Detection of gyrA Mutation Among Clinical Isolates of Campylobacter jejuni Isolated in Egypt by MAMA-PCR

Mayar Maged Said,Salwa F Ahmed,John D Klena,Marshall R Monteville,Fawkia M El-Beih,Tharwat F Ismail,David M Rockabrand,Mohamed S Salama,Hanan El-Mohamady

doi:10.3855/jidc.963

Mayar Maged Said, Salwa F Ahmed + Show 7 more

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Campylobacter spp are the major cause of enteritis in humans and more than 90% of reported infections are caused by Campylobacter jejuni. Fluoroquinolones such as ciprofloxacin are the antibiotics of choice for treatment. An increase in the frequency of ciprofloxacin-resistant Campylobacter has been reported globally due to a single base mutation (C-257 to T) in codon 86 of the quinolone resistance determining region (QRDR) of the gyrA gene altering the amino acid sequence from threonine at position 86 to isoleucine (Thr-86 to Ile). Campylobacter spp (n = 118) were selected from a collection of Egyptian isolates spanning 1998 to 2005. The presence of C. jejuni gyrA gene was confirmed in each isolate by a PCR assay amplifying 368 bp portion of the gyrA gene. C to T alteration was detected by the mismatch amplification mutation assay MAMA PCR. The MIC of nalidixic acid (NA) and ciprofloxacin (CIP) was determined by E-test. C. jejuni gyrA gene was detected in 100 of the Campylobacter spp studied; the other 18 isolates were found to be Campylobacter coli by lpxA PCR. The mutation was detected in 89 C. jejuni resistant isolates with MIC values (NA; 8 - >256 μg/ml) and (CIP; 4 - >32 μg/ml). The other 11 sensitive C. jejuni isolates with MIC values (NA; 0.38 - 3 µg/ml) and (CIP; 0.03 - 0.125 µg/ml) were not amplified by the MAMA primers. There was 100% congruence with MAMA PCR, MIC results and gyrA gene sequence analysis. In Egypt the main mechanism for resistance to fluoroquinolones is an alteration in the gyrA QRDR. MAMA PCR provides an economical and rapid means for screening fluoroquinolone resistance.

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Campylobacter spp are the major cause of enteritis in humans and more than 90% of reported infections are caused by Campylobacter jejuni
We examined the genetic background of the isolates using pulsed field gel electrophoresis to determine whether C. jejuni fluoroquinolone resistance was due to clonal expansion of numerous introductions into genetically diverse strains
Fluoroquinolone-resistance of C. jejuni isolates determined by minimum inhibitory concentration (MIC) and MAMA PCR results are congruent

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Campylobacter spp are the major cause of enteritis in humans and more than 90% of reported infections are caused by Campylobacter jejuni. A single nucleotide transition (C-257 to T) in the quinolone resistance determining region (QRDR) of the C. jejuni and C. coli gyrA alters the amino acid sequence of GyrA at position 86 from threonine to isoleucine (Thr-86 to Ile); this alteration is always associated with high MIC values for fluoroquinolones [6]. Various methods have been used to detect the gyrA C-257 to T transition, including denaturing gradient gel electrophoresis, [7] DNA sequencing of the target gene, [6] restriction fragment length polymorphism, Said et al - Egypt Campylobacter gyrA mutations [8] non-radioisotopic single-strand confirmatory polymorphism and a fluorogenic PCR assay [9] These methods are time-consuming, may require multiple steps, and often involve expensive equipment or reagents limiting their use in routine surveillance studies. Introduction of a second mismatch immediately adjacent to the initial mismatch abolishes amplification of any DNA template [10,11]

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