GENOME SEQUENCES crossm Hybrid Assembly from a Pathogenic Methicillin- and Multidrug-Resistant Staphylococcus pseudintermedius Strain Isolated from a Case of Canine Otitis in Spain Joaquim Viñes,a,b Anna Cuscó,b Olga Francinoa a Servei Veterinari de Genètica Molecular (SVGM), Universitat Autònoma de Barcelona, Bellaterra, Cerdanyola del Vallès, Barcelona, Spain b Vetgenomics SL, Bellaterra, Cerdanyola del Vallès, Barcelona, Spain ABSTRACT Here we report the genome assembly, using a hybrid approach with Il- lumina and Nanopore sequencing, of a pathogenic Staphylococcus pseudintermedius strain isolated from a case of canine otitis. Genome assembly confirmed the antimi- crobial resistance profile (disk diffusion testing) with specific genes and mutations. S taphylococcus pseudintermedius is a coagulase-positive Staphylococcus species within the Staphylococcus intermedius group (1), which is formed by S. intermedius, S. pseudintermedius, S. delphini, and S. cornubiensis (2). This bacterium is a commensal in pets’ microbiota, typically related to dogs and primarily associated with skin, fur, and mucocutaneous sites. However, it has opportunistic behavior, causing several types of infections related mostly to the skin, such as wound infections, pyoderma, and otitis, among others. S. pseudintermedius colonizes 90% of dogs, and methicillin-resistant S. pseudintermedius (MRSP) demonstrates high prevalence worldwide, e.g., 70% in Japan, 50% in China, and 30% in Europe. The report of zoonotic infections due to MRSP highlights its One Health threat. Sequence type 71 (ST71) is the most prevalent in Europe (3–8). Downloaded from https://journals.asm.org/journal/mra on 20 January 2022 by 79.158.116.11. Swab extension of the sample and further Diff-Quick staining revealed the presence of a few cocci in the left ear of a 6-year-old Yorkshire terrier with otitis externa. The S. pseudintermedius G3C4 strain was isolated by overnight culture at 37°C on blood agar. Different antibiotics were tested in a disk diffusion inhibitory assay, with the sensitivity range criteria shown at Table 1 for aminoglycosides, fluoroquinolones, tetracyclines, macrolides, beta-lactams, lincosamides, phenicols, rifamycins, fusidic acid, and co- trimoxazole. DNA from S. pseudintermedius G3C4 was extracted using a DNA microprep kit Citation Viñes J, Cuscó A, Francino O. 2020. (ZymoBIOMICS), following the manufacturer’s instructions, and DNA quality was as- Hybrid assembly from a pathogenic methicillin- and multidrug-resistant sessed by measurements with a Qubit fluorimeter (Invitrogen). The library for Nanopore Staphylococcus pseudintermedius strain isolated sequencing was prepared by transposase fragmentation using a rapid barcoding kit from a case of canine otitis in Spain. Microbiol (product number RBK-SQK004; Oxford Nanopore Technologies), according to the man- Resour Announc 9:e01121-19. https://doi.org/ 10.1128/MRA.01121-19. ufacturer’s instructions. The final library was loaded and sequenced in a MinION Editor Irene L. G. Newton, Indiana University, FLO-MIN106 flow cell v9.4. Bloomington An Illumina (San Diego, CA, USA) library was prepared by enzymatic fragmentation Copyright © 2020 Viñes et al. This is an open- and double indexing using an NGSgo kit (GenDx, Utrecht, Netherlands), according to access article distributed under the terms of the Creative Commons Attribution 4.0 the manufacturer’s instructions. The indexed libraries were pooled, denatured, and International license. diluted to a final concentration of 4 nM. The pooled library was sequenced on the Address correspondence to Joaquim Viñes, MiSeq system (Illumina) with a 300-cycle MiSeq reagent kit v2. joaquim.vines@vetgenomics.com, or Olga The fast5 files generated by Nanopore sequencing were base called and demulti- Francino, olga.francino@uab.cat. Received 4 October 2019 plexed (sorted by barcode) using Albacore v2.3.1, yielding fastq files. A second round Accepted 22 November 2019 of demultiplexing was performed with Porechop (9) (by default), in which barcodes that Published 2 January 2020 agreed with Albacore were kept and the others were removed. Porechop was also used Volume 9 Issue 1 e01121-19 mra.asm.org 1 Viñes et al. TABLE 1 Summary of the antibiotic resistance determined by disk diffusion testing and sequencing Disk diffusion testing results Sequencing results Zone of Sensitivity range Antibiotica inhibition (mm) (␮g/ml) Susceptibilityb Gene(s) associated Mutation associated Location Aminoglycosides GEN 12 ⬍12 to ⬎15 R aac(6=)-Ie-aph(2⬙)-Ia, Genome TOB 17 ⬍17 to ⬎19 R aph(3=)-IIIa, aad(6) AMK 25 ⬍14 to ⬎17 S Fluoroquinolones CIP 9 ⬍20 to ⬎22 R Point mutations in gyrA Genome MARBO 0 ⬍14 to ⬎20 R (positions 12, 251, PRADO 15 ⬍19 to ⬎24 R 2023, and 2140) ORBI 0 ⬍17 to ⬎23 R Tetracyclines TET 10 ⬍18 to ⬎23 R tet(K) Plasmid DOX 14 ⬍20 to ⬎25 R MIN 18 ⬍19 to ⬎24 R Macrolide ERY 0 ⬍13 to ⬎23 R ermB Genome Beta-lactams OXA 0 ⬍16 to ⬎18 R mecA, blaZ Genome FOX 30 ⬍34 to ⬎36 R Lincosamide CLI 0 ⬍14 to ⬎21 R ermB Genome Phenicols CHL 34 ⬍12 to ⬎18 S FFC 30 ⬍12 to ⬎18 S Rifamycin RIF 44 ⬍16 to ⬎20 S Fusidane FD 40 ⬍23 to ⬎25 S SXT 0 ⬍10 to ⬎16 R dfrG Genome Downloaded from https://journals.asm.org/journal/mra on 20 January 2022 by 79.158.116.11. a GEN, gentamicin; TOB, tobramycin; AMK, amikacin; CIP, ciprofloxacin; MARBO, marbofloxacin; PRADO, pradofloxacin; ORBI, orbifloxacin; TET, tetracycline; DOX, doxycycline; MIN, minocycline; ERY, erythromycin; OXA, oxacillin; FOX, cefoxitin; CLI, clindamycin; CHL, chloramphenicol; FFC, florfenicol; RIF, rifampin; FD, fusidic acid; SXT, co-trimoxazole. b R, resistant; S, sensitive. 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 N50 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 60⫻ coverage and lengths of 2.72 Mb and 4.4 kb, respectively. The 5 other contigs had coverage between 1.71⫻ and 1.83⫻. The assembled genome of this S. pseudintermedius isolate (63⫻ coverage) has a size of 2,717,621 bp, with a G⫹C content of 37.50% and 2,548 coding sequences, 59 tRNAs, Volume 9 Issue 1 e01121-19 mra.asm.org 2 Microbiology Resource Announcement 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 G⫹C content of 30.07% and 64⫻ 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 beta- lactam resistance, aac(6=)-Ie-aph(2⬙)-Ia, aph(3=)-IIIa, and aad(6) for aminoglycoside resis- tance, ermB for erythromycin and clindamycin resistance, and dfrG for trimethoprim resis- tance. 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 meth- icillin 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 previ- ously. 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 acces- sion 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. ACKNOWLEDGMENTS The S. pseudintermedius isolate was kindly donated by LETIPharma. Downloaded from https://journals.asm.org/journal/mra on 20 January 2022 by 79.158.116.11. This study was supported by a grant awarded by the Generalitat de Catalunya (Industrial Doctorate Program, grant 2017DI037) and by Vetgenomics. REFERENCES 1. Fitzgerald JR. 2009. The Staphylococcus intermedius group of bacterial ylococcus pseudintermedius ST71 in a Finnish veterinary teaching pathogens: species re-classification, pathogenesis and the emergence of hospital: from outbreak control to outbreak prevention. PLoS One meticillin resistance. Vet Dermatol 20:490 – 495. https://doi.org/10.1111/ 9:e110084. https://doi.org/10.1371/journal.pone.0110084. j.1365-3164.2009.00828.x. 7. Dmitrenko OA, Balbutskaya AA, Skvortsov VN. 2016. Ecological features, 2. 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