Occurrence of Point Mutations in gyrA and parC Genes of Ciprofloxacin- Resistant Pseudomonas aeruginosa Isolated from Burn Infections

The spread of antibiotic resistant bacteria is a worldwide problem. Due to the importance of P. aeruginosa as a multidrug resistant bacterium, this study aimed, through molecular techniques, to detect point mutations in chromosomal genes responsible for the quinolones class of antibiotics resistance. A total of 52 isolates from burn infections were identified using specific primers for P. aeruginosa 16S rDNA. Ciprofloxacin minimum inhibitory concentrations (MIC) were estimated using the agar dilution assay. DNA sequences of the quinolone resistancedetermining regions of gyrA and parC were determined for detecting the mutations found in these genes and the relations among the isolates by constructing phylogenetic trees. The results revealed that only 43 (82.7%) of isolates were P. aeruginosa, of which 31 (72.06%) were resistant to different concentrations of ciprofloxacin, ranging between 4 and >32 μg/ml. Twenty six isolates were selected for sequencing, including sensitive, intermediately resistant, and highly resistant to ciprofloxacin. The ciprofloxacin sensitive isolates did not exert any amino acid alterations in gyrA or parC genes; however, a single intermediately resistant isolate had a single mutation at each gene. Of the total resistant isolates (20), 6 isolates had no mutations at different MIC levels, While 14 isolates had Thr-83-Ile substitution in gyrA and Ser-87-Leu substitution in parC, only five isolates had a second mutation, namely Asp-87-Asn, in gyrA. The phylogenetic analysis of the studied groups showed divergence from the P. aeruginosa PAO1 and PAO1OR reference strains due to increased mutations and polymorphisms in studied isolates. In conclusion, P. aeruginosa occurrence was increased in burn infections and the fluoroquinolones in current use are not as effective as before; the main resistance mechanism in local clinical isolates of P. aeruginosa is mutations, where the main target of fluoroquinolones is gyrA gene. Keyword: P. aeruginosa, gyrA, parC, Mutation, Phylogenetic tree. تانيج يف ةيطقن تا رفط ثودح gyrA و parC ايرتكب نيب Pseudomonas aeruginosa ةلوزعم نيسادكولفوربدلل ةمواقملا قورحلا تاباهتلا نم ندحلا دبع يلع نوصغ , ريملاا دبع يداه رجاه * نسق مولع ،ةايحلا ةيلك ،مولعلا ةعهاج دادغب ، لا قارع . ةصلاخلا ايختكب ةيسهأ ببدبو ،ةيسلاع ةمكذم ةيؾيحلا تادازسمل ةمواقسلا ايخيتكبلا راذتنا لثسي P. aeruginosa ةيظقشلا تا خفظلا فاذتكا ىلإ ةسا رجلا هحه تفجه ، ةدجعتسلا ةيودلأل ةمواقم ايخيتكب اهرابتعاب ISSN: 0067-2904 Hadi and Ali Iraqi Journal of Science, 2021, Vol. 62, No. 10, pp: 3457-3466 3458 تايشقتلا للاخ ؽم ، ةيؾيحلا تادازسمل ةمواقسلا تانؾلؾشيكلا فشص ؽع ةلوؤدسلا تامؾسؾموخكلا تاشيج يف ىمع لؾرحلا ؼت .ةيئيدجلا 52 ؽيجل ةررختم ئداؾب ماجختساب اهشع فذكو قوخحلا ىوجع ؽم ةلدع 16S rDNA ايختكب يف P. aeruginosa ؾمفوخبيس ؽم ىندلاا طبثسلا ديكختلا ؼييقت ؼت . صحف قيخط ؽع ؽيسادك ةيظقشلا تا خفظلا ؽع فذكمل ةمواقسلا تانؾلؾشيكلا ةقظشسل يوؾشلا ضسحلا لدمدت جيجحت ؼت .فيفختلا راجأ تاشيج يف ةدؾجؾسلا gyrA و parC رؾظتلاو ءؾذشلا راجشأ ءاشب للاخ ؽم تلادعلا ؽيب ةقلاعلا داجياو . نأ جئاتشلا تخهعأ 43 ( 82.7 تناك تلادعلا ؽم طقف )٪ ت ايختكبل دؾع P. aeruginosa ػلحكو 31 ( 72.06 ؽيب حوا ختت ؽيسادكؾمفوخبيس ؽم ةفمتخم تا ديكختل ةمواقم تناك تلادعلا هحه ؽم )٪ 4 >ىلإ 32 تخيتخا .لم/ما خغوخكيم 26 ؽيسادكؾمفوخبدلا دازسل ةمواقم اهبمغاو ةظسؾتمو ةسادح اهزعب ةلدع ةفخعسل ؼل ثيح اهل ةيشيجوختشلا جعاؾقلا لدمدت يف تا خيغت ؽيسادكؾمفوخبيدمل ةسادحلا تلادعلا ؽم يأ خهغت تاشيج يف ةيشيملأا ضاسحلأا gyrA وأ parC ةظسؾتم ةلدع يف ةجحاو ةخفط تخهض ػلذ عم ؽكل ، ( ةمواقسلا تلادعلا عؾسجم ؽيب ؽم .ةمواقسلا 20 ىجل ؽكي ؼل ،) 6 تايؾتدسب اهشم تلادع MIC يأ ةفمتخم تا خفط نأ ؽيح يف ، 14 ل ةلدع لاجبتسا ةخفط اهيج Thr-83-Ile يف gyrA و Ser-87-Leu يف parC ، 5 ةيناث ةخفط اهيجل طقف تلادع Asp-87-Asn يف gyrA نا ةيشيجلا ةخجذلا ليمحت خهعا . ةسا رجلا يف ةمجختدسلاو تاشيجلا ػشب ؽم ةذؾخأسلا ةيسايقلا تلالادلا ؽع جعابتت ةسورجسلا عيماجسلا يهو PAO1 , PAO1OR ةسورجسلا تلادعلا يف لاكشلأا دجعتو تا خفظلا جيا دت ببدب نا جاتشتسلاا ؼت جق و . ةباصا ةبدن P. aeruginosa ةلاعف جعت ؼل ايلاح ةمجختدسلا تلاؾشيؾكلا تادازم و ,دادزا جق حوخجلا جسخل سلا تلادعلا ؽيب تلاؾشيؾكلا ةمواقسل ةيساسلاا ةيللاا يه ةخفظلا ؛تناك اسك ايختكبل ةيمح P. aeruginosa و , نا gyrA .تلاؾشيؾكمل يديئخلا فجهلا ؾه Introduction Burn injury is a worldwide public health issue due to increasing nosocomial infections [1,2]. Particularly in developing countries, Pseudomonas aeruginosa pathogen can cause severe nosocomial infections, especially in catheterized, immunocompromised, or burned patients [3, 4] resulting from its containment of intrinsic and acquired resistance to many antibiotic groups [5]. For effective oral treatment, fluoroquinolones are the only available antibiotics for infections caused by this organism [6]. In the treatment of P. aeruginosa infections, ciprofloxacin and levofloxacin are commonly used amongst the fluoroquinolones group [7]. None of the new fluoroquinolones after the third generation retains activity against ciprofloxacin-resistant isolates [8]. Fluoroquinolones work by inhibiting two enzymes involved in bacterial DNA synthesis, both of which are DNA topoisomerases that human cells lack and that are essential for bacterial DNA replication [9], thereby enabling these agents to be both specific and bactericidal [10]. A doubled increase in ciprofloxacin resistance was found among Gram -ve bacilli isolated from hospital's intensive care units in 1990s, because of raised (40%) fluoroquinolones consumption [11]. The main mechanisms of fluoroquinolones resistance in P. aeruginosa are resulting from mutations in DNA gyrase and topoisomerase IV [12], decreased permeability of the cell wall, multidrug efflux pump [13], and plasmid mediated resistance [14]. Most bacteria have both enzymes; however, gyrase is more sensitive to inhibition by quinolones than is topoisomerase IV in Gram -ve bacteria, whereas, in Gram +ve) bacteria, topoisomerase IV is usually the main target and gyrase is interestingly less susceptible [15]. Changes in topoisomerase IV in G-ve bacteria are alleged to have a secondary contribution in resistance development of parC or parE mutations, which confer resistance only in the existence of concomitant DNA gyrase mutations [16]. Resistance develops due to amino acid substitutions in regions in GyrA or ParC called the quinolone resistance determining regions (QRDRs) [17]. Mutations can enhance resistance to antibiotics by controlling enzyme expression or efficacy. Regulatory mutations provide limited resistance while higher resistance is provided by structural mutations via the improvement of the binding affinity between the resistance proteins and antibiotic [18]. The spread of antibiotic resistant bacteria is considered as a global problem. Due to the importance of P. aeruginosa as a multidrug resistant bacterium, this study aimed to detect mutations in chromosomal genes (gyrA and parC) responsible for resistance to the quinolones class of antibiotics. In addition, we aimed at constructing a phylogenetic tree for both genes to observe the effects of mutations on divergence between isolates. Hadi and Ali Iraqi Journal of Science, 2021, Vol. 62, No. 10, pp: 3457-3466 3459 Materials and Methods Isolation of Bacteria Fifty-two isolates from burn and wound infections were gathered from several hospitals in Baghdad, Iraq, which were, based on conventional (cultural and biochemical) tests considered to be P. aeruginosa. 16SrDNA for the Identification of P. aeruginosa DNA was extracted from fifty-two P. aeruginosa isolates by utilizing a commercial kit (G-spin extraction kit, Intron, Korea), depending on the instructions of the production company. Specific primers were used for the identification of 16SrDNA from P. aeruginosa [19]. The PCR mixture consisted of premix (i-taq, Intron, Korea), 1 μl forward and reverse primers, and 3 μl DNA template. The volume was completed with 20 μl sterile distilled water. After optimization, the program was carried out with an initial denaturation , 25 cycles of denaturation at , annealing at , , and Determination of Minimal Inhibitory Concentration The agar dilution method was used to determine the minimal inhibitory concentration of ciprofloxacin (Hexal, Germany) against identified P. aeruginosa, as recommended by the Clinical and Laboratory Standards Institute CLSI [20]. The antibiotic dilution range was prepared to be between 0.0625 and 32 mg/L. The antibiotic solution was dispensed into universal containers containing 20 ml of Muller Hinton agar (Himedia, India) that have been allowed to The bacterial culture was set to 0.5 McFarland (1.5×10 8 CFU/ml), of which inoculums of 0.2 μL were applied to the agar surface with a micropipette. Plates were inverted and incub 3  ̊ 18-24 hr. The lowest concentration of antimicrobial agent that completely inhibits the growth was considered to be the minimal inhibitory concentration. Amplification and sequencing of gyrA and parC The two investigated genes, i.e. gyrA (417 bp) for detecting topoisomerase II subunit [21] and parC (303 bp) for detecting topoisomerase IV subunit of P. aeruginosa [17], were amplified. PCR mixture was composed of premix (i-taq, Intron, Korea), 1 μl of primers (for gyrA, F 5 ́GACGGCCTGAAGC CGGTGCAĆ3 and R 5 ́GCCCACGGCGATACCGCTGGÁ3, while for parC, 1 μl of F 5 ́CGAGCAGGCCTATCTGAACTAT́3 and R 5 ́GAAGGACTTG GGATCGTCCGGÁ3), and 3 μl of DNA template, then the volume was completed with sterile D.W. After optimization, the program was carried out with initial denaturation 3 min), 29 cycles of denaturation at 40 sec), annealing at 64.1C (30 sec) for gyrA 3 parC, 30 sec), and Gel electrophoresis was employed using agarose gel (1.5%) with RedSafe dye for the confirmation of the presence and size of bands in gel under UV Transilluminator. The conventional PCR products of 28 isolates for gyrA and parC were sent to Macrogen Company, USA, for sequencing by using forward primers of gyrA and parC on the genetic analyzer (Applied Biosystems) in addition to Sanger sequencing. Homology search and mutations detection were performed via the online Basic Local Alignment Search Tool (BLAST) program using blastn algorithms, which are available online at the National Center for Biotechnology Information (NCBI) and CDS feature . The Bioedit program (V.7.2) was used for multiple sequence alignment in Clustal W, as well as to calculate the similarity matrix between bacterial isolate groups. Phylogenetic analysis was performed using MEGA7 software, employing the method of neighborhood-joining (NJ). For estimating the nod branching probability, 1000 bootstraps were used. Statistical Analysis Data were presented as percentage and/ or mean ± standard error. The statistical analysis system IBM SPSS Statistics 23 (2015) was used to analyse the effects of different factors in this study. Chiχ was employed to analyse the differences between study groups and assays. Levene's test was used to test homogeneity of variance and Spearman's correlation test was used to analyse other data. A p value of ≤ y g Results and Discussion Bacterial identification with 16S rDNA DNA of fifty-two isolates was extracted and conventional PCR was performed to amplify the PASS gene. 43 (82.7%) isolates were identified as P. aeruginosa. These results corresponded with the result Hadi and Ali Iraqi Journal of Science, 2021, Vol. 62, No. 10, pp: 3457-3466 3460 of local studies [22, 23], where P. aeruginosa was isolated from different clinical specimens and identified using 16S rDNA. This test gives 90% higher accuracy than other methods, allowing the differentiation of this species from other Pseudomonas species and other pathogens. The genotypic identification differs from the phenotypic one, being influenced by the presence or absence of nonconserved genes or characters expression variation; therefore, 16S rDNA gene provides precise identification of isolates [24]. Minimal inhibitory concentration Forty-three isolates of P. aeruginosa were tested to determine the minimal inhibitory concentration for ciprofloxacin by the agar dilution method. 31 (72.09%) isolates showed resistance to ciprofloxacin, 30.23% of which had resistance rate that ranged 4-32 μg/ml, while the rest (41.86%) were resistant at concentrations higher than 32 μg/ml. In addition, 8 (18.60%) isolates showed intermediate resistance at a concentration of 2 μg/ml, while 4 (9.30%) isolates were sensitive at 1 μg/ml. Significant differences were found among the different MIC concentrations [tobt (199.56) = 16.16, p ≤ ] The result of 72.09% resistance obtained here was higher than that of 9% reported by a previous local study in Baghdad [25] and that of 31% from different clinical sources [26]. However, non-local studies showed more similar results to ours. Thirty-eight clinical isolates of P. aeruginosa isolated from different clinical sources in north Lebanon showed 57.89% resistance to ciprofloxacin [17], whereas 64% of 100 isolates from different clinical specimens in Iran were resistant [12] and 100% (81 isolates) were resistant in Tunisia [27]. Differences in resistance rates could be attributed to isolation from different departments, patient populations, and surely the differences in the amount of this antibiotic used by different hospitals. However, the resistance rate is increasing year after year due to the misuse by both hospitals and community. Amplification and sequencing of gyrA and parC PCR was conducted over 43 isolates to amplify the constitutional genes gyrA and parC, where bands were confirmed with gel electrophoresis (Figure 1). All the isolates were positive for both the gyrA and parC genes, as these are essential genes that are constitutively expressed at a relatively constant level and their absence means cell death [28]. This result is consistent with that reported by a previous study [29]. The PCR products f were sequenced or both genes from 26 isolates, some of which being sensitive, intermediate, or resistant. The results were analyzed and compared using BLAST with the reference strains available in the GenBank of the NCBI database. The sequencing results demonstrated 98-100% compatibility with the reference strains PAO1 (AE004091.2) and PAO1OR (LN871187.1). Figure 1Gel electrophoresis of P. aeruginosa amplified genes; A: gyrA (417bp) and B: parC (303bp). Agarose gel (1.5%) stained with RedSafe stain, 80 V/cm for 1hrs and visualized by UV transilluminator. M: 1000 bp DNA ladder. Hadi and Ali Iraqi Journal of Science, 2021, Vol. 62, No. 10, pp: 3457-3466 3461 DNA sequencing of gyrA and parC was used to detect mutations related to the resistance of P. aeruginosa to the fluoroquinolone antibiotic (Figure 2). Transversion mutations showed a frequency of 19 for gyrA and parC, while transition mutations had a frequency of 83 in all the isolates. From 107 mutation, 71 were shown to be silent mutations in all the isolates while 36 were missense mutation. Figure 2Sequence alignments and CDS subunits of gyrA (A) and parC (B) of P. aeruginosa local isolates with PAO1 NCBI reference strain AE004091.2. Black color indicates conserved bases and residues, red color indicates altered bases, pink color indicates amino acid alterations; Thr-83-Ile substitution in gyrA and Ser-87-Leu substitution in parC. Three amino acid substitutions were found in this study; 15 (57.7%) and 5 (19.2%) isolates had Thr-83Ile and Asp-87Asn substitutions, respectively in gyrA, while 15 (57.7%) isolates had Ser-87Leu substitution in parC. All isolates that showed Thr-83-Ile substitution in gyrA also had Ser-87-Leu substitution in parC (Table 1). The association of amino acid substitutions mentioned above with fluoroquinolone resistance in P. aeruginosa was reported in several investigations [30-32]. These amino acid substitutions were found as the main types of replacements [21, 33-35], thereby matching the results in this study. Furthermore, mutations that included Thr-83-Ile (92.44%) and Asn-87-Asp (0.44%) were present as single substituions, whereas those of Thr-83-Ile and Asn-87-Asp (5.78%) were present as double substitutions [36]. Another study found that 100% of isolates resistant to ciprofloxacin had Thr-83-Ile substitution in gyrA and 68.75% had a further alteration in parC [12]. Amino acid alteration at positions Glu-91, Leu-95 [17], Pro-83, Pro-88, and Gly-85 [37] in parC were not detected in this study. Relationship between Ciprofloxacin MIC and Mutations in QRDRs Table 1 illustrates the Ciprofloxacin MIC for 26 isolates and their relationships to mutations in gyrA and parC genes. As observed, no mutations were found in the sensitive isolates. Isolates that had one gyrA (Thr-83-Ile) substitution and one parC substitution (Ser-87Leu) revealed MIC values ranging 2 >32 mg/mL. Moreover, five isolates with dual gyrA alterations (Thr-83-Ile and Asp-87Asn) and one parC alteration (Ser-87-Leu) had MIC values ranging 4>32 μg/ml, with most isolates having MIC >32 μg/ml. Table 1The MIC values of ciprofloxacin for 26 isolates and their relationships to mutations in gyrA and parC genes. Amino acid (codon) encoded by indicated gene at position: *Isolate no. MIC value parC gyrA 87(Ser) 87(Asp) 83(Thr) ---------B-PA22 1 Sensitive ---------B-PA23


Introduction
Burn injury is a worldwide public health issue due to increasing nosocomial infections [1,2]. Particularly in developing countries, Pseudomonas aeruginosa pathogen can cause severe nosocomial infections, especially in catheterized, immunocompromised, or burned patients [3,4] resulting from its containment of intrinsic and acquired resistance to many antibiotic groups [5]. For effective oral treatment, fluoroquinolones are the only available antibiotics for infections caused by this organism [6]. In the treatment of P. aeruginosa infections, ciprofloxacin and levofloxacin are commonly used amongst the fluoroquinolones group [7]. None of the new fluoroquinolones after the third generation retains activity against ciprofloxacin-resistant isolates [8]. Fluoroquinolones work by inhibiting two enzymes involved in bacterial DNA synthesis, both of which are DNA topoisomerases that human cells lack and that are essential for bacterial DNA replication [9], thereby enabling these agents to be both specific and bactericidal [10]. A doubled increase in ciprofloxacin resistance was found among Gram -ve bacilli isolated from hospital's intensive care units in 1990s, because of raised (40%) fluoroquinolones consumption [11]. The main mechanisms of fluoroquinolones resistance in P. aeruginosa are resulting from mutations in DNA gyrase and topoisomerase IV [12], decreased permeability of the cell wall, multidrug efflux pump [13], and plasmid mediated resistance [14]. Most bacteria have both enzymes; however, gyrase is more sensitive to inhibition by quinolones than is topoisomerase IV in Gram -ve bacteria, whereas, in Gram +ve) bacteria, topoisomerase IV is usually the main target and gyrase is interestingly less susceptible [15]. Changes in topoisomerase IV in G-ve bacteria are alleged to have a secondary contribution in resistance development of parC or parE mutations, which confer resistance only in the existence of concomitant DNA gyrase mutations [16]. Resistance develops due to amino acid substitutions in regions in GyrA or ParC called the quinolone resistance determining regions (QRDRs) [17]. Mutations can enhance resistance to antibiotics by controlling enzyme expression or efficacy. Regulatory mutations provide limited resistance while higher resistance is provided by structural mutations via the improvement of the binding affinity between the resistance proteins and antibiotic [18]. The spread of antibiotic resistant bacteria is considered as a global problem. Due to the importance of P. aeruginosa as a multidrug resistant bacterium, this study aimed to detect mutations in chromosomal genes (gyrA and parC) responsible for resistance to the quinolones class of antibiotics. In addition, we aimed at constructing a phylogenetic tree for both genes to observe the effects of mutations on divergence between isolates.

Materials and Methods Isolation of Bacteria
Fifty-two isolates from burn and wound infections were gathered from several hospitals in Baghdad, Iraq, which were, based on conventional (cultural and biochemical) tests considered to be P. aeruginosa. 16SrDNA for the Identification of P. aeruginosa DNA was extracted from fifty-two P. aeruginosa isolates by utilizing a commercial kit (G-spin extraction kit, Intron, Korea), depending on the instructions of the production company. Specific primers were used for the identification of 16SrDNA from P. aeruginosa [19]. The PCR mixture consisted of premix (i-taq, Intron, Korea), 1 µl forward and reverse primers, and 3 µl DNA template. The volume was completed with 20 µl sterile distilled water. After optimization, the program was carried out with an initial denaturation , 25 cycles of denaturation at , annealing at , , and

Determination of Minimal Inhibitory Concentration
The agar dilution method was used to determine the minimal inhibitory concentration of ciprofloxacin (Hexal, Germany) against identified P. aeruginosa, as recommended by the Clinical and Laboratory Standards Institute CLSI [20]. The antibiotic dilution range was prepared to be between 0.0625 and 32 mg/L. The antibiotic solution was dispensed into universal containers containing 20 ml of Muller Hinton agar (Himedia, India) that have been allowed to The bacterial culture was set to 0.5 McFarland (1.5×10 8 CFU/ml), of which inoculums of 0.2 µL were applied to the agar surface with a micropipette. Plates were inverted and incub 3 ˚ 18-24 hr. The lowest concentration of antimicrobial agent that completely inhibits the growth was considered to be the minimal inhibitory concentration.

Amplification and sequencing of gyrA and parC
The two investigated genes, i.e. gyrA (417 bp) for detecting topoisomerase II subunit [21] and parC (303 bp) for detecting topoisomerase IV subunit of P. aeruginosa [17], were amplified. PCR mixture was composed of premix (i-taq, Intron, Korea), 1 µl of primers (for gyrA, F 5´-GACGGCCTGAAGC CGGTGCAC-´3 and R 5´-GCCCACGGCGATACCGCTGGA-´3, while for parC, 1 µl of F 5´-CGAGCAGGCCTATCTGAACTAT-´3 and R 5´-GAAGGACTTG GGATCGTCCGGA-´3), and 3 µl of DNA template, then the volume was completed with sterile D.W. After optimization, the program was carried out with initial denaturation 3 min), 29 cycles of denaturation at 40 sec), annealing at 64.1C (30 sec) for gyrA 3 parC, 30 sec), and Gel electrophoresis was employed using agarose gel (1.5%) with RedSafe dye for the confirmation of the presence and size of bands in gel under UV Transilluminator. The conventional PCR products of 28 isolates for gyrA and parC were sent to Macrogen Company, USA, for sequencing by using forward primers of gyrA and parC on the genetic analyzer (Applied Biosystems) in addition to Sanger sequencing. Homology search and mutations detection were performed via the online Basic Local Alignment Search Tool (BLAST) program using blastn algorithms, which are available online at the National Center for Biotechnology Information (NCBI) and CDS feature .
The Bioedit program (V.7.2) was used for multiple sequence alignment in Clustal W, as well as to calculate the similarity matrix between bacterial isolate groups. Phylogenetic analysis was performed using MEGA7 software, employing the method of neighborhood-joining (NJ). For estimating the nod branching probability, 1000 bootstraps were used.

Statistical Analysis
Data were presented as percentage and/ or mean ± standard error. The statistical analysis system IBM SPSS Statistics 23 (2015) was used to analyse the effects of different factors in this study. Chiχ was employed to analyse the differences between study groups and assays. Levene's test was used to test homogeneity of variance and Spearman's correlation test was used to analyse other data. A p value of ≤ y g Results and Discussion Bacterial identification with 16S rDNA DNA of fifty-two isolates was extracted and conventional PCR was performed to amplify the PASS gene. 43 (82.7%) isolates were identified as P. aeruginosa. These results corresponded with the result

Hadi and Ali
Iraqi Journal of Science, 2021, Vol. 62, No. 10, pp: 3457-3466 3460 of local studies [22,23], where P. aeruginosa was isolated from different clinical specimens and identified using 16S rDNA. This test gives 90% higher accuracy than other methods, allowing the differentiation of this species from other Pseudomonas species and other pathogens. The genotypic identification differs from the phenotypic one, being influenced by the presence or absence of nonconserved genes or characters expression variation; therefore, 16S rDNA gene provides precise identification of isolates [24].

Minimal inhibitory concentration
Forty-three isolates of P. aeruginosa were tested to determine the minimal inhibitory concentration for ciprofloxacin by the agar dilution method. 31 (72.09%) isolates showed resistance to ciprofloxacin, 30.23% of which had resistance rate that ranged 4-32 µg/ml, while the rest (41.86%) were resistant at concentrations higher than 32 µg/ml. In addition, 8 (18.60%) isolates showed intermediate resistance at a concentration of 2 µg/ml, while 4 (9.30%) isolates were sensitive at 1 µg/ml. Significant differences were found among the different MIC concentrations [t obt (199.56) = 16.16, p ≤ ] The result of 72.09% resistance obtained here was higher than that of 9% reported by a previous local study in Baghdad [25] and that of 31% from different clinical sources [26]. However, non-local studies showed more similar results to ours. Thirty-eight clinical isolates of P. aeruginosa isolated from different clinical sources in north Lebanon showed 57.89% resistance to ciprofloxacin [17], whereas 64% of 100 isolates from different clinical specimens in Iran were resistant [12] and 100% (81 isolates) were resistant in Tunisia [27]. Differences in resistance rates could be attributed to isolation from different departments, patient populations, and surely the differences in the amount of this antibiotic used by different hospitals. However, the resistance rate is increasing year after year due to the misuse by both hospitals and community. Amplification and sequencing of gyrA and parC PCR was conducted over 43 isolates to amplify the constitutional genes gyrA and parC, where bands were confirmed with gel electrophoresis (Figure 1). All the isolates were positive for both the gyrA and parC genes, as these are essential genes that are constitutively expressed at a relatively constant level and their absence means cell death [28]. This result is consistent with that reported by a previous study [29].
The PCR products f were sequenced or both genes from 26 isolates, some of which being sensitive, intermediate, or resistant. The results were analyzed and compared using BLAST with the reference strains available in the GenBank of the NCBI database. The sequencing results demonstrated 98-100% compatibility with the reference strains PAO1 (AE004091.2) and PAO1OR (LN871187.1).

Hadi and Ali
Iraqi Journal of Science, 2021, Vol. 62, No. 10, pp: 3457-3466 3461 DNA sequencing of gyrA and parC was used to detect mutations related to the resistance of P. aeruginosa to the fluoroquinolone antibiotic ( Figure 2). Transversion mutations showed a frequency of 19 for gyrA and parC, while transition mutations had a frequency of 83 in all the isolates. From 107 mutation, 71 were shown to be silent mutations in all the isolates while 36 were missense mutation. Three amino acid substitutions were found in this study; 15 (57.7%) and 5 (19.2%) isolates had Thr-83-Ile and Asp-87-Asn substitutions, respectively in gyrA, while 15 (57.7%) isolates had Ser-87-Leu substitution in parC. All isolates that showed Thr-83-Ile substitution in gyrA also had Ser-87-Leu substitution in parC ( Table 1). The association of amino acid substitutions mentioned above with fluoroquinolone resistance in P. aeruginosa was reported in several investigations [30][31][32]. These amino acid substitutions were found as the main types of replacements [21,[33][34][35], thereby matching the results in this study. Furthermore, mutations that included Thr-83-Ile (92.44%) and Asn-87-Asp (0.44%) were present as single substituions, whereas those of Thr-83-Ile and Asn-87-Asp (5.78%) were present as double substitutions [36]. Another study found that 100% of isolates resistant to ciprofloxacin had Thr-83-Ile substitution in gyrA and 68.75% had a further alteration in parC [12]. Amino acid alteration at positions Glu-91, Leu-95 [17], Pro-83, Pro-88, and Gly-85 [37] in parC were not detected in this study. Table 1 illustrates the Ciprofloxacin MIC for 26 isolates and their relationships to mutations in gyrA and parC genes. As observed, no mutations were found in the sensitive isolates. Isolates that had one gyrA (Thr-83-Ile) substitution and one parC substitution (Ser-87-Leu) revealed MIC values ranging 2 ->32 mg/mL. Moreover, five isolates with dual gyrA alterations (Thr-83-Ile and Asp-87-Asn) and one parC alteration (Ser-87-Leu) had MIC values ranging 4->32 µg/ml, with most isolates having MIC >32 µg/ml.

B-PA: Burn-Pseudomonas aeruginosa
A high correlation was found between MIC values of 8->32 µg/ml and the presence of mutations [r= 0.672, P ≤ 0.05]. There was also a correlation between MIC value of 4 µg/ml and mutations [r= 0.454, P ≤ 0.05]. However, no correlation was found between MIC value of 2 µg/mL and mutations [r= 0.255, P > 0.05]. The correlation results confirmed that substitution mutations in this study were generally responsible for ciprofloxacin resistance.
Based on the sequence analysis, the isolates with a mutation in parC hold one or double mutations in gyrA. This observation proved that gyrase was the main target for fluoroquinolone resistance in P. aeruginosa clinical isolates [38]. Yet, isolates possess two mutations in gyrA and one mutation in parC had elevated ciprofloxacin MICs compared with one mutation in gyrA. These findings suggest that the mutation in parC takes place after those occurring in gyrA, resulting in increased resistance to fluoroquinolone in P. aeruginosa [12].
Of twenty resistant isolates, 6 isolates had no mutations at different MIC levels, which suggests resistance by other mechanisms, such as efflux pumps overexpression, reduced membrane permeability, and gyrB and parE mutations, which were previously illustrated in P. aeruginosa fluoroquinolone resistance [17,39]. Sequence similarity matrix and phylogenetic tree of for gyrA and parC in P. aeruginosa For similarity matrix and phylogenetic tree construction, isolates were divided into groups depending on the similarity in their nucleotide sequences. Thus, the isolates divided into eight groups (A, B, C, D, E, F, G and H) for gyrA while for parC the isolates divided to five groups (1, 2, 3, 4 and 5). The sequence similarity matrix for gyrA and parC of 26 P. aeruginosa isolates were estimated by Bioedit program. The results showed that the studied groups (A-H) of gyrA have high sequence similarity, ranging between 97-99%, except for the group H which was observed to have 100% similarity with P. aeruginosa PAO1 strain. The sequence similarity for parC was also high when compared with P. aeruginosa PAO1 strain, ranging 89-99%, and reached to 100% in group 1.
The phylogenetic tree was constructed to determine the relationships among studied groups by comparing the gyrA and parC sequences obtained in this study with GenBank database sequences in NCBI. Figure 3 illustrates a neighbor-joining phylogenetic tree for gyrA sequences, which indicates one cluster that is divided into two subgroups. The first subgroup contained all the local isolates, except those isolates that belonged to groups A and C, which were placed in the second subgroup. Group G showed sister groups with PAO1 (USA) and PAO1OR (France) reference strains which were used for determining the mutations and polymorphisms in the local isolates of this study. Groups A 3463 and C showed divergence with other groups, because these isolates had higher polymorphisms than those isolates of the remaining groups. After phylogenetic analysis of gyrA, P. aeruginosa isolated from pet Chinese stripe-necked turtles was arranged in a distinct clade from clinical, soil and water isolates of P. aeruginosa clades, which means that the gyrA region is genetically more conserved [38]. Phylogenetic tree for parC sequences (Figure 4) indicated one cluster that was divided into two subgroups. Group 1 showed a sister group with PAO1 (USA) reference strain from GenBank. Local isolates in groups 3 and 5 revealed divergences, because they had polymorphisms not found in the strains of GenBank used for sequences comparison in this study.