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ORIGINAL ARTICLE Table of Contents   
Year : 2010  |  Volume : 53  |  Issue : 3  |  Page : 509-512
Novel restriction enzyme SSiI for the detection of mutation in GyrA gene of Salmonella enterica serovar Typhi


1 Department of Microbiology, Armed Forces Medical College, Pune - 411 040, India
2 Department of Pathology, Armed Forces Medical College, Pune - 411 040, India

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Date of Web Publication22-Oct-2010
 

   Abstract 

Aim: Enteric fever is an ongoing problem in the developing nations. Resistance and reduced susceptibility to ciprofloxacin narrows the therapeutic options in enteric fever. The present study was carried out with the objective of determining molecular basis of resistance to fluoroquinolone among the clinical isolates of Salmonella enterica serovar Typhi from different parts of India. Materials and Methods: A total of 60 S.Typhi clinical isolates were subjected to antimicrobial susceptibility testing and determination of minimum inhibitory concentration (MIC) to ciprofloxacin and nalidixic acid. Polymerase chain reaction (PCR) for GyrA gene followed by restriction fragment length polymorphism (RFLP) with restriction enzyme (RE) SSiI was performed to detect mutation at position Ser83. Further confirmation of mutation was done by nucleotide sequencing of GyrA gene. Results: Isolates showed 100% sensitivity to first-line drugs ampicillin, chloramphenicol, and cotrimoxazole. Twelve of the 60 isolates (18%) were susceptible to nalidixic acid (NASST) and the remaining 48 (82%) were resistant to nalidixic acid (NARST). Of these 48 NARST strains, 46 (97.5%) had reduced susceptibility to ciprofloxacin (MIC 0.25-1.0 μg/mL), whereas 2 strains (2.75%) were resistant to ciprofloxacin (MIC 4.0 μg/mL). In RFLP analysis, all the NASST strains showed 3 fragments, whereas all the NARST strains showed 2 fragments due to the loss of 1 restriction site as a result of mutation. All the NARST strains with reduced susceptibility to ciprofloxacin (n = 46) had a single mutation in gyrA gene (Ser 83→Tyr or Ser 83→Phe), whereas double mutations (Ser 83→Phe and Asp 87→Asn) were found in each of the 2 ciprofloxacin-resistant strains. None of the NASST strains (n = 12) revealed any mutation. Conclusion: Our study exemplifies the correlation between nalidixic acid screening test, MIC values, and the detection of mutation in GyrA gene by PCR-RFLP with a novel RE SSiI.This was further confirmed by nucleotide sequencing.

Keywords: Ciprofloxacin resistance, gyrA gene, restriction enzyme SSiI, S. Typhi

How to cite this article:
Agrawal P, Kapila K, Kumar S, Ghosh A N, Maurya AK. Novel restriction enzyme SSiI for the detection of mutation in GyrA gene of Salmonella enterica serovar Typhi. Indian J Pathol Microbiol 2010;53:509-12

How to cite this URL:
Agrawal P, Kapila K, Kumar S, Ghosh A N, Maurya AK. Novel restriction enzyme SSiI for the detection of mutation in GyrA gene of Salmonella enterica serovar Typhi. Indian J Pathol Microbiol [serial online] 2010 [cited 2018 Apr 22];53:509-12. Available from: http://www.ijpmonline.org/text.asp?2010/53/3/509/68299



   Introduction Top


Typhoid fever continues to be a global health problem with an estimated 16 million new cases and 600,000 deaths each year worldwide. [1] The disease is endemic in many developing countries due to poor sanitary conditions, particularly in the Indian subcontinent.  Salmonella More Details enterica serovar Typhi is a highly adapted human-specific pathogen, which has evolved remarkable mechanisms for persistence in its host that help to ensure its survival and transmission. [2]

Multidrug-resistant S.Typhi has been prevalent in India and the neighboring countries since 1989. [3] These strains were resistant to 3 or more antibiotics, such as ampicillin, chloramphenicol, cotrimoxazole, and tetracycline. [4],[5]

Ciprofloxacin has been considered as the drug of choice for the treatment of multidrug-resistant typhoid fever cases for more than a decade. [6] However, since 1995, ciprofloxacin-resistant strains have emerged not only in the Indian subcontinent but globally because of the irrational use of the antibiotic; treatment failures have been reported. [7],[8] In the recent past, there is a remarkable reversal in the resistance pattern of S. Typhi isolates reported from various places in India. [9] Over the years, a significant decrease in resistance to ampicillin, chloramphenicol, and cotrimoxazole has been noticed among S. Typhi isolates. [10]

The primary target of activity of quinolones in Gram-negative bacteria is bacterial DNA gyrase. The DNA gyrase consists of 2 subunits gyrA and gyrB encoded by its respective genes. [6],[11] A single-point mutation in the quinolone resistance determining region (QRDR) of gyrA gene can mediate resistance to the nonfluorinated quinolone nalidixic acid and reduced susceptibility to fluoroquinolones, such as ciprofloxacin (a minimum inhibitory concentration [MIC] of 0.25 μg/mL). [12] In contrast to nalidixic acid-resistant (NARST) strains that select single-step resistant mutants at a high frequency, fluoroquinolone-resistant mutants are not easily selected. Therefore, resistance to fluoroquinolones has been slow to develop. [13] Thus, NARST in vitro acts as an indicator of low level resistance to ciprofloxacin in vivo and forms the basis of nalidixic acid screening test among the S. Typhi isolates. [13],[14]

Resistance mutations of gyrA gene have been clustered in a region of the gene product between amino acids 67 and 122 termed the QRDR. Most frequently observed mutations in NARST strains of S. Typhi in QRDR of gyrA gene are at Ser83 to Phe, Tyr, or Ala and at Asp87 to Gly, Asn, or Tyr. [15]

In India, very few studies have been conducted up to the molecular level of fluoroquinolone resistance. One study from a North Indian hospital has documented mutations at Ser83 and Asp87 of the gyrA gene in NARST S. Typhi isolates, but all these isolates were from one place. [16] In the present study, we have included S. Typhi isolates from different places in India and studied mutations in the gyrA gene responsible for decreased susceptibility to ciprofloxacin.


   Materials and Methods Top


Bacterial Strains

A total of 60 consecutive nonrepeat clinical isolates of Salmonella enterica serovar Typhi were included in the study. All were isolated and identified by conventional microbiological procedures, such as culture, biochemical reactions, and serology, from clinically suspected cases of enteric fever. These isolates were from different geographical regions of India. Armed Forces Medical College is a referral centre for the characterization of Salmonella spp. for various hospitals spread all over the country. Isolates were collected over a period of 1 year (Jan 2006 to Dec 2006).

Antibiotic Susceptibility Tests

Kirby-Bauer disk diffusion method was used to perform antibiotic susceptibility testing for 6 antibiotics-ampicillin (10 μg), chloramphenicol (30 μg), trimethoprim-sulfamethoxazole (1.25-23.75 μg), nalidixic acid (30 μg), ciprofloxacin (5 μg), and ceftriaxone (30 μg). The inhibition zone diameters were measured and interpreted according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. [17] Quality control strain used for the antibiotic susceptibility testing was  Escherichia More Details coli ATCC 25922.

Minimum Inhibitory Concentration

Agar dilution method was used to determine the MIC of nalidixic acid and ciprofloxacin for all the isolates according to the CLSI guidelines. [17] The 2-fold dilutions used for nalidixic acid ranged from 256 to 1.0 μg/mL, whereas for ciprofloxacin it was from 4.0 to 0.016 μg/mL.

Polymerase Chain Reaction

DNA extraction from S. enterica serovar Typhi colonies was performed using the QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany), according to the protocol provided by the kit manufacturer. Extracted DNA was subjected to amplification by polymerase chain reaction (PCR) with primers specific for the QRDR of gyrA as described by Griggs et al. [11] Amplification was performed in a thermal cycler (PerkinElmer) by using the following protocol: Initial denaturation step at 95 C for 5 min, followed by 30 cycles of 1 min of denaturation at 95 C, 1 min of annealing at 55 C, and 1 min of extension at 72 C, with a final extension step of 5 min at 72 C. PCR products were resolved on 1% agarose gel with ethidium bromide dye and visualized under a UV transilluminator.

Restriction Fragment Length Polymorphism

PCR products were purified using QIAquick spin PCR purification kit (QIAGEN). Restriction enzyme (RE) SSiI was used to detect the mutation in GyrA gene at position Ser83. This RE has 2 recognition sites in the amplicon of GyrA gene. Digestion products were resolved on 3% agarose gel with Tris-borate-ethylenediaminetetraacetic acid buffer and ethidium bromide dye. Undigested control was run alongside.

Nucleotide Sequencing

Purified PCR products were subjected to sequencing using ABI Prism 3110 genetic analyzer by Big-Dye terminator cycle sequencing kit (Applied Biosystems).

Submission of Nucleotide Sequences

Sixty sequences of gyrA gene were submitted to GenBank nucleotide sequence database, Bethesda, MD, USA. Sixty accession numbers received are from EF176609 to EF176668.


   Results Top


Antibiotic susceptibility testing by Kirby-Bauer disk diffusion method showed 100% sensitivity to ampicillin, chloramphenicol, cotrimoxazole, and ceftriaxone. Isolates were divided into 3 phenotypic groups on the basis of nalidixic acid and ciprofloxacin susceptibility: Group A (n = 12)-sensitive to both nalidixic acid and ciprofloxacin (nal s cip s ), Group B (n = 46)-resistant to nalidixic acid but sensitive to ciprofloxacin (nal r cip s ), and Group C (n = 2)-resistant to both nalidixic acid and ciprofloxacin (nal r cip r ). Results of MIC of nalidixic acid and ciprofloxacin for 3 phenotypic groups are given in [Table 1]. PCR products visualized on UV transilluminator revealed amplicon size 347 bp [Figure 1].
Table 1: Results of MIC for 3 phenotypic groups

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Figure 1: Amplicons of gyrA gene in Lane 3– 12 corresponding to 347 bp, Lane 1 showing a 100 bp molecular weight marker

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RE digestion products of GyrA gene with SSiI revealed different types of patterns for mutant and nonmutant strains. In the mutant strains, 2 fragments were observed corresponding to 237 and 110 bp, whereas in nonmutant strains, 3 fragments were observed corresponding to 137, 110, and 100 bp. However, the bands of 100 and 110 bp were very close and appeared as a single band [Figure 2].
Figure 2: Restricti on fragment length polymorphism patt erns of gyrA gene amplicons aft er digesti on with SSiI. Lane 1: 50 bp molecular weight marker, Lane 2: undigested control, Lane3– 5 and 7– 9 showing 2 fragments in case of mutant strains and Lane 6 showing 3 fragments of nonmutant strain

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Nucleotide sequencing of GyrA gene revealed no mutation in 12 strains of Group A, single-point mutation (Ser 83→Tyr or Ser 83→Phe) in 46 strains of Group B, and double mutation (Ser 83→Phe and Asp 87→Asn) in each of the 2 strains of Group C (GenBank accession numbers EF176617 and EF176653).


   Discussion Top


Enteric fever is a global public health problem, especially in the developing countries. Proper sanitation, public health education, and vaccination are the long-term preventive measures that would improve this situation. Antibiotic resistant bacteria have emerged due to inappropriate use of antibiotics in treating the infectious diseases.

In the present study, antibiogram of 60 S. Typhi isolates showed 100% sensitivity to first-line drugs ampicillin, chloramphenicol, and cotrimoxazole. Results of nalidixic acid screening and MIC findings were in concordance with NARST strains showing MIC for ciprofloxacin up to 1.0 μg/mL.

RFLP with RE SSiI has successfully detected all the S. enterica serovar Typhi strains having mutation in GyrA gene at position Ser83. Out of the 2 recognition sites (C^CGC) for SSiI, one corresponds with the codon for Ser83 (TC^C) and any change in the nucleotide sequence at this site (eg, TC C→TA C or TC C→TT C coding for tyrosine or phenylalanine, respectively) will result in the loss of one recognition site. Thus, different types of restriction patterns will be produced by mutant and nonmutant strains. Further confirmation of the mutations by nucleotide sequencing showed results in agreement with the RFLP finding.

The RFLP reaction standardized in this study can be easily performed in all clinical microbiology laboratories having basic facilities for molecular techniques, whereas other methods of mutation detection, such as single-strand conformational polymorphism and nucleotide sequencing are more complicated and can only be performed in reference laboratories.

It is already known that mutations in the GyrA gene are mainly responsible for the ciprofloxacin resistance among S. enterica serovar Typhi isolates. [15] In the present study, we have found single mutation in the gyrA gene (Ser 83→Tyr or Ser 83→Phe) responsible for decreased susceptibility to ciprofloxacin (MIC ≤ 1 μg/mL), whereas double mutation leading to ciprofloxacin-resistant isolates (MIC ≥ 4 μg/mL). The substitution of Ser83 with a hydrophobic amino acid has been shown to reduce the level of binding of fluoroquinolones to the gyrase-DNA complex. [13]

All the strains without any mutation in gyrA gene belonged to phenotypic Group A (nal s cip s ), with single mutation belonged to phenotypic Group B (nal r cip s ), and with double mutation belonged to phenotypic Group C (nal r cip r ).

 
   References Top

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4.Anand AC, Kataria VK, Singh W, Chatterjee SK Epidemic multiresistant enteric fever in eastern India. Lancet 1990;335:352.  Back to cited text no. 4      
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6.Giraud E, Brisabois A, Martel JL, Chaslus-Dancla E. Comparative studies of mutations in animal isolates and experimental In vitro and In vivo selected mutants of Salmonella spp. Suggest a counter selection of highly fluoroquinolone resistant strains in the field. Antimicrob Agents Chemother 1999;43:2131-7.  Back to cited text no. 6  [PUBMED]  [FULLTEXT]  
7.Joshi S, Wattal C, Sharma A, Oberoi JK, Prasad KJ. Quinolones - Drug of choice for enteric fever? Indian J Med Microbiol 2004;22:271-2.   Back to cited text no. 7  [PUBMED]  Medknow Journal  
8.Rowe B, Ward LR, Threlfall EJ. Ciprofloxacin-resistant Salmonella Typhi in the UK. Lancet 1995;346:1302.  Back to cited text no. 8  [PUBMED]  [FULLTEXT]  
9.Lakshmi V, Ashok R, Susmita J, Shailaja VV. Changing trends in the antibiograms of Salmonella isolates at a tertiary care hospital in Hyderabad. Indian J Med Microbiol 2006;24:45-8.   Back to cited text no. 9  [PUBMED]  Medknow Journal  
10.Dutta S, Sur D, Manna B, Bhattacharya SK, Deen JL, Clemens JD. Rollback of Salmonella enterica serotype Typhi resistance to chloramphenicol and other antimicrobials in Kolkata, India. Antimicrob Agents Chemother 2005;49:1662-3.  Back to cited text no. 10  [PUBMED]  [FULLTEXT]  
11.Griggs DJ, Gensberg K, Piddock LJ. Mutations in gyrA gene of Quinolone-Resistant Salmonella serotypes isolated from humans and animals. Antimicrob Agents Chemother 1996;40:1009-13.  Back to cited text no. 11  [PUBMED]  [FULLTEXT]  
12.Piddock LJ, Ricci V, McLaren I, Griggs DJ. Role of mutation in the gyrA and parC genes of nalidixic- acid- resistant Salmonella serotypes isolated from animals in the United Kingdom. J Antimicrob Chemother 1998;41:635-41.  Back to cited text no. 12  [PUBMED]  [FULLTEXT]  
13.Hakanen A, Kotilainen P, Jalava J, Siitonen A, Huovinen P. Detection of decreased fluoroquinolone susceptibility in Salmonellas and validation of nalidixic acid screening test. J Clin Microbiol 1999;37:3572-7.  Back to cited text no. 13  [PUBMED]  [FULLTEXT]  
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15.Hirose K, Hashimoto A, Tamura K, Kawamura Y, Ezaki T, Sagara H, et al. DNA sequence analysis of DNA Gyrase and DNA Topoisomerase IV quinolone resistance determining regions of Salmonella enterica serovar Typhi and serovar Paratyphi A. Antimicrob Agents Chemother 2002;46:3249-52.  Back to cited text no. 15  [PUBMED]  [FULLTEXT]  
16.Renuka K, Kapil A, Kabra SK, Wig N, Das BK, Prasad VV, et al. Reduced susceptibility to ciprofloxacin and GyrA gene mutation in North Indian strains of Salmonella enterica serotype Typhi and serotype Paratyphi A. Microb Drug Resist 2004;10:146-9.  Back to cited text no. 16  [PUBMED]  [FULLTEXT]  
17.Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing; Sixteenth Informational Supplement. 9th ed. Wane, Pa: Clinical and Laboratory Standards Institute; 2006. p. M100-S16.  Back to cited text no. 17      

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Correspondence Address:
Parul Agrawal
Department of Microbiology, Armed Forces Medical College, Pune - 411 040, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0377-4929.68299

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