Bacteriological and Molecular Study of Fluoroquinolones Resistance in Pseudomonas aeruginosa Isolated From Different Clinical Sources

The present study was conducted to investigate the resistance of fluoroquinolones (FQs) and the effects of mutations in the resistance gene in clinical isolates of P. aeruginosa isolated from different sources in Al-Hussein Hospital, Al-Samawah city, Iraq. The basic mechanism of the resistant of fluoroquinolones in P. aeruginosa is via mutations occurring in the basic bacterial gyrA gene encoding-subunit A of DNA gyrase . Forty clinical isolates from various sourced (burn 7 (17.5 %), wound 7 (17.5 %), ear 2 (5 %), operation room 12 (30 %), urine 3 (7.5 %), and industrial dialysis center 9 (22.5 %)) were isolated based on bacteriological methods confirmed by 16s rRNA gene using PCR technique. A sensitivity test was conducted to all isolates by Kirby-Pour method using 7 antibiotics of fluoroquinolones. Amongst the 40 clinical isolates, 10 were resistant and 3 were sensitive to all tested antibiotics, while 27 were intermediate, resistant and sensitive to two or more of tested antibiotics, with the resistance being confirmed by the minimum inhibitor concentration (MIC) test. The ten resistant isolates were used to examine the mutations in gyrA gene. A direct sequence method was used and revealed eight mutations in gyrA gene at different positions. In addition, we found that fluoroquinolone activity in the sensitive isolates, after sequencing for these isolates, is a bacteriostatic activity. The results of this study showed the gyrA mutations resulting from the excessive use of antibiotics are one of the mechanisms may be that leading to fluoroquinolone resistance.


Introduction
Pseudomonas aeruginosa (P. aeruginosa) is an ubiquitous bacterium known as a pathogenic microbe responsible for serious opportunistic infections, especially in the immunocompromised subjects [1]. The spread of this organism in health institutions is very dangerous, with infiltrates, via any defect at the basic defense line of the human host, enter the body through the skin, especially in the hospital intensive care units (ICUs) leading to nosocomial infections [1,2]. P.aeruginosa pathogenesis is multifactorial, due to the existence of multiple mechanisms of veritable resistance to most antibiotics, resulting in the production of a variable set of cellular structures and extracellular molecules which play a significant role in increasing pathogenicity [3]. In general, antibiotics are natural products inhibiting the microbes in either bactericidal or bacteriostatic pattern [4]. Fluoroquinolones (FQs) are synthetic antibiotics with bactericidal activity against many species of Gram negative bacterium [4]. Fluoroquinolones are used basically in the treatment of infections generated by P. aeruginosa, but in recent years an increase of fluoroquinolones resistance was observed between clinical isolates at high levels, leading to the failure of the treatment of P. aeruginosa [5]. The basic mechanism of the resistant of fluoroquinolones in P. aeruginosa is mutations occurring in the bacterial gyrA gene encoding subunit A of DNA gyrase [2], thus leading to inhibiting the transcription and blocking DNA replication [6]. The aim of the current study was to detect the occurrence of mutations associated with fluoroquinolones resistance (gyrA gene) using partial fragments from 40 clinical isolates of P. aeruginosa using direct sequencing technique.

Materials and methods Collection of clinical samples
A total of 258 clinical samples were collected from varied clinical sources of patients and teaching laboratories of Al-Hussein Hospital in Al-Samawah city during the period from November 2018 to February 2019. These samples were taken accurately from each source using a sterile swab, which was then placed in a plane tube containing BHI broth and transferred to public health laboratory (bacteriological unit) in Al-Samawah city, where they were incubated at 37 ˚C for 24hr. The main sources of the samples were Burn, Wound, Ear, Operation Room, Urine and Industrial Dialysis Center.

Identification of bacterial isolates
As cultural method, all samples were grown on blood and macconkey agars and incubated-at 37 ˚C for 24hr as an initial diagnostic approach. Additionally, sub-cultures onto macconkey agar were performed to remove bacterial mixed growth and obtain pure colonies of each isolate [7]. For purification, P. aeruginosa was isolated using selective media (Pseudomonas and Cetrimide agars) to confirm the inhibition of other bacterial growth and the production of Pigments [8,9,10]. For microscopic examination, Gram stain was used [11]. Diagnostic tests were performed using a variety set of tests (oxidase, catalase, blood hemolysis, urease, motility, indole, citrate, gelatin, and KIA) [12] with confirmed by VITEK₂ Compact was achieved using Gram-negative card kits (GN ID) through the colorimetric technique [7]. Then, molecular detection of all isolates was performed by the 16s rRNA gene using PCR technique [13].

Minimum inhibitor concentration test
The MICs were performed by agar well dilution method (AWDM) in Mueller-Hinton medium, with following the CLSI 2018 recommendations for ' MIC breakpoints ' and using 7 antibiotics of fluoroquinolones (NA, CIP, NOR, OFX, LOM, LEV, and TRV) [15].

DNA extraction
Genomic DNA was extracted from all isolates using a specific Kit (GenomicDNA Mini Kit, FavorGEN, Korea) in accordance with the user manual of the manufacturer company (specific protocol for gram negative-bacterium). In addition, concentration and purity of DNA for each sample were estimated by a NanoDrop device with a range of 147.8 µg/ml within 1.7 purity to 1236.8 µg/ml within 1.9 purity.

PCR reaction
The PCR technique was used to amplify 16s rRNA and gyrA genes, using specific primers (table 1) [16,17]. A volume of 20 µL was used for amplification in accordance with the user manual of the manufacturer company, which included 10µL of master mix, 1µL of each primer, 3 µL of the DNA template, and 5 µL of nuclease free water. The proper program for this experiment is presented in table 2. Several trails were conducted to determine the optimum conditions to amplify 16s rRNA and gyrA genes in a thermal-cycler device depending on different primers and the DNA template. Afterward, 5 µL was analyzed to detect 16s rRNA and gyrA genes of each PCR product through electrophoresis on 1% agarose gel containing 3 µL of ethidium bromide, and then viewed on UV transilluminator.

DNA sequencing
In the current study, PCR products of the 15 isolates ( 13 for gyrA and 2 for 16s rRNA) were sent to the MacroGenecompany, South Korea, to define the basic nucleotides sequences for the genes. The results of sequencing were then analyzed by BioEdit and MEGA7 software with the NCBI engine.

Results
Amongst the 258 clinical samples, forty P.aeruginosa isolates from different sources were obtained. The isolates of P.aeruginosa distributed among clinical samples based on their source are listed in Table-3. All clinical isolates were identified as P.aeruginosa by a set of a variety of techniques (biochemical tests, cultural, pigment production and VITEK 2 Compact). The results are demonstrated in Table-4.

MIC Test
Amongst the 40 clinical isolates, 10 isolates that were resistant to all tested antibiotics were selected for the MIC test to confirm the resistance of isolates in accordance with the antibiotic sensitivity test, based on recommendations of the CLSI, 2018. The results are listed in the Table-6.

Molecular detection of 16s rRNA and gyrA
The 16s rRNA was used as a molecular marker for the diagnosis of P. aeruginosa. The results of the current study showed that forty isolates of P. aeruginosa contained the 16s rRNA gene, which is considered as the diagnostic gene for this bacteria. Specific primers were used for the amplification of 16s rRNA, with 468 bp by conventional PCR based on 58.8 °C annealing temperature (Figure-2). Whereas, the gyrA gene in the current study was detected by PCR technique in P. aeruginosa isolates associated with fluoroquinolones resistance. Specific primers were used to amplify 287 bp of gyrA gene based on 51.6 °C annealing temperature (Figure-3). 0022 Data analysis using direct sequencing 1. Phylogenetic tree analysis Phylogenetic analysis was conducted through following up the alignment using the BLAST n program for 16s rRNA sequences obtained from the current isolates by Sanger sequencing after trimming by MEGA7 software [19]. The phylogenetic tree (dendrogram) was created by MEGA7 software using the neighbor-joining method as shown in the figure 4 which illustrates the relationships amongst the local isolate of 16s rRNA with the reference strains in GenBank.
In our study, after the sequencing of 10 isolates resistant to all tested antibiotics of fluoroquinolones, we identified 8 mutations in gyrA gene at different positions, of which 3 (37.5 %) were missense-mutations, 4 (50 %) were silent-mutations, and 1 (12.5 %) was a deletion-mutation. Whereas when sequencing for the 3 control isolates (2 sensitive and 1 intermediate), we identified 4 mutations in gyrA gene at different positions, of which 2 (50 %) were missense-mutations, 1 (25 %) was a silent-mutation, and 1 (25 %) was insertion-mutation. Based on this, we identified the fluoroquinolone activity in the sensitive isolates as a bacteriostatic activity, in accordance with the basic tests (antibiotic sensitivity, MIC and sequencing), due to the presence of several mutations which are limiting the use of this class of antibiotics in the future. Nevertheless, all the mutations diagnosed in the gyrA gene were related with all antibiotics (NA, CIP, NOR, OFX, LOM, LEV, and TRV) in accordance with the 10 isolates of P.aeruginosa that were resistant for all tested antibiotics of fluoroquinolones class.

Discussion
Fluoroquinolones are synthetic antibiotics with bactericidal-activity against many species of Gram negative bacterium [20]. In most hospitals, excessive use of a wide spectrum of antibiotics led to the appearance of highly resistant isolates of P. aeruginosa [17]. Based on biochemical, epidemiological, and molecular previous studies, the first target for FQs in P.aeruginosa is DNA gyrase [18]. In the current study, the mutations in gyrA gene of the 13 isolates of P. aeruginosa (10 resistant, 2 sensitive, and 1 intermediate) obtained from the 40 clinical isolates are demonstrated in Table-7) above. To reveal the correlation amongst mutations and isolates associated with fluoroquinolones resistance, a comparison was made for the molecular analysis results of the target gene with MIC test in accordance with the antibiotic sensitivity test. The results in our study showed that, amongst the 40 clinical isolates, 10 were resistant and 3 were sensitive for all tested antibiotics, while 27 were intermediate, resistant and sensitive for two or more of the tested antibiotics. In addition, amongst the 10 isolates of P.aeruginosa resistant to all tested antibiotics of fluoroquinolones, 2 had a single mutation, 1 had a double mutation, and 7 had several mutations. While for the 2 sensitive isolates to fluoroquinolones, 1 had several mutations and 1 had a double mutation, with 1 intermediate isolate that had a double mutation in gyrA gene. These results agree with those of a previous molecular study [17]. However, we identified 9 mutations in gyrA gene in the 13 clinical isolates (10 associated with fluoroquinolones resistant, 2 sensitive, and 1 intermediate) that occurred at different codons (83, 87, 103, 118, 132, 136, 139, and 140). The alterations of nucleic acid in these codons were detected through the change of the amino acid Threonine in the codon 83 to Isoleucine (Thr-83→Ile), which was found in 9 isolates. Additionally, Aspartate in the codon 87 was changed to Asparagine (Asp-87→Asn) in 4 isolates. These results agree with those of a previous reports on clinical isolates of P.aeruginosa [6,17,18,21]. The present analysis also revealed changes of Valine in the codon 103 to Valine (Val-103→Val) in 4 isolates, Alanine in the codon 118 to Alanine (Ala-118→Ala) in 4 isolates, Histidine in the codon 132 to Histidine (His-132→His) in 8 isolates, and Alanine in the codon 136 to Alanine (Ala-136→Ala) inn 4 isolates. These results agree with those of a previous molecular study [2]. In addition, we found changes of Glutamate in the codon 139 to Lysine (Glu-139→Lys) in 2 isolates, Glutamate in the codon 139 to X (deletion G) (Glu-139→X) in 1 isolate, and insertion A in the codon 140 to Lysine (X-139→Lys) in 1 isolate. These results are in disagreement with the results of previous molecular studies [2,17]. In this study, the highest levels of resistant were to Nalidixic Acid (85%), Trovafloxacin (62.5%), Lomefloxacin (45%), Norfloxacin (32.5%), Ciprofloxacin (30%), Levofloxacin (30%), and Ofloxacin (27.5%). The importance of this resistance in P. aeruginosa isolates is that it gradually leads to increased secretion of the virulence factors [2]. Moreover, the highest levels of resistant are usually associated with the existence of gyrA mutations and lead to the failure of the treatment of P. aeruginosa, thus limiting the use of this class of antibiotics in the future.

Conclusions
In conclusion, the results of the present study, in agreement with the previous reports, showed the gyrA mutations resulting from the excessive use of antibiotics are one of the mechanisms may be that leading to fluoroquinolone resistance in P. aeruginosa clinical isolates, where we proved that DNA gyrase encoding gyrA gene is considered as the basic target for fluoroquinolones.