Development of a real-time PCR for detection of Staphylococcus pseudintermedius using a novel automated comparison of whole-genome sequences

Abstract

Staphylococcus pseudintermedius is an opportunistic pathogen in dogs and cats and occasionally causes infections in humans. S. pseudintermedius is often resistant to multiple classes of antimicrobials. It requires a reliable detection so that it is not misidentified as S. aureus. Phenotypic and currently-used molecular-based diagnostic assays lack specificity or are labour-intensive using multiplex PCR or nucleic acid sequencing. The aim of this study was to identify a specific target for real-time PCR by comparing whole genome sequences of S. pseudintermedius and non-pseudintermedius.Genome sequences were downloaded from public repositories and supplemented by isolates that were sequenced in this study. A Perl-script was written that analysed 300-nt fragments from a reference genome sequence of S. pseudintermedius and checked if this sequence was present in other S. pseudintermedius genomes (n = 74) and non-pseudintermedius genomes (n = 138). Six sequences specific for S. pseudintermedius were identified (sequence length between 300–500 nt). One sequence, which was located in the spsJ gene, was used to develop primers and a probe. The real-time PCR showed 100% specificity when testing for S. pseudintermedius isolates (n = 54), and eight other staphylococcal species (n = 43). In conclusion, a novel approach by comparing whole genome sequences identified a sequence that is specific for S. pseudintermedius and provided a real-time PCR target for rapid and reliable detection of S. pseudintermedius.

Description and Comparative Genomics of Macrococcus caseolyticus subsp. hominis subsp. nov., Macrococcus goetzii sp. nov., Macrococcus epidermidis sp. nov., and Macrococcus bohemicus sp. nov., Novel Macrococci From Human Clinical Material With Virulence Potential and Suspected Uptake of Foreign DNA by Natural Transformation

“…CP023497 ). Cas2 exhibits 72% amino acid identity with the CRISPR-associated endonuclease Cas2 of Staphylococcus delphini (Verstappen et al, 2017 ). The whole CRISPR-Cas locus exhibited high nucleotide identity (68%) to the type II-C CRISPR-Cas of S. simulans strain FDAARGOS_383 (GenBank accession no.…”

New Genus Fibralongavirus in Siphoviridae Phages of Staphylococcus pseudintermedius

“…Although the domain structure of the integrases is identical (AP2 binding, SAM-like and catalytic domains), the amino acid sequence is dissimilar, and prophages integrate into different loci in the genome. A prophage that contains an identical integrase to phage QT1 is integrated into tRNA Ser between the genes encoding for the ComK and SdrD proteins in the genome of S. pseudintermedius SL/154 [66]. In contrast, an integrase identical to that of phage 2638A is inserted into the gene for tRNA Arg between genes encoding for the ClpP and WhiA proteins in the genome of S. pseudintermedius 104N (GenBank assembly accession number GCF_002847235).…”

Characterization of Staphylococcus intermedius Group Isolates Associated with Animals from Antarctica and Emended Description of Staphylococcus delphini

“…Assembly accession numbers of genomes retrieved from NCBI genome database are as follows: 8086 (GCF_000308115.1) [25], NCTC 12225 T (GCF_900636325.1), LMG 22219 T (GCF_001792775.2), NCTC 11048 T (GCF_900458545.1) [60], NW1 T (GCF_900183575.1) [16], 14S03309-1 (GCF_002374115.1), 14S03313-1 (GCF_002374125.1), 14S03318-1 (GCF_002369645.1), and 215100905101-2 (GCF_002369695.1) [26].…”

“…The size of the draft genomes of strains P5747 and P6456 was 2.54 and 2.65 Mb, comprised of 47 and 104 contigs, with an average G+C content of 38.2 and 38.1 mol%, respectively (Table S2). The genome assemblies were compared to whole-genome sequences of S. delphini type strain NCTC 12225 T and horse isolates S. delphini 8086 [25] and S. delphini 215100905101-2 [26] (Figure 5). Comparative genomic analysis of predicted genes in the above five genomes identified 2460 gene clusters and 596 singletons.…”

“…Protein profiling through matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) seems to be the most reliable method to differentiate between SIG species [19,20]. Other effective methods for SIG diagnostics are sequencing of the housekeeping genes nuc [21], sodA, and hsp60 [22], and for the discrimination of S. pseudintermedius by means of the pta housekeeping gene [23] or multilocus sequence typing (MLST) [24], as well as whole-genome sequencing [25,26].…”