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Reduction of gastrointestinal tract colonization by Klebsiella quasipneumoniae using antimicrobial protein KvarIa

Reduction of gastrointestinal tract colonization by Klebsiella quasipneumoniae using... Background: Klebsiella quasipneumoniae is an opportunistic pathogen causing antibiotic-resistant infections of the gastrointestinal tract in many clinical cases. Orally delivered bioactive Klebsiella-specific antimicrobial proteins, klebicins, could be a promising method to eradicate Klebsiella species infecting the gut. Methods: Mouse infection model was established based on infection of antibiotic-treated BALB/C mice with K. quasipneumoniae strain DSM28212. Four study groups were used (3 animals/group) to test the antimicrobial efficacy of orally delivered klebicin KvarIa: vehicle-only group (control, phosphate-buffered saline), and other three groups with bacteria, antibiotic therapy and 100 µg of uncoated Kvarla, 100 µg coated KvarIa, 1000 µg coated-KvarIa. Because of the general sensitivity of bacteriocins to gastroduodenal proteases, Kvarla doses were coated with Eudragit , a GMP-certified formulation agent that releases the protein at certain pH. The coating treatment was selected based on measurements of mouse GI tract pH. The quantity of Klebsiella haemolysin gene (khe) in faecal samples of the study animals was used to quantify the presence of Klebsiella. Results: GI colonization of K. quasipneumoniae was achieved only in the antibiotic-treated mice groups. Significant changes in khe marker quantification were found after the use of Eudragit S100 formulated klebicin KvarIa, at both doses, with a significant reduction of K. quasipneumoniae colonization compared to the vehicle-only control group. Conclusions: Mouse GI tract colonization with K. quasipneumoniae can be achieved if natural gut microbiota is sup- pressed by prior antibiotic treatment. The study demonstrates that GI infection caused by K. quasipneumoniae can be significantly reduced using Eudragit -protected klebicin KvarIa. Keywords: Klebsiella quasipneumoniae, Klebicins, KvarIa, Haemolysin gene, Bacteriocins flora of the human mouth or intestines [1, 2]. Patho- Background genic Klebsiella species, such as K. pneumoniae and Klebsiella is a gram-negative and facultative anaerobic K. oxytoca, are the most prevalent infections acquired bacterium of the Enterobacteriaceae family that colo- in hospital (HAI) [2, 3]. Recent studies have identified nizes various environmental niches including normal K. quasipneumoniae as a new species distinguishable from K. pneumoniae. K. quasipneumoniae has been *Correspondence: jurgita.skieceviciene@lsmuni.lt shown to act as an etiological agent in a number of Indre Karaliute and Rima Ramonaite contributed equally to this work clinical Klebsiella-related infection cases, but has often Institute for Digestive Research, Laboratory of Clinical and Molecular been misidentified as K. pneumoniae in HAI [4, 5]. K. Gastroenterology, Lithuanian University of Health Sciences, Mickeviciaus st. 9, 44307 Kaunas, Lithuania quasipneumoniae colonizes the intestinal tract, which Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visithttp:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Karaliute et al. Gut Pathogens (2022) 14:17 Page 2 of 11 can lead to virulent urinary tract and abdominal infec- Methods tions [3–6]. Furthermore, this type of bacteria shows Aim of this study high rates of resistance against antibiotics, and some To investigate the antimicrobial effectiveness of the strains have been characterized as pan-drug resistant, klebicin KvarIa in a mouse model of K. quasipneumo- making these infection extremely difficult to treat [7, niae gastrointestinal (GI) colonization. 8]. For this reason, the development of new antimicro- bial agents is required to mitigate bacterial infections, Mouse models typically acquired by patients while in-hospital. Two different animal study designs were used for K. Colicin-like bacteriocins, produced by gram-neg- quasipneumoniae colonization and KvarIa treatment. ative bacteria, may be a potential alternative to tradi- For both studies, 8–10 weeks old, BALB/c strain, female tional antibiotics [9, 10]. Colicin-like bacteriocins are (n = 8; 19–25  g) and male (n = 16; 22–27  g) mice were a heterogeneous family of proteinaceous toxins, which purchased from the Lithuanian University of Health are capable of killing closely related bacteria, those Sciences vivarium of laboratory animals. All regulated belonging to the same species or, sometimes, the same procedures on living animals were approved by The Lith - genus [11]. This property makes them attractive as uanian Ethics Committee of Biomedical Research (Proto- therapeutics since they offer a more targeted approach col no. G2-119). than conventional antibiotics. Indeed, one of the major issues with conventional antibiotics is the dysbiosis GI model of K. quasipneumoniae (DSM28212) colonization induced by the broad-range killing of bacteria [12, 13]. and KvarIa therapy Most importantly, the mechanisms of bacterial killing Klebsiella quasipneumoniae clinical isolate DSM28212 was by bacteriocins are fundamentally different from those used for GI tract colonization in four different study groups by antibiotics. Consequently, they are active against containing three mice per group (m = 2; f = 1). Vehicle– multi-drug and pan-drug resistant pathogens. only control group was monitored for any changes in the The authors have previously identified and charac- natural host-microbiota without any additional procedures terized several Klebsiella colicin-type bacteriocins, during the period of the experiment. The ability of K. qua - klebicins, which exhibit significant and broad activity sipneumoniae to colonize the GI tract without antibiotic against the pathogenic Klebsiella species. Orally deliv- pre-treatment to disrupt the host-microbiota was tested. In ered klebicins have a potential as an excellent means to order to mimic hospital-acquired infections two groups were eradicate intestinal infections in hospitalized patients given different combinations of antibiotic treatment before that are caused by the multidrug-resistant Klebsiella infection (penicillin (2000 U/ml) + streptomycin (2  mg/ml), strains. However, the proteinaceous nature of bacte- (pen_strep); penicillin (2000 U/ml) + streptomycin (2  mg/ riocins makes them susceptible to quick inactivation ml) + metronidazole (1  g/L) (pen_strep_met)) (study design by gastroduodenal enzymes. Therefore, for the oral in Fig. 1A). These particular antibiotics were chosen because administration of klebicins, they must be encapsulated of their broad mechanism of action against gram-negative or formulated for gastroduodenal protection, for the and gram-positive bacteria. For KvarIa therapy testing the release in the small and large intestine. same composition of antibiotic pre-treatment was used as In this study, the possibility to use klebicins to eradi- in the previously described study. Three groups (A; B; C) cate intestine colonizing Klebsiella was tested in K. 9 received 10   cfu of K. quasipneumoniae orally by pipette quasipneumoniae–KvarIa model. Klebicin KvarIa is feeding once per day. Afterward, ampicillin (500 mg/l) ther- pore forming bacteriocin, highly active against K. qua- apy was used to maintain as low as possible viability of other sipneumoniae [14]. We developed a mouse model of than K. quasipneumoniae bacteria. From day 18th group A K. quasipneumoniae intestinal colonization, tested the was given 100 µg of uncoated KvarIa, groups B and C were pH condition of the mouse GI tract, and established given 100  µg and 1000  µg of Eudragit S100-coated KvarIa a suitable coating for klebicin KvarIa. We also evalu- respectively (detailed study design Fig. 2A). For each mouse, ated the antimicrobial activity of the orally delivered faecal pellets were sampled. Eudragit S100-formulated klebicin in the mouse intes- tinal tract. We confirmed that, even without further Determination of pH of the gastrointestinal tract bacteriocin engineering and improvement, bacterioc- As shown in Fig. 1A, the samples of rectum excreta were col- ins could be employed as oral antimicrobials for effi- lected on six different days during the experiment. Acquired cient control of antibiotic-resistant Klebsiella. samples were homogenized with deionized water (1:10 ratio) K araliute et al. Gut Pathogens (2022) 14:17 Page 3 of 11 and pH was determined using pH METER Mettler Toledo K. quasipneumoniae DSM28212 overnight culture was (Belgium) with the Inlab Ultra-Micro electrode. In addition, equalized to OD = 1.0 in Muller-Hinton medium the pH was measured in the samples taken from the GI tract and diluted 100-fold in 0.8% (w/v) top agar preheated during the laparotomy dissection (the intestinal tract was in a 55 °C water bath. Mixed overlay components were divided into three sections: the stomach, the duodenum, and poured on plates containing solid medium (Muller- the rectum). Hinton containing 1.5% (w/v) agar). Sterile Whatman paper discs (6 mm diameter) were placed on the surface Klebicin production in plants and purification of the soft-agar medium containing bacterial test strain Klebsiella bacteriocin KvarIa was expressed in Nicotiana and 5  µl of protein dilutions were applied to the discs. benthamiana transient expression system and purified as The plates were incubated overnight at 37  °C and the previously described in Denkovskiene et al. [14]. diameter of klebicin inhibition zones was measured. Briefly, plant-produced KvarIa and Eudragit S100- Coating of KvarIa coated KvarIa were mixed with SGF at the recom- 5% Eudragit S100 solution was prepared by dissolv- mended concentration and incubated for up to 60 min, ing 0.5  g Eudragit S100 (Evonik Industries, Germany) in sampling every few minutes and assessing the digestion 10  ml of miliQ H O and by sonication in an ultrasonic of the protein into fragments by SDS-PAGE. Coomassie bath for 30 min at 25 °C. 250 µg of KvarIa was dissolved staining on gels was used to visualize protein decompo- in 200 µg of 5% Eudragit S100. The obtained solution was sition and estimate the MW of peptide products. This lyophilized at − 51 °C for 24 h. method was only used for uncoated KvarIa, as Eudragit S100 distorted protein migration on the SDS-PAGE gel. Simulated gastric digestion and residual KvarIa activity evaluation by soft agar radial diffusion assay Protein samples (KvarIa and Eudragit S100-coated Nucleic acid extraction and synthesis of the cDNA KvarIa) were dissolved in simulated gastric buffer (0.15 M Bacterial DNA and RNA from rectum excrement sam- NaCl, pH 2), at a concentration of 1 mg/ml and incubated ples were extracted using the AllPrep PowerFecal DNA/ at 37  °C with rotation at 200  rpm for 10  min. 0.025  mg RNA Kit. Up to 100 mg of faeces sample were used for (80–113 U) of pepsin from porcine gastric mucosa was the extraction procedures. The quantity and quality of added to 1 mg of protein (pepsin:protein ratio 1:40). Ali- extracted nucleic acids were evaluated by NanoDrop quots of reaction (50  µl) were removed at different time 2000 (Nanodrop Technologies, Wilmington, DE, USA). points (0.5  min, 5  min, 10  min, 20  min, 30  min, and Subsequently, cDNA was synthesized using a High- 60 min after the addition of the pepsin). Digestions were Capacity cDNA Reverse Transcription Kit (Thermo stopped by raising the pH to 6.5 by the addition of 0.5 M Fisher Scientific, Lithuania). 18 ng of cDNA was added ammonium bicarbonate to inactivate pepsin. The pH of into the qualitative real-time PCR (qRT-PCR) reaction. samples was adjusted to 8.0 to get Eudragit S100 coat All processes were completed upon the manufacturer’s dissolved. The dilutions of all samples by ratio 1:2 were instructions. made in distilled water and 5 µL drops of diluted samples were applied on K.quasipneumoniae DSM28212 MHA Quantitative assessment of Klebsiella quasipneumoniae plates for soft agar overlay assay. using Real‑Time–PCR Soft-agar overlay assays were performed as described The haemolysin gene (khe) was chosen as the qualita - by Denkovskiene et  al. [14], with some modifications. tive marker for Klebsiella identification [15, 16]. The (See figure on next page.) Fig. 1 Experimental design of K. quasipneumoniae colonization and pH of GI. A The design of the study of mice gut colonization by K. quasipneumoniae. 1st Vehicle-only control group with natural microbiota (n = 3); 2nd Bacterial control group (n = 3); 3rd, 4th antibiotic pre-treated groups [(i) penicillin, streptomycin (pen_strep group) or (ii) penicillin, streptomycin, and metronidazole (pen_strep_met group)]. Samples were collected on circled days. B The pH values measured in the samples of rectum excreta throughout the layout of the experiment protocol in order to determine the changes in the GI tract using different substances. The control group showed an average of 7.34 pH with the lowest being 6.99 pH and the highest 7.76 pH; the infected mice group without antibiotics showed almost no changes in pH measures through the days (av. 7.27 pH); K. quasipneumoniae infected group was treated with antibiotic therapy (penicillin, streptomycin) and showed stable results (av. 7.7 pH) with th an exception on the 6 day of the experiment (7.94 pH); the last group, which was infected with K. quasipneumoniae showed the most noticeable changes during the combined antibiotic (penicillin, streptomycin, metronidazole) treatment and bacterial colonization with the average of 7.44 pH fluctuating from 6.57 pH to 8.11 pH. No significant differences were found in or between the groups. C Overall differences in values of stomach, duodenum, and rectum of pH measurements between groups. The lowest pH was seen in the stomach (2.34 – 5.46; avg. 3.3). The pH of the small intestine varied between 5.97 and 6.77 (avg. 6.5), with the large intestine showing the highest pH (6.74–8.15; avg. 7.4). A significant difference (p < 0.05) in pH measures was found in the duodenum between 3rd and 4th groups (#), as well as between 1st group and 3rd (*) in rectum Karaliute et al. Gut Pathogens (2022) 14:17 Page 4 of 11 Fig. 1 (See legend on previous page.) K araliute et al. Gut Pathogens (2022) 14:17 Page 5 of 11 Fig. 2 Experimental design of K. quasipneumoniae therapy and assessment of Klebsiella quasipneumoniae colonization. A The design of the study of mice gut colonization by K. quasipneumoniae and KvarIa treatment. All four experimental groups had antibiotic pretreatment, ampicillin therapy, bacterial administration, and different treatment for infection (PBS; uncoated-KvarIa; Eudragit S100-coated KvarIa 100 µg; Eudragit S100-coated KvarIa 1000 µg). Samples were collected on circled days. B Amplification of khe gene in mice faeces after the introduction of K. quasipneumoniae. khe gene was detected by RT-PCR. A lower CT value indicates a higher number of bacteria. The 4th day’s samples were used as a control group because they did not show any bacterial colonization. Statistically significant differences between mice groups (K. quasipneumoniae + Pen_ Strep + Amp vs K. quasipneumoniae + Pen_ Strep_Met + Amp) were not determined. Pen–penicillin, Strep–streptomycin, Met–metronidazole, Amp–ampicillin standard curve was created based on DNA samples of UNG, TaqMan probe (5-6FAM-CGC GAA CTG GAA K. quasipneumoniae (DSM28212) to test the generated GGG CCC G-TAMRA-3), and primers (Forward: 5 -GAT primers’ efficiency. DNA-based standard curve. 10 , GAA ACG ACC TGA TTG CAT TC-3, Reverse: 5 -CCG 5 6 8 9 10 10, 10, 10, 10 , and 10   CFU of K. quasipneumo-GGC TGT CGG GAT AAG -3 (Applied Biosystems, JAV) niae in 200  µl were subjected to DNA extraction with following the manufacturer’s recommendations. The QIAamp Fast DNA Stool Mini Kit (protocol for liquid amplification of the khe gene was determined by ABI sample). Fast 7500 System (Life Technologies, Carlsbad, CA, USA) During this step, the reaction for the qRT-PCR was according to standard protocol. Positive controls for performed using TaqMan Universal Master Mix II with Karaliute et al. Gut Pathogens (2022) 14:17 Page 6 of 11 DNA and RNA were isolated from K. quasipneumoniae colonization of mice gut by K. quasipneumoniae can be and negative—isolated from Esherichia coli. established after eradication of natural gut microflora. Statistical analysis Eudragit S100‑coated KvarIa is partially protected The data were analysed using nonparametric tests. The from digestion by simulated gastric fluid difference between the four protocols groups through - To find out if Eudragit S100-coated KvarIa is resistant to out the layout of the experiment were analysed using pepsin digestion, a simulated gastric digestion experi- Student’s independent t-test. Independent analyses were ment was performed. Exposures of the proteins to Sim- carried out using SPSS Version 19.0 and MiniTab 20.1.2 ulated Gastric Fluid (SGF, commercial acidic pepsin software packages. Results were considered statistically extract) were done using low enzyme-to-substrate ratios significant when p < 0.05 with ± 95% confidence intervals. in order to increase the stringency and relevance of the digestibility assays. Methods were derived from [17–19]. Results It appears, that in conditions used (pepsin:protein Selection of bacteriocin Kvarla coating by pH ratio 1:40), protein coating with Eudragit S100 is able to measurements along the GI tract provide temporal resistance to pepsin digestion. Coated In order to determine the efficient coating and delivery of KvarIa demonstrated still detectable activity in agar diffu - the klebicins in the GI tract, firstly we performed the pH sion assay after 20 min of in vitro gastric digestion, while measurements in faeces and along the GI tract. The pH of uncoated KvarIa was inactivated in simulated gastric the faecal samples is shown in Fig. 1B. The lowest pH was juice very quickly, and completely lost its activity already seen after penicillin, streptomycin, and metronidazole after 0.5  min of digestion (Fig.  3). From the SDS-PAGE treatment. Nonetheless, there was no observation of any profile of uncoated KvarIa digestion products, it is appar - statistically significant changes in GI tract pH between ent that uncoated klebicin in digested by pepsin very groups (average mean ± SD: vehicle-only control group rapidly. 7.3 ± 0.52; bacterial control group 7.5 ± 0.74; pen_strep group 7.7 ± 0.29; pen_strep_met group 7.4 ± 0.62).  The pH levels of the different GI tract sections was also Eudragit S100‑coated KvarIa efficiently reduces K. measured after the mouse decapitation (the stomach quasipneumoniae amount in colon 3.3 ± 0.92, small intestine 6.5 ± 0.34, the large intestine We evaluated the effectiveness of Eudragit S100-coated 7.4 ± 0.42; Fig.  1C) The significant differences in pH KvarIa (100  µg; 1000  µg) in K. quasipneumoniae infec- measures were found between the vehicle-only control tion model. Three different combinations of recom - group and pen_strep group (p = 0.004) in duodenum, binant klebicin were used: uncoated-KvarIa, Eudragit vehicle-only control group and pen_strep_met group S100-coated KvarIa 100  µg, and Eudragit S100-coated (p = 0.04) in rectum. KvarIa 1000  µg. The amplification of the khe marker gene was significantly higher in both Eudragit S100- Colonization of mice gut by K. quasipneumoniae coated KvarIa-treated mice groups than in the control is achieved only after disruption of natural microflora (PBS) and uncoated-KvarIa-treated mice on the last The GI tract infection/colonization model was estab - day of the experiment (22nd day), meaning that bacte- lished in mice using Klebsiella quasipneumoniae. It was rial counts were lower. As shown in Fig.  4 the bacterial designed to reflect bacterial colonization in the host counts were significantly lower after the treatment with after the disruption of natural microflora with antibiot - Eudragit S100-coated KvarIa 100  µg and 1000  µg in ics therapy. There was no colonization of K. quasipneu - contrast with the samples taken on the first day of bac - moniae observed in the vehicle-only control group. The teriocin administration (18th day). The amounts of K. use of antibiotic pre-treatment ((i) penicillin, strepto- quasipneumoniae changed from 6.3 × 10   CFU/50  mg mycin or (ii) penicillin, streptomycin, and metronida- on the 18th day to 3.9 × 10   CFU/50  mg on the 22nd zole), in order to disrupt the host microbiota, resulted day (p = 0.01) in the Eudragit S100-coated KvarIa 100 µg in introduction of K. quasipneumoniae (4th day no bac- group and from 4.0 × 10   CFU/50  mg on the 18th day terial counts were found (0  CFU/70  mg); (i) 8th day— 8 8 to 1.6 × 10   CFU/50  mg on 22nd day (p = 0.009) in the 5.25 × 10  CFU/70 mg, 11th day—3.01 × 10  CFU/70 mg Eudragit S100-coated KvarIa 1000  µg group. No sig- and (ii) 8th day—5.13 × 10   CFU/70  mg and 11th day— nificant changes in bacterial counts were seen in the 3.89 × 10   CFU/70  mg) (Fig.  2). Our data showed that K araliute et al. Gut Pathogens (2022) 14:17 Page 7 of 11 Fig. 3 Evaluation of stability and activity of KvarIa after in vitro gastric digestion assay. A Evaluation of residual activity by agar diffusion assay. Protein samples were incubated at 37 °C, 200 rpm in gastric digestion buffer (pepsin:protein ratio 1:40). Aliquots of reaction (50 µl) were removed at different time points (0.5, 5, 10, 20, 30, and 60 min) and digestion was stopped by the addition of 0.5 M ammonium bicarbonate to inactivate pepsin. The pH of samples with coated KvarIa was adjusted to 8 to get Eudragit 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. B SDS-PAGE of KvarIa after simulated gastric digestion. The 16% TRIS-Tricine gels; unstained marker—PageRuler Unstained Low Range Protein Ladder ( ThermoFisher), 10 µl of each sample loaded per line. Pepsin appears as the stable band between 30 and 42 kDa markers (green arrow). The red arrow indicates the full-size KvarIa. Exposure of KvarIa to pepsin (1:40 pepsin:lysin wt:wt) in acidic SGF results in the rapid breakdown of the protein to lower MW degradation products. Sampling times are shown in minutes vehicle-only control group (PBS) and after the adminis- Discussion tration of uncoated-KvarIa. A rapidly increasing number of antibiotic-resistant and/ or highly virulent bacterial strains is a serious challenge Karaliute et al. Gut Pathogens (2022) 14:17 Page 8 of 11 Fig. 4 Bacterial counts in GI tract after KvarIa therapy. khe gene from DNA templates was determined using RT-PCR. A lower CT value indicates a higher number of bacteria (see Standard Curve). khe has not been detected in all study groups on the 4th day. * Statistically significant differences between 18th and 22nd days in all the experimental groups treated by Eudragit S100-coated KvarIa, p < 0.05. PBS (control) and uncoated-KvarIa (100 µg) treated mice vs Eudragit S100-coated KvarIa (100 µg) treated group on the 22nd day, p < 0.05. PBS (control) and Uncoated KvarIa (100 µg) treated mice vs Eudragit S100-coated KvarIa (1000 µg) treated group on the 22nd day, p < 0.05 faced by today’s healthcare system worldwide. Recent Bacteria were not detected in the bacterial control studies indicate that patients, with hospital-acquired group without prior antibiotic treatment (judged by khe multidrug-resistant K. pneumoniae infection, have a amplification). Similar findings were observed in other significantly higher risk of developing a subsequent Klebsiella mouse models where amoxicillin disruption infection caused by identical bacteria [20–23]. K. qua- of the gut microbiota was accompanied required for gut sipneumoniae were initially thought to be asymptomatic colonization and an enhancement of the virulence of K. carriage isolates until more recent reports highlighted variicola [27]. Other studies illustrated that mouse mod- their potential virulence and increased drug resistance els of K. pneumoniae and treatment with antibiotics led [20–23]. to changes in the host microbiota and the development K. pneumoniae has been extensively studied in many of a transient super-shedder phenotype, which displays different animal models, including models for blood - the enhanced transmission efficiency of bacteria in the stream infections, pneumonia, liver abscess, digestive GI tract [28, 29]. Allegedly, the natural host microbiota and urinary tract infections [24, 25]. On the other hand, activates the defence mechanisms against K. quasip- little is known about closely related species recently sep- neumoniae and inhibits colonization, whereas reduced arated from K. pneumoniae such as K. variicola and K. microbial diversity might promote the ability to infect. quasipneumoniae. There were only limited animal stud - However, the exact mechanisms causing K. quasipneu- ies with K. variicola such as experiments on the bacte- moniae colonization needs further investigation. ria’s ability to colonize the intestinal tract and the host It is known that bacteriocins have antimicrobial immune system response against this opportunistic path- activities against pathogenic microorganisms [30, 31]. ogen [26, 27]. K. quasipneumoniae has been detected in Previous studies have identified various classes of bac - the clinical settings during hospital infections, however, teriocins (e.g.: colicin-like bacteriocins, tailocins, pep- the species has not been tested in animal efficacy mod - tide microcins) and their potential applications in food els, and mechanisms of infection by this bacterium are technology, treatments of infection, and cancer [32–34]. poorly understood. Therefore, the first goal of our study Earlier, we demonstrated the antibacterial efficacy of was to establish an animal model of K. quasipneumo- Klebsiella bacteriocins, klebicins, in  vitro using clini- niae intestinal colonization, in particular, identify the cal Klebsiella isolates. Recombinant pore-forming bac- conditions that allow bacteria to successfully grow in the teriocin KvarIa was identified as one of the most active mouse intestinal tract. We demonstrate here that for suc- klebicins; it showed the highest activity against K. qua- cessful colonization of mice gut by K. quasipneumoniae, sipneumoniae strains and was also tested in  vivo in a the disturbance of natural gut microflora using antibiotic non-mammal animal model, Galleria mellonella larvae, pre-treatment is necessary and sufficient. demonstrating significant antibacterial effect [14]. In this study, we developed a mouse model of intestinal tract K araliute et al. Gut Pathogens (2022) 14:17 Page 9 of 11 infection using K. quasipneumoniae and evaluated the should be studied in validated preclinical animal models effectiveness of Eudragit S100-coated KvarIa treatment to further optimize efficacy of bacteriocins as antibacteri - with the main purpose of investigating the potential of als for intestinal infections. klebicins as a clinical antimicrobials. The obtained results can further be translated to K. pneumoniae and other Conclusions more clinically relevant Klebsiella species. This study demonstrated that successful colonization of The authors determined the most effective coating for the mouse intestinal tract by K. quasipneumoniae can be bacteriocin needed for delivery of the highest concentra- achieved but it requires the eradication of gut resident tions of the klebicin to the large colon. The pH in the GI microbiota with an antibiotic. We also evaluated the anti- tract is a substantial factor, affecting the solubility and microbial activity of the orally delivered Eudragit S100-for- stability of the drug and absorption through the intesti- mulated klebicin in the mouse intestinal tract and show nal tract mucosa. It can vary depending on the diet type, that thus formulated bacteriocins could be employed as fed or fasted states, drugs, microbiota diversity, stress, oral antimicrobials for efficient control of antibiotic-resist - and daily fluid intake. Henceforth, unsuitable pH causes ant Klebsiella. the precipitation of drugs from the solution or the deg- radation of labile compounds [35–37]. Correspondingly, Abbreviations an assessment of pH levels in the GI tract was included PBS: Phosphate-buffered saline; GI: Gastrointestinal; khe: Haemolysin gene; in our study. We distinguished the increased pH level of HAI: Hospital-acquired infection; pen: Penicillin; strep: Streptomycin; met: the rectum content sample in the K. quasipneumoniae Metronidazole; SGF: Sarcoma grow factor; SDS: Sodium dodecyl sulfate; MW: Molecular weight; DNA: Deoxyribonucleic acid; RNA: Ribonucleic acid; cDNA: colonized mice groups treated with antibiotics. However, Complementary Deoxyribonucleic acid; qRT-PCR: Qualitative real-time poly- mice without antibiotics did not show any change in pH merase chain reaction; CFU: Colony-forming unit. levels. Similar results were obtained by Shimizu and col- Acknowledgements leagues in ICR mice housed obtaining specific pathogen- Not applicable. free conditions, there the pH of the cecum and colon increased exceedingly in the experimental groups treated Author contributions IK, RR carried out the experiment, derived the models, analysed samples and with antibiotics [38]. Therefore, the pH measurements of data, interpreted the results, drafted the manuscript, prepared the figures, the GI tract were taken into account when choosing the edited the final version of the manuscript; ED, AM performed the experiments most effective coating for KvarIa delivery. and analyzed the data; JK and JS edited the final version of the manuscript and contributed to the conception and design of the research; JB contributed In this study recombinant bacteriocins KvarIa abil- to the initial part of the experiment related to pharmaceutics; VP contributed ity to eliminate the intestinal tract infection was judged to the animal models and experimental layout; AR and YG contributed to the using khe gene quantification. We identified that both conception and design of the research, interpreted the results, and approved the final version of the manuscript. All authors read and approved the final concentrations (100  µg and 1000  µg) of coated-KvarIa manuscript. significantly reduced the infection in the GI tract of mice models. However, in our study, we did not achieve Funding This work was supported by the Lithuanian Business Support Agency grant full eradication of the K. quasipneumoniae. KvarIa was J05-LVPA-K-03–0011 “Antimicrobial substance for treatment of Klebsiella encapsulated with Eudragit S100 releasing klebicin at infections”. pH above 7 and administered by oral gavage to infected Availability of data and materials (K. quasipneumoniae) mice. Debatably, klebicin activity All data generated during this study are included in this article. could be suppressed or significantly lowered because of the gut microflora disruption or not full eradication, as Declarations well as, dependence on the pH level, which can fluctuate throughout the GI tract for various reasons (e.g. fasting Ethics approval and consent to participate All procedures performed in studies involving animals were in accordance state). Recently, a similar study was published describing with Directive 2010/63/EU and the ethical standards of Lithuanian Univer- the use of encapsulated colicins for the eradication of  E. sity of Health Sciences (approved by State Food and Veterinary Service; No. coli  in mice [36.] Colicins encapsulated into hydrogel G2-119) at which the studies were conducted. This article does not contain any studies with human participants performed by any of the authors. particles were shown to be released from the protective coat at pH above 5 and reduce colonizing E. coli numbers Consent for publication in the gut and in feces, although complete eradication of Not applicable. the pathogen was not achieved [39]. Consequently, fur- Competing interests ther research on klebicin formulation for the most effi - The authors declared that they have no competing interests. cient release in the lower intestinal tract is necessary. Importantly, new  formulations for oral delivery, prefer- ably using approved formulation agents such as Eudragit, Karaliute et al. Gut Pathogens (2022) 14:17 Page 10 of 11 Author details 14. Denkovskienė E, Paškevičius Š, Misiūnas A, Stočkūnaitė B, Starkevič Institute for Digestive Research, Laboratory of Clinical and Molecular U, Vitkauskienė A, Hahn-Löbmann S, Schulz S, Giritch A, Gleba Y, Gastroenterology, Lithuanian University of Health Sciences, Mickeviciaus st. 9, Ražanskienė A. Broad and efficient control of Klebsiella Pathogens by 44307 Kaunas, Lithuania. Department of Drug Technology and Social Phar- peptidoglycan-degrading and pore-forming Bacteriocins Klebicins. Sci macy, Lithuanian University of Health Sciences, Sukileliu Pr. 13, 50161 Kaunas, Rep. 2019. https:// doi. org/ 10. 1038/ s41598- 019- 51969-1. Lithuania. Laboratory of Drug Targets Histopathology, Institute of Cardiology, 15. Whitaker DM, Reichley SR, Griffin MJ, Prager K, Richey CA, Kenelty KV, Lithuanian University of Health Sciences, 50162 Kaunas, Lithuania. Nomads Stevens BN, Lloyd-Smith JO, Johnson CK, Duignan P, Johnson S, Rios UAB, Geležinio vilko 29A, 01112 Vilnius, Lithuania. 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Reduction of gastrointestinal tract colonization by Klebsiella quasipneumoniae using antimicrobial protein KvarIa

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Abstract

Background: Klebsiella quasipneumoniae is an opportunistic pathogen causing antibiotic-resistant infections of the gastrointestinal tract in many clinical cases. Orally delivered bioactive Klebsiella-specific antimicrobial proteins, klebicins, could be a promising method to eradicate Klebsiella species infecting the gut. Methods: Mouse infection model was established based on infection of antibiotic-treated BALB/C mice with K. quasipneumoniae strain DSM28212. Four study groups were used (3 animals/group) to test the antimicrobial efficacy of orally delivered klebicin KvarIa: vehicle-only group (control, phosphate-buffered saline), and other three groups with bacteria, antibiotic therapy and 100 µg of uncoated Kvarla, 100 µg coated KvarIa, 1000 µg coated-KvarIa. Because of the general sensitivity of bacteriocins to gastroduodenal proteases, Kvarla doses were coated with Eudragit , a GMP-certified formulation agent that releases the protein at certain pH. The coating treatment was selected based on measurements of mouse GI tract pH. The quantity of Klebsiella haemolysin gene (khe) in faecal samples of the study animals was used to quantify the presence of Klebsiella. Results: GI colonization of K. quasipneumoniae was achieved only in the antibiotic-treated mice groups. Significant changes in khe marker quantification were found after the use of Eudragit S100 formulated klebicin KvarIa, at both doses, with a significant reduction of K. quasipneumoniae colonization compared to the vehicle-only control group. Conclusions: Mouse GI tract colonization with K. quasipneumoniae can be achieved if natural gut microbiota is sup- pressed by prior antibiotic treatment. The study demonstrates that GI infection caused by K. quasipneumoniae can be significantly reduced using Eudragit -protected klebicin KvarIa. Keywords: Klebsiella quasipneumoniae, Klebicins, KvarIa, Haemolysin gene, Bacteriocins flora of the human mouth or intestines [1, 2]. Patho- Background genic Klebsiella species, such as K. pneumoniae and Klebsiella is a gram-negative and facultative anaerobic K. oxytoca, are the most prevalent infections acquired bacterium of the Enterobacteriaceae family that colo- in hospital (HAI) [2, 3]. Recent studies have identified nizes various environmental niches including normal K. quasipneumoniae as a new species distinguishable from K. pneumoniae. K. quasipneumoniae has been *Correspondence: jurgita.skieceviciene@lsmuni.lt shown to act as an etiological agent in a number of Indre Karaliute and Rima Ramonaite contributed equally to this work clinical Klebsiella-related infection cases, but has often Institute for Digestive Research, Laboratory of Clinical and Molecular been misidentified as K. pneumoniae in HAI [4, 5]. K. Gastroenterology, Lithuanian University of Health Sciences, Mickeviciaus st. 9, 44307 Kaunas, Lithuania quasipneumoniae colonizes the intestinal tract, which Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visithttp:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Karaliute et al. Gut Pathogens (2022) 14:17 Page 2 of 11 can lead to virulent urinary tract and abdominal infec- Methods tions [3–6]. Furthermore, this type of bacteria shows Aim of this study high rates of resistance against antibiotics, and some To investigate the antimicrobial effectiveness of the strains have been characterized as pan-drug resistant, klebicin KvarIa in a mouse model of K. quasipneumo- making these infection extremely difficult to treat [7, niae gastrointestinal (GI) colonization. 8]. For this reason, the development of new antimicro- bial agents is required to mitigate bacterial infections, Mouse models typically acquired by patients while in-hospital. Two different animal study designs were used for K. Colicin-like bacteriocins, produced by gram-neg- quasipneumoniae colonization and KvarIa treatment. ative bacteria, may be a potential alternative to tradi- For both studies, 8–10 weeks old, BALB/c strain, female tional antibiotics [9, 10]. Colicin-like bacteriocins are (n = 8; 19–25  g) and male (n = 16; 22–27  g) mice were a heterogeneous family of proteinaceous toxins, which purchased from the Lithuanian University of Health are capable of killing closely related bacteria, those Sciences vivarium of laboratory animals. All regulated belonging to the same species or, sometimes, the same procedures on living animals were approved by The Lith - genus [11]. This property makes them attractive as uanian Ethics Committee of Biomedical Research (Proto- therapeutics since they offer a more targeted approach col no. G2-119). than conventional antibiotics. Indeed, one of the major issues with conventional antibiotics is the dysbiosis GI model of K. quasipneumoniae (DSM28212) colonization induced by the broad-range killing of bacteria [12, 13]. and KvarIa therapy Most importantly, the mechanisms of bacterial killing Klebsiella quasipneumoniae clinical isolate DSM28212 was by bacteriocins are fundamentally different from those used for GI tract colonization in four different study groups by antibiotics. Consequently, they are active against containing three mice per group (m = 2; f = 1). Vehicle– multi-drug and pan-drug resistant pathogens. only control group was monitored for any changes in the The authors have previously identified and charac- natural host-microbiota without any additional procedures terized several Klebsiella colicin-type bacteriocins, during the period of the experiment. The ability of K. qua - klebicins, which exhibit significant and broad activity sipneumoniae to colonize the GI tract without antibiotic against the pathogenic Klebsiella species. Orally deliv- pre-treatment to disrupt the host-microbiota was tested. In ered klebicins have a potential as an excellent means to order to mimic hospital-acquired infections two groups were eradicate intestinal infections in hospitalized patients given different combinations of antibiotic treatment before that are caused by the multidrug-resistant Klebsiella infection (penicillin (2000 U/ml) + streptomycin (2  mg/ml), strains. However, the proteinaceous nature of bacte- (pen_strep); penicillin (2000 U/ml) + streptomycin (2  mg/ riocins makes them susceptible to quick inactivation ml) + metronidazole (1  g/L) (pen_strep_met)) (study design by gastroduodenal enzymes. Therefore, for the oral in Fig. 1A). These particular antibiotics were chosen because administration of klebicins, they must be encapsulated of their broad mechanism of action against gram-negative or formulated for gastroduodenal protection, for the and gram-positive bacteria. For KvarIa therapy testing the release in the small and large intestine. same composition of antibiotic pre-treatment was used as In this study, the possibility to use klebicins to eradi- in the previously described study. Three groups (A; B; C) cate intestine colonizing Klebsiella was tested in K. 9 received 10   cfu of K. quasipneumoniae orally by pipette quasipneumoniae–KvarIa model. Klebicin KvarIa is feeding once per day. Afterward, ampicillin (500 mg/l) ther- pore forming bacteriocin, highly active against K. qua- apy was used to maintain as low as possible viability of other sipneumoniae [14]. We developed a mouse model of than K. quasipneumoniae bacteria. From day 18th group A K. quasipneumoniae intestinal colonization, tested the was given 100 µg of uncoated KvarIa, groups B and C were pH condition of the mouse GI tract, and established given 100  µg and 1000  µg of Eudragit S100-coated KvarIa a suitable coating for klebicin KvarIa. We also evalu- respectively (detailed study design Fig. 2A). For each mouse, ated the antimicrobial activity of the orally delivered faecal pellets were sampled. Eudragit S100-formulated klebicin in the mouse intes- tinal tract. We confirmed that, even without further Determination of pH of the gastrointestinal tract bacteriocin engineering and improvement, bacterioc- As shown in Fig. 1A, the samples of rectum excreta were col- ins could be employed as oral antimicrobials for effi- lected on six different days during the experiment. Acquired cient control of antibiotic-resistant Klebsiella. samples were homogenized with deionized water (1:10 ratio) K araliute et al. Gut Pathogens (2022) 14:17 Page 3 of 11 and pH was determined using pH METER Mettler Toledo K. quasipneumoniae DSM28212 overnight culture was (Belgium) with the Inlab Ultra-Micro electrode. In addition, equalized to OD = 1.0 in Muller-Hinton medium the pH was measured in the samples taken from the GI tract and diluted 100-fold in 0.8% (w/v) top agar preheated during the laparotomy dissection (the intestinal tract was in a 55 °C water bath. Mixed overlay components were divided into three sections: the stomach, the duodenum, and poured on plates containing solid medium (Muller- the rectum). Hinton containing 1.5% (w/v) agar). Sterile Whatman paper discs (6 mm diameter) were placed on the surface Klebicin production in plants and purification of the soft-agar medium containing bacterial test strain Klebsiella bacteriocin KvarIa was expressed in Nicotiana and 5  µl of protein dilutions were applied to the discs. benthamiana transient expression system and purified as The plates were incubated overnight at 37  °C and the previously described in Denkovskiene et al. [14]. diameter of klebicin inhibition zones was measured. Briefly, plant-produced KvarIa and Eudragit S100- Coating of KvarIa coated KvarIa were mixed with SGF at the recom- 5% Eudragit S100 solution was prepared by dissolv- mended concentration and incubated for up to 60 min, ing 0.5  g Eudragit S100 (Evonik Industries, Germany) in sampling every few minutes and assessing the digestion 10  ml of miliQ H O and by sonication in an ultrasonic of the protein into fragments by SDS-PAGE. Coomassie bath for 30 min at 25 °C. 250 µg of KvarIa was dissolved staining on gels was used to visualize protein decompo- in 200 µg of 5% Eudragit S100. The obtained solution was sition and estimate the MW of peptide products. This lyophilized at − 51 °C for 24 h. method was only used for uncoated KvarIa, as Eudragit S100 distorted protein migration on the SDS-PAGE gel. Simulated gastric digestion and residual KvarIa activity evaluation by soft agar radial diffusion assay Protein samples (KvarIa and Eudragit S100-coated Nucleic acid extraction and synthesis of the cDNA KvarIa) were dissolved in simulated gastric buffer (0.15 M Bacterial DNA and RNA from rectum excrement sam- NaCl, pH 2), at a concentration of 1 mg/ml and incubated ples were extracted using the AllPrep PowerFecal DNA/ at 37  °C with rotation at 200  rpm for 10  min. 0.025  mg RNA Kit. Up to 100 mg of faeces sample were used for (80–113 U) of pepsin from porcine gastric mucosa was the extraction procedures. The quantity and quality of added to 1 mg of protein (pepsin:protein ratio 1:40). Ali- extracted nucleic acids were evaluated by NanoDrop quots of reaction (50  µl) were removed at different time 2000 (Nanodrop Technologies, Wilmington, DE, USA). points (0.5  min, 5  min, 10  min, 20  min, 30  min, and Subsequently, cDNA was synthesized using a High- 60 min after the addition of the pepsin). Digestions were Capacity cDNA Reverse Transcription Kit (Thermo stopped by raising the pH to 6.5 by the addition of 0.5 M Fisher Scientific, Lithuania). 18 ng of cDNA was added ammonium bicarbonate to inactivate pepsin. The pH of into the qualitative real-time PCR (qRT-PCR) reaction. samples was adjusted to 8.0 to get Eudragit S100 coat All processes were completed upon the manufacturer’s dissolved. The dilutions of all samples by ratio 1:2 were instructions. made in distilled water and 5 µL drops of diluted samples were applied on K.quasipneumoniae DSM28212 MHA Quantitative assessment of Klebsiella quasipneumoniae plates for soft agar overlay assay. using Real‑Time–PCR Soft-agar overlay assays were performed as described The haemolysin gene (khe) was chosen as the qualita - by Denkovskiene et  al. [14], with some modifications. tive marker for Klebsiella identification [15, 16]. The (See figure on next page.) Fig. 1 Experimental design of K. quasipneumoniae colonization and pH of GI. A The design of the study of mice gut colonization by K. quasipneumoniae. 1st Vehicle-only control group with natural microbiota (n = 3); 2nd Bacterial control group (n = 3); 3rd, 4th antibiotic pre-treated groups [(i) penicillin, streptomycin (pen_strep group) or (ii) penicillin, streptomycin, and metronidazole (pen_strep_met group)]. Samples were collected on circled days. B The pH values measured in the samples of rectum excreta throughout the layout of the experiment protocol in order to determine the changes in the GI tract using different substances. The control group showed an average of 7.34 pH with the lowest being 6.99 pH and the highest 7.76 pH; the infected mice group without antibiotics showed almost no changes in pH measures through the days (av. 7.27 pH); K. quasipneumoniae infected group was treated with antibiotic therapy (penicillin, streptomycin) and showed stable results (av. 7.7 pH) with th an exception on the 6 day of the experiment (7.94 pH); the last group, which was infected with K. quasipneumoniae showed the most noticeable changes during the combined antibiotic (penicillin, streptomycin, metronidazole) treatment and bacterial colonization with the average of 7.44 pH fluctuating from 6.57 pH to 8.11 pH. No significant differences were found in or between the groups. C Overall differences in values of stomach, duodenum, and rectum of pH measurements between groups. The lowest pH was seen in the stomach (2.34 – 5.46; avg. 3.3). The pH of the small intestine varied between 5.97 and 6.77 (avg. 6.5), with the large intestine showing the highest pH (6.74–8.15; avg. 7.4). A significant difference (p < 0.05) in pH measures was found in the duodenum between 3rd and 4th groups (#), as well as between 1st group and 3rd (*) in rectum Karaliute et al. Gut Pathogens (2022) 14:17 Page 4 of 11 Fig. 1 (See legend on previous page.) K araliute et al. Gut Pathogens (2022) 14:17 Page 5 of 11 Fig. 2 Experimental design of K. quasipneumoniae therapy and assessment of Klebsiella quasipneumoniae colonization. A The design of the study of mice gut colonization by K. quasipneumoniae and KvarIa treatment. All four experimental groups had antibiotic pretreatment, ampicillin therapy, bacterial administration, and different treatment for infection (PBS; uncoated-KvarIa; Eudragit S100-coated KvarIa 100 µg; Eudragit S100-coated KvarIa 1000 µg). Samples were collected on circled days. B Amplification of khe gene in mice faeces after the introduction of K. quasipneumoniae. khe gene was detected by RT-PCR. A lower CT value indicates a higher number of bacteria. The 4th day’s samples were used as a control group because they did not show any bacterial colonization. Statistically significant differences between mice groups (K. quasipneumoniae + Pen_ Strep + Amp vs K. quasipneumoniae + Pen_ Strep_Met + Amp) were not determined. Pen–penicillin, Strep–streptomycin, Met–metronidazole, Amp–ampicillin standard curve was created based on DNA samples of UNG, TaqMan probe (5-6FAM-CGC GAA CTG GAA K. quasipneumoniae (DSM28212) to test the generated GGG CCC G-TAMRA-3), and primers (Forward: 5 -GAT primers’ efficiency. DNA-based standard curve. 10 , GAA ACG ACC TGA TTG CAT TC-3, Reverse: 5 -CCG 5 6 8 9 10 10, 10, 10, 10 , and 10   CFU of K. quasipneumo-GGC TGT CGG GAT AAG -3 (Applied Biosystems, JAV) niae in 200  µl were subjected to DNA extraction with following the manufacturer’s recommendations. The QIAamp Fast DNA Stool Mini Kit (protocol for liquid amplification of the khe gene was determined by ABI sample). Fast 7500 System (Life Technologies, Carlsbad, CA, USA) During this step, the reaction for the qRT-PCR was according to standard protocol. Positive controls for performed using TaqMan Universal Master Mix II with Karaliute et al. Gut Pathogens (2022) 14:17 Page 6 of 11 DNA and RNA were isolated from K. quasipneumoniae colonization of mice gut by K. quasipneumoniae can be and negative—isolated from Esherichia coli. established after eradication of natural gut microflora. Statistical analysis Eudragit S100‑coated KvarIa is partially protected The data were analysed using nonparametric tests. The from digestion by simulated gastric fluid difference between the four protocols groups through - To find out if Eudragit S100-coated KvarIa is resistant to out the layout of the experiment were analysed using pepsin digestion, a simulated gastric digestion experi- Student’s independent t-test. Independent analyses were ment was performed. Exposures of the proteins to Sim- carried out using SPSS Version 19.0 and MiniTab 20.1.2 ulated Gastric Fluid (SGF, commercial acidic pepsin software packages. Results were considered statistically extract) were done using low enzyme-to-substrate ratios significant when p < 0.05 with ± 95% confidence intervals. in order to increase the stringency and relevance of the digestibility assays. Methods were derived from [17–19]. Results It appears, that in conditions used (pepsin:protein Selection of bacteriocin Kvarla coating by pH ratio 1:40), protein coating with Eudragit S100 is able to measurements along the GI tract provide temporal resistance to pepsin digestion. Coated In order to determine the efficient coating and delivery of KvarIa demonstrated still detectable activity in agar diffu - the klebicins in the GI tract, firstly we performed the pH sion assay after 20 min of in vitro gastric digestion, while measurements in faeces and along the GI tract. The pH of uncoated KvarIa was inactivated in simulated gastric the faecal samples is shown in Fig. 1B. The lowest pH was juice very quickly, and completely lost its activity already seen after penicillin, streptomycin, and metronidazole after 0.5  min of digestion (Fig.  3). From the SDS-PAGE treatment. Nonetheless, there was no observation of any profile of uncoated KvarIa digestion products, it is appar - statistically significant changes in GI tract pH between ent that uncoated klebicin in digested by pepsin very groups (average mean ± SD: vehicle-only control group rapidly. 7.3 ± 0.52; bacterial control group 7.5 ± 0.74; pen_strep group 7.7 ± 0.29; pen_strep_met group 7.4 ± 0.62).  The pH levels of the different GI tract sections was also Eudragit S100‑coated KvarIa efficiently reduces K. measured after the mouse decapitation (the stomach quasipneumoniae amount in colon 3.3 ± 0.92, small intestine 6.5 ± 0.34, the large intestine We evaluated the effectiveness of Eudragit S100-coated 7.4 ± 0.42; Fig.  1C) The significant differences in pH KvarIa (100  µg; 1000  µg) in K. quasipneumoniae infec- measures were found between the vehicle-only control tion model. Three different combinations of recom - group and pen_strep group (p = 0.004) in duodenum, binant klebicin were used: uncoated-KvarIa, Eudragit vehicle-only control group and pen_strep_met group S100-coated KvarIa 100  µg, and Eudragit S100-coated (p = 0.04) in rectum. KvarIa 1000  µg. The amplification of the khe marker gene was significantly higher in both Eudragit S100- Colonization of mice gut by K. quasipneumoniae coated KvarIa-treated mice groups than in the control is achieved only after disruption of natural microflora (PBS) and uncoated-KvarIa-treated mice on the last The GI tract infection/colonization model was estab - day of the experiment (22nd day), meaning that bacte- lished in mice using Klebsiella quasipneumoniae. It was rial counts were lower. As shown in Fig.  4 the bacterial designed to reflect bacterial colonization in the host counts were significantly lower after the treatment with after the disruption of natural microflora with antibiot - Eudragit S100-coated KvarIa 100  µg and 1000  µg in ics therapy. There was no colonization of K. quasipneu - contrast with the samples taken on the first day of bac - moniae observed in the vehicle-only control group. The teriocin administration (18th day). The amounts of K. use of antibiotic pre-treatment ((i) penicillin, strepto- quasipneumoniae changed from 6.3 × 10   CFU/50  mg mycin or (ii) penicillin, streptomycin, and metronida- on the 18th day to 3.9 × 10   CFU/50  mg on the 22nd zole), in order to disrupt the host microbiota, resulted day (p = 0.01) in the Eudragit S100-coated KvarIa 100 µg in introduction of K. quasipneumoniae (4th day no bac- group and from 4.0 × 10   CFU/50  mg on the 18th day terial counts were found (0  CFU/70  mg); (i) 8th day— 8 8 to 1.6 × 10   CFU/50  mg on 22nd day (p = 0.009) in the 5.25 × 10  CFU/70 mg, 11th day—3.01 × 10  CFU/70 mg Eudragit S100-coated KvarIa 1000  µg group. No sig- and (ii) 8th day—5.13 × 10   CFU/70  mg and 11th day— nificant changes in bacterial counts were seen in the 3.89 × 10   CFU/70  mg) (Fig.  2). Our data showed that K araliute et al. Gut Pathogens (2022) 14:17 Page 7 of 11 Fig. 3 Evaluation of stability and activity of KvarIa after in vitro gastric digestion assay. A Evaluation of residual activity by agar diffusion assay. Protein samples were incubated at 37 °C, 200 rpm in gastric digestion buffer (pepsin:protein ratio 1:40). Aliquots of reaction (50 µl) were removed at different time points (0.5, 5, 10, 20, 30, and 60 min) and digestion was stopped by the addition of 0.5 M ammonium bicarbonate to inactivate pepsin. The pH of samples with coated KvarIa was adjusted to 8 to get Eudragit 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. B SDS-PAGE of KvarIa after simulated gastric digestion. The 16% TRIS-Tricine gels; unstained marker—PageRuler Unstained Low Range Protein Ladder ( ThermoFisher), 10 µl of each sample loaded per line. Pepsin appears as the stable band between 30 and 42 kDa markers (green arrow). The red arrow indicates the full-size KvarIa. Exposure of KvarIa to pepsin (1:40 pepsin:lysin wt:wt) in acidic SGF results in the rapid breakdown of the protein to lower MW degradation products. Sampling times are shown in minutes vehicle-only control group (PBS) and after the adminis- Discussion tration of uncoated-KvarIa. A rapidly increasing number of antibiotic-resistant and/ or highly virulent bacterial strains is a serious challenge Karaliute et al. Gut Pathogens (2022) 14:17 Page 8 of 11 Fig. 4 Bacterial counts in GI tract after KvarIa therapy. khe gene from DNA templates was determined using RT-PCR. A lower CT value indicates a higher number of bacteria (see Standard Curve). khe has not been detected in all study groups on the 4th day. * Statistically significant differences between 18th and 22nd days in all the experimental groups treated by Eudragit S100-coated KvarIa, p < 0.05. PBS (control) and uncoated-KvarIa (100 µg) treated mice vs Eudragit S100-coated KvarIa (100 µg) treated group on the 22nd day, p < 0.05. PBS (control) and Uncoated KvarIa (100 µg) treated mice vs Eudragit S100-coated KvarIa (1000 µg) treated group on the 22nd day, p < 0.05 faced by today’s healthcare system worldwide. Recent Bacteria were not detected in the bacterial control studies indicate that patients, with hospital-acquired group without prior antibiotic treatment (judged by khe multidrug-resistant K. pneumoniae infection, have a amplification). Similar findings were observed in other significantly higher risk of developing a subsequent Klebsiella mouse models where amoxicillin disruption infection caused by identical bacteria [20–23]. K. qua- of the gut microbiota was accompanied required for gut sipneumoniae were initially thought to be asymptomatic colonization and an enhancement of the virulence of K. carriage isolates until more recent reports highlighted variicola [27]. Other studies illustrated that mouse mod- their potential virulence and increased drug resistance els of K. pneumoniae and treatment with antibiotics led [20–23]. to changes in the host microbiota and the development K. pneumoniae has been extensively studied in many of a transient super-shedder phenotype, which displays different animal models, including models for blood - the enhanced transmission efficiency of bacteria in the stream infections, pneumonia, liver abscess, digestive GI tract [28, 29]. Allegedly, the natural host microbiota and urinary tract infections [24, 25]. On the other hand, activates the defence mechanisms against K. quasip- little is known about closely related species recently sep- neumoniae and inhibits colonization, whereas reduced arated from K. pneumoniae such as K. variicola and K. microbial diversity might promote the ability to infect. quasipneumoniae. There were only limited animal stud - However, the exact mechanisms causing K. quasipneu- ies with K. variicola such as experiments on the bacte- moniae colonization needs further investigation. ria’s ability to colonize the intestinal tract and the host It is known that bacteriocins have antimicrobial immune system response against this opportunistic path- activities against pathogenic microorganisms [30, 31]. ogen [26, 27]. K. quasipneumoniae has been detected in Previous studies have identified various classes of bac - the clinical settings during hospital infections, however, teriocins (e.g.: colicin-like bacteriocins, tailocins, pep- the species has not been tested in animal efficacy mod - tide microcins) and their potential applications in food els, and mechanisms of infection by this bacterium are technology, treatments of infection, and cancer [32–34]. poorly understood. Therefore, the first goal of our study Earlier, we demonstrated the antibacterial efficacy of was to establish an animal model of K. quasipneumo- Klebsiella bacteriocins, klebicins, in  vitro using clini- niae intestinal colonization, in particular, identify the cal Klebsiella isolates. Recombinant pore-forming bac- conditions that allow bacteria to successfully grow in the teriocin KvarIa was identified as one of the most active mouse intestinal tract. We demonstrate here that for suc- klebicins; it showed the highest activity against K. qua- cessful colonization of mice gut by K. quasipneumoniae, sipneumoniae strains and was also tested in  vivo in a the disturbance of natural gut microflora using antibiotic non-mammal animal model, Galleria mellonella larvae, pre-treatment is necessary and sufficient. demonstrating significant antibacterial effect [14]. In this study, we developed a mouse model of intestinal tract K araliute et al. Gut Pathogens (2022) 14:17 Page 9 of 11 infection using K. quasipneumoniae and evaluated the should be studied in validated preclinical animal models effectiveness of Eudragit S100-coated KvarIa treatment to further optimize efficacy of bacteriocins as antibacteri - with the main purpose of investigating the potential of als for intestinal infections. klebicins as a clinical antimicrobials. The obtained results can further be translated to K. pneumoniae and other Conclusions more clinically relevant Klebsiella species. This study demonstrated that successful colonization of The authors determined the most effective coating for the mouse intestinal tract by K. quasipneumoniae can be bacteriocin needed for delivery of the highest concentra- achieved but it requires the eradication of gut resident tions of the klebicin to the large colon. The pH in the GI microbiota with an antibiotic. We also evaluated the anti- tract is a substantial factor, affecting the solubility and microbial activity of the orally delivered Eudragit S100-for- stability of the drug and absorption through the intesti- mulated klebicin in the mouse intestinal tract and show nal tract mucosa. It can vary depending on the diet type, that thus formulated bacteriocins could be employed as fed or fasted states, drugs, microbiota diversity, stress, oral antimicrobials for efficient control of antibiotic-resist - and daily fluid intake. Henceforth, unsuitable pH causes ant Klebsiella. the precipitation of drugs from the solution or the deg- radation of labile compounds [35–37]. Correspondingly, Abbreviations an assessment of pH levels in the GI tract was included PBS: Phosphate-buffered saline; GI: Gastrointestinal; khe: Haemolysin gene; in our study. We distinguished the increased pH level of HAI: Hospital-acquired infection; pen: Penicillin; strep: Streptomycin; met: the rectum content sample in the K. quasipneumoniae Metronidazole; SGF: Sarcoma grow factor; SDS: Sodium dodecyl sulfate; MW: Molecular weight; DNA: Deoxyribonucleic acid; RNA: Ribonucleic acid; cDNA: colonized mice groups treated with antibiotics. However, Complementary Deoxyribonucleic acid; qRT-PCR: Qualitative real-time poly- mice without antibiotics did not show any change in pH merase chain reaction; CFU: Colony-forming unit. levels. Similar results were obtained by Shimizu and col- Acknowledgements leagues in ICR mice housed obtaining specific pathogen- Not applicable. free conditions, there the pH of the cecum and colon increased exceedingly in the experimental groups treated Author contributions IK, RR carried out the experiment, derived the models, analysed samples and with antibiotics [38]. Therefore, the pH measurements of data, interpreted the results, drafted the manuscript, prepared the figures, the GI tract were taken into account when choosing the edited the final version of the manuscript; ED, AM performed the experiments most effective coating for KvarIa delivery. and analyzed the data; JK and JS edited the final version of the manuscript and contributed to the conception and design of the research; JB contributed In this study recombinant bacteriocins KvarIa abil- to the initial part of the experiment related to pharmaceutics; VP contributed ity to eliminate the intestinal tract infection was judged to the animal models and experimental layout; AR and YG contributed to the using khe gene quantification. We identified that both conception and design of the research, interpreted the results, and approved the final version of the manuscript. All authors read and approved the final concentrations (100  µg and 1000  µg) of coated-KvarIa manuscript. significantly reduced the infection in the GI tract of mice models. However, in our study, we did not achieve Funding This work was supported by the Lithuanian Business Support Agency grant full eradication of the K. quasipneumoniae. KvarIa was J05-LVPA-K-03–0011 “Antimicrobial substance for treatment of Klebsiella encapsulated with Eudragit S100 releasing klebicin at infections”. pH above 7 and administered by oral gavage to infected Availability of data and materials (K. quasipneumoniae) mice. Debatably, klebicin activity All data generated during this study are included in this article. could be suppressed or significantly lowered because of the gut microflora disruption or not full eradication, as Declarations well as, dependence on the pH level, which can fluctuate throughout the GI tract for various reasons (e.g. fasting Ethics approval and consent to participate All procedures performed in studies involving animals were in accordance state). Recently, a similar study was published describing with Directive 2010/63/EU and the ethical standards of Lithuanian Univer- the use of encapsulated colicins for the eradication of  E. sity of Health Sciences (approved by State Food and Veterinary Service; No. coli  in mice [36.] Colicins encapsulated into hydrogel G2-119) at which the studies were conducted. This article does not contain any studies with human participants performed by any of the authors. particles were shown to be released from the protective coat at pH above 5 and reduce colonizing E. coli numbers Consent for publication in the gut and in feces, although complete eradication of Not applicable. the pathogen was not achieved [39]. Consequently, fur- Competing interests ther research on klebicin formulation for the most effi - The authors declared that they have no competing interests. cient release in the lower intestinal tract is necessary. Importantly, new  formulations for oral delivery, prefer- ably using approved formulation agents such as Eudragit, Karaliute et al. Gut Pathogens (2022) 14:17 Page 10 of 11 Author details 14. 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Gut Pathogens – Springer Journals

Published: Apr 26, 2022

Keywords: Klebsiella quasipneumoniae; Klebicins; KvarIa; Haemolysin gene; Bacteriocins

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