Bacterial isolates and patients
From 2018 to 2019, the study of 4 linezolid-resistant bacterial strains isolated from patients and associated data was authorized by the ethics committee of the Fourth Affiliated Hospital of Harbin Medical University, China. Before their inclusion in the current study, all patients signed a written informed consent form. Four Staphylococcus capitis isolates resistant to linezolid were identified in the study, all of which were included in further analyses. The strains were first isolated and identified using the VITEK 2 COMPACT system (VITEK 2 Compact, Biomerieux, France) and then confirmed by 16S sequencing. The isolates were recovered from patients with chronic obstructive pulmonary disease (COPD), acute coronary syndrome, rectal cancer, and a male pelvic abscess. Clinical data of the patients harbouring each isolate, including their age, sex, prior exposure to linezolid, and clinical outcome, were obtained retrospectively. The data were kept anonymous.
Antimicrobial susceptibility testing
The tested antimicrobial agents include oxacillin, penicillin, gentamicin, ciprofloxacin, levofloxacin, moxifloxacin, erythromycin, clindamycin, quinupristin/dalfopristin, vancomycin, tetracycline, tigecycline, rifampicin, trimethoprim/sulfamethoxazole, and linezolid, as recommended by the Clinical and Laboratory Standards Institute (CLSI) guidelines . The minimal inhibitory concentration (MIC) (VITEK 2 Compact, Biomerieux, France) of every antibiotic was calculated via the agar dilution MIC method, and the results were interpreted according to the CLSI. The MIC of linezolid was also calculated with the E-test (Biomerieux, France) according to the instructions provided by the manufacturer. S. aureus ATCC 25923 and ATCC 29213 were tested concurrently for quality control (both showed a linezolid MIC of ≤1 μg/ml, Laboratory Department of the Fourth Affiliated Hospital of Harbin Medical University, China).
PCR identification of cfr
PCR was used to determine the existence of cfr in S. capitis. The colonies harvested from agar plates were incubated for 5 minutes in 500 μl of H2O at a temperature of 100 °C. Following 2 minutes of centrifugation, 1 μl of the resultant supernatant was used as a template for PCR conducted using two cfr-specific primers (forward primer: 5′-GAAGCTCTAGCCAACCGTCA-3′, reverse primer: 5′-TCTACCTGCCCTTCGTTTGC-3′, amplicon size: 458 base pairs (bp), overall gene fragment size: 1050 bp, GenBank reference sequence: AM408573). The amplification conditions were as follows: 5 minutes at a temperature of 94 °C; 30 cycles of 94 °C for 30 seconds, 55 °C for 30 seconds, and 72 °C for 1 minute; and a final extension step of 72 °C for 7 minutes. The PCR products were imaged by using a visible light transilluminator (Bioteke, Beijing, China).
Whole-genome sequencing (WGS)
Products of bacterial genomic DNA were sequenced on the Illumina HiSeq (Illumina, America) and PacBio RS (Pacific Biosciences, America) platforms after they were extracted and purified by using a purification kit (TaKaRa, Dalian, China). On the basis of the 16S rRNA nucleotide sequence, a phylogenetic analysis was performed. The obtained 16S and 23S genome sequences are stored in GenBank (accession numbers SUB11152030 and SUB11152217, respectively). The four strains’ representative 16S rRNA nucleotide sequences were compared against the sequences of other Staphylococcus strains stored in GenBank (Supplementary materials). The evolutionary history was inferred using the neighbour-joining method . The optimal tree with a sum of branch length = 0.10605004 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) is shown next to the branches . The tree was drawn to scale with branch lengths in the same units as the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the p-distance method and are presented in units of the number of base differences per site. The analysis involved 27 nucleotide sequences. The included codon positions were 1st + 2nd + 3rd + Noncoding. All positions with less than 50% site coverage were eliminated. That was, fewer than 50% of alignment gaps, missing data, and ambiguous bases were allowed at any position. There were a total of 1496 positions in the final dataset. Phylogenetic inferences were made using the neighbour-joining method within MEGA software (version 7.0.26) .
Genetic environment of the cfr gene
The genetic context of the cfr gene was determined by WGS. The reference sequences of S. capitis pXWZ (GenBank reference sequence MT096435), S. aureus pSR01 (GenBank reference sequence CP048644), S. capitis pcfr-XZ03 (GenBank reference sequence CP077712), and S. xylosus pSX01 (GenBank reference sequence KP890694) were downloaded from the National Center of Biotechnology Information (NCBI). Resistance genes were obtained from the Comprehensive Antibiotic Research Database (CARD) (https://card.mcmaster.ca/). The polished assembly report and annotation information of the obtained genomic sequences of the 4 strains are shown in supplementary tables (Supplementary Tables 1 and 2).