Molecular analysis of CTX-M genes among ESBL producing in Pseudomonas aeru-ginosa isolated from clinical samples by Multiplex-PCR

Abstract

Introduction: Pseudomonas aeruginosa is one of the most common pathogens involved in hospital infections, which today has been considered for resistance to a wide range of antibiotics. The causes of drug resistance in P.aeruginosa isolates are the production of broad-spectrum beta-lactamase enzymes. The aim of this study was to determine the frequency of CTX-M1, CTX-M2, CTX-M3 genes in P.aeruginosa isolated from Hospitals and health centers in Marand city, East Azarbijan. Methods: Pseudomonas aeruginosa isolates were collected from different samples of patients referring to hospi-tals and clinics of Marand city during one year from October 2016 to October 2017 and after determining the phe-notypic identity and antibiogram test, the double-disk test of ESBLs phenotype was evaluated in P.aeruginosa bac-teria. Then bacterial DNA was extracted and evaluated by PCR method using specific primers for CTX-M1,CTX-M2 and CTX-M3 genes. Results: The findings showed of the 85 Pseudomonas aeruginosa bacteria, the highest resistance was to amikacin antibiotics (82.35%), nalidixic acid (80%) and ceftazidime (72.94%), and the highest susceptibility was observed to ampicillin(38.82%) and cefotaxime (38.82%) %). There was no significant relationship between age, sex, and P.ae-ruginosa infections (P> 0.05). The highest gene frequencies of ESBLs are related to the genes of CTX-M1 (29 samples), CTX-M2 (24 samples) and CTX-M3 (11 samples), respectively. Conclusion: The studied genes in this research were all on the chromosome of P.aeruginosa bacteria. Therefore, further examination of ESBL genes such as the CTX-M1 gene seems necessary to control this bacterium.
Keywords: P.aeruginosa, Extended-spectrum beta-lactamases, CTX-M1, CTX-M2, CTX-M3.

Full text

Molecular analysis of CTX-M genes among ESBL producing in Pseudomonas aeru-
ginosa isolated from clinical samples by Multiplex-PCR
Received: Jan 01, 2018; Revised: Feb. 18, 2018; Accepted: Feb. 20, 2018
HOZAN; a Scientific Journal of Environmental Sciences
https://sites.google.com/site/hozanjournalcom/Home
PISSN: 2476-3764; eISSN: 2476-5716
Original article
Abstract
Introduction: Pseudomonas aeruginosa is one of the most common pathogens involved in hospital infections,
which today has been considered for resistance to a wide range of antibiotics. The causes of drug resistance in
P.aeruginosa isolates are the production of broad-spectrum beta-lactamase enzymes. The aim of this study was to
determine the frequency of CTX-M1, CTX-M2, CTX-M3 genes in P.aeruginosa isolated from Hospitals and health
centers in Marand city, East Azarbijan.
Methods: Pseudomonas aeruginosa isolates were collected from different samples of patients referring to hospi-
tals and clinics of Marand city during one year from October 2016 to October 2017 and after determining the phe-
notypic identity and antibiogram test, the double-disk test of ESBLs phenotype was evaluated in P.aeruginosa bac-
teria. Then bacterial DNA was extracted and evaluated by PCR method using specific primers for CTX-M1,CTX-
M2 and CTX-M3 genes.
Results: The findings showed of the 85 Pseudomonas aeruginosa bacteria, the highest resistance was to amikacin
antibiotics (82.35%), nalidixic acid (80%) and ceftazidime (72.94%), and the highest susceptibility was observed to
ampicillin(38.82%) and cefotaxime (38.82%) %). There was no significant relationship between age, sex, and P.ae-
ruginosa infections (P> 0.05). The highest gene frequencies of ESBLs are related to the genes of CTX-M1 (29
samples), CTX-M2 (24 samples) and CTX-M3 (11 samples), respectively.
Conclusion: The studied genes in this research were all on the chromosome of P.aeruginosa bacteria. Therefore,
further examination of ESBL genes such as the CTX-M1 gene seems necessary to control this bacterium.
Keywords: P.aeruginosa, Extended-spectrum beta-lactamases, CTX-M1, CTX-M2, CTX-M3.
This article may be cited as: Jafari-Sales A, Shadi-Dizaji A. Molecular analysis of CTX-M
genes among ESBL producing in Pseudomonas aeruginosa isolated from clinical samples by Mul-
tiplex-PCR. HOZAN J Environment Sci; 2018:2(5):17-29.
Free of Charge
Abolfazl Jafari-Sales *1, Azizeh Shadi-Dizaji 2
1. Department of Microbiology, Kazeroon branch, Islamic Azad University, Kazeroon, Iran.
2. Department of Biotechnology, Ataturk University, Turkey.
(*Corresponding Author: a.jafari_1392@yahoo.com ); Tel: +98(0)914-7611841; Fax: +98(0)414-2274746

2018, vol. 2 (no. 5): page 17-29 HOZAN J Environment Sci
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 a.jafari_1392@yahoo.com
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CTX-M1 CTX-M2CTX-M3
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CTX-M124CTX-M2CTX-M3

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ESBLCTX-M1

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CTX-M1, CTX-M2, CTX-M3


CTX-M

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

   
       
 
     

  
 

 



 -








    

 -




  AmpC 
OprD
   
IIIV

   





     
 




     
ESBLESBL
 
        




CTX-M

 -
  Kluyvera


CTX-M
CTX-M



CTX-M




    







2018, vol. 2 (no. 5): page 17-29 HOZAN J Environment Sci
  
 
 S
2
H
   
DNaseO-F

TSBTryptic soy broth 
Kirby Baur
NCCLS



 



ESBL
  ESBL

      Invitek Stratec
Business    DNA 

CTX-M1
CTX-M2CTX-M3     
PCR
Multiplex PCRMultiplex –PCR
µL PCR µL
dNTPµL  µLTag polymeraseµL
MgCl2µLDNA

mg/mL
 Gel Document USA, Bio Rad 

SPSS
>p

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


±




p >
      



   p>
ESBL

-




CTX-M








ESBL

   
CTX-M1CTX-M2
CTX-
M3 
ESBLESBL

     
ESBL   
ESBL
     -
<p






PCRM: DNA
Ladder 1: CTX-M12: CTX-M23: CTX-M3CTX-M1
54%
46%
 
0
10
20
30
40
50
60


   





2018, vol. 2 (no. 5): page 17-29 HOZAN J Environment Sci








MDR


  
 
   
 

MDR 
    

MDR-


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   
    
   
    
 
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

Wesam



  Rajat Rakesh 
    
Kianpour

    Ahadi

       



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

 -
JemimaVerghese
ESBL
        
ESBLPCR
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

ESBL

ESBL
  
    
0
20
40
60
80
100
CTXM1 CTXM2 CTXM3


ESBL + ESBL -


CTX-M




ESBL
   

       

ESBL


       


     


ESBL



ESBL




ESBL


       VEB,OXA-
10,CTX-M,PER-1,GES-1,OXA-1,OXA-4

ESBLCTX-M
     
CTX-M1
CTX-M1  
CTX-M2  CTX-M3 


CTX-M
 CTX-M1  CTX-M2
CTX-M3
CTX-M1CTX-M2CTX-
M3

ESBL
 CTX-M2
ESBL
 CTX-M1  




CTX-M


      


   



  


-







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
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