ORIGINAL RESEARCH published: 04 April 2019 doi: 10.3389/fmicb.2019.00689 Biodistribution of Liposome-Encapsulated Bacteriophages and Their Transcytosis During Oral Phage Therapy Jennifer Otero 1 , Alba García-Rodríguez 1 , Mary Cano-Sarabia 2 , Daniel Maspoch 2,3 , Ricard Marcos 1,4 , Pilar Cortés 1* and Montserrat Llagostera 1 1 Departament de Genèticai de Microbiologia, Universitat Autònoma de Barcelona, Barcelona, Spain, 2 Catalan Institute of Nanoscience and Nanotechnology (ICN2), CSIC and the Barcelona Institute of Science and Technology, Barcelona, Spain, 3 Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain, 4 Consortium for Biomedical Research in Epidemiology and Public Health (CIBERESP), Carlos III Health Institute, Madrid, Spain Edited by: Pilar García, This study sheds light on the biodistribution of orally administered, liposome- Spanish National Research Council (CSIC), Spain encapsulated bacteriophages, and their transcytosis through intestinal cell layers. Reviewed by: Fluorochrome-labeled bacteriophages were used together with a non-invasive imaging Danish Javed Malik, methodology in the in vivo visualization of bacteriophages in the stomach and intestinal Loughborough University, tract of mice. In those studies, phage encapsulation resulted in a significant increase of United Kingdom Jean-Paul Pirnay, the labeled phages in the mouse stomach, even 6 h after their oral administration, and Queen Astrid Military Hospital, without a decrease in their concentration. By contrast, the visualization of encapsulated Belgium Joana Azeredo, and non-encapsulated phages in the intestine were similar. Our in vivo observations were University of Minho, Portugal corroborated by culture methods and ex vivo experiments, which also showed that the *Correspondence: percentage of encapsulated phages in the stomach remained constant (50%) compared Pilar Cortés to the amount of initially administered product. However, the use of conventional mariapilar.cortes@uab.cat microbiological methods, which employ bile salts to break down liposomes, prevented Specialty section: the detection of encapsulated phages in the intestine. The ex vivo data showed a This article was submitted to higher concentration of non-encapsulated than encapsulated phages in liver, kidney, Antimicrobials, Resistance and Chemotherapy, and even muscle up to 6 h post-administration. Encapsulated bacteriophages were able a section of the journal to reach the liver, spleen, and muscle, with values of 38% ± 6.3%, 68% ± 8.6%, and Frontiers in Microbiology 47% ± 7.4%, respectively, which persisted over the course of the experiment. Confocal Received: 17 December 2018 Accepted: 19 March 2019 laser scanning microscopy of an in vitro co-culture of human Caco-2/HT29/Raji-B cells Published: 04 April 2019 revealed that Vybrant-Dil-stained liposomes containing labeled bacteriophages were Citation: preferably embedded in cell membranes. No transcytosis of encapsulated phages Otero J, García-Rodríguez A, was detected in this in vitro model, whereas SYBR-gold-labeled non-encapsulated Cano-Sarabia M, Maspoch D, Marcos R, Cortés P and Llagostera M bacteriophages were able to cross the membrane. Our work demonstrates the (2019) Biodistribution prolonged persistence of liposome-encapsulated phages in the stomach and their of Liposome-Encapsulated Bacteriophages and Their adherence to the intestinal membrane. These observations could explain the greater Transcytosis During Oral Phage long-term efficacy of phage therapy using liposome-encapsulated phages. Therapy. Front. Microbiol. 10:689. doi: 10.3389/fmicb.2019.00689 Keywords: biodistribution, transcytosis, liposomes, bacteriophages, phage therapy Frontiers in Microbiology | www.frontiersin.org 1 April 2019 | Volume 10 | Article 689 Otero et al. Biodistribution of Liposome-Encapsulated Bacteriophages INTRODUCTION was determined using culture methods. Phage transcytosis was examined using an in vitro model of the human intestinal barrier, With the increasingly frequent appearance and expansion of consisting of a triple co-culture of Caco-2 and HT29 intestinal multidrug-resistant bacteria, there has been a re-evaluation of cells with Raji-B lymphocytes. the therapeutic use of bacteriophages. The advantages of lytic phages over traditional antimicrobials include the ability of phages to self-replicate and their high specificity toward the target MATERIALS AND METHODS bacterium, without affecting the host microbiota. Furthermore, lytic phages do not cause human allergies, nor do they change Bacterial Strains and Bacteriophage the structure, odor or flavor of food products (Hagens and Lysates Offerhaus, 2008). These characteristics make bacteriophages of Salmonella enterica serovar Typhimurium LB5000 (SGSC181; interest for use in veterinary and human medicine, as well as in University of Calgary) was grown in Luria-Bertani (broth with the food industry (Vandenheuvel et al., 2015; Young and Gill, shaking or on Luria-Bertani agar plates for 18 h at 37◦ C and 2015; Cooper et al., 2016; Górski et al., 2016; Pelfrene et al., 2016; was used to propagate and quantify bacteriophage UAB_Phi20. Abedon et al., 2017; Cortés and Llagostera, 2017). This bacteriophage, a member of the Podoviridae family, has Among the different routes of bacteriophage administration, an icosahedral head and non-contractile tail (60 ± 2.7 nm the oral route is likely to be the most appropriate for and 13 ± 0.7 nm, respectively) (Bardina et al., 2012, 2016). phage therapy in humans and animals (Zelasko et al., Its genome has been fully sequenced (Bardina et al., 2016). 2017). Its advantages compared to other routes of parenteral Phage lysates were prepared following a previously described administration include the relative ease delivery, a potentially method (Colom et al., 2015) and filtered through 0.45-µm and low immunogenicity, and greater patient comfort, among others 0.22-µm-pore-size polyethersulfone (PES) membranes (Millex - R (Zelasko et al., 2017). However, oral phage therapy poses several HP; RRID:SCR_008983), followed by ultracentrifugation at challenges. Phages typically lack stability in the extremely acidic 68.584 × g for 2 h (90Ti rotor; OptimaTM × PN-100, environment of the stomach (Jonczyk et al., 2011), and their Beckman coulter, RRID:SCR_008940). The resulting lysate was residence time in the intestine is very short. In a previous additionally purified using an Amicon filtration device (Ultra- study, we tested a method aimed at overcoming these potential 15 Centrifugal 100 kDa MWCO; Millipore, RRID:SCR_008983) drawbacks, by examining the efficacy of phages encapsulated and centrifugation at 5,000 × g for 15 min (Eppendorf 5810R; in cationic lipid envelopes (liposomes) (Colom et al., 2015). Eppendorf, RRID:SCR_000786). The phages were then washed The resulting phage-containing nanocapsules (mean diameter several times with 10 mM MgSO4 under the same conditions. of 309–326 nm) were significantly more stable than non- The final concentration of the purified phage lysate was 1– encapsulated bacteriophages in simulated gastric fluid (pH 2.8). 3 × 1012 pfu/mL. In addition, in broiler chickens, the administration of liposome- encapsulated bacteriophages resulted in phage detection after Cell Lines and Culture Conditions 72 h in 38.1% of the treated animals. By contrast, only 9.5% The human colorectal adenocarcinoma cell lines Caco-2 HT29 of the chickens retained the non-encapsulated bacteriophages. and the human B lymphocyte line Raji were used in a co-culture We also demonstrated the enhanced efficacy of liposome- to mimic the intestinal barrier. Caco-2 cells were kindly provided encapsulated phage therapy in the prolonged protection of by Dr. Isabella Angelis (Istituto Superiore di Sanità, Italy), poultry against Salmonella (Colom et al., 2015). Based on those HT29 (ATCC HTB-38TM ) and the Raji-B cell line (ATCC R R results, we hypothesized that the liposomes could adhere to the CCL-86TM ) were purchased from the American Type Culture intestinal epithelium and the encapsulated bacteriophages could Collection (ATCC, RRID:SCR_001672). All cell lines were undergo transcytosis. However, we found no published studies maintained in Dulbecco’s modified Eagle’s high-glucose medium reporting either of these phenomena, although the adherence of without pyruvate (DMEM w/o pyruvate; Life Technologies, non-encapsulated bacteriophages to the intestinal barrier, their RRID:SCR_008817). The medium was supplemented with 10% diffusion across the mucosal surface, and their translocation fetal bovine serum (FBS), 1% non-essential amino acids (NEAA; through intestinal cells have been described (Barr et al., 2015; PAA Laboratories GmbH, Pasching, Austria) and 2.5 mg Barr, 2017; Shan et al., 2018). Other studies demonstrated the plasmocin (InvivoGen, San Diego, CA, United States)/mL. The capacity of non-encapsulated bacteriophages to migrate into the cell cultures were incubated at 37◦ C in a humidified atmosphere blood and enter internal organs (Dabrowska et al., 2005; Górski of 5% CO2 and 95% air. Routinely, the Caco-2 and HT29 cell et al., 2006; Hamzeh-Mivehroud et al., 2008; Oliveira et al., 2009; lines were sub-cultured once a week with 1% trypsin-EDTA (PAA Górski et al., 2016). Laboratories GmbH) at 7.5 × 105 cells/75 cm2 flask and 4 × 105 Thus, in the present work we studied the adherence of cells/75 cm2 flask, respectively. liposome-encapsulated phages to the intestinal barrier and the ability of those phages to cross the barrier. Specifically, we Phage Encapsulation investigated the in vivo and ex vivo biodistribution of orally Liposome encapsulation of purified UAB_Phi20 (1 × 1011 administered, fluorochrome-stained, liposome-encapsulated pfu/mL) was carried out using the film-hydration method. bacteriophages using a non-invasive imaging methodology. The lipid mixture consisted of 1,2-dilauroyl-rac-glycero- In addition, the presence of bacteriophages in ex vivo organs 3-phosphocholine (DLPC), cholesteryl polyethylene glycol Frontiers in Microbiology | www.frontiersin.org 2 April 2019 | Volume 10 | Article 689 Otero et al. Biodistribution of Liposome-Encapsulated Bacteriophages 600 sebacate (Chol-PEG600), cholesterol (Chol), and (PerkinElmer RRID:SCR_012163), and the images and RE signal cholesteryl 3β-N (dimethylaminoethyl) carbamate hydrochloride were registered and analyzed using Living Image 4.5 software (cholesteryl) in a molar ratio of 1:0.1:0.2:0.7. The mixing process (PerkinElmer RRID:SCR_012163). The mean FLI intensity, was previously described (Colom et al., 2015; Cortés et al., and corresponding standard error of the mean (SEM) or 2018). The resulting suspension of large multilamellar vesicles standard deviation (SD) were determined. All analyses and graph was homogenized using an extruder (Lipex Biomembranes, construction were performed using GraphPad Prism 5 software Vancouver, Canada) and a polycarbonate membrane (pore (GraphPad Prism, RRID:SCR_002798). size, 400 nm) to obtain unilamellar vesicles. The particle- size distributions and zeta potential of the nanocapsules were determined in a ZetaSizer Nano ZS apparatus, by In vivo Biodistribution of Bacteriophage measuring the electrophoretic mobility and using a dynamic in Murine Model light scattering (DLS) analyzer combined with non-invasive The biodistribution of liposome-encapsulated and non- backscatter technology (Colom et al., 2015; Cortés et al., encapsulated labeled UAB_Phi20 bacteriophage was evaluated 2018). One mL of each sample was measured without in vivo and ex vivo in 5-week-old athymic nude female mice dilution. The mean diameter was the median of three (Mus musculus, strain Hsd:Athymic Nude-Foxn1; ENVIGO, different measurements. Santa Perpètua de la Mogoda, Spain). The mice were housed in The percentage of bacteriophages encapsulated by this method quarantine rooms during an acclimatization period of 8 days was calculated as follows: percentage (%) = 100-(Cfree /Ctotal )-100 and inspected by a veterinarian, who confirmed the health of (Colom et al., 2015), where Cfree is the titration of appropriate the mice. The mice were then randomly housed under specific- dilutions of the encapsulation mixture directly onto strain pathogen-free (SPF) conditions in autoventilated racks. Food LB5000 agar plates using the double agar layer method. The total and water were supplied ad libitum. phage concentration (Ctotal ) was determined by mixing 0.5 ml of Both liposome-encapsulated and non-encapsulated stained the liposome-phage dilutions with 0.5 ml (50 mM) of bile salts bacteriophages were orally administered by oral gavage of a (Sigma-Aldrich, RRID:SCR_008988) to disrupt the liposomes single dose of 1.3 × 1013 pfu/kg mouse body weight; the and then plating the resulting suspensions on agar plates using dosing volume was 22.4 mL/kg. For each group (8 animals), the double agar layer method. The encapsulation efficiency was a non-treated mouse was included as an autofluorescence obtained from three independent encapsulation experiments, control of background tissue-fluorescence levels. At 2.5 and and in each experiment, the values come from triplicate plates 5.5 h post-administration, the whole-body biodistribution of of each dilution. the bacteriophages was measured non-invasively in the mice by VTS-750 fluorescence image monitoring, acquiring ventral and dorsal views. In addition, 30 min later, at 3 h and 6 h Fluorescence Labeling of the UAB_Phi20 after phage administration, stomach, intestine, spleen, liver, Bacteriophage kidney, muscle, and blood samples were collected from four Purified UAB_Phi20 bacteriophage (1 × 1012 pfu/mL) was animals and bacteriophage accumulation in the respective tissues stained with the fluorochrome Vivo-Tag S 750 (PerkinElmer, MA, was determined by ex vivo VTS-750 fluorescence monitoring, United States) by adding 100 µl of fluorochrome per 1 mL of acquiring ventral, and dorsal views of the organs. Thereafter, all phage, followed by a 1-h incubation at room temperature in the tissues were transferred to MgSO4 buffer (10 mM). The animals dark. The bacteriophages were then diluted 10-fold in MgSO4 were euthanized by cervical dislocation while still anesthetized, 10 mM and washed by ultrafiltration using an Amicon Ultracel following standard procedures for euthanasia. 10 KDa (Millipore, RRID:SCR_008983) filtration unit at 5000 × g The bacteriophage concentration was determined in whole at 20◦ C for 5 min. The fluorochrome:bacteriophage ratio was organs (stomach, intestine, liver, spleen, and kidney), a dorsal calculated by measuring the absorbance at 750 and 280 nm using portion of muscle and the volume of blood sampled by cardiac a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, puncture to obtain plasma. All samples were weighed and RRID:SCR_008452) according the manufacturer’s instructions. then resuspended in 2 mL of MgSO4 (10 mM). Afterward, The stained-bacteriophage concentration was also calculated by the samples were mechanically homogenized for 15 min and titration with test strain LB5000. Stained bacteriophage was serial dilutions of the homogenates were plated using the encapsulated in the liposome mixture as described for the in vivo double agar layer method. The concentration of encapsulated distribution study. bacteriophages was also determined in the tissues of mice treated To measure in vitro fluorescent light production by the non- with liposome-encapsulated phages, following the procedure encapsulated and liposome-encapsulated labeled bacteriophage, described above. In addition, the presence or absence of serial half-dilutions ranging from 5.8 × 1011 to 1.1 × 109 bacteriophages was checked in all tissues, including those of pfu/mL were prepared in a 96-well black plate. The fluorescence control mice not treated with bacteriophages, using enrichment signal (FLI) was qualitatively evaluated as the radiant efficiency protocols (Guttman et al., 2005). (RE, fluorescence emission radiance per incident excitation In vivo and ex vivo FLI was determined using the IVIS power) and the values for each well were plotted with respect Spectrum imaging system (PerkinElmer RRID:SCR_012163), as to the phage concentration. VTS-750 fluorescence imaging described above. Ex vivo FLI values were calculated by measuring was carried out using the IVIS Spectrum imaging system the RE of both the dorsal and ventral view of each organ and then Frontiers in Microbiology | www.frontiersin.org 3 April 2019 | Volume 10 | Article 689 Otero et al. Biodistribution of Liposome-Encapsulated Bacteriophages FIGURE 1 | In vivo fluorescence images of the biodistribution in mice of VTS-750-stained UAB_Phi20: non-encapsulated phages (A,C) and the products of phage encapsulation (PPE) (B,D). The ventral side of each mouse was imaged 2.5 h (A,B) and 5.5 h (C,D) after oral administration of the phage preparations (n = 4 mice per group). The pseudocolor scale bars are consistent for each corresponding view and show the relative changes over time. considering the weight of each organ, transforming the RE values differentiate for 21 days, until a monolayer of polarized cells to the RE per gram of tissue. had formed. The culture medium was changed every 2 days The in vivo experiments were performed using ICTS (García-Rodríguez et al., 2018). “NANBIOSIS,” specifically, the CIBER-BBN in vivo experimental After 21 days of incubation, the transepithelial electrical platform of the Functional Validation & Preclinical Research resistance (TEER) of the cell monolayers was measured using (FVPR) area1 and Laboratory Animal Service (LAS) of Vall an ohmmeter (Millicell ERS-2 Voltohmmeter; Merck Millipore, d’Hebron Institut de Recerca (VHIR; Barcelona). The animals Darmstadt, Germany). Only values >300 /cm2 were accepted were treated in compliance with the guidelines of the Ethics in the analysis. UAB_Phi20 bacteriophage (0.5 mL, 1 × 1010 Commission (Comité Ético de Experimentación Animal and 1 × 107 pfu/mL) in DMEM with FBS medium was [CEEA]) of the VHIR, Barcelona. The study was approved and inoculated on the apical side of the Transwell. At 2, 6, 24, assigned the authorization number 69/15. and 48 h post-inoculation, bacteriophages from the apical and basolateral sides of the Transwell were quantified on lawns Phage Transcytosis in in vitro Model of of strain LB5000, as previously described. Accordingly, the Intestinal Barrier basolateral volume (1.5 mL) was removed and replaced with fresh FBS-containing DMEM. The TEER was measured after each Bacteriophage transcytosis was assayed on Caco-2 and HT29 transcytosis test to ensure the integrity and polarization of the cells seeded in 12-well culture plates using a polyethylene cell cultures. Liposome-encapsulated UAB_Phi20 bacteriophage terephthalate Transwell (PET) with a 1-µm pore size and an R (5 × 109 pfu/well) was assayed at the same time points and area of 1.12 cm2 (Merck Millipore, Darmstadt, Germany) as under the same conditions but in DMEM without FBS because a support chamber to establish apical and basolateral sides. the presence of FBS resulted in aggregation of the liposomes. Briefly, 1.7 × 105 Caco-2 and HT29 cells clones were mixed in DMEM with FBS and seeded onto the apical compartment of the Transwell at a cell ratio of 90:10, respectively. On Transmission Electron Microscopy (TEM) day 14 after seeding, Raji-B lymphocytes were added to the Liposome-encapsulated phage was examined by cryogenic basolateral side of the Transwell to promote differentiation transmission electron microscopy (cryo-TEM) using a of the Caco-2 cells to M-cells. The co-cultures were left to JEOL JEM-1400 microscope (JEOL, Japan). The samples were prepared as detailed in previous publications 1 http://www.nanbiosis.es/portfolio/u20-in-vivo-experimental-platform/ (Colom et al., 2015; Cortés et al., 2018). Frontiers in Microbiology | www.frontiersin.org 4 April 2019 | Volume 10 | Article 689 Otero et al. Biodistribution of Liposome-Encapsulated Bacteriophages FIGURE 2 | Ex vivo fluorescence of bacteriophages and PPE in the mouse stomach (A) and intestine (B) 3 and 6 h post-administration. The dorsal view of both organs is shown. Pseudocolor scale bars are located on the right for each corresponding set of images. (C) Fluorescence accumulation (RE) per tissue weight (g) in the stomach and intestine 3 h (solid) and 6 h (dotted) after the administration of non-encapsulated phages (white) or PPE (gray). Values of RE/g of PPE were obtained after taking into account the fluorescence attenuation (3.4-fold) observed in the in vitro experiment. The significance of the RE/g values of the encapsulated vs. non-encapsulated phage is shown: ∗∗∗ P < 0.001 and ∗ P < 0.05. Cell monolayers incubated with bacteriophages for 48 h within microscope (Leica Microsystems, RRID:SCR_008960). In this the apical compartment of the Transwell were aspirated, washed case, the liposomes were not extruded through polycarbonate with phosphate-buffered saline (PBS 1 X), and fixed with 2.5% membrane, as the final particles were below the resolution limit glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) at 4◦ C for of the optical microscope (Colom et al., 2015). 2 h. The fixed cells were then washed four times with MiliQ- In addition, both non-encapsulated and liposome- grade water and dehydrated with increasing concentrations of encapsulated bacteriophages (0.5 mL; 1 × 1010 pfu/mL) acetone. After the cells had been embedded in polymerized were transferred to the apical compartment of the Transwell for Epon 812 resin for 48 h, ultrathin sections (60–70 nm) 2 h at 37◦ C in the dark. Afterward, the cell monolayers were were cut using an ultracut microtome (Leica Microsystems, washed and fixed with 4% paraformaldehyde (Sigma-Aldrich, RRID:SCR_008960) and counterstained first with uranyl acetate MO, United States) for 15 min at 37◦ C, washed with PBS 1 X, for 30 min and then with Reynolds solution lead citrate for and maintained overnight at 4◦ C. The cell nuclei and plasma 5 min. The cell monolayers were observed in a JEOL JEM-1400 membranes were then stained for 15 min at room temperature microscope (JEOL, Japan). with Hoechst 33342 and CellmaskTM Deep Red plasma (Thermo Fisher Scientific, RRID:SCR_008452), respectively. After staining, the cell layers were left to stand face down in 35-mm Confocal Laser Scanning Microscopy glass-bottom culture dishes with 14-mm microwells (MatTek (CLSM) Corp., MA, United States). The cell monolayers were imaged Bacteriophage and liposomes were stained with SYBR Gold and on a Leica TSC SP5 confocal microscopy (Leica Microsystems, Vybrant DiI (Thermo Fisher Scientific, MA, United States), RRID:SCR_008960) using the PL APO 63 × /1.4-0.6 oil CS UV respectively, as previously described (Colom et al., 2015). objective (Leica Microsystems, RRID:SCR_008960). The images Confirmation of the liposome encapsulation of the phages were processed with Fiji (Schindelin et al., 2012) and IMARIS R was carried out by CLSM using a Leica TCS SP5 confocal (Bitplane, Zurich, Switzerland) software to visualize the confocal Frontiers in Microbiology | www.frontiersin.org 5 April 2019 | Volume 10 | Article 689 Otero et al. Biodistribution of Liposome-Encapsulated Bacteriophages stacks and to obtain three-dimensional cross-section images Prior to the in vivo experiments, the RE of dilutions of with merged labels. VTS-750-stained UAB_Phi20 (5.8 × 1011 to 1.1 × 109 pfu/mL) was measured using the IVIS Spectrum imaging system. R Statistical Analysis Measurements of the same dilutions of PPE and VTS-750-stained UAB_Phi20 showed that, although a quenching effect occurred The results are expressed as the mean and SD. A Kolmogorov- at higher bacteriophage concentrations, the correlations between Smirnov test was used to check the normality of the samples, fluorescence and the bacteriophage concentration were linear and a repeated measure two-way ANOVA and Tukey’s multiple between 4.5 × 109 and 3.6 × 1010 pfu/mL for UAB_Phi20 phage comparisons test were performed as appropriate, using GraphPad and between 2.3 × 109 and 1.8 × 1010 pfu/mL for PPE. The Prism version 6.00 (GraphPad Prism, RRID:SCR_002798). FLI of the bacteriophage was higher (3.4 ± 1.1 times) than that of PPE, indicating that liposome encapsulation resulted in fluorescence attenuation, which was considered in subsequent RESULTS in vivo measurements. In the in vivo experiments, single oral doses In vivo Biodistribution of Bacteriophages (1.3 × 1013 pfu/kg) of bacteriophage UAB_Phi20 and of in Mice PPE were administered to two groups of mice. Both preparations Data on the encapsulation of VTS-750-stained UAB_Phi20 were well tolerated and did not cause significant body weight phage indicated that the final product was a mixture containing loss or adverse side effects in the mice. In vivo fluorescence ∼46% encapsulated and 54% non-encapsulated UAB_Phi20 imaging showed that, by 2.5 h post-administration, UAB_Phi20 phages (Supplementary Table S1). The size of the liposomes had mainly accumulated in the gastrointestinal tract (stomach as estimated by DLS was 341.6 ± 8.6 nm with a zeta potential and intestinal system), decreasing slightly in these organs of + 34 ± 5.0 mV. Encapsulation was also confirmed by by 5.5 h post-administration (Figure 1). Similar results were visualization of the nanocapsules on the cryo-TEM images achieved following PPE administration. In either case, the and the three-dimensional (3D) spatial superimposition of the presence of phages in other tissues was not detected by in vivo CLSM images showing the fluorescence of both the phages and fluorescence imaging. liposomes (Supplementary Figure S1). The term “the product The ex vivo FLI results confirmed those obtained in vivo, of phage encapsulation” (PPE) is used in the following to refer as they revealed the accumulation of UAB_Phi20 fluorescence to this mixture. (RE) in the stomach (Figure 2A) and intestine (especially the cecum) in both groups of animals (Figure 2B). Calculations of the RE/g of PPE (Figure 2C) took into account the TABLE 1 | Bacteriophage concentrations in ex vivo organs obtained from mice attenuation of the fluorescence observed in the in vitro orally administered bacteriophages and the products of phage measurements (3.4-fold). Thus, 3 and 6 h after treatment, encapsulation (PPE). the bacteriophages were mainly present in the stomach, and the FLI (RE/g) of PPE was significantly higher than that of Organ Time (h) Bacteriophage PPE the non-encapsulated phage (Figure 2C). In the intestine, FLI Concentration (log10 pfu/g) a was maintained over time, with slightly larger numbers of Concentration (log10 Percentage pfu/g)a,b (%) a bacteriophages present in mice treated with UAB_Phi20 than with PPE, both at 3 and 6 h post-administration (Figure 2C). Stomach 3 7.6 ± 0.2 10.0 ± 0.2d 50 ± 8.2 In the spleen, liver, and kidney, as well as in muscle, the 6 7.4 ± 0.6 9.9 ± 0.2d 53 ± 9.4 FLI in mice treated with UAB_Phi20 and PPE indicated Intestine 3 9.0 ± 0.2 9.2 ± 0.1 0 ± 0.0 a generally low and variable accumulation of the phages 6 9.4 ± 0.2 9.2 ± 0.2 0 ± 0.0 among replicates, attributable to the limit of detection of Liver 3 7.2 ± 0.4 6.6 ± 0.2 38 ± 6.3 the imaging technique. Thus, at a concentration of stained 6 7.3 ± 0.6c 4.8 ± 0.7 23 ± 1.6 bacteriophages ≤ 7 log10 pfu/g, the imaging methodology Spleen 3 6.6 ± 0.6c 4.4 ± 0.1 68 ± 8.6 did not allow discrimination between the autofluorescence of 6 5.4 ± 0.5 5.1 ± 0.9 69 ± 7.6 the mouse tissues and the fluorescence of the stained phages Kidney 3 7.6 ± 0.3c 5.7 ± 0.2 0 ± 0.0 (data not shown). 6 6.5 ± 0.3c 4.0 ± 0.1 0 ± 0.0 After the ex vivo FLI measurements, the tissues were weighed, Muscle 3 6.1 ± 0.2 4.9 ± 0.7 47 ± 7.4 homogenized in MgSO4 (10 mM), and the bacteriophage 6 6.4 ± 0.6c 4.6 ± 0.4 47 ± 6.6 concentration was determined, including the concentration of Plasma 3 4.9 ± 0.5 3.7 ± 0.9 21 ± 9.3 encapsulated bacteriophages present in the tissues of PPE-treated 6 5.1 ± 0.1 4.9 ± 0.3 17 ± 7.2 mice. The latter assessment was accomplished by titration of a Each value is the average of four samples ± standard deviation. b Values obtained the phages in untreated tissues and in tissues treated with in samples treated with bile salts. c Statistically significant difference between non- bile salts, as previously described. This allowed calculation of encapsulated and encapsulated phages (P < 0.001 at 3 h in the spleen and kidney the percentage of encapsulated phages present in each tissue and at 6 h in the liver and kidney; P < 0.05 at 6 h in muscle). d Statistically significant difference between encapsulated and non-encapsulated phage (P < 0.001). The (Table 1). The results in the organs and tissues harvested difference in the percentage of encapsulation at 3 and at 6 h was not significant. 3 and 6 h post-administration showed that the encapsulated Frontiers in Microbiology | www.frontiersin.org 6 April 2019 | Volume 10 | Article 689 Otero et al. Biodistribution of Liposome-Encapsulated Bacteriophages phages remained or had accumulated in the spleen (68 ± 8.6% (Table 1). All tissues from the untreated animals were negative and 69 ± 7.6%, respectively), stomach (50 ± 8.2% and for bacteriophages. 53 ± 9.4%, respectively), and muscle (47 ± 7.4% and 47 ± 6.6%, respectively). The corresponding percentages in the liver were lower (38 ± 6.3% and 23 ± 1.6%). By contrast, liposome- Transcytosis of Bacteriophages Across encapsulated bacteriophages were not found in either the the in vitro Intestinal Barrier Model intestine or the kidney, and the percentage in plasma was the The TEER of the intestinal epithelial cell monolayers was lowest measured (Table 1). measured before and after (48 h) bacteriophage inoculation. The total phage concentrations in the organs and tissues of All TEER values were >300 /cm2 , confirming the valid use the two groups of animals are shown in Table 1. Throughout of this method in the experiments. In addition, epithelial the study, phage concentrations in the stomach were significantly cell monolayer polarity and integrity were assessed by TEM, higher in PPE-treated mice than in mice treated with non- which revealed that the bacteriophages did not alter either encapsulated phages, whereas the differences in the intestinal the membrane stability of Caco-2 cells or the robustness of concentrations of the two phage preparations was not significant their tight junctions (Supplementary Figure S2). Transcytosis (Table 1). In other tissues and organs, the phage concentrations of the bacteriophages applied to the apical side of the intestinal were higher in mice administered non-encapsulated phages. epithelial cell monolayers was quantified at 2, 6, 24, and 48 h Specifically, the non-encapsulated phage concentration in the by plating the apical and basolateral contents onto LB5000 agar liver at 3 and 6 h was 7.2 ± 0.4 and 7.3 ± 0.6 log10 pfu/g, plates. The results showed that, after 2 and 48 h, bacteriophage respectively. These levels were maintained over time, whereas in transcytosis in DMEM containing FBS ranged from 4.5 ± 0.0 the PPE-treated mice the concentration in the liver decreased to 5.5 ± 0.1 log10 pfu/mL and from 3.7 ± 0.3 to 4.2 ± 0.3 from 6.6 ± 0.2 log10 pfu/g at 3 h to 4.8 ± 0.7 log10 pfu/g log10 pfu/mL at starting concentrations of 1 × 1010 pfu/mL at 6 h. In the kidney, the accumulation of non-encapsulated and 1 × 107 pfu/mL, respectively (Figure 3). Because the phages and PPE decreased between 3 and 6 h of administration, amount of transcytosis was low, only the higher starting dose whereas a decrease in the spleen occurred only in mice treated (1 × 1010 pfu/mL) was used in the PPE experiment. Furthermore, with the former preparation. The phage concentration increased the experiments were conducted in serum-free DMEM because between 3 and 6 h post-administration in the spleen of the PPE- FBS caused liposomal aggregation (data not shown). Prior to treated mice and in the plasma of both groups. In muscle, phage the transcytosis experiments, we demonstrated that incubation accumulation was maintained over time in all of the treated mice of the PPE in DMEM at 37◦ C for 72 h did not modify either FIGURE 3 | Transcytosis of non-encapsulated phage (A–C) and PPE (D) across an intestinal epithelial monolayer. The bacteriophage concentration was quantified 2, 6, 24, and 48 h post-inoculation by titration of the contents of the apical (black , ) and basolateral (white , ) compartments. DMEM supplemented with FBS was used to study the non-encapsulated bacteriophages (A,B) and serum-free DMEM to compare the transcytosis of non-encapsulated phages (C) and PPE (D). The concentrations applied in the apical chamber were 1 × 1010 pfu/mL (circles) and 1 × 107 pfu/mL (squares). Scatter plots show the means; error bars represent the 95% confidence intervals. The phage concentration in the basolateral chamber is the sum of the values obtained at each time point. Frontiers in Microbiology | www.frontiersin.org 7 April 2019 | Volume 10 | Article 689 Otero et al. Biodistribution of Liposome-Encapsulated Bacteriophages the titer or the percentage of encapsulation (around 46% of the applied dose). Thus, under this condition, the transcytosis of UAB_Phi20 phages was similar that described above for a starting dose of 1 × 1010 pfu/mL. Accordingly, bacteriophage encapsulation did not significantly modify transcytosis at either 2 or 48 h, based on values of 4.6 ± 0.5 and 4.8 ± 0.4 log10 pfu/mL, respectively (Figure 3). It should be noted that encapsulated bacteriophages were not found in the basolateral compartment at any of the sampling times. Furthermore, the percentage of encapsulated phages in the apical section did not change during the experiment, regardless of the initially applied concentration (data not shown). To visualize the transcytosis of bacteriophages through the cell monolayer, bacteriophage UAB_Phi20 was fluorescently labeled using SYBR gold and encapsulated in liposomes labeled with Vybrant Dil. Both the labeled phages and the labeled liposomes containing labeled phages were incubated with intestinal cell monolayers for 2 h. Figure 4 shows the CLSM images depicting the fluorescent signals corresponding to the cells (Figure 4A), phages (Figure 4B), and PPE (Figure 4C) stratified across the barrier thickness, as revealed by the x, y, and z scans of the intestinal cell layer. In addition, three-dimensional images of a cross-section of the cell layer, created using the Imaris R software, showed the merging of the different fluorophores used for staining the cell membrane, nucleus, phages, and liposomes (Figure 5). Non-encapsulated bacteriophages were seen inside the intestinal cells (Figure 5A) and phages from the labeled PPE were present at high density on the cell surface and inside the cells (Figures 5B–D). The latter observation reflected adherence of the liposomes containing the labeled phages, which was corroborated by the coincidence of the fluorescence emitted by the stained phages and liposomes. In addition, aggregation of the liposomes resulted in the formation of very large (µm) bacteriophage- containing masses (Figure 5C). These aggregates were mainly embedded in the cell membrane or remained on the cell surface, but some were also seen inside the cells (Figure 5C). Figure 5D shows the presence of liposomes inside the cells with a detail of the basolateral side of the cell culture (sited in the upper side of the image) and bacteriophages released from the liposomes are also seen in this figure. DISCUSSION The purpose of this work was to investigate the adherence of encapsulated phages in liposomes to the intestinal barrier and the ability of the phages to cross this barrier. The behavior of the encapsulated phages was studied in an athymic nude mouse model using a non-invasive methodology based on the visualization of fluorophore-labeled bacteriophages administered FIGURE 4 | Confocal images of the x, y, and z scans of the in vitro orally in a single dose. Although our original intent was to Caco-2/HT29/Raji-B lymphocytes co-culture model. The results obtained with the non-treated (A), bacteriophage-treated (B) and PPE (C) treated cultures determine the biodistribution of encapsulated phages in chickens, at 2 h are shown. Cell nuclei were stained with Hoechst 33242 (blue), technical problems related to the autofluorescence of the down of bacteriophages with SYBR gold (green), and liposomes with Vybrant Dil (red). the chicks prevented the development of this model. The plasma membrane was stained with CellMask DeepRed (gray). Red In the in vivo study, the emitted fluorescence provided a clear squares indicate the regions where the stained phages (B) or PPE (C) were visualization of the bacteriophages in the mouse gastrointestinal visualized. Scale bars, 20 µm. tract (stomach and intestine) (Figure 1). Ex vivo analysis of Frontiers in Microbiology | www.frontiersin.org 8 April 2019 | Volume 10 | Article 689 Otero et al. Biodistribution of Liposome-Encapsulated Bacteriophages FIGURE 5 | Three-dimensional images of a cross-section of the intestinal barrier model based on a Caco-2/HT29/Raji-B lymphocytes co-culture treated with bacteriophages (A) and PPE (B–D). Cell nuclei were stained with Hoechst 33242 (blue), bacteriophages with SYBR gold (green), and liposomes with Vybrant Dil (red). The plasma membrane was stained with CellMask DeepRed (gray). (A) Non-encapsulated phages (green) are seen inside the cells. (B,C) Stained PPE in different phases. (B) Only encapsulated phages (green) are shown inside the cells (gray, membrane and blue, cell nuclei). (C) Merging of all labels with the liposomes (red) covering the encapsulated phages detailed in (B). The white square indicates the non-encapsulated phages (A), the encapsulated phages (B), and liposome capsule (C). (D) A detail of the basolateral side of the intestinal barrier (sited in the upper side of the image) shows the presence of liposomes (red) inside the cells. The enlarged image shows phages released from the liposomes. Scale bars, 8 µm. the fluorescently labeled bacteriophages in organs removed from the capsules. While liposome-encapsulated phages were from the mice further revealed an increase in the phage detected 72 h after the administration of a single dose to concentration in the stomach, but not in the intestine, 3 h non-infected chickens, non-encapsulated bacteriophages were after PPE administration (Figure 2C). Moreover, accumulation scarcely present (Colom et al., 2015). We thus hypothesized a in the stomach was maintained for at least an additional 3 h, high retention capacity of encapsulated bacteriophages in the although the phage concentration decreased under all conditions stomach that enabled the slow delivery and continuous presence (Figure 2C). The concentration of labeled bacteriophage in the of the bacteriophages in the intestine, thereby contributing organs examined ex vivo using conventional microbiological to prolonged and successful oral phage therapy. The in vivo techniques confirmed the effect of encapsulation on phage and ex vivo data presented herein provide further support for accumulation in the stomach at both 3 and 6 h; that is, this hypothesis. the absence of a decrease in the bacteriophage concentration Fluorophore-labeled bacteriophages could not be observed, (Table 1). While 50% of the phages present in the stomach either in vivo or ex vivo, in organs or tissues other than were encapsulated, no encapsulated phages were detected in those above-mentioned, because the phage concentrations were the intestine (Table 1). In a previous study in a chicken below the limit of detection of the IVIS imaging system (∼7 model, we showed that the cationic lipid mixture used in log10 pfu/g). Further studies using a different fluorophore, a encapsulation protected the bacteriophages against the acidic higher phage concentration, or a more powerful visualization pH of simulated gastric conditions and that encapsulated system are therefore needed to improve phage detection. To our bacteriophages incubated with cecal contents were released knowledge, the ex vivo or in vivo visualization of bacteriophages Frontiers in Microbiology | www.frontiersin.org 9 April 2019 | Volume 10 | Article 689 Otero et al. Biodistribution of Liposome-Encapsulated Bacteriophages in living systems has been achieved in only a few studies and ruled out because homogenization of the intestinal tissue they were carried out using labeled/modified-phages to deliver in MgSO4 , as performed in the ex vivo experiments, breaks pharmaceuticals by intravenous injection (Kelly et al., 2006; the phage capsules in a process mediated by the intestinal Kaźmierczak et al., 2014). Similar to our observations, the results bile salts of the host mice. Nonetheless, encapsulated of those studies demonstrated the utility of molecular imaging to bacteriophages were found in internal organs, including the track phages within living systems. spleen, liver, and muscle. The presence and persistence of non-encapsulated phages in The ability of phages encapsulated in liposomes to overcome the liver, spleen, and kidney and in the blood have been described the intestinal barrier was further studied in an in vitro model by several authors (Dabrowska et al., 2005; Górski and Weber- based on the co-culture of Caco-2 and HT29 intestinal cells Dabrowska, 2005; Górski et al., 2006; Hamzeh-Mivehroud et al., with Raji-B lymphocytes. This model approximates the intestinal 2008; Oliveira et al., 2009; Mi˛edzybrodzki et al., 2017). In barrier and allows comparisons of the permeation capacity of this work, we asked whether encapsulated bacteriophages could nanoparticles with the findings obtained in in vivo studies cross the intestinal barrier to reach internal organs and tissues, (Antunes et al., 2013). Our data showed that the presence as already observed for non-encapsulated bacteriophages. The of the phages did not affect the integrity of intestinal cell liposome-encapsulated phages prepared for use in this work had junctions (Supplementary Figure S2) and that bacteriophage a size of 341.6 ± 8.6 nm, a zeta potential of + 34 ± 5.0 mV, and transcytosis occurred at a low level (Figures 3A–C). However, a capsule formulation that included cholesterol and cholesteryl CLSM revealed the presence of bacteriophages in the cytosol polyethylene glycol 600 Sebacate (Colom et al., 2015). Thus, of intestinal cells (Figures 4B,C). It should also be noted the liposomes were of a size (<500 nm) that allowed their that with a phage starting dose of 1 × 107 pfu/mL our cellular uptake (Jung et al., 2000; Li et al., 2016; Neves et al., transcytosis results agreed with those obtained using P22 in 2016) while the cholesterol or PEG improved their stability MDCK cells (Nguyen et al., 2017). Thus, the model used in the circulation, reducing the likelihood of their removal by in this study can be employed in other investigations of the reticuloendothelial system (Bozzuto and Molinari, 2015; phage transcytosis. Sercombe et al., 2015). Moreover, the hydrophobicity and positive The percentage of bacteriophages that crossed the membrane charge of the capsules favored their mucoadhesion, resulting following PPE administration was also low (Figure 3D) and in their enhanced cellular uptake and lymphatic delivery (Jung encapsulated bacteriophages could not be detected using et al., 2000; Sercombe et al., 2015; Ahn and Park, 2016). Our conventional microbiological methods (data not shown). ex vivo data showed that the number of phages that reached the Nevertheless, in this model, liposome capsules were seen liver, kidney, and even muscle within 6 h post-administration adhering to the cell surface as aggregates, embedded in the cell was significantly higher in mice administered non-encapsulated membrane, and even present inside the cells (Figures 5C,D). phages than in the PPE-treated mice (Table 1). Only in the spleen, These observations and the detection of bacteriophages inside at 3 h post-administration, was there a significant difference the liposomes together suggest that liposomes protect phages between the two groups of mice. This result suggested that the against their elimination from the intestinal tract by excretion. intestinal barrier is more easily crossed by non-encapsulated Following the eventual release of these phage from the liposomes, than by encapsulated phages. In addition, the lipid coat of the they may undergo transcytosis. Unfortunately, however, these phages seemed to favor accumulation in certain organs and results could not be compared with those obtained in our tissues, as suggested by the percentage of encapsulated phages in vivo model because the Transwell membrane obviously does present in the spleen (68.0 ± 8.6), liver (38.4 ± 6.3), and muscle not reproduce the open system of the intestinal tract, among (47.0 ± 7.4) of the mice 3 h after PPE administration. By other differences. contrast, no encapsulated phages were detected in the kidney Taken together, our results suggest that orally administered and hardly any in the plasma (Table 1). To our knowledge, liposome-encapsulated phages remain in the stomach and that ours is the first study to track orally administered, liposome- encapsulation protects the phages until their release. Moreover, encapsulated phages in different organs, although the detection of when encapsulated phages reach the intestine, adherence to the liposome-entrapped phages in the lungs, liver, kidney, and spleen intestinal wall temporarily protects them from both the action of BALB/c mice after 6 h was previously reported (Singla et al., of bile salts and elimination via excretion. We hypothesize 2016). However, in that work, the phages were administered by that liposomes and the phages they contain most likely reach intraperitoneal injection, which may account for the differences internal organs through the duodenum, the segment of the small compared to our study. intestine with the highest absorption rate (Oliveira et al., 2009; Li The in vivo and ex vivo results obtained in our mouse et al., 2016; Van Den Abeele et al., 2017). This region contains model indicated the preferential accumulation of encapsulated M-cells, whose characteristics (high transcytosis capacity, few bacteriophages in the stomach, at least during the first 6 h lysosomes, thinner mucous glycocalyx) (Kou et al., 2013; He post-administration, and that after reaching the intestine et al., 2018) favor the access, uptake, and transport of both they translocated to other organs and tissues. Translocation the positively charged liposome capsules and the phages to the seems to be a rapid process because encapsulated phages underlying lymphoid tissues, albeit by different mechanisms were not found in the intestine as early as 3 h after their (des Rieux et al., 2006; Kou et al., 2013; Li et al., 2016; Yu oral administration. However, the continued presence of et al., 2016), thus enhancing the systemic availability of the encapsulated bacteriophages in the intestine cannot be bacteriophages. The accumulation of encapsulated phages in Frontiers in Microbiology | www.frontiersin.org 10 April 2019 | Volume 10 | Article 689 Otero et al. Biodistribution of Liposome-Encapsulated Bacteriophages the stomach and their adherence to the intestinal wall would cell culture, and statistical and imaging analysis. AG-R explain our previous observations (Colom et al., 2015) of the assisted in establishing and maintaining the intestinal cell greater long-term efficacy of phage therapy using liposome- cultures. MC-S assisted in bacteriophage encapsulation. encapsulated phages. DM and RM reviewed the manuscript. PC and ML contributed to the design of the study, analyses of the results, coordination of the study, and drafting the CONCLUSION manuscript. All authors read and approved the final version of the manuscript. This study is the first to demonstrate the biodistribution and transcytosis of orally administered, liposome-encapsulated bacteriophages. In a murine model based on a non-invasive FUNDING methodology, labeled phages were visualized in the mouse stomach and intestine. Moreover, conventional culture methods This work was supported by the Ministerio de revealed the additional presence of liposome-encapsulated Economía, Industria y Competitividad in Spain grant phages in the stomach and other internal organs, including the BIO2016–77011-R. JO and AG-R received predoctoral spleen and liver, and in muscle. The adherence of liposome- fellowships from the UAB. ICN2 is supported by the containing phages to human intestinal cells, their embedding Severo Ochoa program from Spanish MINECO (Grant within the cells, and the transcytosis of the phages was evidenced No. SEV-2017-0706). by CLSM. Our results contribute to the development of treatment options based on oral phage therapy. Nonetheless, further efforts are required to improve bacteriophage labeling or in vivo ACKNOWLEDGMENTS imaging in order to track the dissemination of liposome- encapsulated phages. We are grateful to the Servei de Microscòpia of the UAB for their support. We thank J. Colom for his assistance in carrying out preliminary experiments in the in vivo biodistribution ETHICS STATEMENT studies, and S. Escribano and J. Ruiz for their excellent technical assistance. The in vivo experiments were performed by in the ICTS “NANBIOSIS,” specifically, by the CIBER-BBN’s in vivo experimental platform of the Functional Validation & Preclinical SUPPLEMENTARY MATERIAL Research (FVPR) area (http://www.nanbiosis.es/portfolio/u20- in-vivo-experimental-platform/) and Laboratory Animal Service The Supplementary Material for this article can be found (LAS) of Vall d’Hebron Institut de Recerca (VHIR; Barcelona). online at: https://www.frontiersin.org/articles/10.3389/fmicb. The animals were treated in agreement with the guidelines of the 2019.00689/full#supplementary-material Ethics Commission [Comité Ético de Experimentación Animal (CEEA)] of the VHIR, Barcelona. The study was approved and FIGURE S1 | (A) Cryo-TEM image of liposome-encapsulated UAB_Phi20; (B) 3D assigned the authorization number 69/15. confocal images of SYBR gold-labeled UAB_Phi20 encapsulated into fluorescent Dil-labeled liposome (red). 3D image of liposome surface is shown on the left and its cross-section images on the right. Scale bars, 5 µm. AUTHOR CONTRIBUTIONS FIGURE S2 | Epithelial cell monolayer TEM images after 48 h of incubation with the non-encapsulated bacteriophage. Tight junctions are indicated by a white JO executed most of the bacteriophage experiments, including ellipse. 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EP2750519. doi: 10.1016/S0939-6411(00)00084-9 Kaźmierczak, Z., Piotrowicz, A., Owczarek, B., Hodyra, K., Miernikiewicz, P., The remaining authors declare that the research was conducted in the absence of Lecion, D., et al. (2014). Molecular imaging of T4 phage in mammalian tissues any commercial or financial relationships that could be construed as a potential and cells. Bacteriophage 4:e28364. doi: 10.4161/bact.28364 conflict of interest. Kelly, K. A., Waterman, P., and Weissleder, R. (2006). In vivo imaging of molecularly targeted phage. Neoplasia 8, 1011–1018. doi: 10.1593/neo.06610 Copyright © 2019 Otero, García-Rodríguez, Cano-Sarabia, Maspoch, Marcos, Cortés Kou, L., Sun, J., Zhai, Y., and He, Z. (2013). The endocytosis and intracellular fate and Llagostera. This is an open-access article distributed under the terms of of nanomedicines: implication for rational design. ıAsian. J. Pharm. Sci. 8, 1–10. the Creative Commons Attribution License (CC BY). The use, distribution or doi: 10.1016/j.ajps.2013.07.001 reproduction in other forums is permitted, provided the original author(s) and the Li, H., Chen, M., Su, Z., Sun, M., and Ping, Q. (2016). Size-exclusive effect of copyright owner(s) are credited and that the original publication in this journal nanostructured lipid carriers on oral drug delivery. Int. J. Pharm. 511, 524–537. is cited, in accordance with accepted academic practice. No use, distribution or doi: 10.1016/j.ijpharm.2016.07.049 reproduction is permitted which does not comply with these terms. Frontiers in Microbiology | www.frontiersin.org 12 April 2019 | Volume 10 | Article 689