Detection of bla _CTX-M gene for ESBL Production Among  Clinical Isolates of Escherichia coli

 

Sandra Sagar¹, Dr.Gopinath P²

¹BDS 2^nd Year, Saveetha Dental College, Saveetha University, Chennai

²Senior Lecturer, Department Of Microbiology, Saveetha Dental College, Saveetha University, Chennai

 

ABSTRACT:

Widespread spread of CTX-M type extended-spectrum beta-lactamases (ESBL) in E. coli strains which are resistant to extended-spectrum cephalosporins is the most remarkable example for rapid and global spread of plasmid mediated antimicrobial resistance in bacteria. The incessant spread and global prevalence of ESBL resistance poses a serious threat to the human health, as it is responsible for the narrowing of therapeutic options pertaining to infections. A sum of 20 uropathogenic isolates of E. coli were subjected for the antibiotic susceptibility pattern using different antibiotics and followed by the detection of bla _CTX-M gene by PCR. By which, we found increased percentage of isolates were resistant to different antibiotics and 30% of them were having this gene. This indicates the prevalence of such antibiotic resistance gene in our region. Proper antibiotic policies would promote the patients health condition better.

 

 

 

 

INTRODUCTION

Extended Spectrum Beta Lactamase (ESBL)-mediated antibiotic resistance is the rapidly growing mode of resistance observed in most of the clinically relevant Gram-negative pathogens. Very importantly, the TEM and SHV type of ESBLs are known to be wide spread and causing several infections all over the world^.[1] In the beginning of the antibiotic resistance era, there has been a massive distinction in the distribution of ESBLs by the emergence of CTX-M, a new ESBL type. CTX-M was found to be different from other classical TEM and SHV, as this gene originated in a clinically insignificant genus Kluyvera spp., present in the environment^.[2] From its earliest appearance, CTX-M has spread rapidly all across the globe to the extent of even replacing the other ESBL types.

 

Widespread spread of CTX-M type extended-spectrum beta-lactamases (ESBL) in E. coli strains which are resistant to extended-spectrum cephalosporins is the most remarkable example for rapid and global spread of plasmid mediated antimicrobial resistance in bacteria^.[3] The incessant spread and global prevalence of ESBL resistance poses a serious threat to the human health, as it is responsible for the narrowing of therapeutic options pertaining to infections.

 

MATERIALS AND METHODS:

Bacterial Isolates:

A total of 20 non repetitive urinary isolates of E. coli were collected from Saveetha Medical College and Hospitals, Chennai. They were processed for a battery of standard biochemical tests and confirmed. Isolates were preserved in semisolid trypticase soy broth stock and were stored at 4 ºC until further use.

 

Antibiotic Susceptibility Testing

Antibiotic susceptibility test was determined for these isolates to routinely used antibiotics such as ampicillin, amoxicillin, amikacin, norfloxacin, ceftazimide, cefotaxime, ciprofloxacin and gentamicin, imipenem as by Kirby Bauer disc diffusion method ^[4].

 

Detection of bla _CTX-M gene in E. coli:

E.coli isolates were detected for the presence of bla _CTX-M  gene by PCR analysis. Detection of the gene was carried out using primer as depicted in table 2. Bacterial DNA was extracted by boiling lysis method. 1 µL of DNA extract was used as template for PCR reaction. The reaction mixture contained 1mM of Mgcl₂0.2mM dNTP mix and 0.8µM of bla _CTX-M gene with 0.5U of Taq polymerase (New England Biolabs) in a 1x PCR buffered reaction. A positive control of E.coli with bla _CTX-M gene was also included in this study. PCR amplification was carried out using thermal cycler (Eppendorf) with the following cycling condition. Initial denaturation at 95°C for 3 minutes, 30 cycles of denaturation at 95°C for 1 minute, primer annealing at 52°C for 40 seconds and primer extension at 72°C for 1 minute and final extension at 72°C for 5 minutes were used. PCR products were resolved in 1.5% agarose gel. A 100bp ladder was including in all the gel analysis. ^[5]

 

 

RESULTS:

Sample Wise Distribution of Clinical Isolates of E.coli:

Of the 20 clinical isolates of E.coli, 12/20 (60%) were from acute urinary tract infections and 8/20 (40%) were from chronic urinary tract infections. Figure 1 depicts the sample wise distribution of clinical isolates of E.coli.     

 

Figure 1: Sample Wise Distribution of Urinary Isolates of E.coli

 

Antibiotic Susceptibility Testing:

In our isolates, we have found increased percentage 14/20 (70%) of isolates showed sensitivity to amikacin followed by gentamicin, which showed sensitivity of 9/20 (45%). 80- 90% of E.coli isolates showed resistance to cephalosporin group of drugs. 6/20 (30%) were found to be resistant to imipenem. However, we have observed an elevated level of resistance to other routinely used antibiotics. The detailed resistant pattern of E.coli isolates were showed in table 1.

 

Table 1: showing antibiotic sensitivity pattern of E.coli

Antibiotics	Sensitivity (20)(%)	Intermediate (20) (%)	Resistant (20)(%)
Ampicillin	5	0	95
Amoxicillin	5	0	95
Ceftazidime	10	10	80
Cefotaxime	5	5	90
Amikacin	70	10	20
Gentamicin	45	20	35
Norfloxacin	15	15	70
Ciprofloxacin	20	5	75
Imipenem	70	0	30

 

Result of bla _CTX-M gene in E.coli:

Of 20 clinical isolates of E. coli, we have observed 6/20 (30%) isolates were found to harbor bla _CTX-M gene by PCR.

 

L1           L2           L3           L4

 

 

 

Figure 2: Shows The Gel Picture of The Multiplex Pcr of Group Specific Ctx-M

L1-100bp; L3-CTX-M positive; L2 and L4-negative

 

DISCUSSION:

ESBL genes are plasmid mediated which can hydrolyze and inactivate a wide variety of beta-lactam antibiotics. These enzymes are generally found in enterobacteriaceae family. The commonly identified ESBL gene types are TEM and SHV. The TEM and SHV enzymes only confer high resistance to early penicillins and first generation cephalosporins.

 

A study by Farshadzadeh et al., 2014 had reported the presence of ESBL genes in Pseudomonas such as bla_PER and bla_CTX-M for about 1.04% and 54.16% respectively ^[6]. Whereas, the present study had reported 30% of isolates showed the presence of bla_CTX-M. But we did not detect the bla_PER gene in our study. Similarly, the study conducted by Bokeian et al., 2015 have also showed the contrast result to the present study, that is they have reported bla_CTX-M gene in none of the Pseudomonas isolates and the 6.6% of phenotypic ESBL positive Pseudomonas isolates ^[7].

 

Similar kind of study conducted by George and his coworkers from Vellore in 2015 have reported 91.8% of  bla_CTX-M gene positivity by PCR, which is inconstant with our results as we found only very lesser positivity.

Another study^[8] from south India reported 79.4% of positive nosocomial isolates positive for CTX-M genes by phenotypic method, of which 63.7% were E. coli and all were positive for the CTX-M-1 group. These reports document evidence of the high prevalence of CTX-M type ESBL in almost all parts of India and highlight the escalating antimicrobial resistance and the likely switch over to CTX-M-15 type ESBL in India.

 

CONCLUSION:

In our study, we conclude that, bla_CTX-M  gene is one of the important gene present among other ESBL genes in E-Coli. This indicates the judicial usage of antibiotics and following the antibiotic policies to overcome the spread of resistant threat among other clinical pathogens. This would help the clinicians to accelerate the patient’s health status with better outcome.

 

ACKNOWLEDGMENT:

We thank Dr. Kalyani, Professor and Head of the Department of Microbiology, Saveetha Medical College, Chennai for kindly providing the clinical isolates to carry out our research work fruitfully.

 

REFERENCES:

1.        Paterson DL, Bonomo RA. Extended spectrum beta-lactamases: A clinical update. Clin Microbiol Rev.18; 2005 : 657-86.

2.        Bonnet R. Growing group of extended spectrum beta-lactamases: The CTX-M enzymes. Antimicrob Agents Chemother. 48; 2004:1-14.

3.        Ibrahim O, Ahamed A, El-Hady M, Ahmediman Z, Ahmed. Detection of bla SHV and bla CTX-M genes in ESBL producing Klebshiella pneumonia isolated from the Egyptian patients with suspected noscomial infections; 14(3); 2013:277-83.

4.        Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Disk Tests; Approved Standards; Document M2-A9, 9^th ed., Vol 26. Wayne, PA: CLSI; 2015.

5.        George EA, Sankar S, Jesudasan MV, Sudandiradoss C, Nandagopal B, Molecular characterization of CTX-M type Extended Spectrum Beta Lactamase producing E-Coli isolated from humans and the environment. Indian. J. Med. Microbiology. 33(Supplement I); 2015: S73-79.

6.        Farshadzaeh Z, Khosaravi A D,Alavi SM, Parhizgari N, Hoveizavi H, Spread of Extended Spectrum Beta Lactamase genes of bla OTX-10,blaPER-1 and blaCTZ-M in Pseudomonas aueroginosa strains isolated from burn patients. Burns, 40(8);2015:1575-80.

7.        Bokaeian M, Zahedani SS, Bajgiran MS, Moghaddam AA, Frequency of PER,VEB,SHV,TEM and CTX-M genes in Resistant strains of Pseudomonas aerigonosa producing Extended spectrum Beta Lactamases; Jundishapur. J. Microbiology, 8(1); 2015.e13783.

8.        Mohamudha Parveen R, Manivannan S, Harish BN, Parija SC. Study of CTX-M type of Extended Spectrum β-lactamase among Nosocomial Isolates of Escherichia coli and Klebsiella pneumoniae in South India. Indian J Microbiol .52;2015:35-40.

 

 

Accepted on 15.07.2016           © RJPT All right reserved

Research J. Pharm. and Tech 2016; 9(10):1593-1595.