Computational protocol: Draft Genome Sequences of 12 Clinical and Environmental Methicillin-Resistant Strains of Staphylococcus pseudintermedius Isolated from a Veterinary Teaching Hospital in Washington State

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Protocol publication

[…] Methicillin-resistant Staphylococcus pseudintermedius (MRSP) is an opportunistic canine pathogen that causes infections of the skin and soft tissue, such as superficial or deep pyoderma, wounds, urinary tract, and other body sites, including otitis media or externa, and is often associated with surgical site infections (, ). MRSP is also commonly isolated from cats () and occasionally from rats, cows, and horses (). Hospitalization and antimicrobial treatment are major risk factors for MRSP colonization or infection, making this an important pathogen associated with veterinary hospital-acquired infections (). The number of MRSP/total S. pseudintermedius infections in hospitalized animals at the Washington State University Veterinary Teaching Hospital increased from 1/17 in 2009 to 11/46 in 2017, peaking at 22/71 in 2015; this increase is consistent with recent increases reported worldwide (). The potential for zoonotic transmission to humans (, ), multidrug resistance (MDR), and concern that MRSP could be mistaken for methicillin-resistant Staphylococcus aureus (MRSA) (, ) suggest the need for improved detection and molecular subtyping tools for epidemiological source tracing of this pathogen, especially during outbreaks. Here, we report draft genome sequences of 12 MDR-MRSP clinical and environmental strains isolated from a veterinary teaching hospital in Washington State (). A single colony of each MDR-MRSP strain was grown overnight at 37°C in brain heart infusion (BHI) broth (Difco). DNA was extracted using zirconia beads and the Qiagen DNeasy tissue kit (Qiagen, USA), with the exception that additional 70% ethanol washes and RNase incubations were added. Paired-end sequencing libraries (2 × 150 bp) were prepared using NEBNext Ultra II kit (NEB, UK), according to the manufacturer’s protocol, and size selected in the range of 422 to 502 bp (average size, ∼458 bp). All strains were sequenced using the Illumina HiSeq 4000 platform (Illumina, Inc., USA). Sequences were trimmed using BBDuk and de novo assembled using Velvet 1.2.10, with the k-mer length set at 99 (). Contigs were reorganized by aligning to the genome sequence of the reference S. pseudintermedius strain 081661 (GenBank accession no. NZCP016073) () using progressiveMauve (). Automated annotation of the assembled contigs was performed using the NCBI Prokaryotic Genome Annotation Pipeline (https://www.ncbi.nlm.nih.gov/genome/annotation_prok/). Whole-genome multilocus sequence typing was performed using the Staphylococcus pseudintermedius MLST database (https://pubmlst.org/spseudintermedius/), as described previously (). All of these strains displayed multidrug resistance (resistance to >3 classes of antibiotics) when MICs were measured according to current Clinical and Laboratory Standards Institute (CLSI) protocols (). The corresponding resistance genes were identified using ResFinder version 3.0 (). All strains contained staphylococcal cassette chromosome mec elements (SCCmec), as identified by ResFinder version 3.0. These genome sequences will aid in the development of improved molecular diagnostics and subtyping methods for epidemiological source tracing of MRSP outbreaks. The detailed comparative genomics analysis of these strains is currently ongoing and will be published independently. […]

Pipeline specifications

Software tools BBTools, Velvet, Mauve, PGAP
Applications Phylogenetics, Nucleotide sequence alignment
Organisms Staphylococcus pseudintermedius, Canis lupus familiaris
Chemicals Methicillin