Materials and Methods

Phylogenetic Distribution of CRISPR-Cas Systems inStaphylococcus lugdunensis

MATERIALS AND METHODSIsolation and identification of S. lugdunensis.A total of 199 S. lugdunensis isolates collected from 2009 to 2014 from Chang Gung Memorial Hospital (Taoyuan, Taiwan) were included in the study. All information associated with the 199 isolates (e.g., specimen type and year of isolation) was collected. Staphylococcus lugdunensis isolates were initially identified by Gram staining, biochemical methods (catalase-positive, coagulase-negative, pyrrolidonyl arylamidase-positive, and ornithine decarboxylase-positive results), and rapid PCR detection (18). All the S. lugdunensis isolates were also confirmed using a matrix-assisted laser desorption ionization-time of flight mass spectrometry system (MALDI-TOF MS; Bruker Biotyper, database 2.0). The isolates were stored in tryptic soy broth containing 20% glycerol at −80°C until use.CRISPR-Cas system identification and sequencing.The Crispr-IIIA-F/Crispr-IIIA-R and Crispr-IIC-F/Crispr-IIC-R primers were designed to specifically detect the cas1 gene for CRISPR-Cas types IIIA and IIC, respectively (Fig. 1B and Table S1). The primers were designed based on S. lugdunensis whole-genome sequences deposited in the National Center for Biotechnology Information (NCBI) database, including sequences for type IIIA-positive strain VISLISI_33 (accession no. {"type":"entrez-nucleotide","attrs":{"text":"CP020769.1","term_id":"1182096454","term_text":"CP020769.1"}}CP020769.1) and type IIC-positive strain C_33 (accession no. {"type":"entrez-nucleotide","attrs":{"text":"CP020768.1","term_id":"1182041651","term_text":"CP020768.1"}}CP020768.1). The PCRs were carried out in a total volume of 20 μl containing 2× master mix (Promega, Madison, WI, United States), 10 pmol of each primer, and 1 μl DNA template. The PCR cycling conditions for CRISPR-Cas system detection were 95°C for 3 min; 30 cycles of 30 s at 95°C, 30 s of annealing at 50°C, and 1 min of extension at 72°C; and a final extension for 3 min at 72°C.The primers Crispr-IIIA-F/Crispr-IIIA-R and Crispr-IIC-F/Crispr-IIC-R were designed for also determining type IIIA and type IIC CRISPR-Cas spacer sequences, respectively (Fig. 1B and Table S2). The PCR cycling conditions for CRISPR-Cas system spacer typing were 95°C for 10 s; 30 cycles of 1 min at 94°C, 1 min of annealing at 55°C, 1 min of extension at 72°C (45 s for CRISPR-Cas IIC), and a final extension for 10 min at 72°C. The Crispr-s-R primer (Table S2) was used to perform downstream sequencing of the type IIIA CRISPR array.Analysis and visualization of CRISPR.All sequences of the CRISPR arrays were characterized to identify CRISPR arrays and extract spacer sequences using CRISPRFinder web software (19) with default settings. Small CRISPRs with evidence level 1 (with three or fewer spacers) were excluded, and only CRISPR arrays with evidence levels 2 to 4 were retained. Level 1 CRISPRs which contained fewer than three spacers were upgraded to level 4 whenever similar direct repeats were found in a level 4 CRISPR. Each unique spacer was assigned a specific spacer number. The spacer sequences were further analyzed to search for known homologies in the NCBI database using BLASTn with an E value cutoff of 0.1. All matches with a bit score above 40 and a query cover above 80% were selected. Only the top 3 hits were shown if multiple hits were found for a given query. Each unique spacer combination within a CRISPR locus was assigned a CT. A new CT number was assigned for CTs not described previously by Argemi et al. (15). The presence (designated “1”) or absence (designated “0”) of each spacer sequence for each strain was summarized using a binary code pattern. The binary patterns of all isolates were uploaded to Phyloviz v. 2.0 to generate a minimum spanning tree using the goeBURST algorithm (20).MLST analysis.MLST was performed for all S. lugdunensis isolates according to a previous study (6). Seven housekeeping genes of each isolate, including aroE, dat, ddl, gmk, ldh, recA, and yqiL, were PCR-amplified and sequenced using the primers described in Table S3. The sequence data were imported to the S. lugdunensis MLST database (http://www.mlst.net) for ST type determination. Sequence types were identified based on the allele profiles. Further analysis was performed using eBURST (http://eburst.mlst.net) to identify clonal complexes (CCs) and founders, as well as to determine the overall population structures. A minimum-evolution (ME) tree of the concatenated sequences (aroE-dat-ddl-gmk-ldh-recA-yqiL) for each ST shared by the S. lugdunensis isolates was generated using Mega X and the Kimura two-parameter model to estimate genetic distances. Statistical support of the nodes in the ME tree was assessed by performing 1,000 bootstrap resamplings.Antimicrobial susceptibility testing.Susceptibility of the S. lugdunensis isolates to oxacillin was determined using an agar dilution assay and interpreted according to the Clinical and Laboratory Standards Institute guidelines (21). Staphylococcus lugdunensis isolates with a MIC ≥ 4 μg/mL were defined as resistant. Staphylococcus aureus ATCC 29213 was used as a control strain. Antimicrobial susceptibility testing was performed in duplicate to ensure reproducibility.SCCmec typing and sequencing.All S. lugdunensis isolates were subjected to SCCmec typing and mecA detection using a multiplex PCR assay to amplify the ccr and mec complexes according to a previous study (22).

Article TitlePhylogenetic Distribution of CRISPR-Cas Systems inStaphylococcus lugdunensis

Abstract

A total of 199S. lugdunensisisolates collected from 2009 to 2014 from Chang Gung Memorial Hospital (Taoyuan, Taiwan) were included in the study. All information associated with the 199 isolates (e.g., specimen type and year of isolation) was collected.Staphylococcus lugdunensisisolates were initially identified by Gram staining, biochemical methods (catalase-positive, coagulase-negative, pyrrolidonyl arylamidase-positive, and ornithine decarboxylase-positive results), and rapid PCR detection (18). All theS. lugdunensisisolates were also confirmed using a matrix-assisted laser desorption ionization-time of flight mass spectrometry system (MALDI-TOF MS; Bruker Biotyper, database 2.0). The isolates were stored in tryptic soy broth containing 20% glycerol at −80°C until use.


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