Hybrid Assembly from a Pathogenic Methicillin- and Multidrug-Resistant Staphylococcus pseudintermedius Strain Isolated from a Case of Canine Otitis in Spain

Here we report the genome assembly, using a hybrid approach with Illumina and Nanopore sequencing, of a pathogenic Staphylococcus pseudintermedius strain isolated from a case of canine otitis. Genome assembly confirmed the antimicrobial resistance profile (disk diffusion testing) with specific genes and mutations.

to trim barcodes and other adapters from the sequences. A total of 93,340 Nanopore reads were retrieved and used for further steps; the median read length was 2,774 bp, the N 50 read length was 4,382 bp, and the median Phred read quality was 14.2. A total of 2,338,855 Illumina reads were generated by the sequencer, with a median Phred read quality of 37.8. De novo genome assembly was performed with data retrieved from Nanopore and Illumina sequencing in a hybrid approach, using Unicycler v0.4.6 (10) (parameters were as follows: R1, Illumina file; R2, Illumina file; l, Nanopore reads). Further analyses included assessment of genome completeness with CheckM v1.0.11 (11) (by default), multilocus sequence typing (MLST) using CGE DTU tools (12), and annotation with Prokka v1.13 (13) (by default); the NCBI Prokaryotic Genome Annotation Pipeline v4.6 was used to determine the number of coding sequences, rRNAs, and tRNAs. We used ABRicate (14) with the CARD and NCBI databases to retrieve antibiotic resistance genes.
Unicycler assembly retrieved 7 contigs. Two of the contigs corresponded to the complete genome and a plasmid, with 60ϫ coverage and lengths of 2.72 Mb and 4.4 kb, respectively. The 5 other contigs had coverage between 1.71ϫ and 1.83ϫ.
The assembled genome of this S. pseudintermedius isolate (63ϫ coverage) has a size of 2,717,621 bp, with a GϩC content of 37.50% and 2,548 coding sequences, 59 tRNAs,

Disk diffusion testing results
Sequencing results

Sensitivity range (g/ml) Susceptibility b Gene(s) associated Mutation associated Location
Ͻ20 to Ͼ22 R Point mutations in gyrA (positions 12, 251, 2023, and 2140) and 19 rRNA copies. CheckM determined completeness of 99.43%. MLST (12) showed that the strain belongs to the most prevalent ST in Europe, ST71, achieving 100% coverage and identity for all of the genes tested (ack, cpn60, fdh, pta, purA, sar, and tuf). A plasmid of 4,439 bp, pSP-G3C4, was also obtained. It has a GϩC content of 30.07% and 64ϫ coverage. In a BLAST search, we obtained a match with Staphylococcus epidermidis ATCC 12228 plasmid pSE-12228-01 (NCBI accession number NC_005008). Table 1 shows the results of disk diffusion susceptibility tests with aminoglycosides, fluoroquinolones, tetracyclines, macrolides, beta-lactams, clindamycin, and co-trimoxazole. Genome analyses with ABRicate revealed the presence of several genes that confer resistance to most of the aforementioned antibiotics, including blaZ and mecA for betalactam resistance, aac(6=)-Ie-aph(2Љ)-Ia, aph(3=)-IIIa, and aad (6) for aminoglycoside resistance, ermB for erythromycin and clindamycin resistance, and dfrG for trimethoprim resistance. We also found sat4, which confers resistance to streptothricin. Point mutations at positions 12, 251, 2032, and 2140 in the gyrA gene (encoding a topoisomerase) explain quinolone resistance (15). The tet(K) gene, conferring resistance to tetracycline, was found in the plasmid.
A SCCmec II-III cassette characteristic of S. pseudintermedius (15) harbors the methicillin resistance gene mecA. Furthermore, aad (6), sat4, and aph(3=)-IIIa genes are located contiguously in the genome, which is an antibiotic resistance gene cluster already described for this species (16). It seems that a fourth gene could be involved in the cluster, namely, ermB, which is located near the triad of genes mentioned previously. Boerlin et al. (17) already reported that there could be a link between macrolide and aminoglycoside resistance in Staphylococcus strains of canine origin.
We confirm that a long-and short-read hybrid approach is an excellent option for sequencing and assembling de novo genomes for in-depth assembly and characterization.
Data availability. The genome sequence of S. pseudintermedius G3C4 has been deposited in the GenBank database with accession number CP032682 and RefSeq accession number NZ_CP032682; the plasmid has been deposited under GenBank accession number MN612109. All raw sequence files can be found under BioProject accession number PRJNA493792.