Molecular Diagnostic Identification of Staphylococcus pseudintermedius

Abstract

We report the first diagnostic test for the identification of Staphylococcus pseudintermedius involving a simple PCR-restriction fragment length polymorphism approach. The method allows discrimination of S. pseudintermedius from the closely related members of the Staphylococcus intermedius group and other important staphylococcal pathogens of humans and dogs.Until recently, Staphylococcus intermedius was considered responsible for most cases of canine pyoderma, a major skin disease of dogs (8). However, using a multilocus sequencing approach, independent research groups have demonstrated that isolates phenotypically identified as Staphylococcus intermedius consist of three distinct species, including S. intermedius, Staphylococcus pseudintermedius, and Staphylococcus delphini, which together represent the S. intermedius group (SIG) (1a, 4). Importantly, it was discovered that S. pseudintermedius, not S. intermedius, is the common canine pyoderma pathogen and that S. delphini, isolated from a variety of different animals, may be more clinically important than was previously thought (1a, 4). The recently identified S. pseudintermedius (5) is occasionally isolated from serious human infections, and the emergence and spread of methicillin-resistant S. pseudintermedius strains are major veterinary and public health issues (1a, 3, 4, 7, 11-13). Sasaki et al. could biochemically differentiate S. intermedius from the other SIG species but was unable to identify phenotypic markers to discriminate S. pseudintermedius, the most clinically relevant species, from S. delphini (12), and DNA sequencing is currently required to identify S. pseudintermedius (1a, 12). In order to address the need for a method of discriminating clinical isolates of S. pseudintermedius, we have developed a rapid, simple, and robust PCR-restriction fragment length polymorphism (RFLP) approach which has been validated independently in laboratories in separate countries.Our previous population genetic study of SIG isolates examined sequence diversity at five gene loci among 104 isolates (1a). In the current study, sequence analysis of one of the loci, pta, which encodes the enzyme phosphoacetyltransferase, revealed the presence of an MboI restriction site in all S. pseudintermedius isolates, which was absent in all S. intermedius and S. delphini isolates examined. Based on this discovery we have designed a simple, robust, and inexpensive PCR-RFLP diagnostic test for the identification of S. pseudintermedius. Staphylococcal genomic DNA isolation was carried out as previously described (1a). PCR amplification of a 320-bp fragment of the pta gene was carried out in a 50-l volume with a 0.2 M concentration of each oligonucleotide primer (pta_f1, AAA GAC AAA CTT TCA GGT AA, and pta_r1, GCA TAA ACA AGC ATT GTA CCG), a 0.2 mM concentration of the de-* Corresponding author. Mailing address:

Methicillin Resistance in Staphylococcus pseudintermedius Isolated from Shelter Dogs in Turkey

“…More and comprehensive studies should be performed to reveal the epidemiology of MRSP infections in companion animals in Turkey, and their role in transmission of antibiotic resistant bacteria between different species. [17] , N: not applicated SAREYYÜPOĞLU, MÜŞTAK CANTEKIN, DİKER…”

“…Staphylococci identified as S. intermedius by Microbact TM Staph 12S system were molecularly investigated for S. pseudintermedius by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the pta gene encoding the enzyme phosphoacetyltransferase [17] . MboI enzyme was used in RFLP to detect S. pseudintermedius isolates harboring this restriction site.…”

Frequency and antimicrobial susceptibility of Staphylococcus pseudintermedius in dogs with otitis externa

“…Samples were plated onto Muller-Hinton agar (Difco Laboratories, EUA) supplemented with 5% equine blood and incubated at 37°C for 24 to 48h. All colonies underwent biochemical evaluation for identification of staphylococci, as previously described by QUINN et al (2011) andBANNOEHR &GUARDABASSI (2012). Biochemical examinations analyzed catalase, oxidase, coagulase, and urease production, carbohydrate fermentation (mannitol, sucrose, and trehalose), arginine production, Voges Proskauer (for acetoin production), and response to polymyxin B.…”

“…Amplicons were separated by electrophoresis on a 1.5% agarose gel with a 1-kb molecular marker (ThermoScientific ® Waltham, Massachusetts, USA). S. pseudintermedius MRSP 3279, a reference strain previously used in other studies (BANNOEHR et al, 2009;BOURGUIGNON et al, 2016) were used as positive controls and amplification of sterile water served as a negative control.…”

Genotypic relatedness and characterization of Staphylococcus pseudintermedius associated with post-operative surgical infections in dogs

“…Identification of the S. pseudintermedius isolates used in the study was performed by Mbo I restriction digests of the pta gene fragment, as described previously (Bannoehr et al, 2009).…”