Detection and sequence analysis of accessory gene regulator genes of Staphylococcus pseudintermedius isolates

Abstract

Background:Staphylococcus pseudintermedius (SP) is the major pathogenic species of dogs involved in a wide variety of skin and soft tissue infections. The accessory gene regulator (agr) locus of Staphylococcus aureus has been extensively studied, and it influences the expression of many virulence genes. It encodes a two-component signal transduction system that leads to down-regulation of surface proteins and up-regulation of secreted proteins during in vitro growth of S. aureus. The objective of this study was to detect and sequence analyzing the AgrA, B, and D of SP isolated from canine skin infections.Materials and Methods:In this study, we have isolated and identified SP from canine pyoderma and otitis cases by polymerase chain reaction (PCR) and confirmed by PCR-restriction fragment length polymorphism. Primers for SP agrA and agrBD genes were designed using online primer designing software and BLAST searched for its specificity. Amplification of the agr genes was carried out for 53 isolates of SP by PCR and sequencing of agrA, B, and D were carried out for five isolates and analyzed using DNAstar and Mega5.2 software.Results:A total of 53 (59%) SP isolates were obtained from 90 samples. 15 isolates (28%) were confirmed to be methicillin-resistant SP (MRSP) with the detection of the mecA gene. Accessory gene regulator A, B, and D genes were detected in all the SP isolates. Complete nucleotide sequences of the above three genes for five isolates were submitted to GenBank, and their accession numbers are from KJ133557 to KJ133571. AgrA amino acid sequence analysis showed that it is mainly made of alpha-helices and is hydrophilic in nature. AgrB is a transmembrane protein, and AgrD encodes the precursor of the autoinducing peptide (AIP). Sequencing of the agrD gene revealed that the 5 canine SP strains tested could be divided into three Agr specificity groups (RIPTSTGFF, KIPTSTGFF, and RIPISTGFF) based on the putative AIP produced by each strain. The AIP of SP contains serine and produce lactone ring structured AIP.Conclusion:Presence of AgrA, B, and D in all SP isolates implies the importance of this regulatory system in the virulence genes expression of the SP bacteria. SP isolates can be typed based on the AgrD auto-inducible protein sequences as it is being carried out for typing of S. aureus isolates. However, further studies are required to elucidate the mechanism of controlling of virulence genes by agr gene locus in the pathogenesis of soft tissue infection by SP.

Detection of Multidrug Resistant Strains in Some Pathogenic Bacteria and Fungi Caused Otitis in Pet Animals

“…Isolation and Identification of Bacteria from were done as the standard methods recommended by Quinn et al (2002). Isolation and Identification of SP was done acc.to AnandaChitra et al (2015).…”

Cytokine response to killed Staphylococcus pseudintermedius antigen in dogs with skin diseases

“…Whole blood of 5 ml was collected in EDTA vial from a total of 24 animals and skin swabs were also collected from the selected dogs so as to confirm the Staphylococcus infection. Isolation and identification of S. pseudintermedius isolates was done as described previously (Anandachitra et al, 2015).…”

Dermatophilus congolensis infection in sheep and goats in Delta region of Tamil Nadu

“…DNA extraction from scab material was performed using tissue DNA extraction spin column kit (Qiagen, Germany) by following the manufacturer’s instruction. DNA from isolated bacteria was extracted by boiling method [ 4 ]. PCR was carried out using the already published primers ( Table-1 ) targeting DC 16S rRNA by Shaibu et al .…”