Synergistic effect of SOS response and GATC methylome suppression on antibiotic stress survival in Escherichia coli

AAC01392-22.R1 Synergistic effect of SOS response and GATC methylome 1 suppression on antibiotic stress survival in Escherichia coli 2 S. Diaz-Diaz1,2,4,*, E. Recacha1,2,4, Marina R. Pulido2,4, María Romero-3 Muñoz2, B. de Gregorio-Iaria2, F. Docobo-Pérez2,3,4, A. Pascual1,2,3,4, 4 J.M. Rodríguez-Martínez2,3,4 5 6 1Unidad de Enfermedades Infecciosas y Microbiología, Hospital Universitario 7 Virgen Macarena, Seville, Spain; 2Departamento de Microbiología, Facultad de 8 Medicina, Universidad de Sevilla, Sevilla, Spain; 3Centro de Investigación 9 Biomédica en Red en Enfermedades Infecciosas (CIBERINFEC), Instituto de 10 Salud Carlos III, Madrid, Spain; 4Instituto de Biomedicina de Sevilla (IBiS), Hospital 11 Universitario Virgen Macarena/CSIC/Universidad de Sevilla, Sevilla, Spain. 12 13 Keywords: antibiotic stress, resistance reversion, quinolones, recA gene, Dam 14 methylation system 15 Running title: Sensitization by targeting dam and recA genes 16 * Corresponding author: Mailing address: Department of Microbiology; 17 University of Seville. Avda Sanchez Pizjuan s/n. 41009 Spain. 18 Phone: +34 954 55 28 63. Fax: +34 954 37 74 13; E-mail: [email protected] 19 Words: Abstract length 231; Text length 3701 20 Tables/Figures/Supplementary data/References number: 2/5/2/4821 AAC01392-22.R1 ABSTRACT 22 Suppression of the SOS response has been shown to enhance in vitro activity 23 of quinolones. Furthermore, Dam-dependent base methylation has an impact 24 on susceptibility to other antimicrobials affecting DNA synthesis. Here, we 25 investigated the interplay between these two processes, alone and in 26 combination, in terms of antimicrobial activity. A genetic strategy was used 27 employing single and double gene mutants for the SOS response (recA gene) 28 and Dam methylation system (dam gene), in both a susceptible and resistant to 29 quinolones isogenic models in Escherichia coli. Regarding the bacteriostatic 30 activity of quinolones, a synergistic sensitization effect was observed when the 31 Dam methylation system and recA gene were suppressed. In terms of growth, 32 after 24h in the presence of quinolones, the ∆dam/∆recA double mutant showed 33 no growth or delayed growth compared to the control strain. In bactericidal 34 terms, spot test showed that the ∆dam/∆recA double mutant was more sensitive 35 than the ∆recA single mutant (about 10-102-fold) and the wild-type (about 103-36 104-fold), both in a susceptible and resistant genetic background. Differences 37 between the wild-type and the ∆dam/∆recA double mutant were confirmed by 38 time-kill assays. The suppression of both systems, in a strain with chromosomal 39 mechanisms of quinolone resistance, prevents the evolution of resistance. This 40 genetic and microbiological approach demonstrated enhanced sensitization of 41 E. coli to quinolones by dual targeting of the recA (SOS response) and Dam 42 methylation system gene, even in a resistant strain model. 43 44 AAC01392-22.R1 INTRODUCTION 45 Antimicrobial resistance has become a major focus of concern in the past few 46 years, as the widespread and indiscriminate use of antimicrobials becomes a 47 serious public health problem (1, 2). Since bacteria are highly adaptive, new 48 resistance mechanisms are continually emerging (3), which means that it is 49 clearly imperative to discover new strategies to combat them. Preventing 50 bacterial adaptation and evolution could be one strategy to combat antimicrobial 51 resistance (4, 5). 52 The DNA of Escherichia coli contains three methylated bases, 6-methyladenine, 53 5-methylcytosine and 4-methylcytosine (6). Methylation is carried out by three 54 enzymes: Hsd, Dam and Dcm methylases. The dam gene is distributed 55 relatively universally among the Enterobacterales (6, 7). 56 The Dam protein plays a role in the regulation and coordination of multiple 57 processes, including DNA replication and transcription, but also has an 58 important role in DNA mismatch repair by catalyzing the transfer of the methyl 59 group from S-adenosyl-L-methionine to the N6 position of adenine in the 5’-60 GATC-3’ sequence (8). Adenosine residues in these sequences are methylated 61 on both strands of the DNA, with the exception of those recently synthesized, at 62 which point replication errors, mainly A-C and G-T mismatches or 63 deletion/insertions of up to four nucleotides, are corrected by mismatch repair 64 (9). In dam null mutants, increased mutability, hyper-recombination and 65 increased susceptibility to DNA-damaging agents have been observed (10–12). 66 When strand methylation does not occur, there is no difference between the 67 template strand and the daughter strand and MutH causes double-strand 68 AAC01392-22.R1 breaks, triggering recombination repair (13), which induce expression of recA 69 gene and, thus, SOS response activation (14). 70 Fluoroquinolones are broad-spectrum antimicrobials that are used for targeted 71 therapy in infections caused by E. coli (15). Their mechanism of action is related 72 to the inhibition of DNA gyrase and topoisomerase type IV, two enzymes 73 essential for bacterial viability due to their involvement in DNA synthesis. The 74 mechanism of action of fluoroquinolones consists of the formation of an 75 antibiotic/enzyme/DNA complex in which double-strand DNA breaks occur (16). 76 Resistance to these antimicrobial agents is mainly caused by target mutations 77 in the topoisomerases (17). Double-strand breaks give rise to single-stranded 78 DNA, which induce the SOS response (18). Hence, the SOS response can be 79 induced by some antimicrobials in different molecular mechanisms, such as 80 DNA damage (18–20), which is the case with fluoroquinolones (21). The SOS 81 response is a coordinated cellular response to genotoxic damage that can 82 contribute to the evolution of resistance (20). When damage occurs, a single 83 strand of DNA is generated, activating the RecA protein. Nucleofilaments then 84 form around the RecA protein, which induces autoproteolysis of another protein, 85 the LexA protein. The LexA protein represses the SOS regulon, which includes 86 more than 50 genes, many of them associated with repair and recombination 87 mechanisms, such as the homologous recombination process, and low-fidelity 88 polymerases such as DinB, UmuC and UmuD (18, 21–23). Other antimicrobial 89 agents such as trimethoprim, which is usually used in combination with 90 sulfamethoxazole, are also capable of activating the SOS response. 91 Trimethoprim inhibits dihydrofolate reductase, blocking the incorporation of 92 thymine into DNA (24–26), which leads indirectly to activation of the SOS 93 AAC01392-22.R1 response (27). Trimethoprim treatment has also been shown to increase the 94 production of reactive oxygen species (ROS), such as hydrogen peroxide, 95 which contributes to trimethoprim toxicity (28). 96 We hypothesized for this study that targeting both systems (recA and 97 dam genes), at the same time, would lead to synergistic sensitization to stress 98 agents such as antimicrobials, which is still uncertain in the literature. We show 99 that the activity and bactericidal action of fluoroquinolones such as ciprofloxacin 100 are enhaced when recA gene, involved in homologous recombination and DNA 101 repair mechanisms, and the dam gene, related to DNA replication, gene 102 expression and DNA mismatch repair, are deleted. This effect was also 103 observed in an E. coli strain with chromosomal mechanisms of resistance to 104 quinolones. 105 106 107 108 109 110 AAC01392-22.R1 RESULTS 111 Enhanced quinolone sensitization by suppression of recA and Dam 112 methylation system 113 We generated a collection of E. coli BW25113 mutants displaying recA 114 gene deletion in combination with suppressed Dam methylation system gene 115 (Δdam) in order to evaluate the interplay of both DNA repair and Dam-116 dependent base methylation on the response to antimicrobial drugs (Table 1, 117 Table S1). We also generated a collection of mutants in E. coli BW15 strain, 118 which presented amino acid changes in GyrA, GyrB, ParE proteins and a 119 deletion in marR gene. 120 In terms of bacteriostatic activity of ciprofloxacin, a sensitization effect 121 was observed in our collection of single and double mutants (MIC values are 122 shown in Table 1). Approximately 2-fold reductions were observed for the Dam 123 methylation system gene knockout (Δdam) and 3-4-fold reductions for the 124 ΔrecA mutant with respect to the wild-type (BW25113). Furthermore, increased 125 sensitization was observed when both, the SOS response and the Dam 126 methylation system were suppressed (Δdam/ΔrecA), obtaining 8-fold reductions 127 compared to the wild-type (BW25113). Interestingly, the same sensitization 128 effect was observed for the ciprofloxacin-resistant strain (BW15). It is 129 noteworthy also that this process of sensitization was similar or even higher for 130 all the antibiotics tested (levofloxacin, moxifloxacin, norfloxacin and ofloxacin), 131 showing a high degree of agreement between the two strains (full susceptible 132 and low-level resistance to quinolones) and the various antimicrobials. In the 133 case of nalidixic acid, the sensitization effect was observed only for the 134 BW25113 group. As expected, nalidixic acid MIC values for BW15 were so high 135 AAC01392-22.R1 (>256µg/mL) that no effect was observed. recA gene and dam gene 136 complementation using the pCA24N and pTP166 plasmids, respectively, 137 showed increased MIC to ciprofloxacin (similar to MIC of the wild-type) in 138 mutants lacking the dam and recA genes (data not shown). 139 The modified FIC values indicated additive sensitization effects for 140 ciprofloxacin and norfloxacin, synergistic effects for levofloxacin and ofloxacin, 141 and indifferent effect for moxifloxacin, nalidixic acid, trimethoprim and 142 trimethoprim-sulfamethoxazole in the BW25113Δdam/ΔrecA strain. For the 143 BW15Δdam/ΔrecA strain, however, additive sensitization effects were observed 144 for ciprofloxacin, norfloxacin and ofloxacin, synergistic effects for levofloxacin, 145 moxifloxacin, trimethoprim and trimethoprim-sulfamethoxazole and an 146 indifferent effect for nalidixic acid (Table 2). 147 MIC was also determined for other families of antimicrobials such as 148 penicillins (amoxicillin and amoxicillin-clavulanic acid), cephalosporins 149 (ceftazidime and cefepime), carbapenems (imipenem and meropenem), and 150 aminoglycosides (amikacin, tobramycin and gentamicin). Although for some of 151 these antimicrobials a slight reduction in MIC of the double mutant 152 (Δdam/ΔrecA) compared to wild-type strains was observed, FIC index showed 153 no synergistic effect (Table S1). 154 155 Decreased bacterial growth in vitro in the presence of quinolones when 156 recA and the Dam methylation system were suppressed 157 In terms of growth, at a ciprofloxacin concentration of 0.002µg/mL 158 (1/8xMIC of wild-type BW25113), the optical densities (OD) of BW25113, 159 BW25113∆dam, BW25113∆recA and BW25113∆dam/∆recA at 24h were 0.7, 160 AAC01392-22.R1 0.5, 0.4 and no growth, respectively (Figure 1A). At a concentration of 161 0.25µg/mL (approximately 1/8xMIC of wild-type BW15), optical densities (OD) 162 of BW15, BW15∆dam, BW15∆recA and BW15∆dam/∆recA at 24h were 0.7, 163 0.6, 0.4 and 0.2, respectively (Figure 1C). AUC comparisons showed significant 164 decreased bacterial growth (p<0.05). 165 In terms of bacteriostatic activity, these data support the effectiveness of 166 combining recA gene inactivation (SOS response) and Dam methylation system 167 suppression (Δdam) in preventing bacterial growth in the presence of sublethal 168 concentrations of ciprofloxacin over longer periods of time (24h). 169 170 recA gene and Dam methylation system suppression enhances 171 ciprofloxacin bactericidal activity 172 The survival of E. coli mutant bacteria was evaluated using spot test 173 assays with multiple inocula (Figure 2). In terms of susceptibility, after 174 ciprofloxacin treatment, no differences were observed between mutants with 175 Dam methylation system suppression (BW25113Δdam and BW15Δdam) 176 compared to the wild-type strains (BW25113 and BW15), whereas, suppression 177 of the SOS response (BW25113ΔrecA and BW15ΔrecA) was more sensitive 178 than wild-type strains (BW25113 and BW15) (about 102-fold). Interestingly, the 179 double mutants with combined SOS response and Dam methylation system 180 suppression (BW25113Δdam/ΔrecA and BW15Δdam/ΔrecA) were more 181 sensitive than the single mutants (BW25113ΔrecA and BW15ΔrecA) (about 10-182 102-fold) and the wild-types (BW25113 and BW15) (about 103-104-fold). Spot 183 test assays performed with other fluoroquinolones, norfloxacin and levofloxacin, 184 showed a similar effect compared to ciprofloxacin (Figure S1). 185 AAC01392-22.R1 To show the impact of the inactivation of recA and dam genes in terms of 186 bacterial viability, time-kill curves were obtained for each isogenic group (Figure 187 3A-3B). At a fixed concentration of 8xMIC relative to each strain, and in a short 188 time of 4 hours, a marked reduction of viable bacteria was observed when both 189 genes were inactivated compared to wild-type strains for each isogenic group 190 (p<0.05). At 4 hours, drop of 4Log was observed for BW25113 and the single 191 mutant BW25113Δdam, whereas drops of 5Log and 6Log were observed for 192 single mutant BW25113ΔrecA and double mutant BW25113Δdam/ΔrecA, 193 respectively (Figure 3A). Besides, at 4 hours, drop of 3Log was observed for 194 BW15 and BW15Δdam, whereas drop of 5Log was observed for BW15ΔrecA 195 and BW15Δdam/ΔrecA mutants (Figure 3B). Therefore, a bactericidal effect 196 (drop of > 3Log) was observed for all strains, with the highest differences for 197 double mutants (BW25113Δdam/ΔrecA and BW15Δdam/ΔrecA) compared to 198 wild-type (BW25113 and BW15) (p<0.05). In a long time of 24 hours, regrowth 199 of BW25113Δdam and BW25113Δdam/ΔrecA was observed. 200 These data support the hypothesis that recA gene and Dam methylation 201 system suppression enhances ciprofloxacin bactericidal activity in short times 202 for a strain without mechanisms of resistance to quinolones and in short and 203 long times for a strain with mechanisms of resistance to quinolones. 204 205 Role of suppression of recA gene and Dam methylation system in the 206 evolution of ciprofloxacin resistance 207 To evaluate the response to gradual antimicrobial pressure, we studied 208 antibiotic resistance in experimental evolution assays (Figure 4), in which the 209 ciprofloxacin concentration was gradually increased each day. The BW25113 210 AAC01392-22.R1 MATERIALS AND METHODS 335 Strains, growth conditions and antimicrobial agents 336 The starting strain for all constructs was wild-type E. coli BW25113 337 (Table 1). E. coli BW25113 (wild-type) single-gene deletion mutants (ΔrecA and 338 Δdam) were selected from the KEIO collection (42). Double-gene deletion 339 mutants (Δdam/ΔrecA) were generated by P1vir phage transduction and 340 removal of the kanamycin resistance cassette using plasmid pCP20 expressing 341 Flp recombinase (34, 43). In addition, ciprofloxacin was used to evolve E. coli 342 BW25113 into a resistant strain [BW15, gyrA (D87G), gyrB (E465D), parE 343 (K390N) and ∆marR] (32). Single (ΔrecA and Δdam) and double mutants 344 (Δdam/ΔrecA) were then generated. recA gene and dam gene complementation 345 was performed using the pCA24N and pTP166 plasmids, respectively (44, 45). 346 Liquid or solid Luria Broth (LB) medium and Mueller-Hinton broth (MHB) 347 were used. Strains were grown at 37ºC. 348 349 Minimal Inhibitory Concentrations (MIC) 350 For each bacterial strain, MICs were determined in triplicate, using the gradient 351 strip technique, following EUCAST recommendations (46). The antimicrobials 352 used were quinolones (ciprofloxacin, levofloxacin, moxifloxacin, norfloxacin, 353 ofloxacin and nalidixic acid), sulfonamides (trimethoprim and trimethoprim-354 sulfamethoxazole), and other antimicrobials (amoxicillin, amoxicillin-clavulanic 355 acid, ceftazidime, cefepime, imipenem, meropenem, amikacin, tobramycin, 356 gentamicin) (Liofilchem, Spain). 357 358 359 AAC01392-22.R1 Modified FIC index analysis 360 The interplay between SOS response and Dam methylation system and their 361 impact on sensitization was quantified using a modified estimation of the 362 fractional inhibitory concentration (FIC) index as below described. The FIC 363 index for double-gene deletion mutants was calculated as follows: 364 𝐹𝐼𝐶 𝑖𝑛𝑑𝑒𝑥 =  (  )  (   ) +  (  )  (   ) . 365 The combination of the two systems was considered synergistic when the FIC 366 index was ≤0.5, additive when it was 0.5 to ≤1, indifferent when it was 1–4, and 367 antagonistic when it was >4, following the criteria of MacKay et al. (47). 368 369 Bacterial growth curves 370 Bacterial growth curves were performed with each isogenic group, based on 371 RecA inactivation, Dam methylation system suppression and combinations 372 using an initial inoculum of 5x103 CFU/mL of exponential cells was used in all 373 experiments. Transparent 96-well flat-bottom plates containing 200μL of 374 sublethal concentrations (1/8xMIC of BW25113 and 1/16xMIC of BW15) of 375 ciprofloxacin in MHB were prepared as previously described. Optical density 376 was quantified using an Infinite 200 PRO plate reader (Tecan, Madrid, Spain) 377 during 24h (31). At least three biological replicates were measured for each 378 condition in at least two independent assays. Finally, area under the curve 379 (AUC) was calculated. 380 381 Spot test 382 In addition, the survival under antibiotic pressure was also evaluated by 383 spot test assays. Fresh overnight cultures were diluted to an OD625nm of 0.1 (ca. 384 AAC01392-22.R1 108 CFU/mL) in LB, then serially diluted in saline solution (1%). A 5μL volume of 385 each dilution was spotted onto LB agar plates with 1/16xMIC (0.001µg/mL) of 386 ciprofloxacin, 1/4xMIC (0.006µg/mL) of levofloxacin and 1/4xMIC (0.024µg/mL) 387 of norfloxacin relative to BW25113 and 1/8xMIC (0.38µg/mL) of ciprofloxacin, 388 1/2xMIC (3µg/mL) of levofloxacin and 1/4xMIC (4µg/mL) of norfloxacin relative 389 to BW15. After 20h of incubation at 37°C, the spots were checked for growth 390 and compared with spots on the control LB agar plates (32). 391 392 Time-kill assays 393 Bacterial viability was evaluated at ciprofloxacin concentrations normalized to 394 MICs (8xMIC relative to the MIC of each strain), therefore, the absolute 395 concentrations of ciprofloxacin were 0.125µg/mL (BW25113), 0.06µg/mL 396 (BW25113Δdam), 0.03µg/mL (BW25113ΔrecA), 0.015µg/mL 397 (BW25113Δdam/ΔrecA), 24µg/mL (BW15), 16µg/mL (BW15Δdam), 8µg/mL 398 (BW15ΔrecA) and 3µg/mL (BW15Δdam/ΔrecA). Growth in drug-free broth was 399 evaluated in parallel as a control. Cultures were incubated at 37ºC, with shaking 400 at 180 rpm. An initial inoculum of 106 CFU/mL was used in all experiments and 401 viable cells were determined each hour during 24 hours by colony counting after 402 removing the antimicrobial before plating. 403 404 Experimental Evolution of Antibiotic Resistance 405 The evolution experiment under antibiotic pressure, as previously 406 described (48), was started by inoculating 2μL of the selected strains, cultured 407 overnight in LB at 37ºC, into 96-well plates with 198μL of LB and a sub-408 inhibitory concentration of the antibiotic under study. To maximize the chances 409 AAC01392-22.R1 of populations acquiring resistance mutations, we started with 6.25x10-5μg/mL 410 (approximately 1/256xMIC BW25113) of ciprofloxacin for the BW25113 group, 411 and 0.01μg/mL (approximately 1/256xMIC BW15) of ciprofloxacin for the BW15 412 group. The plates were incubated for 20h at 37ºC. The OD595nm of each 413 population was measured daily using Infinite 200 PRO plate reader before 414 being transferred to new 96-well plates at twice the antibiotic concentration of 415 the previous day, and the plates were then incubated for 20h at 37°C. In 416 parallel, we performed control populations of each strain in the same conditions 417 but without antibiotics. Optical density values lower than 0.1 indicated 418 population extinction. At least sixteen biological replicates were measured. 419 420 Statistical analysis 421 All statistical analyses were performed using Graphpad Prism 6 software 422 (https://www.graphpad.com). The Student’s t-test was used for statistical 423 evaluation when two groups were compared. The log-rank (Mantel-Cox) test 424 was also used for statistical evaluation in the experimental evolution of antibiotic 425 resistance assays. Differences were considered significant when P values were 426 <0.05. 427 428 AAC01392-22.R1 FUNDING 429 This work was supported by the Plan Nacional de I+D+i 2013‐2016 and 430 the Instituto de Salud Carlos III (projects PI17/01501 and PI20/00239), 431 Subdirección General de Redes y Centros de Investigación Cooperativa, 432 Ministerio de Economía, Industria y Competitividad, Spanish Network for 433 Research in Infectious Diseases (REIPI; RD16/0016/0001 and REIPI 434 RD16/0016/0009)‐co‐financed by European Development Regional Fund “A 435 way to achieve Europe”, Operative program Intelligent Growth 2014‐2020. Sara 436 Diaz-Diaz is supported by a PFIS Grant from the Instituto de Salud Carlos III 437 (FI18/00086), co-funded by ESF "Investing in your future"; Esther Recacha is 438 supported by a Juan Rodés Grant from the Instituto de Salud Carlos III 439 (JR21/00030), co-funded by ESF "Investing in your future"; Marina R. Pulido is 440 supported by a VIPPI-US fellowship from the University of Seville; and María 441 Romero-Muñoz is supported by the Fondo Social Europeo (EJ5-91, Programa 442 Iniciativa de Empleo Juvenil). 443 444 445 AAC01392-22.R1 TRANSPARENCY DECLARATIONS 446 None to declare. 447 448 AAC01392-22.R1 References 449 1. Ferri M, Ranucci E, Romagnoli P, Giaccone V. 2017. Antimicrobial 450 resistance: A global emerging threat to public health systems. Crit Rev 451 Food Sci Nutr 57:2857–2876. 452 2. Witte W. 1998. Medical consequences of antibiotic use in agriculture. 453 Science 279:996–997. 454 3. De Oliveira DMP, Forde BM, Kidd TJ, Harris PNA, Schembri MA, Beatson 455 SA, Paterson DL, Walker MJ. 2020. Antimicrobial Resistance in ESKAPE 456 Pathogens. Clin Microbiol Rev 33. 457 4. Diaz-Diaz S, Recacha E, García-Duque A, Docobo-Pérez F, Blázquez J, 458 Pascual A, Rodríguez-Martínez JM. 2022. 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DNA Res 12:291–299. 583 Figure 4 Table 1. Genotypes and antimicrobial (CIP: Ciprofloxacin; LVX: Levofloxacin; MFX: Moxifloxacin; NFX: Norfloxacin; OFX: Ofloxacin; NA: Nalidixic acid; TMP: Trimethoprim; TMP-STX:Trimethoprim-Sulfamethoxazole) susceptibility (by gradient strips) of isogenic strains. Straina Mechanism of quinolone resistanceb SOS/Dam methylation system MIC (Fold)c Source or reference gyrA gyrB parC parE marR CIP LVX MFX NFX OFX NA TMP TMPSTX BW25113 - - - - - WTd 0.015 0.023 0.047 0.094 0.047 4 0.38 0.094 - BW25113dam - - - - - ∆dam 0.008 (1.9) 0.016 (1.4) 0.023 (2) 0.064 (1.5) 0.047 2 (2) 0.064 (5.9) 0.016 (5.8) This study BW25113recA - - - - - ∆recA 0.004 (3.8) 0.008 (2.9) 0.002 (23.5) 0.032 (2.9) 0.016 (2.9) 0.38 (10.5) 0.094 (4) 0.032 (2.9) This study BW25113dam/recA - - - - - ∆dam∆recA 0.002 (7.5) 0.002 (11.5) 0.003 (15.7) 0.016 (5.9) 0.004 (11.8) 0.5 (8) 0.047 (8) 0.016 (5.8) This study BW15 D87G E465D - K390N G414_C430del - 3 6 12 16 16 >256 1.5 0.19 This study BW15dam D87G E465D - K390N G414_C430del ∆dam 2 (1.5) 3 (2) 8 (1.5) 8 (2) 8 (2) >256 1 (1.5) 0.094 (2) This study BW15recA D87G E465D - K390N G414_C430del ∆recA 1 (3) 2 (3) 3 (4) 4 (4) 4 (4) >256 1 (1.5) 0.125 (1.5) This study BW15dam/recA D87G E465D - K390N G414_C430del ∆dam∆recA 0.38 (7.9) 0.5 (12) 1 (12) 2 (8) 1.5 (10.7) >256 0.25 (6) 0.023 (8.2) This study a Genotype. Strains are isogenic to E. coli BW25113 or E. coli BW15 and carry only chromosomal modifications, SOS dysfunction [recA deletion] and/or Dam methylation system system dysfunction. Resistance-associated mutations located in the GyrA and ParC proteins have been defined as resistance mechanisms that alter the target site. b Amino acid changes [GyrA, GyrB, ParC and ParE proteins] and nucleotidic changes [marR]. c MIC (µg/mL) by gradient strips (Number of fold reduction in MIC compared to wild-type strain for each isogenic subgroup). dWild-type. Table 2. FIC index (CIP: Ciprofloxacin; LVX: Levofloxacin; MFX: Moxifloxacin; NFX: Norfloxacin; OFX: Ofloxacin; NA: Nalidixic acid; TMP: Trimethoprim; TMP-STX:Trimethoprim-Sulfamethoxazole) of isogenic strains. S (Synergistic), A (Additive), I (Indifferent). Strainsa FIC index (CIP) FIC index (LVX) FIC index (MFX) FIC index (NFX) FIC index (OFX) FIC index (NA) FIC index (TMP) FIC index (TMP-STX) BW25113 Δdam/ΔrecA 0,75 (A) 0,38 (S) 1,63 (I) 0,75 (A) 0,34 (S) 1,57 (I) 1,23 (I) 1,5 (I) BW15 Δdam/ΔrecA 0,57 (A) 0,42 (S) 0,42 (S) 0,75 (A) 0,56 (A) 2 (I) 0,5 (S) 0,43 (S) a Genotype. Strains are isogenic to E. coli BW25113 or E. coli BW15 and carry only chromosomal modifications, SOS dysfunction [recA deletion] and Dam methylation system dysfunction.