Serotyping and MLST for K. pneumoniae isolates
Isolates were collected from a previous study . Serotyping K1 and K2 was performed by a rapid testing cassette and PCR [15, 16]. For nonserotype K1/K2 from rapid testing, serotyping was performed by using wzi and wzc sequencing [17, 18] and capsule-specific primers for serotyping . MLST with the seven housekeeping genes (gapA, infB, mdh, pgi, rpoB, phoE and tonB) was performed via PCR using the corresponding primer pairs . A total of 9 isolates were selected for this study.
Virulent gene profiling and the string test
Seven genes, entB, iroN, iucA, iutA, clbA, rmpA and rmpA2, were selected for detection of the K. pneumoniae isolate genes. The primers used for these virulence genes are listed in Supplementary Table 1 [2, 21, 22]. Bacterial DNA was prepared by suspending one loop of fresh colonies in 50 µl of DNAzol (DNAzol® DIRECT, Molecular Research Center, Inc. Cincinnati, OH) and heating the mixture at 95°C for 10 min. AmaR OnePCR (amaR OnePCR, GeneDireX®, Vegas, NV) was used as the PCR mixture, and the amplification procedure was followed by the manufacturer’s protocol.
The string test was performed as a phenotypic method to assess virulence . Isolates were streaked on a blood agar plate and kept to culture overnight. Criteria of hypermucoviscosity were defined as previously described [7, 11, 23]. The colony was stretched with an inoculation loop to measure the visible string, and more than 5 mm was considered a hypermucoviscosity phenotype.
Generate aerobactin and capsule knockout mutants by in-frame deletion
In-frame deletion mutagenesis was used to generate mutants for virulence studies . In brief, the primer sets iucA-AR and iucA-BF were used for iucA deletion construction (Supplementary Table 2), and these primer sets were complementary to each other. The PCR fragment was digested with Xbal and Sacl and then cloned into the puTkmy-MCS plasmid for 799 and 794 strains and the puTkmy-MCS-zeocin plasmid for 3016 strain. These plasmids are suicide vectors harbouring the E.coli sacB gene. When this gene is expressed on the plasmid, a sucrose-sensitive phenotype was present to enable positive selection with sucrose and detect the loss vector. The single-crossover strains were selected from green inositol-nitrate-deoxycholate (BIND) plates. The green plate was coated with kanamycin (50 µg/ml) for 799 and 794 strains and zeocin (25 µg/ml) to prevent the growth of non-Kp contaminants. The transconjugant was selected and verified with PCR via primer sets  (Supplementary Table 2).
In vitrovirulence assessment by neutrophil phagocytosis and the serum resistance assay
A neutrophil phagocytosis assay was performed as previously described . Isolation of neutrophils and pooled serum from healthy volunteers received ethics approved from the Research Ethics Committee of the National Health Research Institute (number: EC1061212-E)
The serum bactericidal assay was modified and described by Podschun’s study and from our previous study [24, 27]. In brief, the bacteria were streaked on Mueller-Hinton agar (MHA) plates overnight to collect a single colony to embed in brain heart infusion (BHI) broth and measured with optical density at 600 nm (OD600) to 0.4. After dilution with phosphate-buffered solution (PBS) from 108 to 106, the bacteria were combined with 750 µl of human serum and incubated for 0, 1, 2, and 3 hours. The mixture of each time frame was serially diluted, and 10-3, 10-4, and 10-5 were streaked on the MHA plate for visual bacterial counts.
Mouse lethality test
Male BALB/c mice aged 6-8 weeks (n=6/strain) were used in the lethality testing. They were purchased from the National Laboratory Animal Center, Taiwan, and housed in the National Defense Medical Center Laboratory Animal Center. The animal protocols of this study were reviewed and approved by the Institutional Animal Care and Use Committee of the National Defense Medical Center (IACUC-19-271) and National Health Research Institute (NHRI) (NHRI-IACUC-107009-A).
Strains were analysed by the acute lethality test in the mouse model. The day before the experiment, the bacteria were streaked on MHA plates and grown in the incubator overnight. The bacteria were transferred to BHI broth and kept in the incubator until OD600 of 0.9. After centrifugation and serial dilutions with PBS, bacteria were randomly intraperitoneally (i.p.) injection to the mice (0.1 ml/mouse, acute injection). Within 14 days of observation, the lethality and virulence of the bacterium were observed and detected by the mouse survival rate. Food and water were provided ad libitum, and cages with new bedding were changed once a week. This experiment was duplicated to confirm the virulence degree of the bacterium. The LD50 was calculated using SigmaPlot version 7.0 from SPSS Inc. (Chicago, IL).