Parallel Evolution to Elucidate the Contributions of PA0625 and parE to Ciprofloxacin Sensitivity in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a ubiquitous pathogen that causes a wide range of acute and chronic infections. Ciprofloxacin, one of the first-line fluoroquinolone class antibiotics, is commonly used for the treatment of P. aeruginosa infections. However, ciprofloxacin-resistant P. aeruginosa is increasingly reported worldwide, making treatment difficult. To determine resistance-related mutations, we conducted an experimental evolution using a previously identified ciprofloxacin-resistant P. aeruginosa clinical isolate, CRP42. The evolved mutants could tolerate a 512-fold higher concentration of ciprofloxacin than CRP42. Genomic DNA reference mapping was performed, which revealed mutations in genes known to be associated with ciprofloxacin resistance as well as in those not previously linked to ciprofloxacin resistance, including the ParER586W substitution and PA0625 frameshift insertion. Simulation of the ParER586W substitution and PA0625 frameshift insertion by gene editing in CRP42 and the model strain PAO1 demonstrated that while the PA0625 mutation does contribute to resistance, mutation in the ParER586W does not contribute to resistance but rather affects tolerance against ciprofloxacin. These findings advance our understanding of ciprofloxacin resistance in P. aeruginosa.


Introduction
Pseudomonas aeruginosa is a Gram-negative opportunistic human pathogen with high prevalence in hospitals. It is a common cause of acute and chronic infections in individuals with cystic fibrosis (CF) or hospitalized in intensive care units [1]. It is recognized as the critical priority tier of antibiotic-resistant pathogens by the World Health Organization for the research and development of new antibiotics [2]. It is also included in the group of "ESKAPE" pathogens, which comprise Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, P. aeruginosa, and Enterobacter species, for which novel therapeutic options are urgently needed [3].
Ciprofloxacin is one of the first-line fluoroquinolone class antibiotics used to treat a wide range of infections by P. aeruginosa [4]. However, an increasing proportion of clinical P. aeruginosa isolates have been found to be resistant to ciprofloxacin [5]. Multiple mechanisms of ciprofloxacin resistance have been demonstrated in P. aeruginosa, mainly including (i) mutations in the ciprofloxacin target-encoding genes gyrA/gyrB (encoding DNA gyrase) or parC/parE (encoding topoisomerase IV) to reduce the affinity of targets for ciprofloxacin [6,7]; (ii) mutations in the regulatory genes resulting in overexpression of efflux pumps to increase the expulsion of ciprofloxacin, including mexS (repressor of mexEF-oprN), nfxB (repressor of mexCD-oprJ), mexZ (repressor of mexXY), and mexR (repressor of mexAB-oprM) [4,8]; and (iii) acquisition of ciprofloxacin modifying genes through plasmidmediated gene transfer [9]. Novel mutations and genes associated with ciprofloxacin resistance were discovered using a laboratory experimental evolution approach to generate Microorganisms 2023, 11, 13 2 of 8 ciprofloxacin-resistant mutants from sensitive strains of P. aeruginosa in combination with whole-genome sequencing [10,11]. Although how these mutations contribute to resistance against ciprofloxacin in P. aeruginosa has not been determined, these studies reveal the high potential of parallel evolution to uncover previously unknown resistance genes.
In this study, we employed experimental evolution and genomic DNA reference mapping to identify resistance genes for ciprofloxacin in P. aeruginosa. We found mutations in genes known to be associated with ciprofloxacin resistance. Our results also revealed mutations in two genes not previously linked to ciprofloxacin resistance, including the ParE R586W substitution and PA0625 frameshift insertion.

Bacterial Strains, Plasmids, Primers, and Culture Conditions
The bacterial strains, plasmids, and primers used in the present study are shown in Tables S1 and S2. The P. aeruginosa strain CRP42 was isolated from the sputum sample of a patient [8]. Bacterial cells were cultured in Luria-Bertani (LB) broth (5 g/L yeast extract, 5 g/L NaCl, and 10 g/L tryptone) or on LB agar plates (supplemented with 15 g/L agar) at 37 • C. When needed, concentrations of tetracycline were used at 10 µg/mL in E. coli and 50 µg/mL in P. aeruginosa. The minimum inhibitory concentration (MIC) of ciprofloxacin was determined by a 2-fold serial dilution method with modifications [12]. Briefly, each well of a 96-well microtiter plate was filled with 100 µL LB broth with serially diluted concentrations of ciprofloxacin. One hundred microliters of bacterial culture (10 4 CFU) were inoculated into each well. Growth was assessed visually after 20 h of incubation at 37 • C. All strains were tested in triplicate.

Parallel Evolution to Select for Ciprofloxacin-Resistant Mutants
The continuous ciprofloxacin evolution experiment with three repeats was carried out in LB broth containing increasing concentrations of ciprofloxacin. On day 1, overnight cultures of CRP42 were inoculated (100-fold dilution) into four tubes containing 2, 4, 8, and 16 µg/mL ciprofloxacin, which correspond to 0.25×, 0.5×, 1×, and 2× MIC for CRP42, respectively. After 24 h of aerobic culture at 37 • C, cells from each population with the highest concentration of ciprofloxacin that allowed bacterial growth to an optical density at 600 nm above 1 were measured for the MIC and reinoculated into fresh medium as before, containing 0.25×, 0.5×, 1×, and 2× MIC (obtained one day earlier). This serial passaging was repeated until the MIC of the evolved strains rose to 4096 µg/mL. Another three repeats were passaged in LB broth medium serving as controls.
To delete the PA0625 gene, a 762 bp fragment immediately upstream of the PA0625 gene and a 670 bp fragment downstream of the PA0625 gene were amplified using PAO1 or CRP42 genomic DNA as templates (primers in Table S2). These two fragments were digested with EcoRI-BamHI and BamHI-HindIII and then ligated into pEX18Tc, resulting in pEX18-PA0625-1 (PAO1 genomic DNA as template) and pEX18-PA0625-2 (CRP42 genomic DNA as template). Gene deletion in PAO1 or CRP42 was performed with pEX18-PA0625-1 or pEX18-PA0625-2 as described above.

Genomic DNA Isolation and Reference Mapping
Genomic DNAs of R1, R2, and R3, as well as control strains C1, C2, and C3, were extracted from overnight cultures of bacterial cells using a DNA purification kit (Tiangen Biotec, Beijing, China). Genomic DNA reference mapping was carried out by GENEWIZ Life Sciences (Suzhou, China) as described in our previous studies [8,14]. The raw sequence data have been deposited in the NCBI database with accession number PRJNA875020.

Ciprofloxacin Susceptibility Assay
Overnight cultures of P. aeruginosa strains were diluted 50-fold into LB broth medium and grown at 37°C to an OD 600 of 1.0. The bacterial cells were treated with ciprofloxacin at a final concentration of 16 µg/mL (for CRP42 and CRP42parE R586W ) or 0.25 µg/mL (for PAO1 and PAO1parE R586W ) and then further cultured at 37°C with agitation (200 rpm) for up to 6 h. The number of viable bacterial cells was determined by serial dilution and plating at the indicated time. The bacterial survival% = (live bacterial number after ciprofloxacin treatment) × 100/(initial inoculum bacterial number).

Statistical Analysis
GraphPad Prism 7.0 software was used to conduct the statistical analyses. P values were calculated using the two-tailed unpaired Student's t test. Differences were considered statistically significant when the P value was below 0.05.

Development of Resistance to Ciprofloxacin by the Clinical Isolate CRP42
To uncover novel ciprofloxacin resistance-related genes and understand the evolutionary characteristics of clinically resistant P. aeruginosa, we utilized strain CRP42, a ciprofloxacin-resistant clinical isolate due to the GyrA D87G mutation and overexpression of the MexEF-OprN efflux pump [8], to perform parallel evolution. The development of ciprofloxacin resistance was carried out by the daily passage of three biologically replicate populations of CRP42 in LB broth medium containing increasing concentrations of ciprofloxacin. After 17-34 days of evolution, the MICs for the three groups reached 4096 µg/mL (a 512-fold increase compared to CRP42 in Figure S1), whereas no change in MIC was detected for the three control groups that were evolved in the antibiotic-free LB broth for the same period. To validate that the resistance is caused by stable mutations, we streaked the population of each replicate that reached the highest MICs at the evolutionary end on LB agar plates. A random colony was isolated from each of the plates and designated R1, R2, and R3. The strains were cultured in LB broth medium for two sequential passages, and the MICs were determined with the 2-fold dilution method. The MICs of these strains were the same as those of the corresponding populations ( Table 1), suggesting that increased resistance to ciprofloxacin was due to stable mutations.

Candidate Mutations Related to Ciprofloxacin Resistance in the Evolved Strains
To determine the genetic events that contribute to the increased ciprofloxacin resistance in the evolved mutants, we conducted genomic DNA reference mapping for the R1, R2, and R3 strains, as well as the control strains C1, C2, and C3. Table 2 shows the candidate mutations related to ciprofloxacin resistance in the evolved strains that were not present in the C1, C2, or C3 strains (all mutated genes are listed in Table S3). The threonine at position 83 of GyrA was substituted with isoleucine, alanine, and valine in the R1, R2, and R3 strains, respectively (Table 2). It has been reported that T83 plays a key role in the binding of ciprofloxacin by GyrA of P. aeruginosa [4]. GyrA variants with T83I and T83A substitutions have been associated with ciprofloxacin resistance in P. aeruginosa clinical isolates and in vitro-evolved resistant mutants [15,16]. In addition to GyrA, mutations were Microorganisms 2023, 11, 13 4 of 8 also observed in GyrB (a proline insertion after codon A458 in R2), ParC (substitutions E91K in R1 and S87L in R3), and ParE (EV insertion before codon D450 in R1, VD insertion before codon G451 in R2, and R586W substitution in R3). All three strains also carried mutations in the nfxB gene (stop codon changed to cysteine in R1 and R2, and G180S substitution in R3), which encodes a repressor for the efflux pump MexCD-OprJ [17]. G180S substitution and stop codon loss mutation (stop codon changed to arginine) have been reported to impair NfxB repressor activity, leading to ciprofloxacin resistance in the strain PAO1 [18]. In line with a previous study [18], our simulated replacements of nfxB increased the MIC of ciprofloxacin in both CRP42 and CSP18mexS CRP42 , a strain with MexEF-OprN efflux pump overproduction [8] (Table 1). In addition, strain R3 carried a frameshift insertion in PA0625, which encodes a possible lytic system of R-type pyocin [19,20].  T83I  T83A  T83V - hypothetical protein --L659fs --a same as that in PAO1 (www.pseudomonas.com, accessed on 6 January 2021).

The ParE R586W Substitution Is Not Responsible for Resistance, but Tolerance to Ciprofloxacin in P. aeruginosa
Mutations in the genes encoding GryA/B and ParC/E are major contributors to ciprofloxacin resistance in P. aeruginosa [4]. The alterations in the quinolone resistance determining region (QRDR) in GyrA/B and ParC/E caused by mutations have been reported to contribute to ciprofloxacin resistance in P. aeruginosa [6,21]. All the mutations in GyrA/B and ParC/E in the R1, R2, and R3 strains, except for the ParE R586W mutation in R3, are located in the QRDR [21]. Therefore, we wanted to investigate whether the R586W substitution in ParE plays a role in ciprofloxacin resistance. The chromosomal parE gene of the parental strain CRP42 was replaced by parE R3 , and the MIC of ciprofloxacin was measured. As shown in Table 1, the replacement of parE with parE R3 had no impact on the MIC of CRP42. The R586W mutation was further generated in ParE of the model strain PAO1. Consistent with the unchanged MIC of CRP42parE R586W , the PAO1parE R586W strain displayed the same MIC against ciprofloxacin as its parental strain PAO1 (Table 1). These results suggested that the ParE R586W substitution is not responsible for ciprofloxacin resistance in P. aeruginosa.
Next, we examined the role of the ParE R586W mutation in the tolerance of P. aeruginosa against ciprofloxacin using the killing assay. Ciprofloxacin concentrations at 2 × MICs for CRP42 and CRP42parE R586W (16 µg/mL) or PAO1 and PAO1parE R586W (0.25 µg/mL) were used. As shown in Figure 1, the ParE R586W substitution significantly increased the bacterial survival rate in both the PAO1 and CRP42 strains.
determining region (QRDR) in GyrA/B and ParC/E caused by mutations have been reported to contribute to ciprofloxacin resistance in P. aeruginosa [6,21]. All the mutations in GyrA/B and ParC/E in the R1, R2, and R3 strains, except for the ParE R586W mutation in R3, are located in the QRDR [21]. Therefore, we wanted to investigate whether the R586W substitution in ParE plays a role in ciprofloxacin resistance. The chromosomal parE gene of the parental strain CRP42 was replaced by parER3, and the MIC of ciprofloxacin was measured. As shown in Table 1, the replacement of parE with parER3 had no impact on the MIC of CRP42. The R586W mutation was further generated in ParE of the model strain PAO1. Consistent with the unchanged MIC of CRP42parE R586W , the PAO1parE R586W strain displayed the same MIC against ciprofloxacin as its parental strain PAO1 (Table 1). These results suggested that the ParE R586W substitution is not responsible for ciprofloxacin resistance in P. aeruginosa.
Next, we examined the role of the ParE R586W mutation in the tolerance of P. aeruginosa against ciprofloxacin using the killing assay. Ciprofloxacin concentrations at 2 × MICs for CRP42 and CRP42parE R586W (16 µg/mL) or PAO1 and PAO1parE R586W (0.25 µg/mL) were used. As shown in Figure 1, the ParE R586W substitution significantly increased the bacterial survival rate in both the PAO1 and CRP42 strains.

PA0625 Mutation Contributes to Ciprofloxacin Resistance in P. aeruginosa
The region from PA0613 to PA0648 encodes bacteriophage-like pyocins [22]. Expression of the genes in this region, including PA0625, was induced by ciprofloxacin [22]. Mutations in some genes in this region have been found to increase resistance to ciprofloxacin in both PAO1 and PA14 strains [22,23]. Our reference mapping and sequencing analysis for the PA0625 amplicon of the R3 strain revealed that R3 carries a frameshift insertion in PA0625. However, the contribution of PA0625 to ciprofloxacin resistance has not been established in P. aeruginosa. To test whether the PA0625 mutation increased the resistance to ciprofloxacin in P. aeruginosa, we replaced the PA0625 gene with PA0625 R3 and deleted PA0625 in both the CRP42 and PAO1 strains using gene recombination. As shown in Table 1, the deletion or replacement of PA0625 with PA0625 R3 resulted in a 1.2-fold and a 2-fold increase in the MIC of ciprofloxacin in CRP42 and PAO1, respectively. These results demonstrated that PA0625 is involved in ciprofloxacin resistance in P. aeruginosa.

Discussion
P. aeruginosa can cause serious nosocomial infections, and treatment is challenging. Ciprofloxacin, one of the first-line fluoroquinolone class antibiotics, has become an important tool in treating P. aeruginosa infections, but increasing resistance threatens its efficacy. Demonstrating novel resistance-related mutations during the development of bacterial resistance to ciprofloxacin may provide clues for novel strategies to suppress resistance evolution. In this study, we combined an in vitro evolution assay with genomic DNA reference mapping to determine resistance-related mutations. We found that mutations occur in genes known to be associated with ciprofloxacin resistance. Our results also revealed that the PA0625 mutation contributes to ciprofloxacin resistance in P. aeruginosa, while the ParE R586W substitution confers P. aeruginosa tolerance to ciprofloxacin.
parE encodes the ParE subunit of topoisomerase IV in P. aeruginosa. ParE variants with amino acid changes of D419N, E459D, A473V, and S457R have been shown to be associated with fluoroquinolone resistance in P. aeruginosa [4,6,7]. Mutations in the parE gene for fluoroquinolone resistance are rare compared to those in gyrA and parC, perhaps because alterations in the sequence of ParE confer lower-level resistance against fluoroquinolone [4,7]. In this study, we demonstrated that the ParE R586W substitution, which was located away from the QRDR, does not contribute to resistance but confers tolerance against ciprofloxacin in P. aeruginosa. Since antibiotic tolerance mutations facilitate the rapid evolution of resistance in E. coli [24], it is possible that the ParE R586W mutation paves the way for the subsequent evolution of ciprofloxacin resistance in P. aeruginosa.
Pyocins are bacteriocins that are synthesized by more than 90% of P. aeruginosa strains [19]. R-type pyocins are able to kill other Gram-negative bacteria in a bacterial niche [19]. Production and release of pyocins lead to lysis of the producer cells [20]. Pyocin biosynthesis genes contribute to bacterial susceptibility to ciprofloxacin because mutations in some of those genes increase resistance to ciprofloxacin and other fluoroquinolones in P. aeruginosa [22]. Our previous studies showed that upregulation of pyocin biosynthesis genes contributes to increased susceptibility to ciprofloxacin [25,26], while downregulation of pyocin biosynthesis genes increases ciprofloxacin resistance in P. aeruginosa [27]. Here, we showed that the PA0625 mutation conferred increased ciprofloxacin resistance in P. aeruginosa. Given the importance of cell lysis in R-type pyocin-mediated susceptibility to ciprofloxacin and the fact that PA0625 likely encodes a lytic system similar to those of bacteriophages [20,22], it is possible that a mutation in PA0625 disabled bacterial cell lysis by R-type pyocin in the presence of ciprofloxacin in P. aeruginosa.
In summary, we discovered two novel mutations that contribute to P. aeruginosa resistance to ciprofloxacin, including PA0625, which directly contributes to resistance, and the ParE R586W substitution, which contributes to tolerance against ciprofloxacin. These findings advance our understanding of ciprofloxacin resistance in P. aeruginosa.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/microorganisms11010013/s1. Figure S1: Development of resistance against ciprofloxacin by clinical isolate CRP42. MICs of experimentally evolved mutants to ciprofloxacin. Three lineages were passaged in the presence of ciprofloxacin for experimental evolution. The MICs of ciprofloxacin were measured daily. Table S1: Bacterial strains and plasmids used in this study. Table S2: Primers used in this study. Table S3: Gene mutations identified by genome reference mapping with elimination of SNVs with heterozygous mutations, synonymous mutations, and the same mutations between evolved strains and control strains. Reference [28] is cited in the supplementary materials.  2021YFE0101700, and 82061148018). The funders had no role in the study design, data collection and interpretation, or the decision to submit the work for publication.

Data Availability Statement:
The raw sequence data of genomic DNA reference mapping have been deposited in the NCBI with accession number, PRJNA875020 (www.ncbi.nlm.nih.gov/sra/?term= PRJNA875020+, accessed on 30 August 2022).

Conflicts of Interest:
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.