Rapid detection of gyrA and parC mutations in quinolone-resistant Salmonella enterica using Pyrosequencing® technology
Fluoroquinolones are broad-spectrum antimicrobials highly effective in the treatment of a wide variety of clinical infections. Salmonella gastroenteritis is usually only treated with fluoroquinolones when the patient is elderly or immunocompromised. Fluoroquinolones are also used for the treatment o...
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Published in | Journal of microbiological methods Vol. 68; no. 1; pp. 163 - 171 |
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Main Authors | , , |
Format | Journal Article |
Language | English |
Published |
Shannon
Elsevier B.V
2007
Elsevier Science |
Subjects | |
Online Access | Get full text |
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Summary: | Fluoroquinolones are broad-spectrum antimicrobials highly effective in the treatment of a wide variety of clinical infections. Salmonella gastroenteritis is usually only treated with fluoroquinolones when the patient is elderly or immunocompromised. Fluoroquinolones are also used for the treatment of systemic
Salmonella infection or for long-term salmonella carriage. Resistance to quinolones is commonly mediated by point mutations within the topoisomerase genes
gyrA and
parC. Pyrosequencing® technology is a DNA sequencing method using ‘sequencing by synthesis’ and is suitable for the rapid detection of single nucleotide polymorphisms (SNPs).
One hundred and ten
Salmonella enterica isolates, representing 18 different serotypes, were used in this study. One hundred and four isolates had ciprofloxacin MICs of 0.25–32 μg/mL; the remaining six were ciprofloxacin-sensitive (ciprofloxacin MIC
≤
0.125 μg/mL). PCR amplification of the quinolone resistance-determining regions of
gyrA and
parC was performed and the resulting amplicons subjected to a Pyrosequencing protocol using cyclic dispensation of nucleotides.
Amino acid substitutions at S83 and D87 were detected in
gyrA and at T57 and S80 in
parC. Silent mutations were also identified at G81 of
gyrA and at V67, H75 and H77 of
parC.
This is the first report of a Pyrosequencing assay being used to detect mutations in genes conferring quinolone resistance. The Pyrosequencing technology is a rapid and reliable alternative to current methods for identification of
gyrA and
parC mutations in
S. enterica. |
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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
ISSN: | 0167-7012 1872-8359 |
DOI: | 10.1016/j.mimet.2006.07.006 |