Detection of blaSHV-1 for ESBL production among clinical strains of Escherichia coli

 

Kayalvili Sanmugam¹, Dr. Gopinath P²

¹BDS  2nd year, Saveetha Dental College, Saveetha University, Chennai.

² Senior  Lecturer, Department of Microbiology, Saveetha Dental College, Saveetha University, Chennai.

 

ABSTRACT:

Plasmid-mediated β-lactamase gene is SHV-1 (sulfhydryl variable), which is chromosomally encoded in the majority of isolates of K. pneumoniae but is usually plasmid-mediated in E. coli. Over the years, many new β-lactam antibiotics have been developed; however, with each new class of antibiotic, a new β-lactamase emerged that caused resistance to that particular class of antibiotic. Presumably, the selective pressure imposed by the use and overuse of new antibiotics in the treatment of patients has resulted in the emergence for new variants of β-lactamase enzymes. A sum of 20 urinary isolates of E. coli were subjected to antibiotic susceptibility testing and detected for bla_SHV-1 gene by PCR. We found increased percentage of isolates were shown to be resistant to different antibiotics and 70% of them were resistant to imipenem drug. 20% of them were harboring bla_SHV-1 gene by PCR. Since ESBL-positive isolates show false susceptibility to expanded-spectrum cephalosporins in standard phenotypic test, it is difficult to relay on it. Specific detection methods recommended by CLSI have to be adopted. Those isolates need to be confirmed by multiplex PCR with sets of resistance genes for ESBLs are recommended where the availability and resources are available for prompt and accurate therapy.

 

 

 

 

 

 

 

 

INTRODUCTION:

Resistant bacteria are emerging as a threat to favorable outcome in the treatment of common infections in community and hospital settings globally^.[1] Among the wide array of antibiotics, β-lactams are the most extensively used. The most common cause of resistance to β-lactam antibiotics is the production of β-lactamases. Emergence of resistance to β-lactam antibiotics started even before the first β-lactam, penicillin had been developed. The first plasmid-mediated β-lactamase TEM-1 was isolated from blood culture of a patient named Temoniera in Greece, in the early 1960s.^[2] TEM-1 are plasmid and transposon mediated resistant gene has facilitated its spread to other species of bacteria.

Another common plasmid-mediated β-lactamase gene is SHV-1 (sulfhydryl variable), which is chromosomally encoded in the majority of isolates of K. pneumoniae but is usually plasmid-mediated in E. coli. Over the years, many new β-lactam antibiotics have been developed; however, with each new class of antibiotic, a new β-lactamase emerged that caused resistance to that particular class of antibiotic. Presumably, the selective pressure imposed by the use and overuse of new antibiotics in the treatment of patients has resulted in the emergence for new variants of β-lactamase enzymes.

 

The introduction of third-generation cephalosporins into clinical practice in the early 1980s was considered as a major breakthrough to combat with β-lactamase-mediated bacterial resistance to antibiotics. Soon after the introduction, the first report of plasmid-encoded β-lactamase capable of hydrolyzing the extended-spectrum cephalosporins was published in 1983 from Germany.^[3] Hence these new β-lactamases were termed as extended-spectrum β-lactamases (ESBLs). So far, the total number of ESBLs characterized exceeds 200 types. There is no consensus on the precise definition of ESBLs. A commonly used working definition is that the ESBLs are β-lactamases capable of conferring bacterial resistance to the penicillins; first, second and third generation cephalosporins; and aztreonam (but not the cephamycins and carbapenems) by hydrolysis of these antibiotics and which are inhibited by β-lactamase inhibitors such as clavulanic acid, tazobactam.^[4] With this background, we have undertaken this study to detect for blaSHV-1 gene by PCR.

 

MATERIALS AND METHODS:

Bacterial isolates:

A total of 20 non repetitive urinary isolates of Escherichia 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.^[5]

 

Detection of bla_SHV-1 gene in E.coli:

Escherichia coli isolates were detected for the presence of bla_SHV-1 gene by PCR analysis. Detection of the gene was carried out using primer as depicted in table 1. 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_SHV-1 gene with 0.5U of Taq polymerase (New England Biolabs) in a 1x PCR buffered reaction. A positive control of E.coli with bla_SHV-1 gene was also included in this study. PCR amplification was carried out using thermal cycler (Eppendorf) with the following cycling condition. Initial denaturation atm  98^oC for 6 min and 35 cycles for 30s, 70^oC for 30s and 69^o C for 60s, followed by a final extension of 5 min at 75^oC. PCR products were resolved in 1.5% agarose gel. A 100bp ladder was including in all the gel analysis.^[6]

 

Table 1: Gene sequencing of blaSHV-1  gene

Primer	Primer sequence	Product size
blaSHV-1	CTGGGAAACGGAACTGAAT CGATTTGCTGATTTCGCTC	341 bp

 

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 is shown in table 2.

 

Table 2: 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_SHV-1 gene in E.coli:

4/20 (20%) clinical isolate of urinary isolates of E.coli was found to harbour bla_SHV-1  gene.

	L1     L2    L3    L4    L5   L6     L7
			341bp

 

 

 

L2-100bp ladder; L3,L4- bla_SHV-1 gene.

Figure 2: Representative gel picture showing presence of bla_SHV-1 gene.

DISCUSSION:

ESBLs are encoded by transferable conjugative plasmids which often code resistant determinants to other antibiotics. The plasmid-mediated resistance against cephalosporins can disseminate among related and unrelated gram-negative bacteria. ESBLs are mostly the products of point mutations at the active site of TEM and SHV enzymes.^[7] Nosocomial outbreaks of infections caused by ESBL-producing gram negative bacteria have also been reported in several regions, which are mainly the result of extensive and inappropriate use of third-generation cephalosporins. Majority of ESBL-producing organisms are E. coli and K. pneumoniae. Minorly include Enterobacter spp., Salmonella spp., Morganella, Proteus mirabilis, Serratia marcescens, and Pseudomonas aeruginosa. The major risk factors implicated to get such resistance strains are long-term exposure to antibiotics, prolonged ICU stay, nursing home residency, severe illness, instrumentation, or catheterization.^[8]

 

In the present study, we have observed increased percentage of resistance to penicillin (95%) group of drugs followed by cephalosporins (80-90%), fluroquinolones (70-75%). Better sensitivity was observed to imipenem 70% against our E. coli isolates. The marked resistance showed against cephalosporins was analyzed by detecting the blaSHV-1 by PCR. Of the 20 urinary isolates of E. coli, 4/20 (20%) of them were showing positive for this gene.

 

Previous studies from India have reported ESBL production ranging from 6% - 87%.^[9,10] One reason for such variability may be the very low number of samples studied. In our study also we have used very less isolates. In recent years, a significant increase in ESBL producers was reported from USA,^[11] Canada,^[12] China,^[13] and Italy.^[14] A recent large survey of 1610 Escherichia coli and 785 Klebsiella pneumoniae isolates from 31 centers in 10 European countries found that the prevalence of ESBL in these organisms ranged from as low as 1.5% in Germany to as high as 39-47% in Russia, Poland, and Turkey.^[15]

 

In 2009, a community-based study demonstrated that the most prevalent ESBL gene was bla_SHV-1 among ESBL-producing Enterobacteriaceae.^[16] Also, bla_SHV-1 is known to be a predominant genotype in other conducted studies,^[17] whereas in our study bla_SHV-1 had the lowest frequency among the identified types. Study conducted by Kargar and coworkers in 2014, reported 29.4% of E.coli isolates were positive for bla_SHV-1 gene,^[18] similar to that of his study we also found 20% of positivity.

 

CONCLUSION:

Since ESBL-positive isolates show false susceptibility to expanded-spectrum cephalosporins in standard phenotypic test, it is difficult to relay on it. Specific detection methods recommended by CLSI have to be adopted. Those isolates need to be confirmed by multiplex PCR with sets of resistance genes for ESBLs are recommended where the availability and resources are available for prompt and accurate therapy.

 

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.

 

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17.     Shahcheraghi F, Moezi H, Feizabadi MM. Distribution of TEM and SHV beta-lactamase genes among Klebsiella pneumoniae strains isolated from patients in Tehran. Med Sci Monit 2007;13:BR247-50

18.     Kargar M, Jahromi MZ , Najafi A , Ghorbani-Dalini S. Molecular detection of ESBLs production and antibiotic resistance patterns in Gram negative bacilli isolated from urinary tract infections. Indian J Path Microbiol. 2014: 57(2);244-248.

 

 

Accepted on 20.07.2016        © RJPT All right reserved

Research J. Pharm. and Tech 2016; 9(9):1447-1450.