The influence of oxygen and oxidative stress on de novo acquisition of antibiotic resistance in E. coli and Lactobacillus lactis

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Jonker, Lisa Teichmann, Meike Wortel, Benno H. ter Kuile This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2932862/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 02 Oct, 2023 Read the published version in BMC Microbiology → Version 1 posted 9 You are reading this latest preprint version Abstract Background : Bacteria can acquire resistance through DNA mutations in response to exposure to sub-lethal concentrations of antibiotics. According to the radical-based theory, reactive oxygen species (ROS), a byproduct of the respiratory pathway, and oxidative stress caused by reactive metabolic byproducts, play a role in cell death as secondary killing mechanism. Results: To investigate whether oxygen and ROS affect de novo acquisition of antibiotic resistance, evolution of resistance was compared in E. coli wildtype and Δ oxyR strains under aerobic and anaerobic conditions. Since Lactococcus lactis ( L. lactis ) does not have an active electron transport chain (ETC) even in the presence of oxygen, and thus forms much less ROS, resistance development in L. lactis was to distinguish between oxygen and ROS. The resistance acquisition in E. coli wildtype under aerobic and anaerobic conditions did not differ much. However, the aerobically grown Δ oxyR strain gained resistance faster than the wildtype or anaerobic Δ oxyR. Inducing an ETC by adding heme increased the rate at which L. lactis acquired resistance. Whole genome sequencing identified crucial mutations involved in the acquisition of resistance. These mutations were specific for each antibiotic. The lexA mutation in Δ oxyR strain under aerobic conditions indicated that the SOS response was involved in resistance acquisition. Conclusions: The concept of hormesis can explain the beneficial effects of low levels of ROS and reactive metabolic byproducts, while high levels are lethal. DNA repair and mutagenesis may therefore expedite development of resistance. Taken together, the results suggest that oxygen as such barely affects resistance development. Nevertheless, non-lethal levels of ROS stimulate de novo acquisition of antibiotic resistance. Reactive oxygen species de novo resistance Antimicrobial resistance Whole genome sequencing Reactive metabolic byproducts Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Background Bacteria can acquire antibiotic resistance by adapting cellular physiology, DNA mutations, and horizontal transfer of resistance genes [ 1 ]. DNA mutations can occur as a result of exposure to non-lethal concentrations of antimicrobials [ 2 ]. These mutations can modify the cellular targets of antibiotics, activate antibiotic efflux pumps, generate enzymes that disable antibiotics, and reduce the permeability of membranes to antibiotics to make bacteria resistant [ 3 ]. While the role of mutations in development of de novo resistance has been documented to some extent [ 4 ][ 5 ][ 6 ], the driving factors for mutations in the bacterial DNA are less well described. According to the radical-based theory, exposure to bactericidal antibiotics results in the formation of reactive oxygen species (ROS) and reactive metabolic byproducts as a secondary effect that hastens bacterial cell death [ 7 ][ 8 ][ 9 ]. The overproduction of ROS damages the DNA, proteins, lipids, and nucleotides pool, and in particular causes the oxidation of guanine to 8-oxo-guanine [ 10 ]. Besides being lethal, ROS can also enhance mutation rates [ 11 ]. In fact, the overall effect of ROS and reactive metabolic byproducts may be hormetic, as low concentrations enable rapid adaptation, while high level are lethal [ 12 ]. Sub-inhibitory doses of ciprofloxacin generate a resistant mutant subpopulation through ROS formation and sigma-S general stress response activity [ 13 ]. Furthermore, multidrug resistance induced by sublethal levels of antibiotics correlates with ROS-induced mutagenesis [ 14 ]. ROS has been described as a key factor in antibiotic-induced SOS mutagenesis, and treatment with the antioxidant N-acetylcysteine reduces ROS and blocks SOS-mediated mutagenesis [ 15 ]. Based on the above considerations we hypothesized that the oxidative stress caused by ROS plays a central role in de novo acquisition of antibiotic resistance. Escherichia coli is a facultative anaerobic procaryote, commonly found in the human gastrointestinal tract [ 16 ]. The gene oxyR in E. coli codes for an oxidative stress regulator, mitigating levels of hydrogen peroxide under aerobic conditions [ 17 ]. Because E. coli is well described and thoroughly studied in many aspects, it is the primary model organism used in this study. The lactic acid bacterium Lactococcus lactis is a fermentative bacterium, that can also grow in the presence of oxygen, but even then does not possess a complete electron transport chain (ETC) [ 18 ]. However, when both heme and oxygen are present, L. lactis can establish an ETC, resulting in NADH oxidation and aerobic respiration [ 19 ] and hence the formation of ROS. It is therefore used in several experiments to separate the respective roles of oxygen and ROS generated by the ETC in development of de novo antimicrobial resistance. The hypothesis that ROS is a driving factor for de novo acquisition of resistance was investigated by comparing resistance development in E. coli MG1655 and Δ oxyR mutant strains grown aerobically and anaerobically. The rate of adaptation to increasing concentrations of antibiotics of the wildtype and the mutant did not differ much under anaerobic conditions, indicating a limited role of oxygen as such. The faster resistance development in Δ oxyR mutant E. coli and in L. lactis with added heme indicates that moderately increased levels of ROS stimulate resistance acquisition, possibly by increasing mutation rates. A hormetic effect that is related to the radical-based killing mechanism was observed. High levels of ROS or reactive metabolic byproducts were lethal, while moderate levels of stress stimulate development of resistance. When radical-based stress levels are low, a moderate increase in stress accelerates resistance development. Results Acquisition of resistance under aerobic and anaerobic conditions FIG 1 Acquisition of resistance to amoxicillin (A, E, I), enrofloxacin (B, F, J), kanamycin (C, G, K), and tetracycline (D, H, L) of E. coli wild-type MG1655 (blue: anaerobic/black: aerobic) and the Δ oxyR knockout (green: anaerobic/red: aerobic) strains. The top 4 panels (A-D) show wild-type MG1655, the second set of panels (E-H) show the Δ oxyR mutant, and the third set of panels (I-L) compares the resistance concentrations of each strain reached at day 20. In order to evaluate the effect of oxygen on the development of resistance by E. coli exposed to sub-lethal levels of antibiotics, initially fully susceptible cells were grown in the presence of stepwise increasing concentrations of four antimicrobials. The evolution of resistance to the bactericidal antibiotics amoxicillin, enrofloxacin, kanamycin, and the bacteriostatic antibiotic tetracycline under aerobic and anaerobic conditions was compared (Fig. 1). To provide additional insight into the role of cellular systems that induce stress caused by reactive oxygen species (ROS), two E. coli strains were used in these experiments: The wild-type MG1655 and the Δ oxyR single gene deletion strain derived from it. OxyR is a transcriptional dual regulator of antioxidant gene expression in response to oxidative stress. We assume that the cell will produce more ROS when oxyR is knocked out. In approximately 30 days, resistance against bactericidal antibiotics (amoxicillin, enrofloxacin, and kanamycin) reached a high concentration (512–2048 µg/mL), while resistance against the bacteriostatic tetracycline was limited to around 32 µg/mL (Fig. 1). There were only minor differences in the final concentrations reached by the wild-type MG1655 under aerobic or anaerobic incubations. The only clear difference was that the final concentration for amoxicillin and kanamycin was double after aerobic growth compared to anaerobic (Fig. 1A and C). In the case of the Δ oxyR mutant, the aerobic incubations reached higher resistance levels and reached them faster, especially in the case of bactericidal antibiotics (Fig. 1E-G). After 20 days, the resistance concentrations reached showed major differences. The Δ oxyR mutant strain under aerobic conditions reached to the highest concentrations compared to the other strains (Fig. 1I-L). Acquisition of resistance in E. coli and L. lactis in rich medium To separate the influence of oxygen itself from that of oxygen derived compounds, such as ROS, the experiments were repeated on L. lactis . As one of very rare microbes L. lactis is homofermentative, both under aerobic and anaerobic conditions, because it lacks the pathway for heme biosynthesis and hence cannot form an endogenous ETC. However, an active respiratory chain is established when the culture is grown in the presence of heme. These features make L. lactis an ideal model organism to address the question of the influence of different levels of activity of the ETC on the development of antibiotic resistance. As L. lactis can only be cultured in rich medium, the experiments were repeated for E. coli grown in rich LB medium as well to facilitate the comparison. After 30 days the resistance to amoxicillin was still in the low range of concentrations (1 or 2 µg/mL) in L. lactis , compared to 1024 µg/mL in E. coli (Fig. 2 A). Because L. lactis is intrinsically resistant to enrofloxacin and kanamycin, these were omitted (Fig. 2 B and C). Instead, moxifloxacin and chloramphenicol were used. During the tetracycline resistance development, tolerated antibiotic concentrations increased from 0.25 µg/mL to 4 µg/mL (16-fold) in L. lactis , from 0.5 µg/mL to 16 µg/mL (32-fold) in E. coli (Fig. 2 D). L. lactis barely acquired amoxicillin resistance even added heme (Fig. 2 E). Only in the case of moxifloxacin resistance did the addition of heme make a noticeable difference (Fig. 2 F), but not on the evolution of resistance to the bacteriostatic antibiotics: chloramphenicol and tetracycline (Fig. 2 G and H). ROS production levels in antibiotic resistant strains The formation of ROS was measured by fluorescent microscopy. Strains of E. coli made resistant to and exposed to the bactericidal antibiotics amoxicillin, enrofloxacin and kanamycin had higher ROS production than strains grown in the presence of the bacteriostatic tetracycline (Fig. 3A). Compared to the MG1655 wild type, the generation of ROS in Δ oxyR strains was significantly enhanced in the presence of enrofloxacin and kanamycin. In L. lactis , only heme added strains that were exposed to the bactericidals amoxicillin and moxifloxacin had noticeable ROS production (Fig. 3B). In the presence of the bacteriostatic antibiotic chloramphenicol and tetracycline, almost no ROS was generated in L. lactis whether heme was added or not. However, with added heme, L. lactis had significantly more ROS generation when exposed to the bactericidal antibiotics amoxicillin and moxifloxacin than the L. lactis strains in the absence of heme. These observations indicate that exposure to bactericidal antibiotics increases ROS production in the oxyR knockout to higher levels than in wildtype E. coli , and similarly in heme added L. lactis compared to regular. Whole genome sequencing to document mutations during resistance evolution To identify mutations that accompany the acquisition of resistance, the genomic DNA of the strains resistant to the highest antibiotic concentrations was sequenced entirely. Variant calling analysis was used to document nucleotide changes as summarized in Fig. 4 . The most frequently observed types of nucleotides changes were AT to CG and CG to GC (Fig. 4 ). Higher numbers of these two types were detected under anaerobic conditions than under aerobic conditions. The MG1655 strain in aerobic conditions had the fewest mutations. L. lactis mainly acquired deletion mutations less of 20 bp (Fig. 4 ). After excluding the same mutations observed in strains grown without antibiotics, the unique mutated genes in each strain were identified and summarized in Venn diagram (Fig. 5 and Fig. 7 A-D). DNA copy number variations are shown in Fig. 6 , Table 1 , and Fig. 7 E-L. In all amoxicillin resistant strains, the genes ampC , frdD , and cpxA were mutated (Fig. 5 A). AmpC is the serine beta-lactamase with substrate specificity for amoxicillin, FrdD is the fumarate reductase subunit D [ 20 ]. CpxA is a membrane-localized sensor kinase that activates CpxR, which promotes efflux complex expression [ 21 ]. The four resistant strains all contained a differential amplification contig that included ampC and frdD (Fig. 6 A-D and Table 1 ). In the resistant strains, some outer membrane porin genes such as ompF and ompC were mutated. These are associated with reduced permeability to antibiotics. Only the Δ oxyR strain under aerobic conditions had a mutation in acrB , which codes for an efflux pump. Strains made resistant to enrofloxacin shared two common mutated genes, DNA gyrase gene, gyrA , and DNA topoisomerase gene, parC , which are well-known quinolone resistance genes [ 22 ] (Fig. 5 B). Other typical quinolone resistance related genes, gyrB and parE were mutated in the Δ oxyR strain under aerobic conditions, parE was mutated as well in the Δ oxyR strain under anaerobic conditions and the MG1655 strain under aerobic conditions. Several DNA or RNA helicase genes were mutated, such as dinG , hrpA , and yoaA . Efflux pump associated genes like acrA , phoQ were mutated in the MG1655 strain under aerobic conditions, and acrR , soxR in the Δ oxyR strains both under aerobic and anaerobic conditions. The MG1655 strain under anaerobic conditions contained a cryptic prophage e14 deletion (Fig. 6 E), which is a well-known mutation associated with quinolone resistance [ 23 ]. In lexA , which inhibits a number of genes involved in the SOS response to DNA damage [ 24 ][ 25 ], a Gly85Ser mutation was observed in the Δ oxyR strain under aerobic conditions. The common mutated gene in kanamycin resistant strains is fusA (Fig. 5 C). During the translation elongation, FusA catalyzes the GTP-dependent ribosomal translocation step [ 26 ]. This mutation may cause antibiotic target alteration. The MG1655 strain under anaerobic conditions had a 5 kb deletion from gene yaiT to yaiW . (Fig. 6 I). This deletion includes sbmA , a peptide antibiotic transporter. The sbmA mutation also occurred in both the MG1655 and Δ oxyR strains under aerobic conditions. There was no common mutated gene in the tetracycline resistant strains (Fig. 5 D). However, a few resistance-related mutated genes were identified. For instance, genes that code for the antibiotic efflux pumps, such as acrA , acrB , acrR , mlaF were mutated. Genes that associated with antibiotic target alteration or protection, like rpoB and rpsJ ; and genes associated with reduced permeability to antibiotics, such as ompF were also mutated. The MG1655 strains under aerobic and anaerobic conditions both contained a 2-fold amplification region from insH1 to insH3 of around 400 kb length (Fig. 6 M and N). The amplified region in the Δ oxyR strain under aerobic conditions started from insH1 as well but stopped in insF1 resulting in a total length of about 800 kb (Fig. 6 P). A roughly 100 kb amplification region was observed in the Δ oxyR strain under anaerobic conditions from gene insH1 to yahH (Fig. 6 O). Table 1 Amoxicillin resistant strains’ amplification contigs Strains Length (kb) Copy numbers (times) Anaerobic MG1655 53 30 Aerobic MG1655 2.5 17 Anaerobic Δ oxyR 5.5 41 Aerobic Δ oxyR 59 9 Mutations in L. lactis with and without added heme. In L. lactis , the number of mutated genes in strains made resistant to bactericidal antibiotics was higher than in strains resistant to bacteriostatic antibiotics (Fig. 7 ). When heme was added to generate a functional ETC L. lactis strains that evolved resistance contained mutations in two genes, purM (Gly250Val), and aroH (Arg168Gly). PurM is involved in purine metabolism [ 27 ]. AroH is involved in the early step of aromatic amino acid biosynthesis [ 28 ]. In amoxicillin resistant L. lactis , the mutated genes, pbp2B and pbpX were observed both with and without added heme. Mutations in these two genes may cause antibiotic action targets modification. The target genes ponA and cdsA were mutated in the heme-added strain as well (Fig. 7 A). The common mutated genes in moxifloxacin resistant strains were gyrA , parC , and llmg1807 . The genes rpoC were mutated in the no heme-added condition, and rpoB in heme-added condition (Fig. 7 B). There was a roughly 45 kb length deletion from llmg1359 to llmg1411 in no heme-added strain (Fig. 7 G). A 42 kb amplification, with around 4-fold copy number increase, from ps301 to ps357 occurred in the heme-added moxifloxacin resistant strain (Fig. 7 H). Only one mutated gene, rplD (Lys68Asn) was detected in no heme-added chloramphenicol resistant L. lactis . RplD, the 50S L4 ribosomal protein, was identified in Neisseria gonorrhoeae as a macrolide resistance protein [ 29 ]. The same gene rplD was mutated but with different amino acid change, Lys68Glu, in the heme-added chloramphenicol resistant L. lactis . The tetracycline resistant L. lactis strains had two common mutated genes, llmg0323 and rpsJ . Llmg0323 is a transcriptional regulator. RpsJ, 30S ribosomal protein S10 is a tetracycline-resistant ribosomal protection protein [ 30 ]. Functional annotation of mutated genes associated with antibiotic resistance. In order to group the mutated genes, they were clustered functionally according to the phylogenetic classification (Fig. 8 ). The IMG/M database of Clusters of Orthologous Groups (COG) was used to annotate all the mutated genes and classify them [ 31 ]. There were no clear differences in which classes of genes were mutated between E. coli wild-type MG1655 and the Δ oxyR strains under aerobic and anaerobic conditions (Fig. 8 A). However, the different antibiotics caused mutations in different classes of genes. The majority of genes mutated after exposure to amoxicillin, enrofloxacin, and kanamycin are involved in cellular processing and signaling, information storage and processing, and metabolism, respectively (Fig. 8 A). In L. lactis , half the number of mutated genes in amoxicillin exposed strains is involved in cellular processes and signaling function. The genes mutated by growth in the presence of moxifloxacin were evenly distributed over information and signaling and metabolism (Fig. 8 B). The number of mutated genes in tetracycline-incubated E. coli , and chloramphenicol, tetracycline-incubated L. lactis were clearly lower than those of cells exposed to bactericidal antibiotics, making the COG comparison problematic. Discussion The role of oxygen in the killing of bacteria by bactericidal antibiotics has been the subject of debate since the “radical based” theory was proposed, which suggests that reactive oxygen species form a secondary killing mechanism in addition to the primary target [ 7 ][ 32 ][ 33 ]. This study examined the role of oxygen in de novo acquisition of resistance by comparing resistance development under aerobic and anaerobic conditions. In the human intestines, E. coli is exposed to anaerobic or micro-aerobic conditions [ 34 ]. Anaerobic growth may therefore be more natural than the aerobic conditions used in most experiments involving E. coli . To test the influence of oxidative stress, resistance adaptation was examined in an E. coli Δ oxyR mutant strain. L. lactis was used to separate between the effects of oxygen and those of ROS, because even under aerobic conditions, it does not possess a complete ETC, unless heme is supplied in the medium [ 35 ]. Hence, it only forms ROS when heme is present in the medium, but not in the absence, even when oxygen is available. Hormesis The de novo acquisition of resistance in wildtype E. coli with and without oxygen was relatively similar. The presence of oxygen seems to have no major influence on resistance development. However, under anaerobic conditions, reactive metabolic byproducts (RMB), particularly reactive electrophilic species, accumulate in antibiotic-treated E. coli [ 9 ]. These RMB contribute to cell death in a similar manner as ROS in aerobic conditions. Possibly ROS and RMB have relatively similar effects on resistance development. Nonetheless, under aerobic conditions, Δ oxyR strain became resistant to bactericidal antimicrobials faster than the wildtype. The oxyR gene codes for a regulatory protein that regulates a system of proteins that protect the cell from ROS and comparable stressors [ 36 ]. The presence of oxygen accelerated the acquisition of resistance in the Δ oxyR strains, indicating that the extra ROS indeed stimulated this process. Hyperproduced ROS damage DNA and the nucleotide pool. The 8-oxo-guanine derived from guanine results in faulty DNA replication, accompanied by the general stress response [ 37 ]. The cell’s repair systems tend to increase rates of mutagenesis [ 38 ]. During antibiotic resistance acquisition, the differences can be observed in the middle and late stages of resistance development, possibly because at that stage increasing numbers of cells accumulate general stress-induced mutations. The observation that heme-added L. lactis gained moxifloxacin resistance faster also points to an effect of ROS (Fig. 2 F), generated by the ETC that was induced by the addition of heme. These observations can be understood in the framework of hormesis[ 39 ][ 12 ]. While mild stress is beneficial in the form of the ability to more rapidly adapt to the presence of antimicrobials, high levels of the same stressors can cause cell death. When radical-based stress levels are low, a relatively moderate increase of the oxidative stress seems to accelerate the acquisition of resistance. Mutations accompanying resistance development Far higher mutation frequencies of AT to CG and CG to GC were observed in anaerobically grown E. coli than in aerobic conditions. A likely cause is the DNA damage induced by acidic fermentation[ 40 ][ 41 ]. However, these kinds of single nucleotide change mutations and the < 20 bp deletion mutations which occurred in the L. lactis were almost never situated in resistance-related genes hot spots. Anaerobically grown untreated E. coli strains also had high mutation frequencies, indicating that this kind of mutation did not influence resistance development. Various E. coli strains which evolved resistance to the bactericidal antibiotics had common mutated genes, for instance, ampC for amoxicillin, gyrA and parC for enrofloxacin, and fusA for kanamycin. These genes are known to play crucial roles in resistance acquisition [ 2 ]. In addition to the mutated genes that are common to all strains with induced resistance, there are mutations in genes that are specific for a certain strain and that follow logically from the characteristics of that strain. Only the amoxicillin resistant E. coli Δ oxyR strain under aerobic conditions had an acrB mutation that may increase amoxicillin efflux. In the aerobically grown Δ oxyR enrofloxacin resistant strain contained a mutation was observed lexA that codes for a transcriptional repressor. Auto-cleavage of LexA triggers the SOS response [ 42 ]. Mutagenic states caused by the SOS stress response may enhance the de novo acquisition of antibiotic resistance [ 43 ][ 44 ]. The Gly85Ser mutation located on the lexA auto-cleavage site [ 45 ], may increase the rate of acquisition of resistance. During kanamycin exposure, SbmA mutations included Phe6fs in the aerobically grown wildtype strain, total deletion in the anaerobically grown wildtype, and Ser372* in the aerobically grown Δ oxyR strain. These mutations block SbmA activity, thus inhibiting kanamycin uptake [ 46 ]. Three of the four cell lines exposed to kanamycin acquired mutations in sbmA that reduced kanamycin uptake. Only the anaerobically grown Δ oxyR strain missed a sbmA mutation, and indeed developed less kanamycin resistance. Mutations in the multidrug efflux pump subunit coding genes acrA , acrB , acrD , and their regulator acrR , emerged in four antibiotic-resistant E. coli strains. Additionally, mutations in the outer membrane porin genes envZ ( ompB ), ompC , and ompF appeared in the amoxicillin, kanamycin, and tetracycline resistant E. coli strains. This could lead to increased resistance when these strains are exposed to other antibiotic treatments due to the cross-resistance [ 47 ]. Comparison with a similar dataset in which E. coli wildtype evolved resistance resistant against the same four antimicrobials showed that apart from the commonly mutated genes described above, there were no other genes mutated in both datasets [ 3 ]. Hence, we must conclude that most mutations that occur during the acquisition of antibiotic resistance were random events that may or may not contribute to the development of resistance. Selection and co-selection afterwards during continued exposure to a specific antimicrobial determine the final list of mutations. Functional distribution of mutations The distribution over functional categories of genes that mutated as a result of exposure to antimicrobials was more consistent and can be related to the mechanisms of action of the particular antibiotic. Both in the dataset on mutations after resistance was built up against two antibiotics [ 3 ] and in the present dataset, a specific antibiotic correlates with specific functions. Mutations accompanying resistance to amoxicillin occur in genes coding for proteins involved in cellular processing and signaling and metabolism. This corresponds to the disruption of cell wall construction by beta-lactam antibiotics [ 48 ]. Enrofloxacin correlates primarily to genes concerning information storage and processing, due to the inhibition of DNA replication [ 49 ], and also to metabolism. Kanamycin mutations are mainly in the area of metabolism, while tetracycline mutations are about evenly divided over cellular processing and signaling and information storage and processing, which also can be understood in the framework of the inhibition of protein synthesis by tetracyclines [ 50 ][ 51 ]. The distribution in L. lactis was similar to that in E. coli . Prolonged exposure to the beta-lactam antibiotic amoxicillin resulted in the amplification of a chromosomal DNA fragment centered around the ampC gene [ 6 ]. The multiplication of a 2.5 kb fragment was observed in the aerobically grown MG1655 strain. The amplified segments were larger in the anaerobically grown MG1655 strain (53 kb) and in the mutant Δ oxyR both under aerobic (59 kb) and anaerobic (5.5kb) conditions. This indicates that the same process was taking place, but not in exactly the same manner. The fragment containing the ampC gene resembled known plasmid-bound beta-lactam resistance genes and could be transferred to a susceptible E. coli strain, that became resistant after this transfer[ 52 ]. These observations suggest that the de novo development of resistance may play a bigger role in spreading of antimicrobial resistance than previously presumed. A similar duplication of part of the chromosome was also observed as a result of tetracycline exposure. In this case, the copy number was only 2-fold at most and it is doubtful that it played a major role in the acquisition of resistance, also because gene amplification is not known as a tetracycline resistance mechanism. Conclusion The effects of ROS and reactive metabolic byproducts on development of antimicrobial resistance in response to exposure to non-lethal concentrations of antibiotics can be understood by the principle of hormesis. High levels of radical-based stress are lethal, while low levels can increase the rate of acquisition of resistance and thus for the survival of the cell. The role of oxygen in de novo acquisition of antibiotic resistance turned out to be indirect. Under anaerobic conditions, the reactive metabolic byproducts seem to function in a similar manner as ROS under aerobic conditions. Still, these stresses caused by reactive compounds remain at a low level when antibiotic concentrations are non-lethal, and the relatively low levels of stress under these conditions accelerate resistance acquisition. Materials and methods Bacterial strains, media and growth conditions The antibiotic-sensitive wildtype strains E. coli MG1655 and L. lactis MG1363 were used throughout the study. The oxyR gene knockout mutant strain JW3933-3 was obtained from the Keio collection [ 53 ], and the kanamycin-resistant cassette was removed using the pCP20 plasmid by FLP Recombination. E. coli strains were grown in LB medium or a phosphate-buffered (100 mM Na 2 H 2 PO 4 ) defined minimal medium containing 55 mM glucose [ 54 ]. L. lactis was grown in 10% Lactose M17 broth medium. E. coli was grown at 37°C, L. lactis was grown at 30°C, and both were shaken at 200 rpm. Anaerobic culture DURAN® bottles with a butyl rubber stopper and an open topped screw cap, were filled with medium, inoculated with a syringe. Resazurin was used as oxygen indicator, and these tubes were autoclaved separately. L. lactis under aerobic respiration conditions was grown with further addition of heme (Sigma) to a final concentration of 2 µg/mL. Amoxicillin, enrofloxacin, kanamycin, tetracycline, moxifloxacin, and chloramphenicol stock solutions (10 mg/mL) were filter sterilized through a 0.2 µM filter and stored at 4°C. Fresh antibiotic solutions were made every 3 days. Evolution experiments Table 2 Initial MICs of E. coli strains E. coli Strains MG1655 MG1655 Δ oxyR Mutant Medium LB Minimal medium Minimal medium Amoxicillin 8/16 4 4 Enrofloxacin 4 0.5 1 Kanamycin 64 16 16/32 Tetracycline 8 2 2/4 Table 3 Initial MICs of L. lactis strains L. lactis Strains MG1363 MG1363 Medium LM17 Heme + LM17 Amoxicillin 0.25/0.5 0.25 Moxifloxacin 4 2 Chloramphenicol 2/4 2 Tetracycline 0.5/1 0.5 In order to induce resistance to each antibiotic and test the susceptibility of the strains, the MICs measurement was performed by serial dilution followed by a determination of the initial antibiotic concentrations (Tables 2 and 3 ). Evolution experiments inducing resistance were performed as described previously [ 55 ]. Briefly, cultures were grown overnight in medium in the presence of sub-lethal levels of antibiotics. The initial antibiotic concentrations were lower than half MICs. When the OD 600 of the culture after 24 hours corresponded to ≥ 75% of the OD 600 of the antibiotic-free or already adapted culture, cells were used and inoculated with double concentrations of the antibiotic. If sufficient growth did not occur, these steps were repeated using cells from tubes without or with a lower antibiotic concentration. Each strain's evolution experiment was independently performed at least twice. In all experiments, cultures without antibiotic exposure were used as controls. MICs was detected three times a week to monitor resistance. MICs was measured in 96-well plates in a spectrophotometer plate reader (Thermo Fisher Scientific) at 37 o C. Each well contains 150 µL final volumes with the OD 600 of 0.05 bacteria, antibiotics concentrations range from 0.25 to 2048 with steps of a factor of two. After overnight culture, the lowest concentration that yielded a final OD 600 < 0.2 was considered the MICs. ROS measurement To determine the formation of ROS, overnight cultured E. coli and L. lactis strains made resistant to a specific antibiotic were diluted to OD 600 of 0.2 and exposed to the highest concentration of these antibiotics that still allowed growth. After administration of the antibiotic, cells were cultured for 3 hours at 37°C ( E. coli ) or 30°C ( L. lactis ) shaking at 200 rpm. Cell cultures were incubated with 5 µL 10 mM H 2 DCFDA (Sigma) fluorescent dye dissolved in DMSO. Culturing tubes were covered with aluminum foil to prevent exposure to light and incubated for 45 minutes at the same temperature. After incubation, 1 mL of culture was spun down at 6000 rpm for 5 min, the cell pellet was dissolved in medium. 1.3 µL cell suspension was loaded on a microscope slide glass with 2% agarose mixed with medium. Images were acquired on a Nikon Eclipse Ti microscope with NIS-elements AR software. Fluorescent signal was detected at excitation/emission wavelength of 488/510 nm and was shown in green. Images were processed using Fiji/ImageJ software. Whole genome sequencing The genomic DNA was isolated from the final stable resistant strains by the DNeasy blood and tissue kit (Qiagen). Genomic DNA libraries were generated using the NEBNext Ultra II FS DNA Library Prep kit for Illumina (New England BioLabs) in combination with NEBNext multiplex oligos for Illumina (96 Unique Dual Index Primer Pairs; New England BioLabs) according to the manufacturer’s instructions. Briefly, 500 ng genomic DNA was used as input with a fragmentation time of 5 min, aiming at an insert size distribution of 275–475 bp by following the corresponding size selection option provided in the protocol. The resulting size distribution of the libraries with indexed adapters was assessed using a 2200 TapeStation System with Agilent D1000 ScreenTapes (Agilent Technologies). The libraries were quantified on a QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific) using the NEBNext Library Quant Kit for Illumina (New England BioLabs) according to the instructions of the manufacturer. The libraries were clustered and sequenced (2 x 150 bp) on a NextSeq 550 Sequencing System (Illumina) using a NextSeq 500/550 Mid Output v2.5 kit (300 cycles) (Illumina). After sequencing, FastQC and MultiQC were used to evaluate the quality of raw reads. BBmerge was used to discover the adapter sequences, which were then imported to Cutadapt to be removed. In order to remove low-quality bases, Trimmomatic was utilized. The removal of optical duplicates was achieved via Clumpify. After mapping the reads to the reference by Bowtie2, GATK was used for marking PCR duplicates. The variant calling was done by Freebayes and used Snpeff to do the variant annotation. Subsequently, the SNPs were selected with IGV from the output vcf files. The mutated genes were selected from the resistant strains compared with the no-antibiotic exposure control strain, and the repeat mutated genes were eliminated. IMG/M was used to classify the mutated genes into clusters of orthologous groups (COG) categories. To do the copy number analysis, the cn.MOPS was used to identify larger genomic alterations that result in an abnormal number of copies of one or more genes. Abbreviations ROS Reactive oxygen species RMB Reactive metabolic byproducts ETC Electron transport chain COG Clusters of orthologous groups Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Availability of data and materials The binary alignment/map (bam) files of the sequenced strains have been deposited in the NCBI database and can accessed at BioProject PRJNA954686 and PRJNA954732. Competing interests We declare that no competing interests exist. Funding This study was financed by the Netherlands Food and Consumer Product Safety Authority (NVWA). The NVWA was not involved in design of the experiments, analysis of the data or writing the manuscript. Authors' contributions WQ and BtK conceived the project. WQ performed experiments. MJ performed the bioinformatic analysis. WQ and BtK wrote the manuscript. LT proved the knockout strain. MW and LT contributed to the final manuscript. All authors critically reviewed the manuscript and approved the final version. Acknowledgements We thank S. Brul for stimulating discussions and suggestions for improvement of an earlier version of the manuscript. Author information Laboratory for Molecular Biology and Microbial Food Safety, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands Wenxi Qi, Lisa Teichmann, Meike Wortel & Benno H. Ter Kuile RNA Biology & Applied Bioinformatics, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands Martijs J. Jonker Netherlands Food and Consumer Product Safety Authority, Office for Risk Assessment, Utrecht, The Netherlands Benno H. ter Kuile References MacLean RC, Millan AS. “The evolution of antibiotic resistance,” Science (80-.). , vol. 365, no. 6458, pp. 1082–1083, Sep. 2019, doi: 10.1126/SCIENCE.AAX3879 . Händel N, Schuurmans JM, Feng Y, Brul S, Kuile BH. “Interaction between Mutations and Regulation of Gene Expression during Development of De Novo Antibiotic Resistance,” Antimicrob. Agents Chemother. , vol. 58, no. 8, p. 4371, 2014, doi: 10.1128/AAC.02892-14 . Hoeksema M, Jonker MJ, Brul S, Ter Kuile BH. “Effects of a previously selected antibiotic resistance on mutations acquired during development of a second resistance in Escherichia coli,” BMC Genomics , vol. 20, no. 1, pp. 1–14, Apr. 2019, doi: 10.1186/s12864-019-5648-7 . 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Cite Share Download PDF Status: Published Journal Publication published 02 Oct, 2023 Read the published version in BMC Microbiology → Version 1 posted Editorial decision: Major revision 08 Aug, 2023 Reviews received at journal 19 Jul, 2023 Reviewers agreed at journal 10 Jul, 2023 Reviewers agreed at journal 07 Jul, 2023 Reviewers invited by journal 06 Jul, 2023 Editor invited by journal 12 Jun, 2023 Editor assigned by journal 12 Jun, 2023 Submission checks completed at journal 08 Jun, 2023 First submitted to journal 14 May, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2932862","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":210304131,"identity":"4bb30b07-dcf4-4000-8872-a62513e26a3c","order_by":0,"name":"Wenxi Qi","email":"","orcid":"","institution":"University of Amsterdam","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Wenxi","middleName":"","lastName":"Qi","suffix":""},{"id":210304132,"identity":"e365006c-dd30-427d-8669-ae4dbbe4cafe","order_by":1,"name":"Martijs J. 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The top 4 panels (A-D) show wild-type MG1655, the second set of panels (E-H) show the Δ\u003cem\u003eoxyR\u003c/em\u003e mutant, and the third set of panels (I-L) compares the resistance concentrations of each strain reached at day 20.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-2932862/v1/275c1cec1596d4bf4e877df2.png"},{"id":39792791,"identity":"08bc40a8-e31f-4574-9ff5-057339e98907","added_by":"auto","created_at":"2023-07-10 13:56:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":5191583,"visible":true,"origin":"","legend":"\u003cp\u003eAcquisition of resistance by \u003cem\u003eE. coli \u003c/em\u003eMG1655 to amoxicillin (A), enrofloxacin (B), kanamycin (C), tetracycline(D) in rich medium (LB), and \u003cem\u003eL. lactis\u003c/em\u003e MG1363 to amoxicillin (A), tetracycline (D) in rich medium (M17). Acquisition of resistance to amoxicillin (E), moxifloxacin (F), chloramphenicol (G), and tetracycline (H) by \u003cem\u003eL. lactis\u003c/em\u003e and Heme-added \u003cem\u003eL. lactis\u003c/em\u003ein M17 medium.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-2932862/v1/ef223b15a986454b8a8f10ab.png"},{"id":39794322,"identity":"039ffea2-aa06-4a04-b2c8-26f78cfd7802","added_by":"auto","created_at":"2023-07-10 14:04:38","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4624127,"visible":true,"origin":"","legend":"\u003cp\u003eROS measurement in antibiotic resistant \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eL. lactis \u003c/em\u003eunder fluorescent microscopy. Resistant strains were treated with the highest concentrations of antibiotics that still allowed growth. The fluorescent dye H\u003csub\u003e2\u003c/sub\u003eDCFDA was used to detect the ROS. Cells with ROS production were counted with ImageJ, \u003cem\u003eE. coli\u003c/em\u003e (A), \u003cem\u003eL. lactis\u003c/em\u003e (B). Means ± SD, statistical significance was investigated using a one-way ANOVA *p \u0026lt; 0.05, **p \u0026lt; 0.001, ***p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-2932862/v1/0b5108727afa93e012c127f0.png"},{"id":39792784,"identity":"8c8628f5-0783-4224-9708-8f5a18bb049e","added_by":"auto","created_at":"2023-07-10 13:56:38","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2534463,"visible":true,"origin":"","legend":"\u003cp\u003eHeat map of the types of nucleotide changes in MG1655 and Δ\u003cem\u003eoxyR \u003c/em\u003eunder aerobic and anaerobic conditions (A); \u003cem\u003eL. lactis\u003c/em\u003e, heme-added \u003cem\u003eL. lactis\u003c/em\u003e and \u003cem\u003eE. coli \u003c/em\u003ein rich medium (B). N: no treatment control, A: amoxicillin, E: enrofloxacin, K: kanamycin, T: tetracycline, M: moxifloxacin, C: chloramphenicol. Ins: insertion, del: deletion, dup: duplication.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-2932862/v1/a881d364163fb2bed6005a80.png"},{"id":39794956,"identity":"c3c82383-75fb-4b94-9ee0-28f45fdb88cb","added_by":"auto","created_at":"2023-07-10 14:12:38","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":6231221,"visible":true,"origin":"","legend":"\u003cp\u003eOverlap of mutated genes in the anaerobic wild-type MG1655 (blue oval), the aerobically grown wild-type MG1655 (yellow oval), the anaerobic\u003cem\u003e \u003c/em\u003emutant Δ\u003cem\u003eoxyR \u003c/em\u003e(green oval), and the aerobic mutant Δ\u003cem\u003eoxyR \u003c/em\u003e(red oval) made resistant to amoxicillin (A), enrofloxacin (B), kanamycin (C), and tetracycline(D).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-2932862/v1/1804fd5a1e0924876f2ed1c5.png"},{"id":39794320,"identity":"2122e2d4-a050-4c6e-b6a2-e35c229a10f3","added_by":"auto","created_at":"2023-07-10 14:04:38","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":530062,"visible":true,"origin":"","legend":"\u003cp\u003eVariation of DNA copy numbers in WGS of the anaerobically grown wild-type MG1655 (A, E, I, M), aerobic wild-type MG1655 (B, F, J, N), anaerobic\u003cem\u003e \u003c/em\u003emutant Δ\u003cem\u003eoxyR \u003c/em\u003e(C, G, K, O), and aerobic mutant Δ\u003cem\u003eoxyR\u003c/em\u003e (D, H, L, P) made\u003cem\u003e \u003c/em\u003eresistant to amoxicillin (A-D), enrofloxacin (E-H), kanamycin (I-L), and tetracycline(M-P).\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-2932862/v1/48d24984198f89a62c0b1169.png"},{"id":39792790,"identity":"2cfcde2c-2ab6-4ecd-b1ce-5b7872287971","added_by":"auto","created_at":"2023-07-10 13:56:38","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":6583850,"visible":true,"origin":"","legend":"\u003cp\u003eMutated genes in wild-type\u003cem\u003e L. lactis\u003c/em\u003e and heme-added \u003cem\u003eL. lactis\u003c/em\u003e after evolution of resistance to amoxicillin (A), moxifloxacin (B), chloramphenicol (C), and tetracycline (D). DNA copy number of WGS of \u003cem\u003eL. lactis\u003c/em\u003e resistant to amoxicillin (E, F), moxifloxacin (G, H), chloramphenicol (I, J), and tetracycline (K, L) in no heme-added \u003cem\u003eL. lactis\u003c/em\u003e (E, G, I, K) is compared to heme-added\u003cem\u003e L. lactis\u003c/em\u003e (F, H, J, L).\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-2932862/v1/03506d6ae37b3417e99fdcc5.png"},{"id":39792787,"identity":"05248ee9-3d60-430b-b29e-c586c84d66a3","added_by":"auto","created_at":"2023-07-10 13:56:38","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":6154317,"visible":true,"origin":"","legend":"\u003cp\u003eCluster of orthologous groups (COG) classifications of mutated genes. A: amoxicillin, E: enrofloxacin, K: kanamycin, T: tetracycline, M: moxifloxacin, C: chloramphenicol; AN: anaerobic condition, AE: aerobic condition. Panel A: \u003cem\u003eE. coli\u003c/em\u003e; Panel B: \u003cem\u003eL. lactis\u003c/em\u003e. The IMG/M was used to classify the genes (https://img.jgi.doe.gov/).\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-2932862/v1/fcc772605a83bd3b88074b9a.png"},{"id":44302440,"identity":"65af4a60-f390-41c7-a5a7-c22711f907c7","added_by":"auto","created_at":"2023-10-09 15:10:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2917586,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2932862/v1/c44701a8-fdd0-4a14-bc46-ccdc9d40461c.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The influence of oxygen and oxidative stress on de novo acquisition of antibiotic resistance in E. coli and Lactobacillus lactis","fulltext":[{"header":"Background","content":"\u003cp\u003eBacteria can acquire antibiotic resistance by adapting cellular physiology, DNA mutations, and horizontal transfer of resistance genes [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. DNA mutations can occur as a result of exposure to non-lethal concentrations of antimicrobials [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. These mutations can modify the cellular targets of antibiotics, activate antibiotic efflux pumps, generate enzymes that disable antibiotics, and reduce the permeability of membranes to antibiotics to make bacteria resistant [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. While the role of mutations in development of \u003cem\u003ede novo\u003c/em\u003e resistance has been documented to some extent [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e][\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e][\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], the driving factors for mutations in the bacterial DNA are less well described.\u003c/p\u003e \u003cp\u003eAccording to the radical-based theory, exposure to bactericidal antibiotics results in the formation of reactive oxygen species (ROS) and reactive metabolic byproducts as a secondary effect that hastens bacterial cell death [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e][\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e][\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The overproduction of ROS damages the DNA, proteins, lipids, and nucleotides pool, and in particular causes the oxidation of guanine to 8-oxo-guanine [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Besides being lethal, ROS can also enhance mutation rates [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In fact, the overall effect of ROS and reactive metabolic byproducts may be hormetic, as low concentrations enable rapid adaptation, while high level are lethal [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Sub-inhibitory doses of ciprofloxacin generate a resistant mutant subpopulation through ROS formation and sigma-S general stress response activity [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Furthermore, multidrug resistance induced by sublethal levels of antibiotics correlates with ROS-induced mutagenesis [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. ROS has been described as a key factor in antibiotic-induced SOS mutagenesis, and treatment with the antioxidant N-acetylcysteine reduces ROS and blocks SOS-mediated mutagenesis [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Based on the above considerations we hypothesized that the oxidative stress caused by ROS plays a central role in \u003cem\u003ede novo\u003c/em\u003e acquisition of antibiotic resistance.\u003c/p\u003e \u003cp\u003e \u003cem\u003eEscherichia coli\u003c/em\u003e is a facultative anaerobic procaryote, commonly found in the human gastrointestinal tract [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The gene \u003cem\u003eoxyR\u003c/em\u003e in \u003cem\u003eE. coli\u003c/em\u003e codes for an oxidative stress regulator, mitigating levels of hydrogen peroxide under aerobic conditions [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Because \u003cem\u003eE. coli\u003c/em\u003e is well described and thoroughly studied in many aspects, it is the primary model organism used in this study. The lactic acid bacterium \u003cem\u003eLactococcus lactis\u003c/em\u003e is a fermentative bacterium, that can also grow in the presence of oxygen, but even then does not possess a complete electron transport chain (ETC) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. However, when both heme and oxygen are present, \u003cem\u003eL. lactis\u003c/em\u003e can establish an ETC, resulting in NADH oxidation and aerobic respiration [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] and hence the formation of ROS. It is therefore used in several experiments to separate the respective roles of oxygen and ROS generated by the ETC in development of \u003cem\u003ede novo\u003c/em\u003e antimicrobial resistance.\u003c/p\u003e \u003cp\u003eThe hypothesis that ROS is a driving factor for \u003cem\u003ede novo\u003c/em\u003e acquisition of resistance was investigated by comparing resistance development in \u003cem\u003eE. coli\u003c/em\u003e MG1655 and Δ\u003cem\u003eoxyR\u003c/em\u003e mutant strains grown aerobically and anaerobically. The rate of adaptation to increasing concentrations of antibiotics of the wildtype and the mutant did not differ much under anaerobic conditions, indicating a limited role of oxygen as such. The faster resistance development in Δ\u003cem\u003eoxyR\u003c/em\u003e mutant \u003cem\u003eE. coli\u003c/em\u003e and in \u003cem\u003eL. lactis\u003c/em\u003e with added heme indicates that moderately increased levels of ROS stimulate resistance acquisition, possibly by increasing mutation rates. A hormetic effect that is related to the radical-based killing mechanism was observed. High levels of ROS or reactive metabolic byproducts were lethal, while moderate levels of stress stimulate development of resistance. When radical-based stress levels are low, a moderate increase in stress accelerates resistance development.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eAcquisition of resistance under aerobic and anaerobic conditions\u003c/b\u003e \u003cb\u003eFIG 1\u003c/b\u003e Acquisition of resistance to amoxicillin (A, E, I), enrofloxacin (B, F, J), kanamycin (C, G, K), and tetracycline (D, H, L) of \u003cem\u003eE. coli\u003c/em\u003e wild-type MG1655 (blue: anaerobic/black: aerobic) and the Δ\u003cem\u003eoxyR\u003c/em\u003e knockout (green: anaerobic/red: aerobic) strains. The top 4 panels (A-D) show wild-type MG1655, the second set of panels (E-H) show the Δ\u003cem\u003eoxyR\u003c/em\u003e mutant, and the third set of panels (I-L) compares the resistance concentrations of each strain reached at day 20.\u003c/p\u003e \u003cp\u003eIn order to evaluate the effect of oxygen on the development of resistance by \u003cem\u003eE. coli\u003c/em\u003e exposed to sub-lethal levels of antibiotics, initially fully susceptible cells were grown in the presence of stepwise increasing concentrations of four antimicrobials. The evolution of resistance to the bactericidal antibiotics amoxicillin, enrofloxacin, kanamycin, and the bacteriostatic antibiotic tetracycline under aerobic and anaerobic conditions was compared (Fig.\u0026nbsp;1). To provide additional insight into the role of cellular systems that induce stress caused by reactive oxygen species (ROS), two \u003cem\u003eE. coli\u003c/em\u003e strains were used in these experiments: The wild-type MG1655 and the Δ\u003cem\u003eoxyR\u003c/em\u003e single gene deletion strain derived from it. OxyR is a transcriptional dual regulator of antioxidant gene expression in response to oxidative stress. We assume that the cell will produce more ROS when \u003cem\u003eoxyR\u003c/em\u003e is knocked out. In approximately 30 days, resistance against bactericidal antibiotics (amoxicillin, enrofloxacin, and kanamycin) reached a high concentration (512\u0026ndash;2048 \u0026micro;g/mL), while resistance against the bacteriostatic tetracycline was limited to around 32 \u0026micro;g/mL (Fig.\u0026nbsp;1).\u003c/p\u003e \u003cp\u003eThere were only minor differences in the final concentrations reached by the wild-type MG1655 under aerobic or anaerobic incubations. The only clear difference was that the final concentration for amoxicillin and kanamycin was double after aerobic growth compared to anaerobic (Fig.\u0026nbsp;1A and C). In the case of the Δ\u003cem\u003eoxyR\u003c/em\u003e mutant, the aerobic incubations reached higher resistance levels and reached them faster, especially in the case of bactericidal antibiotics (Fig.\u0026nbsp;1E-G). After 20 days, the resistance concentrations reached showed major differences. The Δ\u003cem\u003eoxyR\u003c/em\u003e mutant strain under aerobic conditions reached to the highest concentrations compared to the other strains (Fig.\u0026nbsp;1I-L).\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAcquisition of resistance in \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eL. lactis\u003c/em\u003e in rich medium\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo separate the influence of oxygen itself from that of oxygen derived compounds, such as ROS, the experiments were repeated on \u003cem\u003eL. lactis\u003c/em\u003e. As one of very rare microbes \u003cem\u003eL. lactis\u003c/em\u003e is homofermentative, both under aerobic and anaerobic conditions, because it lacks the pathway for heme biosynthesis and hence cannot form an endogenous ETC. However, an active respiratory chain is established when the culture is grown in the presence of heme. These features make \u003cem\u003eL. lactis\u003c/em\u003e an ideal model organism to address the question of the influence of different levels of activity of the ETC on the development of antibiotic resistance. As \u003cem\u003eL. lactis\u003c/em\u003e can only be cultured in rich medium, the experiments were repeated for \u003cem\u003eE. coli\u003c/em\u003e grown in rich LB medium as well to facilitate the comparison.\u003c/p\u003e \u003cp\u003eAfter 30 days the resistance to amoxicillin was still in the low range of concentrations (1 or 2 \u0026micro;g/mL) in \u003cem\u003eL. lactis\u003c/em\u003e, compared to 1024 \u0026micro;g/mL in \u003cem\u003eE. coli\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Because \u003cem\u003eL. lactis\u003c/em\u003e is intrinsically resistant to enrofloxacin and kanamycin, these were omitted (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and C). Instead, moxifloxacin and chloramphenicol were used. During the tetracycline resistance development, tolerated antibiotic concentrations increased from 0.25 \u0026micro;g/mL to 4 \u0026micro;g/mL (16-fold) in \u003cem\u003eL. lactis\u003c/em\u003e, from 0.5 \u0026micro;g/mL to 16 \u0026micro;g/mL (32-fold) in \u003cem\u003eE. coli\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). \u003cem\u003eL. lactis\u003c/em\u003e barely acquired amoxicillin resistance even added heme (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Only in the case of moxifloxacin resistance did the addition of heme make a noticeable difference (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eF), but not on the evolution of resistance to the bacteriostatic antibiotics: chloramphenicol and tetracycline (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eG and H).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003eROS production levels in antibiotic resistant strains\u003c/b\u003e \u003c/h2\u003e \u003cp\u003eThe formation of ROS was measured by fluorescent microscopy. Strains of \u003cem\u003eE. coli\u003c/em\u003e made resistant to and exposed to the bactericidal antibiotics amoxicillin, enrofloxacin and kanamycin had higher ROS production than strains grown in the presence of the bacteriostatic tetracycline (Fig.\u0026nbsp;3A). Compared to the MG1655 wild type, the generation of ROS in Δ\u003cem\u003eoxyR\u003c/em\u003e strains was significantly enhanced in the presence of enrofloxacin and kanamycin. In \u003cem\u003eL. lactis\u003c/em\u003e, only heme added strains that were exposed to the bactericidals amoxicillin and moxifloxacin had noticeable ROS production (Fig.\u0026nbsp;3B). In the presence of the bacteriostatic antibiotic chloramphenicol and tetracycline, almost no ROS was generated in \u003cem\u003eL. lactis\u003c/em\u003e whether heme was added or not. However, with added heme, \u003cem\u003eL. lactis\u003c/em\u003e had significantly more ROS generation when exposed to the bactericidal antibiotics amoxicillin and moxifloxacin than the \u003cem\u003eL. lactis\u003c/em\u003e strains in the absence of heme. These observations indicate that exposure to bactericidal antibiotics increases ROS production in the \u003cem\u003eoxyR\u003c/em\u003e knockout to higher levels than in wildtype \u003cem\u003eE. coli\u003c/em\u003e, and similarly in heme added \u003cem\u003eL. lactis\u003c/em\u003e compared to regular.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eWhole genome sequencing to document mutations during resistance evolution\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo identify mutations that accompany the acquisition of resistance, the genomic DNA of the strains resistant to the highest antibiotic concentrations was sequenced entirely. Variant calling analysis was used to document nucleotide changes as summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003e. The most frequently observed types of nucleotides changes were AT to CG and CG to GC (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Higher numbers of these two types were detected under anaerobic conditions than under aerobic conditions. The MG1655 strain in aerobic conditions had the fewest mutations. \u003cem\u003eL. lactis\u003c/em\u003e mainly acquired deletion mutations less of 20 bp (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003e). After excluding the same mutations observed in strains grown without antibiotics, the unique mutated genes in each strain were identified and summarized in Venn diagram (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-D). DNA copy number variations are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003e, Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, and Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e7\u003c/span\u003eE-L.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn all amoxicillin resistant strains, the genes \u003cem\u003eampC\u003c/em\u003e, \u003cem\u003efrdD\u003c/em\u003e, and \u003cem\u003ecpxA\u003c/em\u003e were mutated (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). AmpC is the serine beta-lactamase with substrate specificity for amoxicillin, FrdD is the fumarate reductase subunit D [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. CpxA is a membrane-localized sensor kinase that activates CpxR, which promotes efflux complex expression [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The four resistant strains all contained a differential amplification contig that included \u003cem\u003eampC\u003c/em\u003e and \u003cem\u003efrdD\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-D and Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In the resistant strains, some outer membrane porin genes such as \u003cem\u003eompF\u003c/em\u003e and \u003cem\u003eompC\u003c/em\u003e were mutated. These are associated with reduced permeability to antibiotics. Only the Δ\u003cem\u003eoxyR\u003c/em\u003e strain under aerobic conditions had a mutation in \u003cem\u003eacrB\u003c/em\u003e, which codes for an efflux pump.\u003c/p\u003e \u003cp\u003eStrains made resistant to enrofloxacin shared two common mutated genes, DNA gyrase gene, \u003cem\u003egyrA\u003c/em\u003e, and DNA topoisomerase gene, \u003cem\u003eparC\u003c/em\u003e, which are well-known quinolone resistance genes [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Other typical quinolone resistance related genes, \u003cem\u003egyrB\u003c/em\u003e and \u003cem\u003eparE\u003c/em\u003e were mutated in the Δ\u003cem\u003eoxyR\u003c/em\u003e strain under aerobic conditions, \u003cem\u003eparE\u003c/em\u003e was mutated as well in the Δ\u003cem\u003eoxyR\u003c/em\u003e strain under anaerobic conditions and the MG1655 strain under aerobic conditions. Several DNA or RNA helicase genes were mutated, such as \u003cem\u003edinG\u003c/em\u003e, \u003cem\u003ehrpA\u003c/em\u003e, and \u003cem\u003eyoaA\u003c/em\u003e. Efflux pump associated genes like \u003cem\u003eacrA\u003c/em\u003e, \u003cem\u003ephoQ\u003c/em\u003e were mutated in the MG1655 strain under aerobic conditions, and \u003cem\u003eacrR\u003c/em\u003e, \u003cem\u003esoxR\u003c/em\u003e in the Δ\u003cem\u003eoxyR\u003c/em\u003e strains both under aerobic and anaerobic conditions. The MG1655 strain under anaerobic conditions contained a cryptic prophage e14 deletion (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003eE), which is a well-known mutation associated with quinolone resistance [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In \u003cem\u003elexA\u003c/em\u003e, which inhibits a number of genes involved in the SOS response to DNA damage [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e][\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], a Gly85Ser mutation was observed in the Δ\u003cem\u003eoxyR\u003c/em\u003e strain under aerobic conditions.\u003c/p\u003e \u003cp\u003eThe common mutated gene in kanamycin resistant strains is \u003cem\u003efusA\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). During the translation elongation, FusA catalyzes the GTP-dependent ribosomal translocation step [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. This mutation may cause antibiotic target alteration. The MG1655 strain under anaerobic conditions had a 5 kb deletion from gene \u003cem\u003eyaiT\u003c/em\u003e to \u003cem\u003eyaiW\u003c/em\u003e. (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003eI). This deletion includes \u003cem\u003esbmA\u003c/em\u003e, a peptide antibiotic transporter. The \u003cem\u003esbmA\u003c/em\u003e mutation also occurred in both the MG1655 and Δ\u003cem\u003eoxyR\u003c/em\u003e strains under aerobic conditions.\u003c/p\u003e \u003cp\u003eThere was no common mutated gene in the tetracycline resistant strains (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). However, a few resistance-related mutated genes were identified. For instance, genes that code for the antibiotic efflux pumps, such as \u003cem\u003eacrA\u003c/em\u003e, \u003cem\u003eacrB\u003c/em\u003e, \u003cem\u003eacrR\u003c/em\u003e, \u003cem\u003emlaF\u003c/em\u003e were mutated. Genes that associated with antibiotic target alteration or protection, like \u003cem\u003erpoB\u003c/em\u003e and \u003cem\u003erpsJ\u003c/em\u003e; and genes associated with reduced permeability to antibiotics, such as \u003cem\u003eompF\u003c/em\u003e were also mutated. The MG1655 strains under aerobic and anaerobic conditions both contained a 2-fold amplification region from \u003cem\u003einsH1\u003c/em\u003e to \u003cem\u003einsH3\u003c/em\u003e of around 400 kb length (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003eM and N). The amplified region in the Δ\u003cem\u003eoxyR\u003c/em\u003e strain under aerobic conditions started from \u003cem\u003einsH1\u003c/em\u003e as well but stopped in \u003cem\u003einsF1\u003c/em\u003e resulting in a total length of about 800 kb (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003eP). A roughly 100 kb amplification region was observed in the Δ\u003cem\u003eoxyR\u003c/em\u003e strain under anaerobic conditions from gene \u003cem\u003einsH1\u003c/em\u003e to \u003cem\u003eyahH\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003eO).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAmoxicillin resistant strains\u0026rsquo; amplification contigs\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStrains\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLength (kb)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCopy numbers (times)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAnaerobic MG1655\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAerobic MG1655\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAnaerobic Δ\u003cem\u003eoxyR\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e41\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAerobic Δ\u003cem\u003eoxyR\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eMutations in\u003c/b\u003e \u003cb\u003eL. lactis\u003c/b\u003e \u003cb\u003ewith and without added heme.\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn \u003cem\u003eL. lactis\u003c/em\u003e, the number of mutated genes in strains made resistant to bactericidal antibiotics was higher than in strains resistant to bacteriostatic antibiotics (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e7\u003c/span\u003e). When heme was added to generate a functional ETC \u003cem\u003eL. lactis\u003c/em\u003e strains that evolved resistance contained mutations in two genes, \u003cem\u003epurM\u003c/em\u003e (Gly250Val), and \u003cem\u003earoH\u003c/em\u003e (Arg168Gly). PurM is involved in purine metabolism [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. AroH is involved in the early step of aromatic amino acid biosynthesis [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In amoxicillin resistant \u003cem\u003eL. lactis\u003c/em\u003e, the mutated genes, \u003cem\u003epbp2B\u003c/em\u003e and \u003cem\u003epbpX\u003c/em\u003e were observed both with and without added heme. Mutations in these two genes may cause antibiotic action targets modification. The target genes \u003cem\u003eponA\u003c/em\u003e and \u003cem\u003ecdsA\u003c/em\u003e were mutated in the heme-added strain as well (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e7\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eThe common mutated genes in moxifloxacin resistant strains were \u003cem\u003egyrA\u003c/em\u003e, \u003cem\u003eparC\u003c/em\u003e, and \u003cem\u003ellmg1807\u003c/em\u003e. The genes \u003cem\u003erpoC\u003c/em\u003e were mutated in the no heme-added condition, and \u003cem\u003erpoB\u003c/em\u003e in heme-added condition (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). There was a roughly 45 kb length deletion from \u003cem\u003ellmg1359\u003c/em\u003e to \u003cem\u003ellmg1411\u003c/em\u003e in no heme-added strain (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e7\u003c/span\u003eG). A 42 kb amplification, with around 4-fold copy number increase, from \u003cem\u003eps301\u003c/em\u003e to \u003cem\u003eps357\u003c/em\u003e occurred in the heme-added moxifloxacin resistant strain (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e7\u003c/span\u003eH). Only one mutated gene, \u003cem\u003erplD\u003c/em\u003e (Lys68Asn) was detected in no heme-added chloramphenicol resistant \u003cem\u003eL. lactis\u003c/em\u003e. RplD, the 50S L4 ribosomal protein, was identified in \u003cem\u003eNeisseria gonorrhoeae\u003c/em\u003e as a macrolide resistance protein [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The same gene \u003cem\u003erplD\u003c/em\u003e was mutated but with different amino acid change, Lys68Glu, in the heme-added chloramphenicol resistant \u003cem\u003eL. lactis\u003c/em\u003e. The tetracycline resistant \u003cem\u003eL. lactis\u003c/em\u003e strains had two common mutated genes, \u003cem\u003ellmg0323\u003c/em\u003e and \u003cem\u003erpsJ\u003c/em\u003e. Llmg0323 is a transcriptional regulator. RpsJ, 30S ribosomal protein S10 is a tetracycline-resistant ribosomal protection protein [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eFunctional annotation of mutated genes associated with antibiotic resistance.\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn order to group the mutated genes, they were clustered functionally according to the phylogenetic classification (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e8\u003c/span\u003e). The IMG/M database of Clusters of Orthologous Groups (COG) was used to annotate all the mutated genes and classify them [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. There were no clear differences in which classes of genes were mutated between \u003cem\u003eE. coli\u003c/em\u003e wild-type MG1655 and the Δ\u003cem\u003eoxyR\u003c/em\u003e strains under aerobic and anaerobic conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). However, the different antibiotics caused mutations in different classes of genes. The majority of genes mutated after exposure to amoxicillin, enrofloxacin, and kanamycin are involved in cellular processing and signaling, information storage and processing, and metabolism, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). In \u003cem\u003eL. lactis\u003c/em\u003e, half the number of mutated genes in amoxicillin exposed strains is involved in cellular processes and signaling function. The genes mutated by growth in the presence of moxifloxacin were evenly distributed over information and signaling and metabolism (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). The number of mutated genes in tetracycline-incubated \u003cem\u003eE. coli\u003c/em\u003e, and chloramphenicol, tetracycline-incubated \u003cem\u003eL. lactis\u003c/em\u003e were clearly lower than those of cells exposed to bactericidal antibiotics, making the COG comparison problematic.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe role of oxygen in the killing of bacteria by bactericidal antibiotics has been the subject of debate since the \u0026ldquo;radical based\u0026rdquo; theory was proposed, which suggests that reactive oxygen species form a secondary killing mechanism in addition to the primary target [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e][\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e][\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. This study examined the role of oxygen in \u003cem\u003ede novo\u003c/em\u003e acquisition of resistance by comparing resistance development under aerobic and anaerobic conditions. In the human intestines, \u003cem\u003eE. coli\u003c/em\u003e is exposed to anaerobic or micro-aerobic conditions [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Anaerobic growth may therefore be more natural than the aerobic conditions used in most experiments involving \u003cem\u003eE. coli\u003c/em\u003e. To test the influence of oxidative stress, resistance adaptation was examined in an \u003cem\u003eE. coli\u003c/em\u003e Δ\u003cem\u003eoxyR\u003c/em\u003e mutant strain. \u003cem\u003eL. lactis\u003c/em\u003e was used to separate between the effects of oxygen and those of ROS, because even under aerobic conditions, it does not possess a complete ETC, unless heme is supplied in the medium [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Hence, it only forms ROS when heme is present in the medium, but not in the absence, even when oxygen is available.\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eHormesis\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003ede novo\u003c/em\u003e acquisition of resistance in wildtype \u003cem\u003eE. coli\u003c/em\u003e with and without oxygen was relatively similar. The presence of oxygen seems to have no major influence on resistance development. However, under anaerobic conditions, reactive metabolic byproducts (RMB), particularly reactive electrophilic species, accumulate in antibiotic-treated \u003cem\u003eE. coli\u003c/em\u003e [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. These RMB contribute to cell death in a similar manner as ROS in aerobic conditions. Possibly ROS and RMB have relatively similar effects on resistance development. Nonetheless, under aerobic conditions, Δ\u003cem\u003eoxyR\u003c/em\u003e strain became resistant to bactericidal antimicrobials faster than the wildtype. The \u003cem\u003eoxyR\u003c/em\u003e gene codes for a regulatory protein that regulates a system of proteins that protect the cell from ROS and comparable stressors [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The presence of oxygen accelerated the acquisition of resistance in the Δ\u003cem\u003eoxyR\u003c/em\u003e strains, indicating that the extra ROS indeed stimulated this process. Hyperproduced ROS damage DNA and the nucleotide pool. The 8-oxo-guanine derived from guanine results in faulty DNA replication, accompanied by the general stress response [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The cell\u0026rsquo;s repair systems tend to increase rates of mutagenesis [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDuring antibiotic resistance acquisition, the differences can be observed in the middle and late stages of resistance development, possibly because at that stage increasing numbers of cells accumulate general stress-induced mutations. The observation that heme-added \u003cem\u003eL. lactis\u003c/em\u003e gained moxifloxacin resistance faster also points to an effect of ROS (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eF), generated by the ETC that was induced by the addition of heme. These observations can be understood in the framework of hormesis[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e][\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. While mild stress is beneficial in the form of the ability to more rapidly adapt to the presence of antimicrobials, high levels of the same stressors can cause cell death. When radical-based stress levels are low, a relatively moderate increase of the oxidative stress seems to accelerate the acquisition of resistance.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMutations accompanying resistance development\u003c/h2\u003e \u003cp\u003eFar higher mutation frequencies of AT to CG and CG to GC were observed in anaerobically grown \u003cem\u003eE. coli\u003c/em\u003e than in aerobic conditions. A likely cause is the DNA damage induced by acidic fermentation[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e][\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. However, these kinds of single nucleotide change mutations and the \u0026lt;\u0026thinsp;20 bp deletion mutations which occurred in the \u003cem\u003eL. lactis\u003c/em\u003e were almost never situated in resistance-related genes hot spots. Anaerobically grown untreated \u003cem\u003eE. coli\u003c/em\u003e strains also had high mutation frequencies, indicating that this kind of mutation did not influence resistance development.\u003c/p\u003e \u003cp\u003eVarious \u003cem\u003eE. coli\u003c/em\u003e strains which evolved resistance to the bactericidal antibiotics had common mutated genes, for instance, \u003cem\u003eampC\u003c/em\u003e for amoxicillin, \u003cem\u003egyrA\u003c/em\u003e and \u003cem\u003eparC\u003c/em\u003e for enrofloxacin, and \u003cem\u003efusA\u003c/em\u003e for kanamycin. These genes are known to play crucial roles in resistance acquisition [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In addition to the mutated genes that are common to all strains with induced resistance, there are mutations in genes that are specific for a certain strain and that follow logically from the characteristics of that strain. Only the amoxicillin resistant \u003cem\u003eE. coli\u003c/em\u003e Δ\u003cem\u003eoxyR\u003c/em\u003e strain under aerobic conditions had an \u003cem\u003eacrB\u003c/em\u003e mutation that may increase amoxicillin efflux. In the aerobically grown Δ\u003cem\u003eoxyR\u003c/em\u003e enrofloxacin resistant strain contained a mutation was observed \u003cem\u003elexA\u003c/em\u003e that codes for a transcriptional repressor. Auto-cleavage of LexA triggers the SOS response [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Mutagenic states caused by the SOS stress response may enhance the \u003cem\u003ede novo\u003c/em\u003e acquisition of antibiotic resistance [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e][\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. The Gly85Ser mutation located on the \u003cem\u003elexA\u003c/em\u003e auto-cleavage site [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e], may increase the rate of acquisition of resistance. During kanamycin exposure, SbmA mutations included Phe6fs in the aerobically grown wildtype strain, total deletion in the anaerobically grown wildtype, and Ser372* in the aerobically grown Δ\u003cem\u003eoxyR\u003c/em\u003e strain. These mutations block SbmA activity, thus inhibiting kanamycin uptake [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Three of the four cell lines exposed to kanamycin acquired mutations in \u003cem\u003esbmA\u003c/em\u003e that reduced kanamycin uptake. Only the anaerobically grown Δ\u003cem\u003eoxyR\u003c/em\u003e strain missed a \u003cem\u003esbmA\u003c/em\u003e mutation, and indeed developed less kanamycin resistance.\u003c/p\u003e \u003cp\u003eMutations in the multidrug efflux pump subunit coding genes \u003cem\u003eacrA\u003c/em\u003e, \u003cem\u003eacrB\u003c/em\u003e, \u003cem\u003eacrD\u003c/em\u003e, and their regulator \u003cem\u003eacrR\u003c/em\u003e, emerged in four antibiotic-resistant \u003cem\u003eE. coli\u003c/em\u003e strains. Additionally, mutations in the outer membrane porin genes \u003cem\u003eenvZ\u003c/em\u003e (\u003cem\u003eompB\u003c/em\u003e), \u003cem\u003eompC\u003c/em\u003e, and \u003cem\u003eompF\u003c/em\u003e appeared in the amoxicillin, kanamycin, and tetracycline resistant \u003cem\u003eE. coli\u003c/em\u003e strains. This could lead to increased resistance when these strains are exposed to other antibiotic treatments due to the cross-resistance [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Comparison with a similar dataset in which \u003cem\u003eE. coli\u003c/em\u003e wildtype evolved resistance resistant against the same four antimicrobials showed that apart from the commonly mutated genes described above, there were no other genes mutated in both datasets [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Hence, we must conclude that most mutations that occur during the acquisition of antibiotic resistance were random events that may or may not contribute to the development of resistance. Selection and co-selection afterwards during continued exposure to a specific antimicrobial determine the final list of mutations.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eFunctional distribution of mutations\u003c/h2\u003e \u003cp\u003eThe distribution over functional categories of genes that mutated as a result of exposure to antimicrobials was more consistent and can be related to the mechanisms of action of the particular antibiotic. Both in the dataset on mutations after resistance was built up against two antibiotics [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] and in the present dataset, a specific antibiotic correlates with specific functions. Mutations accompanying resistance to amoxicillin occur in genes coding for proteins involved in cellular processing and signaling and metabolism. This corresponds to the disruption of cell wall construction by beta-lactam antibiotics [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Enrofloxacin correlates primarily to genes concerning information storage and processing, due to the inhibition of DNA replication [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e], and also to metabolism. Kanamycin mutations are mainly in the area of metabolism, while tetracycline mutations are about evenly divided over cellular processing and signaling and information storage and processing, which also can be understood in the framework of the inhibition of protein synthesis by tetracyclines [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e][\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. The distribution in \u003cem\u003eL. lactis\u003c/em\u003e was similar to that in \u003cem\u003eE. coli\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eProlonged exposure to the beta-lactam antibiotic amoxicillin resulted in the amplification of a chromosomal DNA fragment centered around the \u003cem\u003eampC\u003c/em\u003e gene [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The multiplication of a 2.5 kb fragment was observed in the aerobically grown MG1655 strain. The amplified segments were larger in the anaerobically grown MG1655 strain (53 kb) and in the mutant Δ\u003cem\u003eoxyR\u003c/em\u003e both under aerobic (59 kb) and anaerobic (5.5kb) conditions. This indicates that the same process was taking place, but not in exactly the same manner. The fragment containing the \u003cem\u003eampC\u003c/em\u003e gene resembled known plasmid-bound beta-lactam resistance genes and could be transferred to a susceptible \u003cem\u003eE. coli\u003c/em\u003e strain, that became resistant after this transfer[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. These observations suggest that the \u003cem\u003ede novo\u003c/em\u003e development of resistance may play a bigger role in spreading of antimicrobial resistance than previously presumed. A similar duplication of part of the chromosome was also observed as a result of tetracycline exposure. In this case, the copy number was only 2-fold at most and it is doubtful that it played a major role in the acquisition of resistance, also because gene amplification is not known as a tetracycline resistance mechanism.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe effects of ROS and reactive metabolic byproducts on development of antimicrobial resistance in response to exposure to non-lethal concentrations of antibiotics can be understood by the principle of hormesis. High levels of radical-based stress are lethal, while low levels can increase the rate of acquisition of resistance and thus for the survival of the cell. The role of oxygen in \u003cem\u003ede novo\u003c/em\u003e acquisition of antibiotic resistance turned out to be indirect. Under anaerobic conditions, the reactive metabolic byproducts seem to function in a similar manner as ROS under aerobic conditions. Still, these stresses caused by reactive compounds remain at a low level when antibiotic concentrations are non-lethal, and the relatively low levels of stress under these conditions accelerate resistance acquisition.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eBacterial strains, media and growth conditions\u003c/h2\u003e \u003cp\u003eThe antibiotic-sensitive wildtype strains \u003cem\u003eE. coli\u003c/em\u003e MG1655 and \u003cem\u003eL. lactis\u003c/em\u003e MG1363 were used throughout the study. The \u003cem\u003eoxyR\u003c/em\u003e gene knockout mutant strain JW3933-3 was obtained from the Keio collection [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e], and the kanamycin-resistant cassette was removed using the pCP20 plasmid by FLP Recombination. \u003cem\u003eE. coli\u003c/em\u003e strains were grown in LB medium or a phosphate-buffered (100 mM Na\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e) defined minimal medium containing 55 mM glucose [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. \u003cem\u003eL. lactis\u003c/em\u003e was grown in 10% Lactose M17 broth medium. \u003cem\u003eE. coli\u003c/em\u003e was grown at 37\u0026deg;C, \u003cem\u003eL. lactis\u003c/em\u003e was grown at 30\u0026deg;C, and both were shaken at 200 rpm. Anaerobic culture DURAN\u0026reg; bottles with a butyl rubber stopper and an open topped screw cap, were filled with medium, inoculated with a syringe. Resazurin was used as oxygen indicator, and these tubes were autoclaved separately. \u003cem\u003eL. lactis\u003c/em\u003e under aerobic respiration conditions was grown with further addition of heme (Sigma) to a final concentration of 2 \u0026micro;g/mL. Amoxicillin, enrofloxacin, kanamycin, tetracycline, moxifloxacin, and chloramphenicol stock solutions (10 mg/mL) were filter sterilized through a 0.2 \u0026micro;M filter and stored at 4\u0026deg;C. Fresh antibiotic solutions were made every 3 days.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eEvolution experiments\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eInitial MICs of \u003cem\u003eE. coli\u003c/em\u003e strains\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e Strains\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMG1655\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMG1655\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eΔ\u003cem\u003eoxyR\u003c/em\u003e Mutant\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMedium\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMinimal medium\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMinimal medium\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmoxicillin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8/16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEnrofloxacin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKanamycin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16/32\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTetracycline\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2/4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eInitial MICs of \u003cem\u003eL. lactis\u003c/em\u003e strains\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eL. lactis\u003c/em\u003e Strains\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMG1363\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMG1363\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMedium\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLM17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHeme\u0026thinsp;+\u0026thinsp;LM17\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmoxicillin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.25/0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMoxifloxacin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChloramphenicol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2/4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTetracycline\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.5/1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eIn order to induce resistance to each antibiotic and test the susceptibility of the strains, the MICs measurement was performed by serial dilution followed by a determination of the initial antibiotic concentrations (Tables\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eEvolution experiments inducing resistance were performed as described previously [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Briefly, cultures were grown overnight in medium in the presence of sub-lethal levels of antibiotics. The initial antibiotic concentrations were lower than half MICs. When the OD\u003csub\u003e600\u003c/sub\u003e of the culture after 24 hours corresponded to \u0026ge;\u0026thinsp;75% of the OD\u003csub\u003e600\u003c/sub\u003e of the antibiotic-free or already adapted culture, cells were used and inoculated with double concentrations of the antibiotic. If sufficient growth did not occur, these steps were repeated using cells from tubes without or with a lower antibiotic concentration. Each strain's evolution experiment was independently performed at least twice. In all experiments, cultures without antibiotic exposure were used as controls. MICs was detected three times a week to monitor resistance.\u003c/p\u003e \u003cp\u003eMICs was measured in 96-well plates in a spectrophotometer plate reader (Thermo Fisher Scientific) at 37\u003csup\u003eo\u003c/sup\u003eC. Each well contains 150 \u0026micro;L final volumes with the OD\u003csub\u003e600\u003c/sub\u003e of 0.05 bacteria, antibiotics concentrations range from 0.25 to 2048 with steps of a factor of two. After overnight culture, the lowest concentration that yielded a final OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.2 was considered the MICs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eROS measurement\u003c/h2\u003e \u003cp\u003eTo determine the formation of ROS, overnight cultured \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eL. lactis\u003c/em\u003e strains made resistant to a specific antibiotic were diluted to OD\u003csub\u003e600\u003c/sub\u003e of 0.2 and exposed to the highest concentration of these antibiotics that still allowed growth. After administration of the antibiotic, cells were cultured for 3 hours at 37\u0026deg;C (\u003cem\u003eE. coli\u003c/em\u003e) or 30\u0026deg;C (\u003cem\u003eL. lactis\u003c/em\u003e) shaking at 200 rpm. Cell cultures were incubated with 5 \u0026micro;L 10 mM H\u003csub\u003e2\u003c/sub\u003eDCFDA (Sigma) fluorescent dye dissolved in DMSO. Culturing tubes were covered with aluminum foil to prevent exposure to light and incubated for 45 minutes at the same temperature. After incubation, 1 mL of culture was spun down at 6000 rpm for 5 min, the cell pellet was dissolved in medium. 1.3 \u0026micro;L cell suspension was loaded on a microscope slide glass with 2% agarose mixed with medium. Images were acquired on a Nikon Eclipse Ti microscope with NIS-elements AR software. Fluorescent signal was detected at excitation/emission wavelength of 488/510 nm and was shown in green. Images were processed using Fiji/ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eWhole genome sequencing\u003c/h2\u003e \u003cp\u003eThe genomic DNA was isolated from the final stable resistant strains by the DNeasy blood and tissue kit (Qiagen). Genomic DNA libraries were generated using the NEBNext Ultra II FS DNA Library Prep kit for Illumina (New England BioLabs) in combination with NEBNext multiplex oligos for Illumina (96 Unique Dual Index Primer Pairs; New England BioLabs) according to the manufacturer\u0026rsquo;s instructions. Briefly, 500 ng genomic DNA was used as input with a fragmentation time of 5 min, aiming at an insert size distribution of 275\u0026ndash;475 bp by following the corresponding size selection option provided in the protocol. The resulting size distribution of the libraries with indexed adapters was assessed using a 2200 TapeStation System with Agilent D1000 ScreenTapes (Agilent Technologies). The libraries were quantified on a QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific) using the NEBNext Library Quant Kit for Illumina (New England BioLabs) according to the instructions of the manufacturer. The libraries were clustered and sequenced (2 x 150 bp) on a NextSeq 550 Sequencing System (Illumina) using a NextSeq 500/550 Mid Output v2.5 kit (300 cycles) (Illumina).\u003c/p\u003e \u003cp\u003eAfter sequencing, FastQC and MultiQC were used to evaluate the quality of raw reads. BBmerge was used to discover the adapter sequences, which were then imported to Cutadapt to be removed. In order to remove low-quality bases, Trimmomatic was utilized. The removal of optical duplicates was achieved via Clumpify. After mapping the reads to the reference by Bowtie2, GATK was used for marking PCR duplicates. The variant calling was done by Freebayes and used Snpeff to do the variant annotation. Subsequently, the SNPs were selected with IGV from the output vcf files. The mutated genes were selected from the resistant strains compared with the no-antibiotic exposure control strain, and the repeat mutated genes were eliminated. IMG/M was used to classify the mutated genes into clusters of orthologous groups (COG) categories. To do the copy number analysis, the cn.MOPS was used to identify larger genomic alterations that result in an abnormal number of copies of one or more genes.\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eROS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eReactive oxygen species\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRMB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eReactive metabolic byproducts\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eETC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eElectron transport chain\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCOG\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eClusters of orthologous groups\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003eEthics approval and consent to participate\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003eConsent for publication\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003eAvailability of data and materials\u003c/p\u003e\n\u003cp\u003eThe binary alignment/map (bam) files of the sequenced strains have been deposited in the NCBI database and can accessed at BioProject PRJNA954686 and PRJNA954732.\u003c/p\u003e\n\u003cp\u003eCompeting interests\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe declare that no competing interests exist.\u003c/p\u003e\n\u003cp\u003eFunding\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis study was financed by the Netherlands Food and Consumer Product Safety Authority (NVWA). The NVWA was not involved in design of the experiments, analysis of the data or writing the manuscript.\u003c/p\u003e\n\u003cp\u003eAuthors' contributions\u003c/p\u003e\n\u003cp\u003eWQ and BtK conceived the project. WQ performed experiments. MJ performed the bioinformatic analysis. WQ and BtK wrote the manuscript. LT proved the knockout strain. MW and LT contributed to the final manuscript. All authors critically reviewed the manuscript and approved the final version.\u003c/p\u003e\n\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eWe thank S. Brul for stimulating discussions and suggestions for improvement of an earlier version of the manuscript.\u003c/p\u003e\n\u003cp\u003eAuthor information\u003c/p\u003e\n\u003cp\u003eLaboratory for Molecular Biology and Microbial Food Safety, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands\u003c/p\u003e\n\u003cp\u003eWenxi Qi, Lisa Teichmann, Meike Wortel \u0026amp; Benno H. Ter Kuile\u003c/p\u003e\n\u003cp\u003eRNA Biology \u0026amp; Applied Bioinformatics, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands\u003c/p\u003e\n\u003cp\u003eMartijs J. Jonker\u003c/p\u003e\n\u003cp\u003eNetherlands Food and Consumer Product Safety Authority, Office for Risk Assessment, Utrecht, The Netherlands\u003c/p\u003e\n\u003cp\u003eBenno H. ter Kuile\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMacLean RC, Millan AS. \u0026ldquo;The evolution of antibiotic resistance,\u0026rdquo; \u003cem\u003eScience (80-.).\u003c/em\u003e, vol.\u0026nbsp;365, no. 6458, pp.\u0026nbsp;1082\u0026ndash;1083, Sep. 2019, doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1126/SCIENCE.AAX3879\u003c/span\u003e\u003cspan address=\"10.1126/SCIENCE.AAX3879\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eH\u0026auml;ndel N, Schuurmans JM, Feng Y, Brul S, Kuile BH. \u0026ldquo;Interaction between Mutations and Regulation of Gene Expression during Development of De Novo Antibiotic Resistance,\u0026rdquo; \u003cem\u003eAntimicrob. 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Drug Resist.\u003c/em\u003e, vol.\u0026nbsp;17, no. 2, pp.\u0026nbsp;141\u0026ndash;147, Jun. 2011, doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1089/MDR.2010.0101\u003c/span\u003e\u003cspan address=\"10.1089/MDR.2010.0101\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Reactive oxygen species, de novo resistance, Antimicrobial resistance, Whole genome sequencing, Reactive metabolic byproducts","lastPublishedDoi":"10.21203/rs.3.rs-2932862/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2932862/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e: Bacteria can acquire resistance through DNA mutations in response to exposure to sub-lethal concentrations of antibiotics. According to the radical-based theory, reactive oxygen species (ROS), a byproduct of the respiratory pathway, and oxidative stress caused by reactive metabolic byproducts, play a role in cell death as secondary killing mechanism. \u003cbr\u003e\n \u003cstrong\u003eResults:\u003c/strong\u003e To investigate whether oxygen and ROS affect \u003cem\u003ede novo\u003c/em\u003eacquisition of antibiotic resistance, evolution of resistance was compared in \u003cem\u003eE. coli\u003c/em\u003e wildtype and Δ\u003cem\u003eoxyR\u003c/em\u003e strains under aerobic and anaerobic conditions. Since \u003cem\u003eLactococcus lactis\u003c/em\u003e (\u003cem\u003eL. lactis\u003c/em\u003e) does not have an active electron transport chain (ETC) even in the presence of oxygen, and thus forms much less ROS, resistance development in \u003cem\u003eL. lactis\u003c/em\u003e was to distinguish between oxygen and ROS. The resistance acquisition in \u003cem\u003eE. coli\u003c/em\u003e wildtype under aerobic and anaerobic conditions did not differ much. However, the aerobically grown Δ\u003cem\u003eoxyR\u003c/em\u003e strain gained resistance faster than the wildtype or anaerobic Δ\u003cem\u003eoxyR.\u003c/em\u003e Inducing an ETC by adding heme increased the rate at which \u003cem\u003eL. lactis \u003c/em\u003eacquired resistance. Whole genome sequencing identified crucial mutations involved in the acquisition of resistance. These mutations were specific for each antibiotic. The\u003cem\u003e lexA\u003c/em\u003e mutation in Δ\u003cem\u003eoxyR\u003c/em\u003e strain under aerobic conditions indicated that the SOS response was involved in resistance acquisition.\u003cbr\u003e\n \u003cstrong\u003eConclusions:\u003c/strong\u003e The concept of hormesis can explain the beneficial effects of low levels of ROS and reactive metabolic byproducts, while high levels are lethal. DNA repair and mutagenesis may therefore expedite development of resistance. Taken together, the results suggest that oxygen as such barely affects resistance development. Nevertheless, non-lethal levels of ROS stimulate \u003cem\u003ede novo\u003c/em\u003e acquisition of antibiotic resistance.\u003c/p\u003e","manuscriptTitle":"The influence of oxygen and oxidative stress on de novo acquisition of antibiotic resistance in E. coli and Lactobacillus lactis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-07-10 13:56:33","doi":"10.21203/rs.3.rs-2932862/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2023-08-08T09:59:51+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2023-07-19T17:45:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"df386fdd-d91d-45fc-ad59-867db277e2e4","date":"2023-07-10T12:38:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"602919ff-aa9d-4a3d-8ad6-e5e9df107a35","date":"2023-07-07T06:45:10+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-07-06T19:14:02+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2023-06-12T18:36:11+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-06-12T05:16:44+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2023-06-08T12:54:52+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Microbiology","date":"2023-05-14T07:46:33+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a1474be2-18a0-4385-bfc2-9a05fdcd32cb","owner":[],"postedDate":"July 10th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2023-10-09T15:09:20+00:00","versionOfRecord":{"articleIdentity":"rs-2932862","link":"https://doi.org/10.1186/s12866-023-03031-4","journal":{"identity":"bmc-microbiology","isVorOnly":false,"title":"BMC Microbiology"},"publishedOn":"2023-10-02 15:02:45","publishedOnDateReadable":"October 2nd, 2023"},"versionCreatedAt":"2023-07-10 13:56:33","video":"","vorDoi":"10.1186/s12866-023-03031-4","vorDoiUrl":"https://doi.org/10.1186/s12866-023-03031-4","workflowStages":[]},"version":"v1","identity":"rs-2932862","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2932862","identity":"rs-2932862","version":["v1"]},"buildId":"ehx78VzkSd0WSzXnipQa-","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}