Identification of appropriate reference genes for qPCR studies in Staphylococcus pseudintermedius and preliminary assessment of icaA gene expression in biofilm-embedded bacteria

Abstract

BackgroundQuantitative PCR is rapidly becoming the standard method for analyzing gene expression in a wide variety of biological samples however it can suffer from significant error if stably expressed reference genes are not identified on which to base the analysis. Suitable reference genes for qPCR experiments on Staphylococcus pseudintermedius have yet to be identified.ResultsThree reference genes in S. pseudintermedius were identified and validated from a set of eight potential genes (proC, gyrB, rplD, rho, rpoA, ftsZ, recA, sodA). Two strains of S. pseudintermedius were used, and primer specificity and efficiency were confirmed and measured. Ranking of the genes with respect to expression stability revealed gyrB, rho and recA as the best reference genes. This combination was used to quantify expression of a single biofilm associated gene, icaA, in logarithmic, stationary and biofilm growth phases, revealing that expression was significantly upregulated in the biofilm growth phase in both strains.ConclusionThree reference genes, gyrB, rho and recA, were identified and validated for use as reference genes for quantitative PCR experiments in S. pseudintermedius. Also, the biofilm associated gene icaA was shown to be significantly upregulated in biofilm samples, consistent with its role in biofilm production.

Comparative RNA-seq-Based Transcriptome Analysis of the Virulence Characteristics of Methicillin-Resistant and -Susceptible Staphylococcus pseudintermedius Strains Isolated from Small Animals

“…MRSP isolates are able to produce biofilm, and MRSP ST71 isolates, in particular, are better biofilm producers than other MRSP clones (5,6). The icaA gene can be significantly upregulated in biofilm samples, suggesting a role in the biofilm production by S. pseudintermedius (7). The ability to form biofilm may play an important role in the pathophysiology of bacterial infections and can be related to survival and persistence of S. pseudintermedius, namely, MRSP, in the environment (5,6).…”

The metalloprotease SepA governs processing of accumulation‐associated protein and shapes intercellular adhesive surface properties in Staphylococcus epidermidis

“…Relative quantification of gene expression was performed on a Light Cycler 480 (Roche Applied Science, Mannheim, Germany). The staphylococcal housekeeping genes gyrB, rho and tpiA, coding for DNA gyrase subunit B, transcription termination factor rho and triosephosphate isomerase tpiA, respectively, were used as internal reference genes (Theis et al, 2007;Crawford et al, 2014;Sihto et al, 2014). The TaqMan Fast Advanced Master Mix 23 (Thermo Fisher Scientific) was used according to the manufacturer's instructions, duplicates of the reaction were set up manually in a reaction volume of 15 ml.…”

Biofilm‐Associated Gene Expression in Staphylococcus pseudintermedius on a Variety of Implant Materials

“…Primers available for reference genes and for icaA from a previous study were used . Gene sequences for icaB , icaC , icaD , spsE , ebpS , mecA , atl , agrA , and agrB were examined in S. epidermidis and S. aureus in previous studies, and analogous sequences were identified in S. pseudintermedius for evaluation in this study.…”

“…Primers available for reference genes and for icaA from a previous study were used. 25 Gene sequences for icaB, icaC, icaD, spsE, ebpS, mecA, atl, agrA, and agrB were examined in S. epidermidis and S. aureus in previous studies, 12,13,15,[26][27][28][29] and analogous sequences were identified in S. pseudintermedius for evaluation in this study. Primers were designed (Table S1) using a combination of GeneRunner software version 3.05 (Hasting Software, Inc, Hasting, NY) and the National Centre for Biotechnology Information online primer designing tool (http://www.ncbi.nlm.nih.gov/tools/ primer-blast/) using gene sequences for 2 strains of S. pseudintermedius (strains ED99 and HKU10-03) available from Genbank (http://www.ncbi.nlm.nih.gov/genbank/).…”

“…A recent study has identified suitable reference genes for use in gene expression quantification in S. pseudintermedius and identified significant upregulation of the intracellular adhesin gene (icaA) associated with biofilm formation in MRSP biofilm samples. 25 These preliminary studies provide the foundation for qPCR gene expression studies in S. pseudintermedius.…”