Development of a caseinase assay for PCR independent detection of Esp gene carriage among Enterococci

Dada Ayokunle Christopher, Asmat Ahmad, Yook Heng Lee, Gires Usup

Research output: Chapter in Book/Report/Conference proceedingConference contribution

Abstract

Currently, there is no known relationship between caseinase and carriage of esp gene. Also, no breakpoints exist for phenotypic assays that are used to infer virulence characteristics among Enterococci. In the present study, caseinase activity was measured by a radial diffusion assay for 113 enterococci isolates. A standard curve with predictive r2 value of 0.939 was produced by dispensing several doubling dilutions of proteinase K into 3% skimmed milk agar wells. Caseinase activity for all tested enterococci was subsequently converted into proteinase K activity, using the obtained chart. Caseinase activity ranged from 1.74 x 10-8 to 4.47 x 10 -7ug/ml and 6.37 x 10-8 to 8.82 x 10-8 ug/ml per colony of environmental and clinical enterocococci tested, proportionate to proteinase K activity. Caseinase activity among environmental strains was five-fold higher than was observed among clinical strains. Fishers exact test revealed significant associations between esp gene carriage and caseinase activity (diameter on skimmed milk, z=8 to 13mm) at p>0.1. However, the probability of association was strongest at z=13 mm (p=0.033) suggesting a range of diameter cutoffs that was exclusive to and may be used to predict the presence of environmental enterococci strains harbouring esp gene. Results obtained from sensitivity analysis showed increasing assay sensitivity from cut-off of 9 mm (61.54%) up to 84.62% (13 mm). Specificity of the caseinase assay slightly decreased from 50% to 42.86% as cut-off increased from 9 to 13 mm. The caseinase assay described here potentially proves useful in preliminary PCR independent screening of environmental enterococci isolates for the detection of strains which carry the esp gene known to increase the severity of enterococcal infections.

Original languageEnglish
Title of host publicationAIP Conference Proceedings
Pages202-207
Number of pages6
Volume1571
DOIs
Publication statusPublished - 2013
Event2013 UKM Faculty of Science and Technology Post-Graduate Colloquium - Selangor
Duration: 3 Jul 20134 Jul 2013

Other

Other2013 UKM Faculty of Science and Technology Post-Graduate Colloquium
CitySelangor
Period3/7/134/7/13

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carriages
genes
cut-off
milk
virulence
infectious diseases
sensitivity analysis
charts
dilution
screening
sensitivity
curves

Keywords

  • Caseinase assay
  • Esp gene detection
  • PCR independent Enterococci

ASJC Scopus subject areas

  • Physics and Astronomy(all)

Cite this

Development of a caseinase assay for PCR independent detection of Esp gene carriage among Enterococci. / Christopher, Dada Ayokunle; Ahmad, Asmat; Lee, Yook Heng; Usup, Gires.

AIP Conference Proceedings. Vol. 1571 2013. p. 202-207.

Research output: Chapter in Book/Report/Conference proceedingConference contribution

Christopher, DA, Ahmad, A, Lee, YH & Usup, G 2013, Development of a caseinase assay for PCR independent detection of Esp gene carriage among Enterococci. in AIP Conference Proceedings. vol. 1571, pp. 202-207, 2013 UKM Faculty of Science and Technology Post-Graduate Colloquium, Selangor, 3/7/13. https://doi.org/10.1063/1.4858655
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abstract = "Currently, there is no known relationship between caseinase and carriage of esp gene. Also, no breakpoints exist for phenotypic assays that are used to infer virulence characteristics among Enterococci. In the present study, caseinase activity was measured by a radial diffusion assay for 113 enterococci isolates. A standard curve with predictive r2 value of 0.939 was produced by dispensing several doubling dilutions of proteinase K into 3{\%} skimmed milk agar wells. Caseinase activity for all tested enterococci was subsequently converted into proteinase K activity, using the obtained chart. Caseinase activity ranged from 1.74 x 10-8 to 4.47 x 10 -7ug/ml and 6.37 x 10-8 to 8.82 x 10-8 ug/ml per colony of environmental and clinical enterocococci tested, proportionate to proteinase K activity. Caseinase activity among environmental strains was five-fold higher than was observed among clinical strains. Fishers exact test revealed significant associations between esp gene carriage and caseinase activity (diameter on skimmed milk, z=8 to 13mm) at p>0.1. However, the probability of association was strongest at z=13 mm (p=0.033) suggesting a range of diameter cutoffs that was exclusive to and may be used to predict the presence of environmental enterococci strains harbouring esp gene. Results obtained from sensitivity analysis showed increasing assay sensitivity from cut-off of 9 mm (61.54{\%}) up to 84.62{\%} (13 mm). Specificity of the caseinase assay slightly decreased from 50{\%} to 42.86{\%} as cut-off increased from 9 to 13 mm. The caseinase assay described here potentially proves useful in preliminary PCR independent screening of environmental enterococci isolates for the detection of strains which carry the esp gene known to increase the severity of enterococcal infections.",
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AB - Currently, there is no known relationship between caseinase and carriage of esp gene. Also, no breakpoints exist for phenotypic assays that are used to infer virulence characteristics among Enterococci. In the present study, caseinase activity was measured by a radial diffusion assay for 113 enterococci isolates. A standard curve with predictive r2 value of 0.939 was produced by dispensing several doubling dilutions of proteinase K into 3% skimmed milk agar wells. Caseinase activity for all tested enterococci was subsequently converted into proteinase K activity, using the obtained chart. Caseinase activity ranged from 1.74 x 10-8 to 4.47 x 10 -7ug/ml and 6.37 x 10-8 to 8.82 x 10-8 ug/ml per colony of environmental and clinical enterocococci tested, proportionate to proteinase K activity. Caseinase activity among environmental strains was five-fold higher than was observed among clinical strains. Fishers exact test revealed significant associations between esp gene carriage and caseinase activity (diameter on skimmed milk, z=8 to 13mm) at p>0.1. However, the probability of association was strongest at z=13 mm (p=0.033) suggesting a range of diameter cutoffs that was exclusive to and may be used to predict the presence of environmental enterococci strains harbouring esp gene. Results obtained from sensitivity analysis showed increasing assay sensitivity from cut-off of 9 mm (61.54%) up to 84.62% (13 mm). Specificity of the caseinase assay slightly decreased from 50% to 42.86% as cut-off increased from 9 to 13 mm. The caseinase assay described here potentially proves useful in preliminary PCR independent screening of environmental enterococci isolates for the detection of strains which carry the esp gene known to increase the severity of enterococcal infections.

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