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Back to Journal »Infection and Resistance» Volume 14

The first study in Saudi Arabia to investigate the resistance of aminoglycosides and sulfonamides carried by plasmids in clinical isolates of Acinetobacter baumannii genotyped by RAPD-PCR: announcing a drug called aac(6ʹ)-SL New allelic variants and three new mutations in the sul1 gene in Acinetobacter(s)

Author El-Badawy MF, Abou-Elazm FI, Omar MS, El-Naggar ME, Maghrabi IA

Published on November 12, 2021, the 2021 volume: 14 pages 4739-4756

DOI https://doi.org/10.2147/IDR.S324707

Single anonymous peer review

Editor who approved for publication: Dr. Héctor M. Mora-Montes

Mohamed F El-Badawy,1 Fatma I Abou-Elazm,2 Mohamed S Omar,3 Mostafa E El-Naggar,4 Ibrahim A Maghrabi5 1 Department of Microbiology and Immunology, School of Pharmacy, Sadat City University, Sadat City , Menofia, 32897, Egypt; 2 Department of Microbiology and Immunology, Faculty of Pharmacy, Misr University of Science and Technology, October 6, Egypt; 3 Department of Chemistry, Faculty of Science, Benha University, Benha, 13508, Egypt; 4 Pharmacology and Department of Toxicology, School of Pharmacy, Sadat City University, Sadat City, Menufia, 32897, Egypt; 5 Department of Clinical Pharmacy, School of Pharmacy, Taif University, Taif, 21974, Saudi Arabia Mailing address: Mohamed F El-Badawy Department of Microbiology and Immunology, School of Pharmacy, Sadat City University, Sadat City, Menofia, 32897 Egypt Phone 20 103-205-9964 Email [email protected] Background: Bowman Acinetobacter (A. baumannii) is one of the most important nosocomial pathogens, which can cause a variety of infections. Purpose: This study aims to investigate the aminoglycoside modifying enzymes (AMEs) encoded by the sul1 gene, 16S rRNA methyltransferase (RMT) and altered dihydropetroleum acid synthase ( DHPS) the presence of plasmid genes. Kingdom of Saudi Arabia (KSA) Taif. Mutations in the aac(6ʹ)-Ib and sul1 genes have also been studied. Methods: The sensitivity of forty clinical isolates of Acinetobacter baumannii to ten antibiotics was studied. In addition to the sul1 gene, plasmid DNA was also extracted and nine genes encoding aminoglycoside resistance were screened. The clonal correlation was determined by random amplified polymorphic DNA (RAPD)-PCR. Mutations in the aac(6ʹ)-Ib and sul1 genes were detected by capillary electrophoresis sequencing (CES). Results: All the isolates were Acinetobacter baumannii, and 42.5% of them showed high levels of aminoglycoside resistance (HLAR). The most common AME and RMT coding genes are aph(3ʹ)-VI, two aac(6ʹ) gene variants [aac(6ʹ)-Ib and aac(6ʹ)-SL], ant(3ʹʹ)-I and armA. 90%, 87.5%, 85% and 45% of the isolates were positive respectively. The aminoglycoside resistance coding genes from other studies, namely aac(3)-II, aac(6ʹ)-II and rmtB, were not detected. Only 15% of isolates contained the sul1 gene. RAPD-PCR divided 40 isolates into three clusters, of which cluster II was the main cluster. DNA sequencing revealed that 34.29% (12/35) of the isolates that tested positive for aac(6ʹ)-Ib were found to have a common missense mutation at position 102, indicating the existence of a new allele named aac(6ʹ)-SL genetic mutation. In addition, DNA sequencing revealed three missense mutations in the sul1 gene. Conclusion: This is the first study in Saudi Arabia to study aminoglycoside and sulfonamide resistance genes carried by plasmids in clinical isolates of Acinetobacter baumannii. In addition to the new mutation in the sul1 gene, a new allelic variant of aac(6ʹ)-Ib was also detected. Keywords: AMEs, armA, RAPD-PCR, Acinetobacter baumannii, 16S rRNA

Acinetobacter baumannii (A. baumannii) is one of the most clinically important non-intestinal gram-negative pathogens, which can cause a wide range of nosocomial infections (NI), especially in frail patients admitted to the intensive care unit (ICU) middle. 1

In the clinical setting, Acinetobacter baumannii is the most pathogenic and common species in the genus Acinetobacter, followed by Acinetobacter calcoacetus (A. calcoacetus) and Acinetobacter lwoffii (A. lwoffii). 2,3

A. baumannii is the only species of Acinetobacter that inherently contains blaOXA-51, which is then used to identify and distinguish A. baumannii from other Acinetobacter species. 4,5

There are multiple drug resistance (MDR), extensive drug resistance (XDR) and pan drug resistance (PDR) patterns in Acinetobacter baumannii because they contain inherent resistance genes, 2 gene mutations and/or horizontal gene transfer (HGT)) Through mobile genetic elements (MGE), such as insert sequence elements (ISE), plasmids and transposons. 6

Due to the limited therapeutic options for the treatment of infections caused by MDR, XDR and PDR strains, the high morbidity and mortality of Acinetobacter baumannii usually occur in the hospital environment1. Therefore, in 2013, MDR Acinetobacter baumannii was called For the Centers for Disease Control and Prevention (CDC) 8 is a “serious threat”, in 2016, carbapenem-resistant Acinetobacter baumannii (CRAB) was listed as “Level I: Key Priority” by the World Health Organization (WHO) Priority Pathogen List Pathogen" .9

Aminoglycosides are broad-spectrum antibiotics, which mainly act by selectively binding to the 30S subunit of bacterial ribosomes and selectively inhibiting bacterial protein synthesis. Destroy the bacterial cell membrane, resulting in the formation of holes in the bacterial cell membrane, leading to the death of the bacterial cell. 11,12

After the emergence of β-lactam and quinolone resistant strains, aminoglycosides have been widely used in clinical settings to eradicate MDR Gram-negative isolates, despite their nephrotoxic and ototoxic complications. 13 According to the aforementioned, aminoglycoside resistant strains have appeared all over the world. 14

The resistance of bacteria to aminoglycosides can be mediated by different mechanisms, such as overexpression of efflux pumps, down-regulation of outer membrane proteins (OMP), ribosomal target modification, and enzyme loss of aminoglycoside modifying enzymes (AME). live. 5

The production of AME is the most important mechanism of aminoglycoside resistance. The spread of AME occurs through MGE. MGE usually has other determinants of resistance to other antibiotics. And easy to spread of aminoglycoside resistant strains in the hospital environment. 15,16

AME is acetylated, phosphorylated and adenylated by aminoglycoside acetyltransferase (ACC), aminoglycoside nucleotide transferase (ANT) and aminoglycoside phosphotransferase (APH) of 2-deoxystrandamine core or sugar moiety- The ability of OH or -NH2, thereby eliminating the activity of aminoglycosides), respectively lead to poor binding of aminoglycosides to bacterial ribosomes, and then unable to function. 18

Aminoglycoside resistance can also be methylated by methyltransferase (MT) such as aminoglycoside-resistant methyltransferase A (armA) 14 and 16S rRNA methyltransferase B (rmtB). Ribosomal target modification is achieved. 13,19

Sulfonamides are the oldest antibacterial agents that have been effectively used to treat bacterial infections since 193220. Because of their structural similarity to P-amino groups, they bind to dihydropteroate synthase (DHPS) to inhibit bacterial folic acid Synthesize 21 to exert antibacterial activity-benzoic acid (PABA), which is used by bacteria for the biosynthesis of folic acid. twenty two

Resistance to sulfa drugs can be chromosome or plasmid mediated. 21 Chromosome-mediated sulfa drug resistance involves mutations in the DHPS gene (called folP gene) or the altered DHPS coding gene (called sul gene 23) obtained through MGE. twenty four

As mentioned above, plasmid-mediated sulfa resistance is achieved through the sul gene, which can be translocated between the plasmid and the chromosome through MGE. 25 Plasmids carrying the sul gene can be transmitted between the same/different bacterial species or different bacterial genera through transformed conjugation. 24

There are three variants of the sul gene, namely: sul1, sul2 and sul3.24. According to reports, the sul1 gene is carried on the large conjugative plasmid and type 1 integron, and the sul2 gene has also been found to be carried on the small non-conjugating plasmid and the large conjugative plasmid. . The 25 sul3 gene was first reported to be carried on a 54-kb conjugative plasmid in E. coli in 2003. 26

Random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) is a rapid molecular technique commonly used in molecular typing and epidemiological tracking, based on the use of randomly combined short oligonucleotides of 8 to 15 nucleotides Utilize primers to distinguish bacterial isolates. To different regions of the entire bacterial genome, many DNA fragments are subsequently amplified, and these fragments migrate to different distances on the agarose gel, resulting in a complex pattern of DNA bands. 27

Due to the lack of published data on the genetic background of clinical isolates of Acinetobacter baumannii in the Kingdom of Saudi Arabia (KSA) that are resistant to aminoglycosides and sulfa drugs, this study used this question to investigate the presence of AMEs and 16S rRNA. The gene encoding the transferase and the altered DHPS plasmid was found in a clinical isolate of Acinetobacter baumannii recovered from a large tertiary healthcare hospital in the western region of Taif, Saudi Arabia. In addition, epidemiological typing using RAPD-PCR was performed to track the spread of the isolates studied between different hospital locations.

The current research is carried out in accordance with the regulations of the Ethics Committee of Taif University in accordance with Ethics Approval No. 43-001, which has been accredited by the National Bioethics Committee (HAO-02-T-105).

The current study is conducted on 40 clinical isolates of Acinetobacter baumannii recovered from patients who were hospitalized or treated in various medical departments in a large tertiary hospital in Taif, Saudi Arabia between October 2016 and May 2017. of. All isolates are recovered from clinical specimens in routine research to the microbiology laboratory as part of routine hospital laboratory procedures. From sputum (n = 20), blood (n = 4), tracheal aspirate (n = 2), urine (n = 4), abdominal fluid (n = 1), wound swab (n = 6) Clinical isolates and catheter tips (n = 3) were recovered from the test. All strains were frozen at -80°C in tryptic soy broth (Scharlau, Spain) containing 15% glycerol for emergency use.

All strains are mainly isolated on blood agar and then purified on Oxoid agar (Oxoid, UK). Through the Vitek 2® system (BioMérieux, France) and API 20NE® (BioMérieux, France), the isolates were temporarily identified at the genus and species level. Species-level molecular confirmation is achieved by the amplification of chromosomal coding genes, namely: blaOXA-51 uses the specific primers previously described (Table 1). 2 Table 1 Primer sequences and cycling conditions used in PCR

Table 1 Primer sequences and cycling conditions used in PCR

The sensitivity of all clinical isolates to ten different antibiotics was tested; gentamicin, amikacin, streptomycin, sulfamethoxazole/trimethoprim, ciprofloxacin, levofloxacin, meropenem, Glycycline, polymyxin, and colistin represent 6 different classes of antibiotics. The sensitivity test was carried out by the broth micro-dilution method as described above, in which Klebsiella pneumoniae (Klebsiella pneumoniae) ATCC 700603 and Pseudomonas aeruginosa (Pseudomonas aeruginosa) 27853 were used as standard control strains . Sensitivity results are interpreted according to the breakpoints of all tested antibiotics of the Clinical Laboratory Standards Institute (CLSI)29, while the breakpoints of streptomycin are interpreted according to Yang et al. 30

When the studied clinical isolates are resistant to three different types of antibiotics except carbapenems, they are considered to have the MDR phenotype, and when the MDR isolates are resistant to meropenem, they are considered to have the XDR table type. If XDR isolates are resistant to colistin and tigecycline, consider the PDR phenotype. 2

According to the definition proposed by Nie et al.,31 Upadhyay et al.32 and Doi et al.33 all isolates with a MIC value of ≥512 µg/mL to gentamicin and amikacin are considered to have high levels of aminoglycoside resistance. Drug (HLAR) phenotype.

Extract the total DNA (chromosome and plasmid DNA) as described above, and use it for PCR reaction, amplification of blaOXA-51 and genotyping of RAPD-PCR technology. The extracted DNA is stored in a sterile DNase-free 0.5 mL tube at -20 °C until use.

Plasmid extraction was performed to study the presence of AME, 16S rRNA methyltransferase encoding gene, and sul1 gene. Each isolate was cultured in 5 mL lysogen broth (LB) (Himedia®, India) to increase plasmid production. Then place the inoculated LB tube in a shaking incubator at 37°C for 16 hours. After that, centrifuge the culture tube at 13,200 rpm for 3 minutes. The bacterial cell pellet is then washed twice with phosphate buffer and any remaining buffer on the pellet is removed. According to the manufacturer's instructions, plasmid DNA was extracted from the washed pellet by GeneJET® Plasmid Miniprep Kit (Thermo Fischer Scientific, USA).

The plasmid DNA from each isolate was used as a template to study the AMEs-encoding genes carried by seven different plasmids: ant(3″)-I, aph(3ʹ)-VI, aac(3)-II, aac(6ʹ) -II, aac(6ʹ)-Ib, aad(2)-Ia, aad(4)-Ia and two 16S rRNA methyltransferase encoding genes namely; armA and rmtB use the specific primers and loops listed in Table 1 Conditions. In addition, the sul1 gene of all isolates was studied.

PCR was performed in a 0.2 DNase-free PCR tube containing 30 μL of the total reaction mixture. The reaction mixture consisted of 6 μL of 5x master mix (Solis BioDyne, Estonia) and 4 μL of DNA template, equivalent to approximately 12-15 ng. 0.9 μL was extracted from each of the forward (10 pmol/μL) and reverse (10 pmol/μL) primers (Table 1).

Complete the volume of the reaction mixture to 30 µL by adding 18.2 µL of sterile DNase-free water. Five strains from the Laboratory Culture Collection provided by the Microbiology Laboratory of Taif University were used as positive controls for the 11 research genes. E. coli ATCC 25922 was used as a negative control strain. For the RAPD-PCR technique, 1.8 μL HLWL74 primer 35 was used.

Amplify the target gene using Mastercycler gradient® (Eppendorf, Germany), oligonucleotide primers (Seoul, Korea) and the cycling conditions listed in Table 1. All amplified PCR products were run on a 1% agarose (Scharlau, Spain) gel containing 500 ng/mL ethidium bromide. Randomly amplified DNA fragments generated by RAPD-PCR are run on a 2.5% agarose gel to achieve band separation.

All PCR products for capillary electrophoresis sequencing (CES) were initially extracted from the agarose gel through a glass fiber membrane using the Gel Extraction SV Kit (MG®, Korea). According to the manufacturer's instructions, the maximum yield was 90%.

A 96-capillary ABI PRISM® 3730XL DNA Analyzer (Applied Biosystems, USA) was used.

All published DNA sequencing ab1 files are manually corrected using FinchTv software 40 version 1.5.0 (Geospiza Inc, USA). 40 Use the blastn service to upload the corrected sequence to the National Center for Biotechnology Information (NCBI) 41, where the query DNA sequence is identified and corrected.

Upload the corrected DNA sequences of aac(6ʹ)-Ib and sul1 genes to the open reading frame (ORF) finder website 42 to obtain the corresponding amino acid sequences using the following search parameters; (i) aac(6ʹ)-Ib and sul1 genes The minimum ORF length of is 300 and 600 respectively; (ii) the genetic code is bacteria and archaea; (iii) the ORF start codon used is only ATG.

Using the blastp service, with sequence identity and query coverage as the selection criteria, in the NCBI database for the corresponding amino acid sequence reported only in Acinetobacter baumannii, the corresponding sequenced aac(6ʹ)-Ib and sul1 genes Search for amino acid sequence. ≥98%, of which AAG33663.1 and WP_063855115 are used as aminoglycoside 6ʹ-N-acetyltransferase-Ib and sulfa-resistant DHPS Sul1 amino acid RefSeq, respectively.

Use Jalview software 43 to use the Fast Fourier Transform (MAFFT) network service to compare the translated query amino acid sequence with multiple sequence alignment (MSA) and the selected reference sequence.

The partial sequences of the mutant aac(6ʹ)-Ib and sul1 genes were submitted to GenBank44 through the submission portal45 on the NCBI website, and each mutant gene was assigned a specific GenBank accession number and a specific protein accession number.

Using BioNumerics® 7.5 software 46 to analyze the amplified DNA fragments generated by RAPD-PCR, among them, the clonal correlation between 40 clinical isolates of Acinetobacter baumannii was based on the generated dendrogram using unweighted paired group method and The arithmetic mean (UPGMA) is determined using the dice coefficient.

This study shows that according to the phenotypic profile obtained from Vitek2 and API 20NE systems, all recovered isolates belong to the genus Acinetobacter, and all isolates have been genetically confirmed to be Acinetobacter baumannii, 100% of which are isolates The chromosome-positive blaOXA-51 gene was detected (Figure 1). Figure 1 PCR product of blaOXA-51 amplified product used for molecular confirmation of Acinetobacter baumannii. L1; 100 bp DNA ladder provided by Solis BioDyne, Estonia; lanes 60-65 and 66-72 represent isolate numbers.

Figure 1 PCR product of blaOXA-51 amplified product used for molecular confirmation of Acinetobacter baumannii. L1; 100 bp DNA ladder provided by Solis BioDyne, Estonia; lanes 60-65 and 66-72 represent isolate numbers.

For tested aminoglycosides, the current study shows that 55% (22/40), 57.5% (23/40) and 95% (38/40) of Acinetobacter baumannii have clinical effects on gentamicin and amidobacteria Carcin and streptomycin resistance, respectively.

Except that all the isolates are sensitive to tigecycline, the drug susceptibility test showed that polymyxin B and colistin are the most effective drugs after tigecycline, with 85% (34/40) and 67.5 respectively. % (27/40) of the isolates are sensitive.

Regarding the other antibiotics tested, meropenem and ciprofloxacin were found to be the least effective drugs, and 97.5% (39/40) of the isolates were resistant to each drug. In addition, current research shows that 70% (28/40) and 77.5% (31/40) of the isolates are resistant to levofloxacin and sulfamethoxazole/trimethoprim, respectively.

Fortunately, none of the isolates showed a PDR pattern, and all of them were at least sensitive to tigecycline or colistin. On the other hand, it was found that 90% (36/40) of the isolates showed an XDR pattern, of which 67.5% (27/40) and 22.5% (9/40) of the isolates had resistance to four and five different antibiotic classes. Resistance, respectively. Only 5% of the isolates showed the MDR pattern, and only two of them were resistant to the three antibiotic classes shown in Tables 2 and 3. Table 2 Sensitivity pattern of clinical isolates of Acinetobacter baumannii

Table 2 Drug susceptibility model of Acinetobacter baumannii clinical isolates

The MIC50 values ​​of the tested aminoglycosides were 16 to 1024 μg/mL, and the MIC50 values ​​of gentamicin, amikacin, and streptomycin were 16, 64, and 1024 μg/mL, respectively. On the other hand, the MIC90 of all tested aminoglycosides was 1024 μg/mL.

It was also found that the MIC50 and MIC90 values ​​of the tested polymyxin were in the range of ≤0.5 to 2 μg/mL and 8 to 32 μg/mL, respectively.

For sulfamethoxazole/trimethoprim, the values ​​of MIC50 and MIC90 were found to be 13.4/256 μg/mL and 53.8/1025 μg/mL, respectively. In addition, the MIC50 and MIC90 values ​​of ciprofloxacin and levofloxacin were significantly increased (Table 2).

The current study showed that 42.5% of the isolates showed HLAR, of which 17 had MIC values ​​for gentamicin and amikacin ≥512 µg/mL, while 52.5% of the isolates showed LLAR, of which 21 had Streptomycin resistance, MIC value <512 µg/mL to gentamicin and amikacin. On the other hand, 5% (2/40) of the isolates were sensitive to all aminoglycosides tested.

Current research shows that aph(3ʹ)-VI and aac(6ʹ)-Ib gene variants [aac(6ʹ)-Ib and aac(6ʹ)-SL] (Figure 2 and Supplementary Figure 1-3) are the most common plasmids Encoding AME, 90% (36/40) and 87.5% (35/40) of the isolates tested positive (Table 3). On the other hand, ant(3″)-I and armA (Figure 2 and Supplementary Figures 4-6) ranked 3rd and 4th among the most common plasmids carrying aminoglycoside resistance coding genes, of which 85% (34 /40) and 45% (18/40) of the isolates were positive respectively. Table 3 Drug resistance and genetic characteristics of clinical isolates of Acinetobacter baumannii Figure 2 PCR products encoded by aminoglycoside and sulfonamide resistance genes 。L1; 100 bp DNA ladder provided by Solis BioDyne, Estonia; lanes 1-5 represent the amplified resistance genes of each PCR product mentioned above.

Table 3 Drug resistance and genetic characteristics of clinical isolates of Acinetobacter baumannii

Figure 2 PCR products of genes encoding aminoglycosides and sulfonamide resistance. L1; 100 bp DNA ladder provided by Solis BioDyne, Estonia; lanes 1-5 represent the amplified resistance genes of each PCR product mentioned above.

The lowest detection gene responsible for aminoglycoside modification by adenylation is aad(4ʹ)-Ia, of which 5% (2/40) of the isolates tested positive. No genes encoding aminoglycoside resistance from other studies were detected, and all isolates tested negative for aad (2ʹʹ)-Ia, aac(6ʹ)-II, aac(3)-II and rmtB.

Regarding the investigation of plasmid-mediated sulfa resistance, the current study showed that 15% (6/40) of the isolates were found to contain the altered DHPS encoding gene; sul1 gene (Figure 2 and Supplementary Figures 7 and 8).

The current study revealed 19 different drug-resistant genotypes among 38 aminoglycoside resistant strains (Table 4), among which the drug-resistant genotype A (blaOXA-51, ant(3″)-I, aph(3ʹ) )-VI, aac(6ʹ)-Ib) are the most common, among which 4-6 kinds of antibiotics are resistant. On the other hand, the resistance genotype B (blaOXA-51, ant(3″)-I, aph(3ʹ )-VI, aac(6ʹ)-SL) and resistance genotype C (blaOXA-51, ant(3″) )-I, aph(3ʹ)-VI, armA, aac(6ʹ)-Ib) are the second A ubiquitous drug-resistant genotype is resistant to 4-6 and 6-9 antibiotics, respectively. Table 4 Comprehensive phenotype and genotype resistance of clinical isolates of aminoglycoside-resistant Acinetobacter baumannii

Table 4 Comprehensive phenotype and genotype resistance of clinical isolates of aminoglycoside-resistant Acinetobacter baumannii

Only 34.29% (12/35) of acc(6ʹ)-Ib positive isolates were found to have a common missense mutation at position 102, in which leucine (hydrophobic) was replaced by serine (neutral polarity) amino acid The substitution position 102 represents a new allelic variant, which is named aac(6ʹ)-SL because there is a serine (S) amino acid in position 102 (L102S) instead of leucine, and it is normal in position 117 Leucine (L) is present (Figure 3). Figure 3 Multiple sequence alignment of the amino acid sequence of 12 mutant aminoglycoside 6'-N-acetyltransferase-Ib [AAC(6')-Ib] encoded by the new aac(6')-SL allelic variant . The code AAG33663.1 represents the accession number of the reference AAC(6')-Ib protein. The code QZB49340-QZB4934051 represents the new login ID assigned by GenBank for the mutant AAC (6')-Ib protein detected in the current study.

Figure 3 Multiple sequence alignment of the amino acid sequence of 12 mutant aminoglycoside 6'-N-acetyltransferase-Ib [AAC(6')-Ib] encoded by the new aac(6')-SL allelic variant . The code AAG33663.1 represents the accession number of the reference AAC(6')-Ib protein. The code QZB49340-QZB4934051 represents the new login ID assigned by GenBank for the mutant AAC (6')-Ib protein detected in the current study.

Sequencing of the sulfa drug-resistant DHPS coding gene; that is, sul1 revealed a common new missense mutation in 66.6% (4/6) of the isolates that tested positive, in which cysteine ​​(neutral polarity) was changed by the 98th Glycine (hydrophobic) substitution at position (C98G). Two other new missense mutations were detected in only one isolate (Acb_96), in which aspartic acid (acidic) replaced asparagine (neutral polarity) at position 40 (D40N), followed by; Alanine (hydrophobic) is replaced by threonine (neutral polarity) in position 233 (A232T), as shown in Figure 4. Figure 4 Multiple sequence alignment of the amino acid sequence of mutant sulfonamide-resistant dihydropteroate synthase (DHPS). The code WP_063855115.1 represents the accession number of the reference protein sequence. The code QZH81639-QZH8163942 represents the new registration number assigned by GenBank for the mutant sulfa-resistant DHPS detected in the current study.

Figure 4 Multiple sequence alignment of the amino acid sequence of mutant sulfa-resistant dihydropteroate synthase (DHPS). The code WP_063855115.1 represents the accession number of the reference protein sequence. The code QZH81639-QZH8163942 represents the new registration number assigned by GenBank for the mutant sulfa-resistant DHPS detected in the current study.

The mutation aac(6ʹ)-Ib gene detected in the current study and named acc(6')SL allelic variant was assigned the following gene accession numbers by GenBank: MZ820065 MZ820066, MZ820067, MZ820071, MZ820072, MZ82802028, MZ820069 , MZ820070, MZ820073 and MZ820074 were used to isolate strain numbers Acb73, Acb75, Acb81, Acb75, Acb80, Acb82, Acb83, Acb90, Acb91, Acb97, Acb100, respectively.

The mutant sul1 gene detected in this study was assigned the following gene accession numbers by Genbank: MZ751055, MZ751056, MZ751057 and MZ751058, which were used to isolate No. Acb62, Acb81, Acb82 and Acb96, respectively.

The amplified fragments generated by RAPD-PCR (Figure 5 and Supplementary Figure 9) were able to divide 40 clinical isolates of Acinetobacter baumannii into three main clusters (Figure 6), of which 92.5% (37/40) isolates They belong to cluster II, and 5% (2/40) of the isolates are related to cluster I, showing the same band profile. Only one isolate is related to cluster III. Figure 5 RAPD-PCR DNA fingerprinting of clinical isolates of Acinetobacter baumannii. L1 and L2 represent 100 bp and 1 kb DNA molecular weight standards, respectively, and they are provided by Solis BioDyne, Estonia. Lanes 94 to 99 and 100 to 110 represent isolate numbers. Figure 6 The clonal correlation between Acinetobacter baumannii clinical isolates is based on the unweighted paired group method constructed by the arithmetic mean (UPGMA) dendrogram of the generated RAPD-PCR band map. The aac(6')-Ib genes detected in isolates Acb_73, Acb_75, Acb_80, Acb_81, Acb_83, Acb_84, Acb_90, Acb_91, Acb_97, Acb_108, Acb_109 are more specifically referred to herein as aac(6')-SL Variant learning. 

Figure 5 RAPD-PCR DNA fingerprinting of clinical isolates of Acinetobacter baumannii. L1 and L2 represent 100 bp and 1 kb DNA molecular weight standards, respectively, and they are provided by Solis BioDyne, Estonia. Lanes 94 to 99 and 100 to 110 represent isolate numbers.

Figure 6 The clonal correlation between Acinetobacter baumannii clinical isolates is based on the unweighted paired group method constructed by the arithmetic mean (UPGMA) dendrogram of the generated RAPD-PCR band map. The aac(6')-Ib genes detected in the isolates Acb_73, Acb_75, Acb_80, Acb_81, Acb_83, Acb_84, Acb_90, Acb_91, Acb_97, Acb_108, Acb_109 are more specifically referred to herein as aac(6')-SL Variant learning. 

Since cluster II is the main cluster related to 37 isolates, it was found that the cluster contained 24 different band characteristics, of which 14 isolates showed 14 different unique characteristics with different RAPD-PCR band patterns. The 23 isolates showed 10 different combination characteristics (including multiple isolates in the same profile) and showed the same specific RAPD-PCR band pattern, for example, isolates Acb_66, Acb_98, Acb_68, Acb_108 are included in A combined profile (Figure 6) shows the same RAPD-PCR band pattern, as well as isolates Acb_110 and Acb_67, Acb_107 and Acb_99, Acb_71 and Acb_73, Acb_90 and Acb_69, Acb_94 and Acb_65, Acb_62 and Acb_63, Acb_70 , Acb_74 and Acb_825, Acb_74 and Acb_825 isolates, showing the same nine specific profiles and the RAPD-PCR band pattern of each Acb_825.

Antibiotic resistance is one of the biggest obstacles facing the health system in most (if not all) countries in the world, because the problem increases the financial burden on the health system because infected patients stay in the hospital for a long time, 34 in particular In the ICU, because patients infected with the MDR bacterial strain usually suffer from life-threatening diseases47, they may need to be admitted to the ICU and placed on a ventilator, leading to increased treatment costs. A. baumannii is one of the most important members of these MDR strains, so the research on A. baumannii is of great significance worldwide, especially in KSA. 2

According to the review of the existing citations and the Saudi review conducted by Yezli et al. 48 and Ibrahim et al. 49 in 2019, there is no report on plasmid-mediated aminoglycoside and sulfonamide resistance in the clinical practice of A. baumannii. The isolate is in the Taif region, so we were prompted to investigate such a subject in an attempt to contribute to the establishment of data on such a problem.

By consulting the cited literature, we found that gentamicin, tobramycin and amikacin were officially introduced into Saudi Arabia in 1975, 1983 and 1984, respectively. 50 According to the data retrieved from the previous literature, it was found that the emergence of gentamicin-resistant strains began to slowly appear in KSA with resistance from low to high levels, as reported by Moaz et al.50 and Memish et al.51. The resistance levels to gentamicin during 1983 and 1984 were 26.6% (254/959) and 31.2% (1930/6189)), which were carried out in clinical isolates of Pseudomonas aeruginosa in Saudi Arabia in 2009, respectively. There is no data on the resistance of Acinetobacter baumannii. 56

It is not surprising that the high incidence of aminoglycoside resistance has been reported in clinical isolates of Acinetobacter baumannii. Recent studies have confirmed the existence of certain inherent aminoglycoside resistance genes on the chromosome of Acinetobacter. For example, in 201752, it was reported that the gene encoding ANT (3″)-II was located on the chromosome of Acinetobacter and was found to be transferred horizontally between Acinetobacter species. Through homologous recombination. According to previous data, AME pairing Intrinsic and acquired resistance of aminoglycosides can easily occur through intrinsic and/or acquired resistance.

The current study shows that 55% of clinical isolates of Acinetobacter baumannii are resistant to gentamicin. This finding is relatively consistent with the results of Haseeb et al.53 and Abdalhamid et al.54, who reported 46% and 54.6 % Of clinical isolates of Acinetobacter baumannii are resistant to gentamicin. The isolates recovered from Mecca and Dammam were resistant to gentamicin, respectively.

Regarding amikacin resistance, our research shows that compared with the recent Saudi study conducted by Almaghrabi et al., the amikacin resistance rate is low, of which 74.5% of clinical isolates of Acinetobacter baumannii are from Saudi Arabia. The Aseer area in the southwest is resistant to amikacin, while only 27.5% of the isolates included in this study are resistant to amikacin, which indicates that the rate of amikacin resistance in the western part of the kingdom is far lower Southwestern region reported at the time of research.

According to the definition proposed by Nie et al.,31 Upadhyay et al.32 and Doi et al.33 considered HLAR in Acinetobacter baumannii when the MIC value for gentamicin and amikacin was ≥512 µg/mL, therefore, This study showed that 42.5% of the isolates showed HLAR, of which 17 isolates had MIC values ​​for gentamicin and/or amikacin ≥512 µg/mL. This finding was lower than that reported by Upadhyay et al. 32. The authors showed that 79.2% of clinical isolates of Acinetobacter baumannii exhibited HLAR.

Since there are few studies on the genetic background of AME in clinical isolates of Acinetobacter baumannii in KSA, in this section, the results of the current study are compared with the results of other studies in countries close to the Kingdom of Saudi Arabia, such as the Gulf countries. , And countries that employ most of the labor force in the Kingdom of Saudi Arabia, such as Egypt, India, and Pakistan.

The current study revealed the high prevalence of genes encoding AME. It was found that all isolates exhibiting HLAR resistance contained at least one plasmid encoding AME and/or 16S rRNA methylase. This finding is consistent with recent findings. The Indian32 study reported that 83.8% of clinical isolates of HLAR A. baumannii contained genes encoding AME and/or 16s methyltransferase.

The molecular study of AME in this study showed that aph (3ʹ)-VI and aac(6ʹ)-Ib are the most common AME-encoding genes, and 90% and 87.50% of the isolates tested positive. A closely related finding is Polotto Et al. 56 reported that aph (3ʹ)-VI and aac(6ʹ)-Ib were the most common AME-encoding genes, which were detected in 55% and 47% of clinical isolates of Acinetobacter baumannii, respectively.

Regarding the prevalence of gene-encoding nucleotide transferases, current research shows that 85%, 5% and none of the isolates were found to contain ant(3′′)-I, aad(4ʹ)-Ia and aad(2ʹ)- Respectively Ia, these findings are highly consistent with those of Nie et al. They reported a high prevalence of ant(3'')-I, of which 95.1% of the isolates tested positive, and all isolates had aad(2ʹ) -Ia and aad(4ʹ)-Ia. These findings are similar to our findings. Our findings indicate that the prevalence of aad(4ʹ)-Ia is low, but we failed to prove aad(2ʹ)-Ia. Based on the aforementioned data, the authors of this study concluded that ant(3'')-I is the most commonly detected variant of the ANT encoding gene in Acinetobacter baumannii.

Contrary to the current research results and the results of Nie et al.31, Jouybari et al.57 recently conducted a study on clinical isolates of Acinetobacter baumannii in Iran and reported a relatively low 33% of ant(3ʹ)-Ia. Prevalence.

Regarding the gene encoding 16S rRNA methylase, only the armA gene was detected in this study, and 45% (18/40) of the isolates tested positive, but rmtB was not detected. The previous Chinese study31 conducted a closely related study, which reported that 59.54% (103/173) of Acinetobacter baumannii clinical isolates tested positive for armA, while no isolates tested positive for rmtB.

Current research shows that, at least in this study, the presence of AMEs encoding genes is not evidence of the incidence of HLAR patterns, among which aph(3ʹ)-VI, aac(6ʹ)-Ib, ant(3')-I, and aad (4ʹ)-Ia detected patterns in 76.19% (16/21), 71.43% (15/21), 90.48% (19/21) and 9.52% (2/21) isolates that did not show HLAR. In addition, 80.95% (17/21) of the isolates that did not exhibit HLAR were found to contain AME encoding 2-4 genes, ensuring that the coexistence of AME is not a condition for the occurrence of HLAR mode.

On the contrary, the current study shows that 94.11% (16/17) of HLAR model isolates were found to contain the armA gene, indicating that there is a close relationship between the presence of the armA gene and the incidence of HLAR. This conclusion is also reported by Doi et al. Made this discovery. 33

In the current study, 19 different genetic aminoglycoside resistance profiles have been detected. In the same context, the Iranian study conducted by Jouybari et al. reported 22 aminoglycoside resistance gene profiles, indicating that clinical isolates of Acinetobacter baumannii from Iran are genetically more diverse than those from Saudi Arabia.

The multiple aminoglycoside genetic profiles of the isolates investigated in the current study indicate that foreign labor recruited from neighboring countries may help to introduce genetically diverse isolates.

In addition to the content mentioned in the previous paragraph, the ease of transmission of AMEs and 16S rRNA methylase encoding genes via plasmids or other MGEs (such as integrons and transposons) is another possibility that can explain the current research The wide diversity of genetic profiles of aminoglycosides detected. All previous possibilities have limited the effectiveness of aminoglycoside antibiotics in the treatment of infections caused by aminoglycoside-resistant strains. Therefore, effective measures should be taken to overcome antibiotics The spread of resistant strains, especially in the hospital environment, is to (i) follow infection control measures, (ii) follow antibiotic management policies, and (iii) avoid unreasonable use of broad-spectrum antibiotics unless they are needed. 58

Since current research shows that ciprofloxacin is the least effective drug and 97.5% of the isolates are resistant to it, the authors decided to sequence the amplified aac(6ʹ)-Ib gene to study the previously reported The aac(6ʹ)-Ib-cr mutation (responsible for the acetylation of ciprofloxacin)59 was the cause of this discovery, but only 34.29% (12/35) of the isolates that tested positive for aac(6ʹ)-Ib were found A new allelic variant is shown, called aac (6ʹ)-SL because leucine (L) is replaced by serine (S) at position 102 (L102S).

The detected aac(6ʹ)-SL variants are considered to be new variants of aac(6ʹ)-Ib, including all previously reported aac(6ʹ)-Ib allelic variants associated with ciprofloxacin resistance 59 The arginine (R) or tryptophan (W) at positions 117 and 102 are different from our findings. Our findings reveal the presence of serine in position 102 and leucine in position 117.

The authors of the current study believe that the newly detected aac(6ʹ)-SL allelic variant may greatly promote 100% ciprofloxacin resistance in isolates that suppress this new allelic variant. It is based on the fact that the wild-type aac(6')-Ib gene that led to the previously reported aac(6')-Ib-cr variant has been shown to acetylate ciprofloxacin, and then ciprofloxacin cannot exert its effects. Effect, leading to ciprofloxacin resistance.

Regarding sulfa resistance, this study showed that although 19.53% (6/31) of the plasmids carried the sul1 gene, 77.5% (31/40) of the study isolates were resistant to sulfamethoxazole/trimethoprim. Sulfamethoxazole/trimethoprim-resistant strains indicate that resistance to sulfa drugs in strains that test negative for the sul1 gene largely contributes to the existence of other determinants of resistance, such as the efflux pump system 60 and/or other sul gene variants, such as sul2 and/or sul3.21

Contrary to the current study showing that 15% of the isolates tested positive for the sul1 gene, according to a recent Iranian study conducted by Tavakol et al., the prevalence of the sul1 gene reported in Acinetobacter baumannii was relatively high 61, he claimed 63.63 % The isolates of Acinetobacter baumannii were found to contain the sul1 gene, indicating that during the study period, the sul1 gene was less prevalent in Acinetobacter baumannii in Saudi Arabia than in Iran.

The RAPD-PCR molecular epidemiological survey showed that 92.5% of the investigated isolates were confirmed to be transmitted in the hospital because they were classified into a cluster with high clonal similarity (cluster II), although these isolates were from different The location of the hospital was recovered in a different location. The approximately 7-month interval from October 19, 2016 to May 14, 2017, confirmed the circulation of these isolates in the hospital environment during the study period.

It is necessary to perform gene cloning and expression experiments on the detected new aac(6ʹ)-SL allelic variants to confirm the role of this new gene variant in ciprofloxacin resistance.

This is the first study in Saudi Arabia to clarify the aminoglycoside resistance of plasmids by screening the plasmid encoding genes of AME and 16S rRNA methyltransferase from clinical isolates of Acinetobacter baumannii. Current research shows that there is a close relationship between the existence of armA genes and the occurrence of HLAR, and the coexistence of one or more AME-encoding genes is not a prerequisite for the occurrence of HLAR mode. aph (3ʹ)-VI and aac(6ʹ)-Ib are the most common AME-encoding genes in the hospital environment, and the armA gene is the only 16S rRNA detected in the clinical isolates of Acinetobacter baumannii that are transmitted in hospitals related to clones RAPD-PCR fingerprinting of the methylase encoding gene. This is the first study to detect the new allelic variant we named aac(6ʹ)-SL and three new mutations in the sul1 gene.

The current work is supported by the project number (TURSP-2020/18) supported by researchers from Taif University, Taif University, Saudi Arabia.

The author declares that there is no conflict of interest in this work.

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