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 in 2016 and 2017. Among them, FK3226, FK3992, FK4003 were isolated from patients with liver abscess, and FK3065, FK3087, FK3170 were isolated from the sterile sites of non-liver abscess patients (ascites, biliary drainage fluid). Identification and antimicrobial susceptibility testing were conducted on all isolates using a VITEK2 system (bioMérieux, Marcy L’Etoile, France). K. pneumoniaeATCC 700603 was served as the control strain.
Growth curves and biofilm formation
Six K. pneumoniae clinical isolates (FK3065, FK3087, FK3170, FK3226, FK3992, FK4003) and K. pneumoniae ATCC 700603 were grown in Luria-Bertani (LB) broth containing 50 μM iron, 30 μM iron, 10 μM iron, no additional iron, and 50 μM iron with 200 μM iron chelating agent (2,2’-Dipyridyl), respectively [9,10]. Samples were collected at 0 h, 2 h, 4 h, 6 h, 8 h, 16 h, 24 h and the absorbance was read at 600nm.
The biofilm assay was performed as described previously. Briefly, six K. pneumoniae clinical isolates and K. pneumoniae ATCC 700603 were grown overnight in LB broth with 50 μM iron, 30 μM iron, 10 μM iron, no additional iron, and 50 μM iron with 200 μM iron chelating agent. The overnight culture of cells was then diluted 1:100 in fresh LB broth with or without 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 negative control. Planktonic cells were removed and the wells were washed twice with sterile water, the wells were stained with 150 μL 0.1% crystal violet for 10 min and wells were rinsed twice with sterile water [11]. Stained biofilms were solubilized with 95% ethanol and quantified by measuring the OD600 using a microplate reader.
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 were listed in Table 1 [12,13]. 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 (G. mellonella) G. mellonella killing assays were carried out on five clinical isolates and K. pneumoniae ATCC 700603 as described previously, with minor modifications [14]. Eight G. mellonella weighing between 200mg–250mg were randomly selected for each strain. Larvae were injected with 10 μL of bacterial suspension (108 CFU/mL) in phosphate-buffered saline (PBS), into the last left proleg using a 25 μL Hamilton precision syringes. 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 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. P<0.05 was considered as statistically significant.
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 isolation, 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. Total RNA was extracted from culture 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 [15]. We selected several isolates to analyze the expression ofiucB, iroB, entB, and irp1 genes according to the gene carrying conditions (Table 3). The rpoB gene was used as an internal control to normalize the data. Each sample was measured in triplicates and mean of Ct values were used for analysis. Gene expression levels were calculated using 2−△△Ct method.