids isolated in broiler production ( 17 ). This suggests that ampC amplification may occur in E. coli developing beta-lactam resistance and that subsequently the fragment is incorporated in ESBL plasmids. In addition to inactivation of the antibiotic through AmpC, various other molecular mechanisms can contribute to beta-lactam resistance, including active efflux pumps, decreased influx, and target site modification ( 18 ). Multiple point mutations were demonstrated to directly contribute to resistance, such as mutations in the efflux pump AcrAB-TolC ( 19 ) and the outer membrane porin OmpC/OmpF ( 20 ). Besides, some mutations that alter metabolism also confer to resistance ( 21 )( 22 ). For example, a mutation in the 2- oxoglutarate dehydrogenase ( sucA ) gene raised carbenicillin resistance through lower basal respiration, thereby avoiding metabolic toxicity and reducing lethality. To understand the competitive, synergistic and epistatic effects of DNA mutations associated with ampC amplification, this study investigates the time course of mutations in the chromosome and ampC amplification. Using the de novo development of amoxicillin resistance in E. coli as a model, this study addresses five questions: 1) What is the time course and pattern of ampC amplification? 2) Is the amplified ampC fragment always the same, or can the length vary? 3) Is the ampC copy number the primary factor determining the AmpC activity? 4) If the ampC gene is removed, does the amplification of the fragment around it still occur? 5) Is the pattern of mutations accompanying development of resistance different in the presence and absence of ampC ? Answering these questions provides insights into the complex dynamics of beta-lactam resistance development and the role of ampC amplification in this process. RESULTS Relationship between AmpC activity and amoxicillin resistance To investigate the role of AmpC in amoxicillin resistance, nine replicates of wild type (WT) and six of ampC knock-out mutants (Δ ampC ) were made highly resistant by growing them at stepwise increasing sublethal amoxicillin concentrations (Fig.1a). The acquisition of resistance by Δ ampC occurs significant slower than by the WT. Correspondingly, the minimal inhibitory concentration (MIC) reached at least 1024 mg/L in WT and only approximately 128 mg/L in Δ ampC (Fig.1b). The AmpC activity was determined using the chromogenic substrate nitrocefin in the presence of intact live cells, as the enzyme functions as an ectoenzyme ( 9 ). Compared to the activity of the naive wildtype, the activities encountered in the evolved WT increased by a factor of 39-156 in the final incubations (Fig.1c). The log 2 transformed fold change of MIC and AmpC activity exhibited a linear relationship with R 2 =0.730 (Fig.1d). This indicates that de novo amoxicillin resistance in E. coli can be largely attributed to the increase in AmpC activity. AmpC gene amplification during the evolution of resistance The ampC gene, coding for a beta-lactamase, can be amplified when the cells are exposed to stepwise increasing non-lethal levels of amoxicillin ( 16 ). In order to determine the moment that this amplification takes place, the copy number of chromosomal ampC was measured at three-day intervals using qPCR in all evolved WT replicates with naive WT as reference (Fig.2a). The first amplifications were observed on day nine. In the first six days, the ampC copy number did not increase in any of the replicates (Fig.2b). After that, in only one replicate did the copy number increased gradually, in all other replicates an initial jump of a factor eight or more was observed. The median time point for amplification was around day 12, the last single copies were seen on day 16, and at days 19 and 22 all copy numbers ranged from 8 to 19. In order to determine the size and composition of the DNA fragment containing ampC that is amplified, whole genome sequencing was performed on all replicates of WT and three replicates of Δ ampC at day 22. Seven different fragments were found, of which five were unique and two fragments were observed twice (Fig.3a). The length of the amplified region ranged from 7.5 kb to 13.4 kb. Five fragments shared the same left terminal, located within the ecnB promoter or reading frame, indicating that a preferred target exists for initiation of the amplification. One termination sequence is shared by 3 fragments, each with a different initiation site. All amplified fragments contain the sugE , blc and ampC genes. SugE codes for efflux transporters in the multidrug resistance family ( 23 ), while blc codes for an outer membrane lipoprotein ( 24 ). Both sugE and blc may contribute to amoxicillin resistance. We tested whether the amplification fragments are connected to each other by designing the forward primer to bind to the start and the reverse primer to bind to the end sequences of the amplified fragments. The junction between two copies of amplification contigs was cloned by PCR and confirmed by Sangar sequencing. If the product shows a connection, either the fragment has become circular, or two or more fragments are connected. Two types of junctions were found (Fig.3b). In four of nine replicates, two amplification contigs directly connect, with 0 to 7 bp overlapping base pairs. In the other five, an IS1 transposon of 768 bp, was inserted in the junctions with also 0 to 7 bp homology base pair between one amplification fragment and the IS1 element. Interestingly, all of replicates with IS1 inserted into amplification junction have one of their flank sites located in the ecnB promoter or reading frame. In evolved Δ ampC , there was no amplification near the original ampC position, indicating that the other genes in the amplification fragment of evolved WT contribute much less or not at all to amoxicillin resistance ( Fig. 3c ). Instead, a large chromosomal duplication or triplication occured in a similar position, between base pairs 387,800 and 1,391,170 on the chromosomal map. Genes in this region that may contribute to amoxicillin resistance include acrA , acrB , ompF , sulA, ftsZ . Download figure Open in new tab Fig. 1 Evolution of amoxicillin resistance. a Amoxicillin concentration in the cultures. b Measurement of MIC for amoxicillin. Data in a, b are shown as means ± SD, statistical significance was determined with Wilcoxon signed-rank test, ****p<0.0001, *p<0.05. c Measurement of AmpC activity. Each point represents the mean of three technical replicates. d Relationship between MIC and AmpC activity. The fold change of MIC and AmpC activity were transformed by log 2 and fitted with linear regression (R 2 =0.730). Download figure Open in new tab Fig. 2 ampC copy number at different time points during the amoxicillin evolution. a The copy number measurement in each biological replicate. b The distribution of ampC copy number at each time point in nine biological replicates. Each point is the mean of three technical replicates. Download figure Open in new tab Fig. 3 Amplification regions in the evolved strains. a The various amplification fragments in evolved WT. b Junctions between fragments after amplification. Two situations are observed. Four replicates’ amplified fragment connected tandemly with 0-7 base pair overlapping. In five replicates’amplified fragments have an IS1 transposon insertion in their connection junctions with 0-7 overlapping base pair between amplified fragment and IS1. c Read counts in whole genome sequencing of evolved Δ ampC strains. Large regions with duplication or triplication occurred. DNA mutations in the evolved WT and Δ ampC To find which genes are mutated in association with the amplification of ampC , whole genome sequencing was performed on DNA isolated at the moment of ampC amplification (AT) and the end of the evolution experiment (FT) in WT strains, as well as the Δ ampC strains at FT. Mutations were identified through aligning to E. coli genome download from NCBI, eliminating those also present in the control and those observed at frequencies below 10%. Except mutations referred to ampC , a total of 99 DNA mutations (84 single-nucleotide polymorphisms, 5 deletions and 10 insertions) were identified in all evolved WT (Table S1, S2). Of these, 32 of point mutations were outside of reading frames. In the evolved Δ ampC strains we identified 97 DNA mutations with the same analysis workflow (96 single- nucleotide polymorphisms and one deletion), 55 of them are outside of gene reading frames (Table S2, S3). The genes of interest are divided in two functional groups, stress response and cell envelope, and are presented in a heatmap ( Fig. 4 ). Download figure Open in new tab Fig. 4 Mutations associated with ampC amplification in amoxicillin evolution . The color intensity indicates the frequency of mutations in the genes indicated. The time points are the ampC duplication (AT) and the end of the experiment inducing resistance (FT). R1 to R9 are evolved replicates of WT, A1 to A3 are evolved replicates of Δ ampC . Download figure Open in new tab Fig. 5 AmpC transcription level in amoxicillin evolution. A Fold-change in ampC expression compared to the native WT. Each point represents the mean of three technical replicates. b The relationship between ampC copy number and ampC mRNA level. The log- transform relative copy number and mRNA were fitted into a linear trendline. Not one mutation is present in all replicates. In the evolved WT, several mutations associated with stress response are identified coding for sigma factors ( rpoA , rpoB , rpoD , and rpoS ). Most mutations are observed in rpoD , that contains mutations in six out of nine replicates. The majority of these mutations show an increasing or sustained frequency from the time point of the first amplification observed to the end of the evolution experiment. In one replicate mutations in rpoD appear to outcompete those in rpoS , as evidenced by a decreased mutation frequency in rpoS and a simultaneous increase in rpoD . The DNA repair related gene polA was mutated in two replicates. Genes related to cellular response to abiotic stimulus ( uspE / phoQ ), stress-induced mutagenesis ( yicC ) and biosynthesis of purine ribonucleotides ( adk ) were mutated occasionally. Four genes ( phoQ / adk / cpxA / envZ ) in the network of phosphorus metabolic processes, which are involved in energy and carbon metabolism, had mutations, suggesting a role of the network in building up amoxicillin resistance. The gene cpxA codes for a membrane sensor for envelope stress response ( 25 ). Its mutations can increase β-lactam resistance in E. coli ( 26 ). Different mutations with high and increasing frequencies occur in cpxA in several replicates, N346K in R2, Q34P in R4, T16_T17 deletion in R5 and K121I in R7. The mutations in membrane related genes correspond to different mechanisms for amoxicillin resistance, such as active efflux and decreased influx ( 18 ). Because amoxicillin blocks cell wall synthesis by inhibiting transpeptidation, the mutations in genes related to cell division ( mreB / mreC / amiC ) may counter the effects of long-term amoxicillin exposure. The genes envZ and ompR code for a two-component system that forms a sensor for outer membrane diffusion pores OmpF and OmpC ( 27 ). The change of both ompC and ompF can alter their substrate specificity and restrict the influx of amoxicillin, causing resistance ( 28 , 29 ). The mutations consisted of P248L in envZ and D33fs in ompC .These mutations occurred in the same replicates at the same times. The mutations frequency in ompC is higher than ompF , although the deletion of ompC causes a lower MIC against many beta-lactam antibiotics, while the deletion of ompF increases them ( 20 ). AcrB is a well-known drug efflux pump that affects E. coli amoxicillin resistance ( 30 ). The mutation in acrB was accumulated in three replicates in AT. However, these mutations were absent at the end of the evolution experiment. Both fdoH and nrdH are involved in the electron transport chain, hence the mutations in these two genes indicate that the ampC amplification occurs in parallel with the fitness alteration. The evolved Δ ampC strains tend to exhibit mostly mutations associated with membrane. All Δ ampC replicates contained mutations in ftsI / acrB / envZ . The inhibition of FtsI activity by binding of beta-lactam antibiotics is lethal, as this is an essential cell division protein ( 31 ). Although the gain-of-function mutations in ftsI is well-known for amoxicillin resistance ( 32 ), this mutation did not occur in evolved WT strains. In addition, in evolved Δ ampC , the proportion of mutations located in envZ / ompC / acrB is relatively higher than in the WT, suggesting an increasing role of other resistance pathways. The mutation in envC , which codes for the efflux pump AcrE, occurred occasionally. Although the acrEF operon is not essential in E. coli when the acrAB operon is expressed, when acrAB become inactive, the acrEF operon complements the function of acrAB under stressful conditions ( 33 ). This suggests AcrE has the ability to cause resistance. Besides, mutation accumulation was observed in the genes coding for the cell division protein FtsE and the peptidoglycan lipid biosynthesis enzyme MraY in Δ ampC, but not in the WT. In regard to the mutations associated with stress response, the mutations in rpoD which have a high frequency in evolved WT were not observed in Δ ampC . However, the mutations in sigma factor subunit rpoA occurred at a higher frequency in Δ ampC . Fewer mutations in stress response genes were observed in evolved Δ ampC strains than in the WT. Only mutations in DNA mismatch repair protein gene mutL and putative methyltransferase gene yfiF were found. Relationship between AmpC mRNA level and ampC copy number To determine the consequences of the observed ampC gene copy number increase, ampC mRNA levels were measured at the same time points that the ampC copy number was ascertained (Fig.5a). Even though there was no gene amplification within the first 6 days of the evolution experiments, a continuous increase of ampC mRNA levels was evident, indicating that other factors also enhance ampC transcription ahead of ampC amplification. During induction of resistance, ampC mRNA levels significantly increased in all replicates, ranging from 85 to 961-fold change compared to the WT. In order to establish the relationship between ampC copy number and mRNA level, the ampC mRNA level and copy number fold-change were transformed by log 2 and fitted in linear function (Fig.5b). The ampC mRNA level and copy number have a positive correlation. However, the linear formula does not fit well. This suggests that other factors, in addition to ampC copy number, also have considerable impact on ampC gene transcription. Trajectory of mutations related to ampC Instigated by the evidence above, we further explored how the other mutations affect ampC transcription. Besides gene dosage, promoter activity is another crucial factor influencing gene transcriptional levels. Therefore, the ampC promoter region was sequenced at 8 different time points during the evolution experiments (Table S4). AmpC promoter mutations were observed as early as day 2, affecting the -10 box of the promoter. As evolution progressed, more mutations emerged. However, not all of them were retained until the final days. The first mutation in the ampC promoter occurs earlier than ampC amplification. Combining this information with the observation that ampC mRNA levels increase by about 10-fold prior to ampC gene amplification, suggests that the mutated ampC promoter is responsible for the initial increase in ampC transcription. The mutations in the ampC promoter occurred in three main elements: the -10 box, the -35 box, and the attenuator. In the nine replicates, the mutations in the -10 box and -35 box were conserved, -11 G>A in -10 box and -32 A>T, but not those in the attenuator. In the attenuator area 5 different mutations were found in different replicates at the end of evolution. Additionally, there were a few mutations in other sites that have not been reported before in the ampC promoter. To uncover a possible influence of changes in the ampC promoter region on the ampC copy number, the trajectory of mutations in the ampC promoter region was documented ( Fig.6 ). Mutations in the -10 box already occurred by day 2. However, the frequency of this mutation decreased after day 3, accompanied by an increase in the frequency of mutations in the -35 box. After the ampC copy number started to increase, mutations in the -10 box and -35 box did not show systematic changes. Mutations in the attenuator and undescribed area were more unpredictable, with their mutation frequency continuously changing throughout the entire evolution process. Download figure Open in new tab Fig. 6 Trajectory of mutations in ampC promoter and copy number change. Mutations frequency was calculated in nine evolved population. P. represents ampC promoter. CN. represents ampC copy number. Undescribed is the mutations in other sites that have not been reported. DISCUSSION Strains with higher AmpC production have a selective advantage in amoxicillin resistance development. The overexpression of the AmpC enzyme can be achieved by the enhancement of ampC gene transcription, both by increasing the ampC promoter strength and the gene copy number. When exposed to increasing concentrations of amoxicillin, E. coli gained resistance by increasing the transcription of ampC by a factor exceeding 100 ( 9 ). This study reports the time course of the amplification of a chromosomal fragment containing ampC and the genetic events accompanying it. The considerable delay before the first amplification events observed in this study, indicates that the mutated ampC promoter causes the initial increase of AmpC activity and amoxicillin resistance. Therefore, the process of amplification is not gradual. Instead, the initial jump in copy number seems to be around 8-fold and occurs after at least 6 days of exposure to increasing amoxicillin levels. The mobile genetic elements could drive the antibiotic resistance genes’ copy number increase ( 15 ). The chromosomal DNA fragment isolated by Darphorn ( 16 ) from E. coli made amoxicillin resistant by de novo evolution, contains the IS1 transposon, which was introduced at the connection of the start and end of the fragments. This fragment can transfer from the amoxicillin evolved E. coli functioning as donor to susceptible E. coli receptor cells. In Proteus mirabilia amplification involving IS1 is based on homology recombination identifying two IS1 copies as homology regions for initial recombination, followed by tandem duplication of the region between IS1 elements ( 34 ). In this IS1-based amplification, the frequency of the initial duplication is 150-fold lower than that of the following amplification to higher copy numbers ( 35 ). If this is the same in E. coli , that would explain the pattern of ampC amplification observed here. All replicates with IS1 inserted in the junction of two amplification fragments had one of their amplifications flanking within the ecnB reading frame or promoter. The extremities of the IS1 sequence in particular are crucial for cointegration ( 36 ). The ecnB promoter and the tail of IS1 share the same 7 bp sequence. This implies that P ecnB contains a region homologous to part of IS1, suggesting a potential function of this sequence as the required homologous region for the recombination event, recruiting IS1 transposon and leading to amplification. The absence of amplification in original position of ampC in evolved Δ ampC strains indicates that none of the other genes in the fragment confer enough resistance advantage for its amplification. It also rules out the already unlikely possibility that the cell would be aware of the location of ampC in the genome and amplify the region around it when exposed to amoxicillin. Instead, a large region, containing more than 1000 genes, was duplicated or triplicated. Similar amplification was also observed in tetracycline evolved E.coli ( 37 ), indicating that the driving factor may be the presence of multi-drug resistance genes within the amplified region. Gene expression involves the coordination of multiple dynamic events subject to multi-level regulation ( 38 ). The positive relationship between AmpC activity and the MIC, ampC copy number and mRNA levels suggest that according to hierarchical control analysis ( 39 ) the genetic control component fully determines levels of expression, which in turn control AmpC activity. Hence, the ampC copy number exerts considerable influence over its expression, but that it is not the only factor. The mutations in the promoter areas are crucial in the initial stages of resistance development. The comparison of gene mutations during evolution in WT and Δ ampC suggests that the deletion of ampC not only reduces the ability of resistance acquisition but also alters the evolutionary trajectory in E. coli . Point mutations in several genes exhibited higher frequency in evolved Δ ampC strains compared with WT, suggesting that additional mutations were needed to compensate for the missing ampC gene. The mutations in gene ftsI and acrB occur in all Δ ampC strains. These genes are known to confer amoxicillin resistance based on pathways associated with target alternation ( 40 ) and efflux pumps ( 30 ), respectively. In contrast, in the WT the mutation in acrB disappeared from the time point of the first amplification observed to the end of the evolution experiment in three replicates, and the mutation in ftsI was not observed. This suggests that these mutations may cause higher fitness costs than ampC amplification. Besides, mutations in envZ may confer resistance by decreasing drug influx through the membrane porins OmpC/OmpF ( 20 )( 41 ). Moreover, these mutations have been shown to increase carbapenem resistance in ompCF -deleted backgrounds ( 42 ). Mutations in the envZ gene occur in all evolved Δ ampC strains and also accumulate in several evolved WT strains, indicating that they synergize with AmpC. Several DNA mutations present in the WT after resistance evolution were not observed in evolved Δ ampC . The mutations in rpoD accumulated in most evolved WT. However, this did not occur in evolved Δ ampC , indicating that rpoD may be important for ampC transcription enhancement. RNA polymerase coded by sigma factor 70 (σ 70 ) gene rpoD is essential for gene transcription ( 43 ). RpoD connects to both the -10 and -35 regions in promoter to initiate transcription ( 44 ). A mutation at a same position in rpoD (Asp445Glu) was recently reported in cefotaxime evolved E. coli ( 32 ). Mutations in the rpoD gene in our study changed amino acids 445 (Asp445Ala, Asp445Val), 447 (Ala447Pro) and 570 (Asp570Gly) in several replicates. These sites are in the conserved regions 2.4 and 4.2 of the σ 70 subunit, which connect to the -10 and -35 motif within the promoter, respectively ( 45 ). As mutations in gene rpoD occur later than ampC duplication and mutations in -10 box and -35 box of ampC promoter, the σ 70 mutants potentially enhance binding affinity between the RNA polymerase and gene promoter and thereby improving the utilization of high gene dosage. Altogether, our study can bring new thought of amoxicillin resistance prevention based on resistance emergence timeline. Also, we propose the DNA mutations which accompany with the ampC overexpression. MATERIALS AND METHODS Strains, culture conditions and adaptive evolution Evans medium pH 6.9 supplemented with 55mM glucose ( 46 ) was used to culture the E. coli MG1655 (WT) and BW25113-Δ ampC (Δ ampC , NBRP E. coli , Keio library. Kanamycin resistance was removed) strains culturing at 37 °C. Stock solutions of amoxicillin (10 mg/mL) were dissolved with MM medium and filter-sterilized and stored at -80 °C. The evolved population everyday was stored with 30 % glycerol in -80 °C. Wild-type E. coli Δ ampC were made amoxicillin evolved through laboratory evolution experiments as described before( 47 ). Nine replicates of WT and three of Δ ampC were used for amoxicillin resistance evolution, and one culture not exposed to antibiotics was used as control. The starting amoxicillin concentration was 1 µg/mL for both WT and Δ ampC , which is a quarter of the WT E. coli MIC (4 µg/mL) and half of Δ ampC MIC (2 µg/mL), and the starting OD 600 was 0.1. After 24 h grown in 10 mL tube with 5 mL Evans medium in 37 °C, 200 rpm incubator, if the OD 600 of the culture in higher concentration was higher than 70 % that in lower concentration, the antibiotic concentration was doubled in the subsequent incubation, otherwise using the same antibiotic concentration. The evolution experiments lasted 22 days. MIC measuring During the evolution process, MIC values were measured every 3 to 4 days in 96-well plates in plate readers (Thermo Scientific Multiskan FC with SkanIt software) according to previous description ( 48 ). Amoxicillin concentration ranged from 2 to 2048 µg/mL increasing by a factor of 2 at each step. The starting OD 600 was 0.05. Plates were incubated at 37 °C for 24 h, with shaking and OD 595 measurements were conducted every 10 min. The MIC was defined as the lowest amoxicillin concentration that reduced the growth to OD 595 less than 0.2 after 24 h. AmpC activity measurement The stored strains were grown in Evans medium overnight from storage tubes kept at -80 °C and diluted 1:100 with fresh Evans medium. Cultures were harvested at late-log phase and washed in 1 M PBS 7.0 buffer. Cells were diluted to OD 600 2 with PBS. 50 µL cell suspension was mixed with 50 µL 10 µg/mL nitrocefin and incubated at 37 °C in a plate reader with pulse shaking. The OD 492 was measured every minute for 10 h. The fold-change of the activity was calculated by dividing the activity of evolved population by that of naive WT. Quantitative PCR Genomic DNA was extracted with the DNeasy Blood and Tissue kit (Qiagen) for copy number measurements and whole genome sequencing. RNA was isolated using RNeasy Protect Bacteria Kit (Qiagen) and reverse transcription was carried out with iScript cDNA Synthesis kit (BIO-RAD). TaqMan™ Universal PCR Mix (ThermoFisher) was used for Quantitative PCR (qPCR), performed with the Applied Biosystems 7300 realtime PCR system (Applied Biosystems) Primers and probes for qPCR (Table S5) were obtained from Integrated DNA Technologies and 6-FAM and TAMRA were used as dye and quencher of probe. A sample of naive WT was used as reference. The cDNA or genomic DNA were diluted to the same concentration (10 ng/µL). Cycle threshold (Ct) values were determined by automated threshold analysis using the ABI Prism 1.0 software. Gene copy numbers or gene relative production were determined using the -ΔΔCt method using GADPH as the reference gene. Sequencing of the ampC promoter AmpC promoter was amplified by Herculase II Fusion DNA Polymerase (Agilent) using isolated genomic DNA as template and F-AmpC Prom, R-AmpC prom (Table S5) as primers. PCR product was purified using MSBSpinRapace kit (Stratec) and sequenced by Sangar (Macrogen Europe). The result was analyzed through Snapgene. Only the highest signal of mutations at each site was recorded. Whole-genome sequencing Whole genome sequencing was conducted utilizing next generation sequencing Illumina (NextSeq 550 system) following established protocol( 47 ). NEBNext Ultra II FS DNA Library Prep Kit for Illumina (New England BioLabs) and NEBNext Multiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs; New England BioLabs) were used for creating a genomic DNA library. After removing the adapter using Cutadapt ( 49 ), the raw data was trimmed ( 50 ) and deduplicated. Then the bam files were aligned to references (NC000913 for WT and CP009273 for Δ ampC ) with Bowtie2 ( 51 ). Freebayes ( 52 ) and Lofreq ( 53 ) were used for allele frequency calculation and variant calling. Snpeff ( 54 )was used for variant annotation. The point mutations with allele frequency lower than 0.1 and those also occurring in the drug-free cultured control were removed. 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