Bacterial isolates and antimicrobial susceptibility testing
During June 1, 2016 to December 31, 2017, a total of 163 KP-PLA cases were collected from the First Affiliated Hospital of Wenzhou Medical University (Wenzhou, China) with an annual admission of more than 160,000 inpatients. The diagnosis of KP-PLA was conducted based on the clinical criteria [7, 28]. Initial strains were isolated from sterile fluid (including pus, blood, and drainage fluid) of KP-PLA patients and identified as K. pneumoniae by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS; bioMérieux, Lyons, France). Antimicrobial susceptibility testing of K. pneumoniae isolates was conducted by bioMerieux VITEK-2 (BioMérieux, Marcy-l’Étoile, France) initially. Multidrug resistant strains were defined as non-susceptible to three or more different antimicrobial categories [29]. A total of 12 MDR K. pneumoniae were found in 163 KP-PLA cases. Meanwhile, an equal number of antimicrobial-susceptible hypervirulent strains were selected as the experimental hypervirulent control strains (isolated from healthy, ambulatory patients with KP-PLA and carried both aerobactin and rmpA genes) and the standard strain ATCC 700603 as the hypovirulent standard strain [1, 30].
The minimum inhibitory concentrations (MICs) of ampicillin, aztreonam, ceftriaxone, ceftazidime, cefepime, imipenem, ciprofloxacin, levofloxacin, gentamicin, tobramycin, sulfamethoxazole/trimethoprim, nitrofurantoin and colistin were confirmed by the agar dilution method and microdilution broth method. The results were interpreted by the latest guidelines published by the Clinical and Laboratory Standards Institute (CLSI; Pittsburgh, PA, USA) and the European Committee on Antimicrobial Susceptibility Testing clinical breakpoints (http://www.eucast.org). Escherichia coli ATCC25922 and Pseudomonas aeruginosa ATCC27853 were served as the quality control strains.
Growth curves
The growth curves of 12 PLA-causing MDR K. pneumoniae isolates were measured by following previous methods [31]. In brief, overnight cultures of selected K. pneumoniae clinical isolates from KP-PLA and K. pneumoniae ATCC 700603 were diluted 1:100 by Luria-Bertani (LB) broth. The cultures were incubated at 37 °C with constant shaking at 200 rpm. Bacteria suspensions were collected at 0, 2, 4, 6, 8, 10, 12, 18, 24 h and the absorbance at 600 nm was determined. Each suspension was measured in triplicates and averages of absorbance values were used for analysis. The growth of PLA-causing MDR K. pneumoniae was evaluated by plotting the values of OD600 against time.
String test and quantification of capsule
The bacterial colonies of K. pneumoniae strain on an agar plate were stretched by an inoculation loop. The string test was considered positive when the strain generates a viscous string with a length of > 5 mm, and this strain was also considered hypermucoviscous [30].
Capsule was quantified as described previously with some modifications [10, 32]. Briefly, 500 µL of cultured bacteria suspensions were resuspended and adjusted to 108 CFU/mL, and 1.2 mL sodium tetraborate in sulfuric acid were added in the resuspensions that placed in ice bath and incubated for 5 min at 100 °C, and then left on ice for 10 min. A 20 µL volume of 1.5 mg/mL m-hydroxyldiphenyl was then added and mixed. After a 5 min incubation at room temperature, the absorbance at 590 nm was measured. The glucuronic acid content was determined from a standard curve of glucuronic acid and expressed as µg/108CFU. Results were presented as the mean of the data of three independent experiments.
Susceptibility to serum killing
Serum bactericidal activity was measured using the method as described previously [6]. Bacteria suspensions in nutrient broth were collected during logarithmic phase and adjusted to 106 CFU/mL of concentration. 25 µL of bacteria suspension was added to 75 µL of pooled human sera in the tube. Tubes were shaken and incubated for 0, 1, 2, or 3 h. An aliquot of each bacterial suspension was removed at the designated time point and diluted corresponding fold by adding Mueller-Hinton broth, and then cultured to determine the number of viable bacteria after exposure to serum. Results were expressed as a percentage of the inoculum and graded, then a strain was considered serum resistant or serum sensitive according to the standards, and each strain was tested at least three times.
Biofilm formation assay
The biofilm formation assay was measured using the method of Wilksch et al. [33]. Briefly, clinical isolates were grown to logarithmic phase in LB broth and diluted 1:100 with fresh LB broth. A total of 200 µL of each dilution were added to a 96-well polystyrene microtiter plate and blank controls were set at the same time, and per strain was set three duplicate wells. Then, the plate was incubated at 37℃ for 24 h. Planktonic cells were removed, and the wells were washed three times with sterile water, and then stained with 250 µL 0.1% crystal violet for 10 min and rinsed three times with sterile water. Stained biofilms were solubilized with 95% ethanol and quantified by measuring the OD600. Each sample was measured in triplicates, and the averages of absorbance values were used for analysis.
Infection model of Galleria mellonella larvae
The model of G. mellonella larvae was carried out on the three PLA-causing MDR isolates (FK3068, FK3228, FK4603) and three control isolates (FK3112, FK3837, FK3914) that were randomly selected and standard strain ATCC 700603 to investigate the virulence and pathogenicity of the strains [34–35]. A serial concentration gradient bacterium suspension of each strain (107,106,105,104 CFU/mL) was prepared in advance. Eight larvae weighing of 200 mg − 250 mg were randomly selected for each strain and each concentration. A 10 µL of bacterial suspension was injected into the last left proleg by using a 25 µL Hamilton precision syringe. Larvae injected with 10 µL phosphate-buffered saline were used as control. And then, the insects were incubated at 37℃ in the dark and observed after 24 h, 48 h, 72 h and 96 h. Larvae were considered dead when they repeatedly failed to respond to physical stimuli.
Polymerase chain reaction (PCR) for capsular serotypes and virulence genes
Crude genomic DNA was extracted from PLA-causing K. pneumoniae isolates. Subsequently, capsular serotype-specific genes (for serotypes of K1, K2, K5, K20, K54, and K57) and virulence genes (aerobactin, rmpA, iroN, kfuBC, wcaG, ybtA, magA, fimH, mrkD, uge, entB, and ureA) were amplified by PCR using specific primes as previously described [24, 36–38]. In addition, strains with these genes determined by PCR and DNA sequencing were selected as positive control for the subsequent PCR experiments.
Multilocus sequence typing (MLST)
In this study, seven housekeeping genes of K. pneumoniae (gapA, mdh, phoE, tonB, infB, pgi and rpoB) were amplified and sequenced to characterize the genotypes of PLA-causing isolates according to the provided protocols (http://bigsdb.pasteur.fr/klebsiella/klebsiella.html/). The alleles and STs were assigned according to the online database of the Pasteur Institute MLST for K. pneumoniae.
Statistical analysis
All statistical analyses were performed using SPSS 22.0 software (IBM, Armonk, NY, USA). Continuous variables were expressed as mean values ± SD or median (25th − 75th percentile), whereas categorical variables were described as the number and percentage of subjects. Comparisons for continuous variables were made using either the Student’s t test or the Mann–Whitney U test, and comparisons for categorical variables using either the Chi-square test or Fisher’s exact test. The mortality of G. mellonella were assessed by Kaplan-Meier analysis and log-rank test.