From January 2019 to May 2019, the study was conducted in the Disinfection & Sterilization Center of the First Affiliated Hospital, School of Medicine, Zhejiang University, where both gastrointestinal and respiratory endoscopes are reprocessed. During the study period, procedures in our institution were performed using bronchoscopes (model BF260) (Olympus, Japan). The cleaning of bronchoscopes was carried out with an enzymatic detergent solution, endozyme. Manual disinfection was performed by soaking the device into 2% glutaraldehyde.
Samples were collected under aseptic conditions from bronchoscopes following clinical procedures and after usual decontamination procedures by flushing thoroughly with 10 mL of sterilized phosphate buffered saline (PBS) as described previously . Collected samples were put in cool-boxes with ice-packs (4-8°C) upon collection and transported in 4 hours to laboratory.
Bacterial isolation and identification
All samples were plated on Mueller-Hinton agar plates (Oxoid, UK) using the sterile swab. The agar plates were incubated for 18–24 hours at 37°C. Single colony was selected from each species per sample. All of the positive cultures were selected for identification. Bacterial identification was conducted by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) (Bruker, Leipzig, Germany), and further checked by PCR and sequencing.
Antimicrobial susceptibility testing
The minimum inhibitory concentrations (MICs) of 9 K. aerogenes isolates was determined using the agar dilution method according to the Clinical and Laboratory Standards Institute (CLSI) standards . 19 antimicrobials were tested as described previously . Antimicrobial susceptibility testing for colistin and tigecycline were performed by the microbroth dilution method as described by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (http://www.eucast.org/). The MIC results were interpreted using the CLSI standards (Third Edition: M45).
Whole genome sequencing (WGS) and in silico analysis
WGS was performed on all K. aerogenes strains identified in this work.
The extracted genomic DNA was evaluated by agarose gel electrophoresis. The concentration and purity of genomic DNA were determined using NanoDrop 2000 (Thermo Scientific, Waltham, USA) and Qubit® version 2.0 fluorometer (Thermo Scientific), respectively. The sequencing library was prepared by using Illumina Nextera XT kit (Illumina, San Diego, USA). A-tailed fragments were ligated with paired-end adaptors and PCR-amplified with a 500-bp insert. WGS was performed with an Illumina NovaSeq 6000 platform (Novogene Co., China). PCR adapter reads and low-quality reads from the paired-end and mate-pair library were filtered using in-house pipeline. Paired reads were then assembled into a number of scaffolds using Velvet version 1.2.10 . Multilocus sequence typing (MLST) analysis was performed as described previously . ARGs were identified using the ResFinder 2.1 database .
Phylogenetic analysis of K. aerogenes isolates
To further characterized the evolutionary relationship among K. aerogenes isolates, we created a core genome-based phylogenetic tree using 7 K. aerogenes genomes sequenced in this study and 51 randomly selected publicly available K. aerogenes genomes (Table S1). The isolate collection includes strains from humans (n = 44), the environment (n = 9) and other sources (n = 5) that were widely distributed over time and geographical locations. All collection genomes were annotated using Prokka  and RAST tool . The core genes in the genomes of K. aerogenes genomes were identified using Prokka  and Roary . Maximum likelihood-based phylogenetic reconstruction was performed with RAxML version 8.2.10 using generalized time reversible (GTR) + Γ nucleotide substitution model . One hundred bootstrap replicates were evaluated to determine branch support. A maximum-likelihood phylogenetic tree based on the core single nucleotide polymorphism alignments was generated using FastTree . Phylogenetic tree visualizations were produced using the Interactive Tree of Life (https://itol.embl.de/).