Aim of this study
To investigate the antimicrobial effectiveness of the klebicin KvarIa in a mouse model of K. quasipneumoniae gastrointestinal (GI) colonization.
Two separate experimental settings of the murine models were used for K. quasipneumoniae colonization and KvarIa treatment. For both models, 8-10 weeks old, BALB/c strain, female (n=8; 19-25g) and male (n=16; 22-27g) mice were purchased from the Lithuanian University of Health Sciences vivarium of laboratory animals. All regulated procedures on living animals were approved by The Lithuanian Ethics Committee of Biomedical Research (Protocol no. G2-119).
GI model of K. quasipneumoniae (DSM28212) colonization and KvarIa therapy
Klebsiella quasipneumoniae clinical isolate DSM28212 was used for GI tract colonization in four different study groups containing three mice per group (m=2; f=1). Vehicle–only control group was monitored for any changes in the natural host-microbiota without any additional procedures during the period of the experiment. The ability of K. quasipneumoniae to colonize the GI tract without antibiotic pre-treatment to disrupt the host-microbiota was tested. In order to mimic hospital-acquired infections two groups were given different combinations of antibiotic treatment before infection (penicillin (2000 U/ml) + streptomycin (2 mg/ml), (pen_strep); penicillin (2000 U/ml) + streptomycin (2 mg/ml) + metronidazole (1 g/L) (pen_strep_met)) (study design in 1A fig.). For KvarIa therapy testing three groups (A; B; C) received 109 cfu of K. quasipneumoniae orally by pipette feeding once per day. From day 18th group A was given 100 µg of uncoated KvarIa, groups B and C were given 100 µg and 1000 µg of Eudragit S100-coated KvarIa respectively (detailed study design 2A fig.). For each mouse, faecal pellets were sampled.
Determination of pH of the gastrointestinal tract: As shown in figure 1A, the samples of rectum excreta were collected on six different days during the experiment. Acquired samples were homogenized with deionized water (1:10 ratio) and pH was determined using pH METER Mettler Toledo (Belgium) with the Inlab Ultra-Micro electrode. In addition, the pH was measured in the samples taken from the GI tract during the laparotomy dissection (the intestinal tract was divided into three sections: the stomach, the duodenum, and the rectum).
Klebicin production in plants and purification
Klebsiella bacteriocin KvarIa was expressed in Nicotiana benthamiana transient expression system and purified as previously described in Denkovskiene et al .
Coating of KvarIa
5% Eudragit S100 solution was prepared by dissolving 0.5 g Eudragit S100 (Evonik Industries, Germany) in 10 ml of miliQ H2O and by sonication in an ultrasonic bath for 30 min at 25°C. 250 µg of KvarIa was dissolved in 200 µg of 5% Eudragit S100. The obtained solution was lyophilized at -51°C for 24 h.
Simulated gastric digestion and residual KvarIa activity evaluation by soft agar radial diffusion assay: Protein samples (KvarIa and Eudragit S100-coated KvarIa) were dissolved in simulated gastric buffer (0.15 M NaCl, pH 2), at a concentration of 1mg/ml and incubated at 37°C with rotation at 200 rpm for 10 min. 0.025 mg (80-113 U) of pepsin from porcine gastric mucosa was added to 1 mg of protein (pepsin:protein ratio 1:40). Aliquots of reaction (50 µl) were removed at different time points (0.5 min, 5 min, 10 min, 20 min, 30 min, and 60 min after the addition of the pepsin). Digestions were stopped by raising the pH to 6.5 by the addition of 0.5 M ammonium bicarbonate to inactivate pepsin. The pH of samples was adjusted to 8.0 to get Eudragit S100 coat dissolved. The dilutions of all samples by ratio 1:2 were made in distilled water and 5 µL drops of diluted samples were applied on K.quasipneumoniae DSM28212 MHA plates for soft agar overlay assay.
Soft-agar overlay assays were performed as described by Denkovskiene et al , with some modifications. K. quasipneumoniae DSM28212 overnight culture was equalized to OD595=1.0 in Muller-Hinton medium and diluted 100-fold in 0.8% (w/v) top agar preheated in a 55°C water bath. Mixed overlay components were poured on plates containing solid medium (Muller-Hinton containing 1.5% (w/v) agar). Sterile Whatman paper discs (6 mm diameter) were placed on the surface of the soft-agar medium containing bacterial test strain and 5 µl of protein dilutions were applied to the discs. The plates were incubated overnight at 37°C and the diameter of klebicin inhibition zones was measured.
Briefly, plant-produced KvarIa and Eudragit S100-coated KvarIa were mixed with SGF at the recommended concentration and incubated for up to 60 min, sampling every few minutes and assessing the digestion of the protein into fragments by SDS-PAGE. Coomassie staining on gels was used to visualize protein decomposition and estimate the MW of peptide products. This method was only used for uncoated KvarIa, as Eudragit S100 distorted protein migration on the SDS-PAGE gel.
Nucleic acid extraction and synthesis of the cDNA
Bacterial DNA and RNA from rectum excrement samples were extracted using the AllPrep PowerFecal DNA/RNA Kit and AllPrep DNA/RNA Mini Kit (Qiagen, Germany). ~100 mg of faeces sample were used for the extraction procedures. The quantity and quality of extracted nucleic acids were evaluated by NanoDrop 2000 (Nanodrop Technologies, Wilmington, DE, USA). Subsequently, cDNA was synthesized using a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Lithuania). 18 ng of cDNA was added into the qualitative real-time PCR (qRT-PCR) reaction. All processes were completed upon the manufacturer’s instructions.
Quantitative assessment of Klebsiella quasipneumoniae using Real-Time – PCR
The haemolysin gene (khe) was chosen as the qualitative marker for Klebsiella identification [15, 16]. The standard curve was created based on DNA samples of K. quasipneumoniae (DSM28212) to test the generated primers’ efficiency. DNA-based standard curve. 103, 105, 106, 108, 109, and 1010 CFU of K. quasipneumoniae in 200 µl were subjected to DNA extraction with QIAamp Fast DNA Stool Mini Kit (protocol for liquid sample).
During this step, the reaction for the qRT-PCR was performed using TaqMan Universal Master Mix II with UNG, TaqMan probe (5-6FAM-CGCGAACTGGAAGGGCCCG-TAMRA-3), and primers (Forward: 5 -GATGAAACGACCTGA TTGCATTC-3, Reverse: 5 -CCGGGCTGTCGGGATAAG-3 (Applied Biosystems, JAV) following the manufacturer’s recommendations. The amplification of the khe gene was determined by ABI Fast 7500 System (Life Technologies, Carlsbad, CA, USA) according to standard protocol. Positive controls for DNA and RNA were isolated from K. quasipneumoniae and negative - isolated from E. coli.
The data were analysed using nonparametric tests. The difference between the four protocols groups throughout the layout of the experiment were analysed using Student’s independent t-test. Independent analyses were carried out using SPSS Version 19.0 and MiniTab 20.1.2 software packages. Results were considered statistically significant when p < 0.05 with ±95% confidence intervals.