Bacterial strains and antimicrobial susceptibility profiling
Six K. pneumoniae clinical isolates (FK3065, FK3087, FK3170, FK3226, FK3992, FK4003) were collected from patients in the First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China during 2016 and 2017. Herein, FK3226, FK3992, FK4003 were isolated from patients with liver abscess, and FK3065, FK3087, FK3170 were isolated from other abdominal invasive infection sites of non-liver abscess patients (ascites, biliary drainage fluid). Identification was conducted on all isolates using VITEK MS system (bioMérieux, Marcy L’Etoile, France). Antimicrobial susceptibility testing was performed by VITEK2 system (bioMérieux, Marcy L’Etoile, France) with AST-GN13 card. K. pneumoniae ATCC 700603 served as the control strain.
Growth curves and biofilm formation
The effect of iron on the growth of K. pneumoniae was measured following previous methods with some modifications [11,12]. In brief, overnight cultures of all K. pneumoniae clinical isolates (FK3065, FK3087, FK3170, FK3226, FK3992, FK4003) and K. pneumoniae ATCC 700603 were diluted 1:100 in Luria-Bertani (LB) broth supplemented with different iron concentrations (50 μM, 30 μM, 10 μM, 0 μM) and with 200 μM iron chelating agent (2,2'-Dipyridyl) +(-) 50 μM iron, respectively. Wherein, strains under LB + 200 μM iron chelating agent condition were used as control and LB + 200 μM iron chelating agent + 50 μM iron was set to produce an iron-restricted condition. The cultures were incubated at 37 °C with constant shaking at 180 rpm. Samples were collected at 0 h, 2 h, 4 h, 6 h, 8 h, 16 h, 24 h and the absorbance at 600nm was determined. Each sample was measured in triplicates and averages of absorbance values were used for analysis. The growth of K. pneumoniae was evaluated by plotting the values of OD600 against time.
The biofilm assay was performed as published with some modifications [13]. Briefly, six K. pneumoniae clinical isolates and K. pneumoniae ATCC 700603 were grown overnight in LB broth. The overnight cultures were then diluted 1:100 in fresh LB broth supplemented with different iron concentrations (50 μM, 30 μM, 10 μM, 0 μM) and with 200 μM iron chelating agent +(-) 50 μM iron. A total of 100μL of each dilution were added to a 96-well polystyrene microtiter plate and incubated at 37 ℃ for 24 h. Wells containing media alone were used as blank. Planktonic cells were removed and the wells were washed twice with sterile water, then the wells were stained with 150 μL 0.1% crystal violet for 10 min and rinsed twice with sterile water. Stained biofilms were solubilized with 95% ethanol and quantified by measuring the OD600 using a microplate reader. Each sample was measured in triplicates and averages of absorbance values were used for analysis.
Detection of virulence genes of isolates
Virulence genes (magA, iucB, iroB, entB, irp1, iroN, kfuBC, rmpA, wcaG, alls, ybtA, ureA, uge, wabG, fimH and mrkD) and capsular serotypes (K1, K2, K5, K20, K54 and K57) of the six K. pneumoniae clinical isolates and K. pneumoniae ATCC 700603 were amplified by Polymerase Chain Reaction (PCR). Primers for the aforementioned genes are listed in Table 1 [14,15]. Positive PCR products were sequenced by Beijing Genomics Institute Technology Co. Ltd (Shanghai, China). Nucleotide sequences were compared using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi).
Infection model of Galleria mellonella larvae
G. mellonella killing assays were carried out on the six clinical isolates and K. pneumoniae ATCC 700603 as described previously, with minor modifications [16]. Eight larvae weighing between 200mg-250mg were randomly selected for each strain. A 10 μL of bacterial suspension (108 CFU/mL) in phosphate-buffered saline (PBS) was injected into the last left proleg using a 25 μL Hamilton precision syringe. The bacterial suspension was prepared by culturing the strains in LB broth containing 50 μM iron, and 50 μM iron with 200 μM iron chelating agent for 24 h. Larvae injected with 10 μl PBS were used as control. The insects were incubated at 37℃ in the dark and observed after 24 h, 48 h and 72 h. Larvae were considered dead when they repeatedly failed to respond to physical stimuli. The primary outcome for the insect model was rapidity and extent of mortality of G. mellonella assessed with Kaplan-Meier analysis and log-rank test.
Quantitative reverse transcription PCR (qRT-PCR)
The effects of iron on the expression levels of K. pneumoniae siderophore genes (iucB, iroB, entB and irp1) were evaluated using quantitative reverse transcription PCR (qRT-PCR). For RNA extraction, K. pneumoniae isolates were grown in fresh LB medium with 50 μM iron, and 50 μM iron with 200 μM iron chelating agent at 37 ℃ for 24h. K. pneumoniae isolates grown in fresh LB medium were used as control. Total RNA was extracted using a RNeasy Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. The extracted RNA samples were stored at −80 ℃. Purified RNA was reverse transcribed into cDNA for qRT-PCR analysis using a cDNA synthesis kit (TaKaRa, Tokyo, Japan) according to the manufacturer’s instructions. Gene expression levels were measured with qRT-PCR using a 7500 RT-PGE system (TOYOBO, Osaka, Japan) and SYBR Green qRT-PCR Kit (TOYOBO) with the specific primers listed in Table 2 [17]. The rpoB gene was used as an internal control to normalize the data. Each sample was measured in triplicates and averages of Ct values were used for analysis. Gene expression levels were calculated using 2−△△Ct method.
Statistical Analysis
All experiments were conducted independently with at least two replicates on different days, and results were expressed as mean ± standard deviation or average. The total area under the curve was calculated for analysis on growth. Unpaired or two-tailed paired t-tests were used to evaluate the significance of differences between two groups. One-way analysis of variance (ANOVA) was performed to analyze the significance among more groups. Statistical significance was determined at P < 0.05. Statistical analyses were performed using SPSS version 17.0 statistical software.