Development of multiplex polymerase chain reaction assay for rapid detection of Staphylococcus aureus and selected antibiotic resistance genes in bovine mastitic milk samples

Abstract

To improve the diagnosis of Staphylococcus aureus mastitis and its potential antibiotic resistance in dairy cattle, a multiplex polymerase chain reaction (PCR) assay was developed for simultaneous species identification and detection of penicillin, erythromycin, and tetracycline resistance genes. The assay was first verified using 85 clinical isolates of S. aureus, and its effectiveness was evaluated by testing 99 mastitic milk samples. The multiplex PCR assay had high detection sensitivity in samples from both groups (100% agreement with single-gene PCR), with detection limits of 100–103 colony-forming units (cfu)/ml. The detection limits of the current assay for nuc, blaZ, erm B, erm C, tet K, and tet M genes were 101, 101, 102, 100, 102, and 103 cfu/ml, respectively. There was a good correlation between genotypic analysis by PCR and phenotypic determination using S. aureus species identification and susceptibility testing methods. High diagnostic sensitivities and specificities were shown for the target resistance genes ( blaZ gene, 97.2% and 92.9%; ERM resistance genes, 100% and 95.6%; TET resistance genes, 100% and 96%). Results suggested that the current PCR assay could be used as an alternative diagnostic method in the routine diagnosis for rapid, sensitive, and specific detection of S. aureus and its associated antibiotic resistance genes in mastitic milk samples.

Detection of Staphylococcus Aureus Among Coagulase Positive Staphylococci from Animal Origin Based on Conventional and Molecular Methods

“…This gene, coding a thermostable nuclease, seems to be highly specific and a unique genetic marker for S. aureus detection confirmed both in our and previous research (21,31,32). However, there is a report about nuc negative S. aureus clade from animals in sub-Saharan Africa (33) harboring a homologue of nuc gene.…”

Cezayir’de İnek Mastitislerinden İzole Edilen Stafilokokların Antibiyotik Direncinin Fenotipik ve Moleküler Yöntemlerle Belirlenmesi

“…Moreover, 76.19% of isolates were phenotypically resistant to erythromycin but all of them were negative for ermA and ermC genes. This finding is in disagreement with those from in northern China [66] . Acquisition of resistance in S. aureus isolates attributed to mutation in gene or due to exchange of genetic material between organisms, since resistance genes carrying mobile genetic elements of S. aureus have exceedingly been explored [68] .…”

“…Furthermore, occurrence of tetracycline-resistant genes among the bovine staphylococci isolates has been previously observed [65] . Tetracycline resistance encoding gene tetM was present in 100% of isolates which is higher than previously detected in china by Gao et al [66] . The proportion of the tetracycline genes is almost similar with the previous reports [65] .…”

Molecular types and antimicrobial resistance profile of Staphylococcus aureus isolated from dairy cows and farm environments

“…Prevalence of antimicrobial resistant genes blaZ, ermB, and tetK was studied by the multiplex PCR assay developed previously (5). PCR primer sequences used for detection of AMR genes are detailed in Table 2.…”