K. pneumoniae strains
All 43 K. pneumoniae strains were isolated from patients with PLA at Department of Infectious Diseases, the First Affiliated Hospital of Zhejiang University in 2017. The specimens included abscess, drainage, and puncture fluid. K. pneumoniae strains were confirmed using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry system (Bruker Daltonics Inc., Fremont, CA, USA). The strains were stored at − 80 °C prior to use. Standard strains K. pneumoniae ATCC 700603 and Escherichia coli ATCC 25922, purchased from the National Centre for Medical Culture Collection of China, were used as controls for strain identification and antimicrobial susceptibility testing (AST).
NTUH-K2044 (Accession number: AP006725.1) is a hypervirulent K. pneumoniae strain typed as K1 and was isolated from Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan [18]. HS11286 (Accession number: CP003200.1) is a hypovirulent K. pneumoniae typed as K47 and containing blaKPC and was isolated from Department of Laboratory Medicine, Huashan Hospital, Fudan University, Shanghai, China [19]. Strains NTUH-K2044 and HS11286 were used as controls for string test and G. mellonella lethality test.
All the strains were non-repetitive. All patients were diagnosed with PLA based on pathological and imaging evidences (B-mode ultrasonography and computed X-ray tomography).
Determination of hypermucoviscous phenotype
The hypermucoviscous phenotype was determined by “string test” as described previously [20]. Formation of a viscous string > 5 mm in length was considered as a positive phenotype.
AST analyses
AST for the 43 K. pneumoniae strains was performed using a bioMérieux VITEK-2 analyzer (bioMérieux Co., Marcy-Etoile, France) and the Kirby-Bauer (K-B) method. The GN337 card included the antibiotics ticarcillin/clavulanate, piperacillin-tazobactam, ceftazidime, cefepime, cefoperazone/tazobactam, aztreonam, imipenem, meropenem, amikacin, tobramycin, ciprofloxacin, levofloxacin, doxycycline, minocycline, tigecycline, chloramphenicol, and trimethoprim-sulfamethoxazole. The K-B method included ampicillin, cefazolin, cefuroxime, cefotaxime, gentamycin, nitrofurantoin, and fosfomycin. AST results were elucidated based on the latest guidelines by the Clinical and Laboratory Standards Institute (CLSI; Pittsburgh, PA, USA), and the latest breakpoint by the European Committee on Antimicrobial Susceptibility Testing (EUCAST; Basel, Switzerland; for tigecycline). MDR strains were characterized as strains non-susceptible to three or more antimicrobial classes [21].
Multilocus sequence typing (MLST)
DNA of all 43 K. pneumoniae strains was extracted using the QIAamp DNA mini kit (QIAGEN Co., Venlo, Netherlands). Seven housekeeping genes (gapA, infB, mdh, pgi, phoE, rpoB, and tonB) [22] were sequenced for STs of the 43 strains according to the K. pneumoniae MLST database given at the website (http://www.pasteur.fr/recherche/genopole/PF8/mlst/Kpneumoniae.html). The primers are shown in Additional file 1.
Polymerase chain reaction (PCR) for serotypes, drug-resistance, and virulence genes
Serotypes (K1, K2, K5, K20, K54, and K57) [23, 24], drug-resistance genes (blaKPC, blaKPC-2, blaCTX-M1, blaCTX-M2, blaCTX-M8, blaCTX-M9, blaOXA48, blaNDM, blaIMP, and blaSHV) and virulence genes (wzy-K1, wzx, wzc, allS, entB, irp2, ybtS, iroB, iroN, iucA, kfu, fimH, mrkD, wabG, uge, rmpA, rmpA2, c-rmpA, p-rmpA, p-rmpA2, terB, peg-344, peg-589 and peg-1631) [12, 25, 26] were all determined by regular PCR using an Applied Biosystems Veriti PCR system (ABI, San Ramon, CA, USA). The primers used are described in Additional file 1. Sequencing of wzi loci was also used to determine serotypes [27] by comparison with the database of Pasteur Institute (https://bigsdb.pasteur.fr/klebsiella/klebsiella.html).
Definitions of putative HvKP, cKP, Hv-CRKP, and carbapenem-resistant HvKP strains
Hypercapsule-associated genes (wzy-K1, c-rmpA, p-rmpA and p-rmpA2) and siderophore genes (entB, irp2, iroB, and iroN) were included for screening HvKP and cKP. HvKP and cKP were putatively defined as described previously [28]. CRKP was defined as K. pneumoniae strains that are non-susceptible to imipenem or meropenem. Hv-CRKP was defined as CRKP (cKP) that acquires key virulence genes that confer hypervirulence. Carbapenem-resistant HvKP was defined as HvKP (serotypes K1, K2, K5, K10, K20, K25, K27, and K57) that acquires carbapenem resistance.
MGE and PFGE analyses
MGE and PFGE analyses were both performed as the reference [29].
G. mellonella lethality test
G. mellonella larvae were used to determine the lethality of K. pneumoniae strains [30]. G. mellonella larvae, weighing approximately 300 mg, were purchased from Tianjin Huiyude Biotech Company, Tianjin, China. Mid-log phase cultures of K. pneumoniae strains were washed with phosphate-buffered saline and further adjusted to a concentration of 1 × 107 CFU/mL. Ten G. mellonella larvae were used per test. Survival analysis was done to compare the lethality of K. pneumoniae strains. All experiments were performed in triplicates.
Statistical analysis
GraphPad Prism 8 (GraphPad Software Inc., USA) was used to perform Chi-square test, Fisher’s exact test and survival analysis. The value of p < 0.05 was regarded as statistically significant.