Acquired resistance of Stenotrophomonas maltophilia to antimicrobials induced by herbicide paraquat dichloride

Stenotrophomonas maltophilia, a ubiquitous environmental bacterium, is an important cause of nosocomial infections. Although banned in some countries, paraquat (PQ) is commonly used to control weeds. In this study, we investigated the effects of increasing concentrations of PQ on S. maltophilia and its antimicrobial resistance. The sequential exposure of S. maltophilia K279a to increasing concentrations of PQ induces the formation of strains with increased resistance to PQ. Among the 400 PQ-resistant isolates tested, 70 clones were resistant to 16 μg/ml ciprofloxacin (CIP), and around 18% of the PQ/CIP-resistant isolates showed increased resistance to all the tested antimicrobials including, the aminoglycosides, quinolones, cephalosporin, chloramphenicol, and co-trimoxazole. The results of the expression analysis of the antimicrobial resistance genes in the five selected PQ/CIP-resistant isolates demonstrated the high expression of genes encoding efflux pumps (smeYZ, smaAB, smaCDEF, smeDEF, smeVWX, and smtcrA) and the enzymes aph(3’)-IIc, blaL1, and blaL2. However, expression of the genes known for PQ resistance (i.e., mfsA and sod) were not altered relative to the wild-type levels. Whole genome sequence analysis identified gene mutations that could account for the antimicrobial resistance, namely, smeT (TetR family regulatory protein), rplA (ribosomal protein L1), and acnA (aconitase A). Ectopic expression of wild-type AcnA partially complemented the fluoroquinolone-resistant phenotype of the mutant with mutated acnA, which suggests the role of aconitase A in antimicrobial susceptibility. Exposure of S. maltophilia to PQ thus induces the development of strains that increase resistance to multiple antimicrobials.


Introduction
Stenotrophomonas maltophilia is a gram-negative aerobic bacterium, which is recognized as one of the major causes of hospital-acquired infections (HAIs) worldwide.The incidence of community-acquired S. maltophilia infections is also increasing [1].HAIs are generally associated with high mortality and increased morbidity, length of hospital stay, and medical treatment costs.Resistance to multiple antimicrobial agents poses a major challenge to the management of nosocomial infections caused by S. maltophilia.This bacterium has developed an array of resistance mechanisms, including the production of antibiotic-modifying and -degrading enzymes and multidrug efflux transporters, a reduction in outer membrane permeability, and the modification of antibiotic targets, which make S. maltophilia intrinsically resistant to multiple antimicrobials [2,3].The resistance of S. maltophilia to antimicrobial agents can be acquired by the acquisition of resistance genes through horizontal gene transfers from microorganisms that reside in the same habitats as well as the mutations of genes associated with antimicrobial resistance [4].
Paraquat dichloride (1,1 0 -dimethyl-4-4 0 -bipyridinium dichloride, PQ), also known as methyl viologen, is an active ingredient of non-selective herbicides.PQ is commonly used to control weeds and grasses in agriculture, even though its use has been banned or restricted in more than 67 countries [5].PQ exerts its herbicidal activity by interfering with photosynthesis pathways.It accepts a free electron from the ferredoxin photosystem I to form a PQ radical cation (PQ �+ ), which immediately reacts with oxygen to form highly reactive superoxide anions while concomitantly generating the parent PQ, which is recycled to continuously produce superoxide anions [6].Extensive cellular damage from excessive reactive oxygen species (ROS) causes cell membrane disruption, which leads to leaf wilting, chlorosis, and eventually, desiccation [7].As a redox cycling substance, PQ is potentially hazardous to non-target organisms, including humans, animals, and microorganisms.
PQ has shown to be very immobile and highly persistent in soil.It does not hydrolyze or photodegrade in aqueous solutions and is resistant to microbial degradation [8].As S. maltophilia is an ubiquitous environmental bacterium, it may be exposed to PQ contamination in soil and aquatic environments.Little is known about the responses of S. maltophilia against PQ.It has previously been demonstrated that the expression of mfsA encodes an efflux transporter for PQ, and fluoroquinolone antibiotics are controlled by SoxR, a superoxide-sensing transcriptional regulator.Without treatment with PQ and other superoxide generators, SoxR slightly represses mfsA expression.Upon exposure to PQ, oxidized SoxR triggers the expression of mfsA, thereby inducing cross-protection to PQ [9].High expression of mfsA also increases resistance to fluoroquinolones [10].PQ exerts its toxicity to living cells primarily through the generation of superoxide anions.The functions of three superoxide dismutase (SOD) enzymes, namely, two manganese-containing SODs (SodA1 and SodA2) and an ironcontaining SOD (SodB), in S. maltophilia have been characterized [11].The S. maltophilia K279a genome contains two more Cu/Zn SODs (Smlt0160 and Smlt0161) [2].The expression of sodA1 is presumably regulated by SoxR because the menadione (superoxide generator)mediated upregulation of sodA1 is abolished in the soxR mutant [11].It is thus likely that SoxR modulates the cross talk between the oxidative stress response and protection against antibiotics.We show herein that the exposure of S. maltophilia K279a to PQ could induce the generation of mutant strains that showed increased resistance to ciprofloxacin (CIP) as well as other quinolones and several classes of antimicrobial agents.

Bacterial growth conditions and selection of PQ and CIP resistance
The clinical isolate S. maltophilia K279a [2] was used to select PQ-resistant strains by applying a serial passage method as previously described [12], with some modifications to the culturing conditions.A single colony of K279a was inoculated into a lysogenic broth (LB) and incubated at 35˚C for 24 h with shaking at 150 rpm.After incubation, bacterial cells were inoculated into an LB that had been supplemented with 500 μg/ml PQ (MIC of 1,024 μg/ml [13]) at a starting OD 600 of 0.2 and the culture was incubated for 24 h under the same growth conditions.The subculturing was repeated 7 consecutive times, starting with an initial OD 600 of 0.2 in fresh LB medium supplemented with increasing concentrations of PQ (750, 1,000, 1,250, 1,500, 2,000, 2,500, and 3,000 μg/ml).The culture was then spread onto the LB plates containing 3,000 μg/ ml PQ, and 400 isolated colonies were tested for their ability to grow on Mueller-Hinton agar plates supplemented with 16 μg/ml CIP.
A disk diffusion assay was also used to determine the levels of resistance against oxidants.The disks were prepared by soaking the blank disks in 10 μl 0.5 M PQ, 2.0 M menadione, and 0.5 M phenazine and placing them on a lawn of cells instead of antimicrobial disks.The data were shown as the mean of the three replications ± standard deviation (SD).

Whole genome sequencing and mutation analysis
Whole genome sequencing was performed by Porcinotec (Bangkok, Thailand).The genomic DNA of the S. maltophilia strains was isolated using a GF-1 Bacterial DNA Extraction Kit (Vivantis, Malaysia) in accordance with the manufacturer's protocols.The quality of the extracted DNA was determined using a DeNovix QFX fluorometer (Wilmington, DE) The library preparation of the genomic DNA was performed using a QIAseq FX DNA Library Preparation Kit (Qiagen, Hilden, Germany).The DNA fragments were labeled with different sequencing adaptors.The DNA libraries were checked on QIAxcel Advanced System (Qiagen, Hilden, Germany) and quantified using the DeNovix QFX fluorometer.The DNA libraries were sequenced using V2 chemistry with 2�250 bp reads on the Illumina MiSeq platform (Illumina, San Diego, CA).The output reads were processed using Trimmomatic version 0.39 [16], with the parameters set to LEAD:28, TRAILING:28, SLIDING WINDOW:4:15, and MINLEN:36 to remove the adapters and filter the low-quality reads with a quality score less than Q30.FastQC version 0.12.1 was used to assess the quality of the trimmed sequencing reads, which the quality score had to be equal to or greater than Q30 [17].
Snippy version 4.6.0[18] was used to identify the single nucleotide polymorphisms (SNPs) between the haploid reference genome (S. maltophilia K279a; accession no: NC_010943.1)and the sequencing reads.The assembled genomic sequences of S. maltophilia K279a variants have been deposited in the NCBI databases under the BioProject (accession number PRJNA1072246).

Real-time reverse transcription PCR
The expression levels of the antibiotic resistance genes were determined using real-time reverse transcription PCR (qRT-PCR), as previously described [19].The list of genes and primers used are shown in Table 1.The exponentially growing cultures of the S. maltophilia strains in the LB medium were harvested for total RNA extraction.The reverse transcription was carried out using 1 μg of DNase I-treated total RNA, a RevertAid Reverse Transcriptase (Thermo Fisher Scientific, Waltham, MA), and random hexamers in line with the manufacturer's recommendations.The cDNA (10 ng) was then used as the DNA templates for amplification of the antibiotic resistance genes using the SYBR Select Master Mix for CFX (Thermo Fisher Scientific) and specific primers (Table 1).The PCRs were run on a StepOnePlus Real-Time PCR System (Thermo Fisher Scientific) for 40 cycles of denaturation at 95˚C for 30 s and annealing at 60˚C for 15 s, with an extension of 30 s at 72˚C.The 16S rRNA, which was amplified with the primers BT2781 and BT2782 (Table 1), was used as the normalized gene.Following PCR, a melting curve analysis was conducted to verify the specificity of the amplified DNA sequences.The melting curves from a representative experiment are displayed in S1 Fig.Then, the results were analyzed based on the normalized gene expression and the cycle threshold method (2 -ΔΔC(t) ) and expressed as fold changes of the expression relative to the level in S. maltophilia wild-type K279a.The experiments were repeated independently three times, and the means ± SDs are provided.All expression data are presented in S1 Table.

Cloning of acnA
The putative acnA gene (smlt3608) encoding aconitase A was PCR amplified from S. maltophilia K279a genomic DNA with the primers BT8277 and BT8278 using Phusion High-Fidelity DNA Polymerase (Thermo Fisher Scientific).A 2,650 bp amplicon was cloned into pBBR1MCS-3 [20], an E. coli/S.maltophilia shuttle plasmid vector, cut with SmaI, which yielded pAcnA.The recombinant plasmid was verified via DNA sequencing and no mutations were observed (S2 Fig) .The pAcnA plasmid was introduced into the KPQC13 mutant using electroporation, as previously described [9].

Statistical analysis
The statistical analyses were performed using SPSS version 23.00 (SPSS Inc., Chicago, IL).One-way ANOVA followed by Dunnett's post-hoc test was used to compare the data pertaining to the S. maltophilia variant strains relative to K279a wild-type.A p-value <0.05 was considered statistically significant.

Selection of PQ/CIP-resistant mutants
After the serial passage of the S. maltophilia K279a culture in the presence of increasing concentrations of PQ, bacterial culture was spread onto LB plates containing 3,000 μg/ml PQ.Four hundred isolated colonies tolerant to 3,000 μg/ml PQ were then randomly selected and tested for their ability to grow on plates containing 16 μg/ml CIP (the MIC of K279a for CIP was 2 μg/ml).Among the tested isolates, 70 PQ-resistant isolates (70/400, 17.5%) could resist the CIP at 16 μg/ml.The antibiotic susceptibility profiles for representative antibiotics from various classes, including those recommended by the CLSI [15] for the treatment of S. maltophilia infections (i.e., quinolones [CIP, levofloxacin, and moxifloxacin], aminoglycosides [amikacin and gentamicin], cephalosporin [ceftazidime], penicillin [amoxicillin/ clavulanic acid], sulfonamide [co-trimoxazole], and chloramphenicol) were determined for all 70 PQ/CIP- resistant isolates using a Kirby-Bauer disk diffusion assay.An isolate that showed a smaller inhibition zone diameter (�5 mm) for each antibiotic than the K279a wild-type was considered to have significantly increased resistance, as demonstrated in a previous study [21].All 70 isolates showed increased resistance to CIP, levofloxacin, and chloramphenicol, as illustrated in Fig 1 .In the case of levofloxacin, all the isolates were categorized as "resistant" as per the CLSI guidelines (inhibition zone diameter �13 mm) [15].Moreover, we found that 85% of the isolates showed increased resistance to amikacin, gentamicin, and amoxicillin/clavulanic acid, while 67% showed increased resistance to ceftazidime.About half the isolates displayed increased resistance to co-trimoxazole, the drug of choice when treating S. maltophilia infection.However, none of the isolates were categorized as intermediate or resistant based on the CLSI guidelines (2022).Increased resistance to all the tested antibiotics was exhibited by 28% of the isolates (Fig 1).

Complementation of KPQC13 with wild-type acnA (smlt3608)
The mutant KPQC13 showed a frameshift mutation of smlt3608 (acnA) encoding aconitase A, which shares a 42% amino acid identity with Escherichia coli AcnA [26].To validate the role of S. maltophilia acnA in antimicrobial susceptibility, a full-length acnA gene from K279a was PCR amplified and cloned into a pBBR1MCS-3 [20] expression plasmid vector, which generated pAcnA.The antibiotic susceptibility profile of KPQC13 harboring pAcnA (KPQC13/ pAcnA) was determined and compared with that of the K279a wild-type and KPQC13 harboring empty vector.The expression of AcnA partially complemented the antimicrobial resistance phenotype of KPQC13 against CIP, levofloxacin, moxifloxacin, and ceftazidime (Fig 2B).

Discussion
In this study, the sequential exposure of S. maltophilia K279a to PQ resulted in the formation of mutants that showed increased resistance to PQ. Around 17.5% of the PQ-resistant isolates could resist CIP (at a concentration of 16 μg/ml) and levofloxacin.As a redox cycling compound, PQ undergoes enzymatic one-electron reduction to generate a transient PQ radical, which can be reoxidized by molecular oxygen (O 2 ) to continuously produce superoxide anion, a primary oxygen radical [9].Superoxide anion is a highly reactive species that is able to trigger a cascade of ROS, such as H 2 O 2 , and hydroxyl radicals, which cause oxidative damage to biomolecules.The nucleotide pool is also a target of ROS, and the incorporation of oxidized nucleotides into DNA leads to an increase in mutation frequency [27].The exposure of S. maltophilia to PQ would therefore induce mutations, which lead to resistance phenotypes to PQ as well as antibiotics.SoxR mediated the interplay between the oxidative stress response and antimicrobial resistance in S. maltophilia.SoxR regulates the genes that alleviate PQ toxicity, including mfsA (MFS efflux transporter), sodA1 (SOD), and those that are involved as multidrug transporters (mfsA and smeVWX) [9,11,28].Mutations of soxR have been reported to be associated with the upregulation of genes in regulons [29].Unexpectedly, neither the mutations of soxR nor the promoter region of soxR were detected in any of the selected PQ/CIP-resistant mutants.Our expression analysis revealed that the levels of mfsA and sodA1 transcription were not altered in any of the PQ/CIP-resistant mutants relative to the K279a wild-type.However, the KPQC29 mutant showed increased levels of smeVWX expression (4.3 ± 2.0 fold).Upregulation of smeVWX is at least in part responsible for increased resistance to quinolones and chloramphenicol in the KPQC29 mutant.Three regulators (i.e., SmeRv, AzoR, and SoxR) are involved in the regulation of smeVWX expression.SmeRv and AzoR directly control the expression of smeVWX as positive and negative regulators, respectively, while oxidized SoxR upregulates smeRv expression [28,30,31].We found that increased smeVWX expression in the KPQC29 mutant was not due to mutations of the smeRv, azoR, and soxR genes or their promoter regions.Nevertheless, changes in the levels of the SmeRv and AzoR proteins in the KPQC29 mutant could not be ruled out.
The expression of the other sod genes (i.e., sodA2, sodB, sodC1 and sodC2) were also unchanged (Table 2).The PQ-resistant phenotype of the mutant thus did not rise due to increased levels of the SODs and MfsA efflux pump.This is supported by the finding that none of the tested PQ/CIP resistance showed any increased resistance against the other redox cycling agents/superoxide generators, including menadione and phenazine (Fig 2B).Mechanisms in S. maltophilia other than SODs and MfsA are therefore responsible for PQ resistance.
Mutations that cause changes in the genes involved in the regulation and expression of functional genes contributing to cell permeability, antibiotic resistance mechanisms, efflux transporters, RNA polymerase subunits, and cell metabolism generally explain the resistance of bacteria to antibiotics [32][33][34].Whole genome sequence analysis of the selected PQ/CIPresistant mutants revealed mutations with potential effects on antibiotic resistance, such as the genes encoding SmeT (smlt4073) the transcriptional regulator in the TetR family, the periplasmic protein TonB (smlt0009), and the 50S ribosomal subunit protein L1 (smlt0895, rplA).SmeT negatively regulates the expression of smeDEF, which encodes the multidrug efflux pumps belonging to the resistance-nodulation-cell-division (RND) family [35].Mutations of SmeT lead to constitutively high expression of smeDEF, which is associated with increased resistance to co-trimoxazole, macrolide erythromycin, chloramphenicol, tetracyclines, and quinolones [36,37].In our study, the expression analysis revealed that the KPQC24 mutant, in which smeT was mutated, expressed elevated levels of smeDEF (5.7 ± 3.9 fold) relative to its parental wild-type K279a.However, KPQC24 was highly resistant to quinolones (nalidixic acid, CIP, levofloxacin and moxifloxacin) and macrolide (azithromycin) but not to co-trimoxazole, and chloramphenicol.
KPQC3 contained a frameshift mutation of smlt0009, which encodes the periplasmic membrane protein TonB.TonB is an energy transducing protein of the Ton complex, which consists of TonB, ExbB, and ExbD.The Ton complex uses the proton motive force at the inner membrane to couple energy to the outer membrane transporters to facilitate the uptake of rare nutrients, including iron and cobalamine, into the periplasm [38].Disruption of TonB has been shown to render S. maltophilia resistant to the siderophore-conjugated antibiotics that require Ton complex-mediated transport, such as β−lactams and fluoroquinolones [39].The increased resistance of KPQC3 to cephalosporins, carbapenems, and fluroquinolone is thus in part due to the mutation of TonB, which results in a lower uptake of antibiotics.
We observed mutations of rplA in KPQC3 and KPQC13 mutants.Mutations of the 50S ribosomal protein L1 (RplA) have been observed in the gentamicin resistance mutants of S. maltophilia K279a, which were selected in a laboratory; mutations of rplA led to the activation of aminoglycoside efflux pump SmeYZ production, which thereby increased resistance to aminoglycosides [40].Similarly, in this study, both mutants expressed high levels of smeYZ (34.4 ± 17.9 for KPQC3 and 44.4 ± 12.2 for KPQC13).Mutations of rplA leading to high expression of SmeYZ would contribute to increased resistance to aminoglycoside antibiotics with KPQC3 and KPQC13 mutants.It has been proposed that aminoglycosides, the drugs that target the A-site of 16S ribosomal RNA of the 30S ribosome, are potential drivers of the selection of strains with overproduced SmeYZ that occurs due to rplA mutations in S. maltophilia [40].A more recent study illustrated that treating K279a with a sublethal level of CIP can also induce rplA mutation [19].Accordingly, mutations of rplA can be induced not only through exposure to aminoglycosides and CIP but also to PQ.This acquired resistance may be one of the important factors rendering S. maltophilia resistant to aminoglycosides because mutations of RplA associated with the overproduction of SmeYZ exist in various clinical isolates [40].It is of note that the contribution of SmeYZ to the virulence of S. maltophilia in a mouse model has been reported [41].Moreover, KPQC29 was also expressed high level of smeYZ (30.5 ± 8.4 fold) without mutations of ribosomal proteins or its transcriptional regulators, two-component regulatory system SmeSyRy (Smlt2200 and Smlt2199) [42] and SmaRS (Smlt2645 and Smlt2646) [21].The reason for upregulation of SmeYZ in KPQC29 is unknown.However, KPQC29 carried two mutations on mfsQ (smlt2678) and smlt2538 encoding hypothetical protein containing ferritin-like domain.MfsQ has been shown to play no roles on antibiotic resistance of S. maltophilia K279a and is regulated by MfsR, a TetR type transcriptional regulator [22,43].Despite containing ferritin-like domain, smlt2538 was not a member of genes contributing to iron homeostasis systems identified in S. maltophilia K279a [44].The physiological function of smlt2538 in both iron homeostasis and antibiotic resistance is under investigation.Notably, the high expression of the SmeYZ aminoglycoside efflux pump in KPQC3, KPQC13, and KPQC29 mutants relative to the K279a wild-type (34.4± 17.9, 44.4 ± 12.2, and 30.5 ± 8.4 fold, respectively) was associated with enhanced resistance to amikacin, gentamicin, and netilmicin.This is in contrast to the lower expression levels observed in KPQC22 and KPQC24 (1.5 ± 0.5 and 2.1 ± 0.7 fold, respectively), as shown in Table 3 and Fig 2.
The KPQC13 mutant showed frameshift mutation of acnA (smlt3608) encoding aconitase A. Aconitase, a [4Fe-4S] cluster containing enzyme, catalyzes the reversible and stereo-specific isomerization of citrate and isocitrate via cis-aconitate in the tricarboxylic acid (TCA) cycle.E. coli produces two aconitases, AcnA. a stationary-phase enzyme, that can be induced by iron and oxidative stress, and AcnB, a major enzyme synthesized during exponential growth [45].Moreover, the AcnA and AcnB apo-proteins function as post-transcription regulator by interacting with the target mRNAs [46].The finding that complementation of KPQC13 with pAcnA plasmid could partially diminish the increased antibiotic resistance of the KPQC13 mutant against fluoroquinolone antibiotics (CIP, levofloxacin, and moxifloxacin) and cephalosporin (ceftazidime) indicates the involvement of AcnA in the antibiotic resistance of S. maltophilia.The linkage between aconitase and antibiotic resistance has recently been reported in Vibrio cholerae, where a lack of AcnB increased its tolerance to the antibiotics that target protein synthesis, including tetracycline, erythromycin, and chloramphenicol [47].It has been reported that ROS plays a role in the antibiotic-mediated killing of bacteria, in particular those belonging to aminoglycosides, quinolones, and β-lactam [48].The TCA cycle increases ROS production by generating the reducing equivalents NADH and FADH 2 , which deliver their electrons to the electron transport chain; this catalytic activity is believed to be triggered by treatment with antibiotics [48].Thus, lower TCA cycle activity, for example, disruption of aconitase genes would reduce the level of post-antibiotic ROS production, thus rendering it less susceptible to the antibiotic [47].The mutation of the aconitase gene acnA therefore contributes partly to the increased resistance to quinolones observed in the KPQC13 mutant.Moreover, the ectopic expression of acnA from pAcnA was unable to fully complement the increased antibiotic resistance phenotypes (fluoroquinolones and ceftazidime) of KPQC13 to wild-type levels.This might be due to the presence of mutations in other genes in KPQC13, such as rplA and opuD2, which may also influence antimicrobial resistance.Alternatively, the use of pBBR1MCS-3, a medium-copy-number plasmid vector, in a complementation experiment could be a contributing factor.High expression of genes on pAcnA, including acnA and tetracycline resistance genes, may adversely affect KPQC13's physiology.Utilizing a singlecopy complementing system might yield more definitive results.
Mutations of opuD2 (smlt3910) encoding the putative glycine betaine transporter were detected in KPQC13 and KPQC22.The glycine betaine transporter belongs to the betaine-choline-carnitine transporter (BCCT) family.It is involved in the uptake of a compatible solute glycine betaine, which is a small, uncharged organic compound that functions as an osmoprotectant to maintain cell permeability and volume during high salinity stress [49].To our knowledge, the role of the glycine betaine transporter in antibiotic resistance is as yet unknown and is being investigated.
KPQC24 had a point mutation of glnA (smlt0151) encoding putative glutamine synthetase type I, an enzyme that catalyzes the reaction that produces glutamine from the condensation of L-glutamate and ammonia.Glutamine is used as a nitrogen donor for synthesizing nitrogen-containing molecules, such as purine and pyrimidine [50].S. maltophilia K279a possesses two coding sequences, smlt0151 and smlt1580, encoding putative glutamine synthetases [2].Metabolite analysis has revealed that glutamine is repressed in multidrug-resistant uropathogenic E. coli [51].Exogenous glutamine potentiates the killing activity of β-lactam, aminoglycoside, quinolone, and tetracycline against E. coli and promotes the influx of ampicillin, which leads to the accumulation of the antibiotic [51].Glutamine-potentiated ampicillinmediated bacterial killing has been observed in other gram negative bacteria, including Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumonia, and Vibrio parahaemolyticus [51].In KPQC24, the point mutation of GlnA occurred at D396N, which is located on the adenylation loop and regulates glutamine synthetase activity via the adenylation of a tyrosine residue.The conserved adenylation loop NLYDLP [52] corresponded to the D 396 LYDLP of S. maltophilia GlnA.The D396N mutation would therefore make the mutant's adenylation loop match the conserved NLYDLP domain perfectly, and it is thus unlikely to affect the enzymatic activity of GlnA.
Altogether, our results indicate that increased antimicrobial resistance seems to be in harmony with the upregulation of the genes responsible for the antibiotic resistance of the S. maltophilia PQ/CIP-resistant mutants.The development of multidrug-resistant mutants due to PQ exposure highlights the risk that improper use of the herbicide PQ can contribute to antibiotic resistance problems.
Furthermore, there are some limitations in this study.Selecting a larger number of PQ/ CIP-resistant mutants may reveal more novel genes contributing to antibiotic resistance in S. maltophilia.We have identified mutations in genes that have never been reported for their involvement in antibiotic resistance in S. maltophilia, such as opuD2, acnA, and glnA.Further investigations are therefore required to elucidate their functions.

Conclusion
In this study, we showed that exposure of S. maltophilia K279a to PQ can induce the development of mutant strains with increased antimicrobial resistance to CIP, clinically important quinolone levofloxacin, and other quinolones.Simultaneously increased resistance of the PQ/ CIP-resistant mutants to aminoglycosides, cephalosporins, carbapenems, chloramphenicol, azithromycin, and co-trimoxazole was observed.Mutations of the SmeT regulator and ribosomal protein L1 led to the increased production of antibiotic resistance proteins, mainly drug efflux pumps.The mutation of the AcnA enzyme in the central metabolism rendered it resistant to antibiotics, including fluoroquinolones and ceftazidime.The extensive use and poor management of the herbicide PQ may stimulate the development of antimicrobial-resistant strains of S. maltophilia in soil and aquatic environments.It is recognized that environments, particularly aquatic ones, act as reservoirs and transmission routes for the dissemination of pathogens to humans [53].This raises concerns that the misuse of PQ could increase the risk of developing antibiotic resistance, especially in S. maltophilia.

Table 3 . Expression profiles of the antimicrobial resistance genes in the S. maltophilia mutants.
Data represent the mean ± SD of fold change relative to the expression of K279a from three replicated experiments.C, chloramphenicol; TE, tetracycline; SXT, cotrimoxazole; ND, no data.Asterisk (*) indicates significant difference compared to K279a according to one-way ANOVA, followed by Dunnett's post-hoc test (p < 0.05).https://doi.org/10.1371/journal.pone.0309525.t003