Class 1,2 Integron Genes Distribution in Pseudomonas aeruginosa Isolated from Clinical Specimens.

 

Haider Qassim Raheem1, Ehasn F. Hussein2, Ahmed Hameed Rasheed 3

1,3DNA Research Center, University of Babylon, Hilla, Babylon, Iraq.

2College of Medicine of Hamorabi, University of Babylon, Hilla, Babylon, Iraq.

*Corresponding Author E-mail: haiderbio412@gmail.com, ehsan.f.hussein@gmail.com, alhly612@gmail.com

 

ABSTRACT:

Pseudomonas aeruginosa, a Gram-negative human pathogen, P. aeruginosa is lone of the furthermost common hospital pathogens also is a chief concern, particularly in immune-compromised patients. The purpose of this study was to identify phenotypic and genotypic antibiotic resistance in Pseudomonas aeruginosa isolated from wound infection The bacterial isolates (30) were obtained from patients admitted to Mirjan Medical City in Babylon, Iraq (burns, wound unit). was identified biochemically and morphologically, and the isolates were subjected to standard bacteriological culturing processes on blood and MacConkey agar plates for 24-48 hours at 37oC for isolation and purification, Viteck 2 compact system confirmed the isolates and antibacterial sensitivity as well. These findings revealed that P.aeruginosa has a high rate of penicillin resistance, with a resistance rate of (100percent ) isolates. Advanced resistance to cephalosporin antibiotics was also found in resistant isolates of Cefoxitin, Ceftriaxone (75%), Ceftazidime, and cefepime (85%). For carbapenem antibiotics, had a high resistance rate (90percent). Aminoglycosides have variable resistance to Amikacin (60percent), Gentamicin (70 percent), and tobramycine resistant (90%). The findings revealed that all Pseudomonas aeruginosa isolates tested positive for Class 1,2 Integron resistance genes. with positive results (35%) for Int1 and 35% for Int2 (25%). This study found that P.aeruginosa has a high rate of resistance to Penicillins, Cephalosporin, Carbapenem and Aminoglycosides antibiotics.

 

KEYWORDS: Pseudomonas aeruginosa, Antibiotic, Integron, and Clinical isolates.

 

 


INTRODUCTION:

Pseudomonas aeruginosa, a Gram-negative human pathogen, the pathogen is one of the most common nosocomial pathogens, It reasons infections in a variety of body part, including the (respiratory, gastrointestinal, and urinary tracts), as well as skin infections.1 This organism is extremely resistant to harsh conditions and can survive in a variety of environments, including hospitals, on therapeutic apparatus for example automated vents, catheters, then in bowls contamination occurs as a result of the stability of these environments.2

 

P.aureginosa devised a number of mechanisms in order to survive antimicrobial resistance is a term used to describe a person's resistance to antimicrobial, the most important B-lactam resistance mechanism is the production of B -lactamase enzymes. Resistance, on the other hand, can be caused by overexpression of efflux systems, (Penicillin-binding proteins) thru little attraction for B-lactams are synthesized, as is membrane permeability, all of these mechanisms can coexist or be isolated in P. aeruginosa.3 One of the most intriguing aspects of P. aeruginosa is how easily it canister develop resistance toward several antibiotics. Alterations, novel gene achievements, up-regulation of countenance of current genes or loss of them, may wholly give to these strains' extensive range resistance. Penicillin, cephalosporin, aminoglycoside, quinolone, and tetracycline are examples of antibiotics used to treat P.aeruginosa infections.4 Integrons are transportable bacterial genetic elements that act as rally points for capturing genes and then joining site-specific recombination mechanisms into exogenous and promoterless open reading frames (ORFs) to convert them into functional genes.5 The term integron was coined to describe a community of seemingly transportable elements, which are usually linked with transposons and conjugative plasmids, confirming their distribution across bacterial species.6,7 Integrons act to production an significant character in antibiotic resistance transformation since they container bind, incorporate, in addition to prompt gene cassettes that encode resistance to antibiotic.8.9

 

MATERIALS AND METHODS:

Isolation and Characterization:

The bacterial isolates (30) were obtained from patients admitted to Mirjan Medical City in Babylon, Iraq (burns and wound unit). was recognized biochemically and morphologically, and the isolates were exposed for isolation and purification to typical bacteriological culturing processes on blood and MacConkey agar plates for 24-48 hours at 37oC.Viteck 2 compact system confirmed the isolates and antibacterial sensitivity as well (Biomrieux).10.11

 

MOLECULAR METHODS:

DNA Extraction and Amplifications:

Genomic DNA was extracted from bacteria using the Favor Prep TM Genomic DNA Mini Kit in accordance with the industrialist's (Favorgen/Taiwan) protocols. The extracted DNA was stored at temperatures ranging from 2 to 8 degrees Celsius. Primers for oligonucleotides were used in training. Traditional PCR thermocyclers used the primer to detect antibiotic resistance. These primers were provided by Alpha Business, Canada, as shown in table (1).

 

Table (1): Primers used in this training.

Genes

Sequence

Ref.

Int1

F 5'-GGTGTGGCGGGCTTCGTG-3'

R 5'-GCATCCTCGGTTTTCTGG-3'

12

Int2

F5'-CACGGATATGCGACAAAAAGGT-3'

R5-'GTAGCAAACGAGTGACGAAATG-3'

 

RESULTS:

Vitek 2 System Antibiotic Profile of P. aeruginosa Isolates:

Thirty P.aeruginosa isolates with antibacterial sensitivity calculated using the Vitek 2 method demonstrated diverse designs of resistance to various antibiotics, with the highest resistance to Penicillins: Piperacillin/Tazobactam, Ticarcillin/Clavulanic Acid, and Ampicillin/Sulbactam with a resistance rate of (100 percent) isolates. Advanced resistance to cephalosporin antibiotics was discovered with cefoxitin, ceftriaxone (75percent), Ceftazidime and cefepime (85%) were used to treat resistant isolates. For carbapenem antibiotics, imipenem and meropenem isolates had a high resistance rate (90percent). Aminoglycosides have variable resistance to Amikacin (60percent). Gentamicin-resistant bacteria (70percent). Isolates that are tobramycine resistant (90%) as illustrated in the table (2). The current study's findings are consistent with, as P.aeruginosa isolates showed high resistance to Cefotaxime (81percent), Cefotaxime (78percent), Piperacillin (76percent), and Ciprofloxacin and Tobramycin (74percent), (72percent). Amikacin and Meropenem were both resistant to both of them (70percent). In another study, P. aeruginosa was isolated from burn swabs in four Baghdad hospitals in a percentage of (31.46percent), and the isolates were 100percent resistant to Cefotaxime, Cephalothin, Gentamycin, and Trimethoprim, while 55percent were recorded for Amikacin and 88 percent for Ciprofloxacin13,14,15


Table (2): Resistance of P. aeruginosa Isolates to Antibiotics by the Vitek 2 System.

Interpretation

MIC

Antibiotic

Susceptible

Intermediate

Resistance

(0%)

0

(100%)

=<64

Ticarcillin/Clavulanic Acid

(0%)

0

(100%)

=<64

Piperacillin/Tazobactam

(0%)

0

(100%)

=<32

Ampicillin/Sulbactam

(15%)

0

(85%)

=<64

Ceftazidime

(25%)

0

75%)

=<64

Ceftriaxone

(20%)

0

80%))

=<32

Cefepime

25%))

0

(75%)

=<64

Cefazolin

(10%)

0

(90%)

=<16

Meropenem

(10%)

0

(90%)

=<16

Imipenem

(15%)

(25%)

(60%)

=<32

Amikacin

(30 %)

0

(70 %)

=<16

Gentamicin

(10%)

0

(90%)

=<16

Tobramycin

 


Molecular Detection of Class 1 and 2 Integron Genes in P.aeruginosa:

In order to identify Class 1,2 Integron resistance genes, two sets of primers were tested in each isolate. Figures depict the results (1 and 2). The study also discovered that integron of class 1,2 was significantly associated with antibiotic resistance, including aminoglycosides, fluoroquinolones, and betalactam compounds. Other studies at Iran's Tabriz's Imam Reza hospital found a measurable percentage of (9), Two P.aeruginosa isolates were found to have a positive MDR pattern. The cla ss 1 integron was found in 74 (92.5percent) of MDR isolates. This study did not uncover the Class2 integron gene. In other studies, class 1integron was found in Acinetobacter baumannii isolates from nine hospitals in Turkey.16The Int1 gene was found in 38.7 percent of the total isolates, but no Int2 integron was found.

 

 

Figure (1): Gel electrophoresis for the product Int1gene (amplified size of 457 bp) using a P.aeruginosa isolate DNA template (1.5 agarose, 70 volts for 60-120min).

 

Figure (2): Gel electrophoresis for the product Int2gene (amplified size of 789bp) using a P.aeruginosa isolate DNA template (1.5 agarose, 70 volts for 60-120min).

 

DISCUSSION:

Because of the presence of this antibiotic-resistant bacteria in hospitalized patients' wounds and burns, wounds and burns can become contaminated with P. aeruginosa bacteria It can be found in tap water, handrails, and toilets, as well as hospital care workers, and thus the bacteria spreads from one patient to the next. Antibiotic resistance may be caused by the coordinated action of multi-drug, efflux pumps thru antibiotic resistance genes then decreased penetrability of bacterial cell covers. Apart from self-resistance, P.aeruginosa can easily acquire resistance through chromosomal mutations in genes encoded by chromosomes or through gene transfer for determinants of resistance. Resistance to aminoglycosides is caused by bacteria P.aeruginosa producing modified enzymes such as Phosphotransferase and N-acetyl-transferase. The plasmidor chromosome contains the genes for these enzymes.17,18 Carbapenems (imipenem), cephalosporins (like ceftazidime and cefepime), aminoglycosides (tobramycin and amikacin), and fluoroquinolones (e.g., ciprofloxacin and levofloxacin) are commonly used as the initial treatment until culture and antibiotic sensitivity testing results are completed in the laboratory. However, due to the appearance of bacterial strains resistant to antibiotic, controlling on it and completing patient treatment became difficult.19

 

Integrons are transportable genetic components of bacteria act as rally points for capturing genes and then joining site-specific recombination mechanisms into exogenous and promoter less open reading frames (ORFs) to convert them into functional genes.5The term integron was coined to describe a community of seemingly transportable elements, which are usually linked with transposons and conjugative plasmids, confirming their distribution across bacterial species.6 Integrons act to production an significant character in antibiotic resistance transformation since they container bind, incorporate, in addition to prompt gene cassettes that encode resistance to antibiotic.7

 

Class 1 integrons are the most prevalent in clinical Gram-negative isolate surveys, accounting for 22% to 59% of all isolates. These have only recently begun to appear in (Gm +ve) bacteria. In UPEC, class 1 and 2 integrons were specified, and they were linked to the carriage of multidrug resistance (MDR).20

 

Nonetheless, these negative integron isolates were antibiotic resistant. Antibiotic resistance genes might have been acquired from these isolates via chromosomal encoded enzymes or other mobile components. The majority of the positive integrin isolates were resistant to aminoglycosides. It may be explained by the fact that many of the gene cassettes that confer aminoglycoside resistance are conceded on class1,2.21 The study also found a high rate of resistance to various beta-lacta m compounds in integron-positive strains, implying that the carriage of several al beta-lactamase encoding genes by Class 1,2 integrom and carbapenemase OXA-type Metallo-beta-lactamase enzymes may have resulted in carbapaenem resistance. The findings revealed no link between the presence of the Class1,2 integron and fluoroquinolone resistance, Perhaps because fluoroquinolone resistance is caused by chromosomal point mutations.22,23

 

CONCLUSION:

This study found that P.aeruginosa has a high rate of resistance to penicillins, with a resistance rate of (100%) isolates. Advanced resistance to cephalosporin antibiotics was also found in resistant isolates of Cefoxitin, Ceftriaxone (75percent), Ceftazidime, and cefepime (85percent). For carbapenem antibiotics, imipenem and meropenem had the highest resistance rate (90%) among isolates. Aminoglycosides have variable resistance to Amikacin (60percent), Gentamicin-resistant bacteria (70percent), Isolates that are tobramycine resistant (90%) The findings revealed that all 20 P.aeruginosa isolates tested positive for Class 1,2 Integron resistance genes. with positive results (35% for Int1 and 35% for Int 2) (25 percent).

 

CONFLICT OF INTEREST:

No conflict of interest.

 

ACKNOWLEDGEMENTS:

I am grateful to the staff of the DNA Research Center/University of Babylon.for providing the completely necessary aptitudes required for fruitful work accomplishment of the current work.

 

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Received on 27.09.2021 Modified on 10.11.2021

Accepted on 14.12.2021 RJPT All right reserved

Research J. Pharm. and Tech. 2022; 15(7):3165-3168.

DOI: 10.52711/0974-360X.2022.00529