Body weight (g) and the organ dimensions (g/kg) of the BALB/c mice in control C (n = 7), treated with ATB for 5 days (DC group, n = 9) and then after 10 days without antibiotic treatment (DC + R group, n = 8).
Decontamination of specific pathogen-free (SPF) mice of BALB/c line was accomplished by administration of amoxicillin per os potentiated with potassium clavulanate at a dose of 387.11 mg/kg body weight and ciprofloxacin administered s.c. at a dose of 18.87 mg/kg body weight every 12 h for 5 days. This resulted in a decreased viability of microorganisms in feces and the cecal content of mice and decreased counts of cultivable microorganisms in the feces, which by day 3 of study declined below the recovery level and to the reduction of animal microbiota to two detected cultivable species, namely Escherichia coli (GenBank KX086704) and Enterococcus sp. (GenBank KX086705). Convalescence of decontaminated animals under gnotobiotic conditions for 10 days prevented restoration of species diversity of mice microbiota and sufficed to return the metabolic, hematological and morphological values to the physiological range. It also restored the fermentative activity of the intestine to the level similar to that observed before antibiotic treatment. Animals subjected to this procedure can be used in further studies. As a result, we created a mouse gnoto model with reduced and controlled microbiota without alteration of the overall health status of the respective animals.
- amoxicillin-clavulanic acid
Autochthonous microbiota in the gastrointestinal tract (GIT) of mammals are a complex, dynamic, spatially and density diverse community of non-pathogenic micro-organisms. They are a metabolically active entity , playing an important role in affecting morphology of the intestine and thus also in its maturation and development, in forming a key barrier against pathogenic bacteria, affecting the immune system through modulation and providing essential products of its metabolism to the host. Accumulating evidence reveals that the gut microbiota plays a major role in promoting health, as a result of which it is often referred to as the “forgotten organ” [2, 3]. These microbiota are key factors in maintaining homeostasis, with functions affecting virtually every organ in the body, such as the regulation of bone mass , brain development and behavior [5, 6, 7], hepatic function , and aspects of adipose tissues  and the cardiovascular system .
In the several past decades, many animal models were used in the studies of dynamically and ecologically diverse community of micro-organisms in gastrointestinal tract (GIT). These micro-organisms are exactly those that help us to understand better the biological complexity of processes underlying their symbiotic relationships with the host. Extensive use of rodents in experiments is related to the fact that these animals can adjust easily to new conditions, multiply quickly, exhibit low nutritional needs and have low requirements on their environment . Like human beings, conventional rodents harbor trillions of bacteria and viruses . The uniformity of microbiota assumed previously in the controlled populations of inbred laboratory animals may not be so high. Some variations may be caused by differences in rearing and handling of animals, and others may result from factors that have not been identified as yet and may affect composition of the microbiota within populations and individuals over time. This should be taken into account when designing experiments involving laboratory animals and interpreting results of such experiments . Despite the fact that only few studies were dealing with systematic comparison of microbiota of highly hygienically standardized mice to those kept in less strict environment, there is sufficient background that allows one to assume limited species complexity in highly microbiologically standardized animals [14, 15]. With increasing use of such rodents, it is reasonable to expect that microbiota of limited diversity alters the known responses of rodents within experimental settings . Using a simplified approach, laboratory animals can be divided to conventional laboratory animals, i.e. those harboring various proportions of other live organisms, and gnotobiotic laboratory animals with accurately defined microbiota. The term germ-free (GF) (axenic) refers to an animal demonstrably free from microbes, including bacteria, viruses, fungi, protozoa, and parasites, throughout its lifetime [17, 18]. GF animals selectively colonized with one or more bacterial species are referred to as gnotobiotic [19, 20]. This term is derived from the Greek “gnotos”, meaning known, and “bios” which means life [17, 21]. Gnotobiotic animals offer a wide range of advantages compared to other animal models when studying the physiology of the digestive tract. This involves particularly the study of mutual interaction of natural microflora and pathogens in the digestive tract and the mechanisms of probiotic effects of microorganisms . Germ-free animal models have been used to explore host-microbiota interactions in entire fields, including lipid metabolism , cardiology , neurogastroenterology [5, 6, 23, 24], reproductive biology [25, 26], and bone homeostasis .
An alternative is a temporary gut sterilization, which may involve absolute or selective elimination of microflora [27, 28]. Some researchers [29, 30] described procedures based on oral administration of antibiotics that allowed them to achieve complete elimination of bacterial flora of rats’ digestive tract and to maintain its bacteria free status. In other studies, various cocktails of antibiotics sufficed to completely or selectively sterilize the gastrointestinal tracts of mice and rats [31, 32, 33, 34]. Administration of oral antibiotic for the purpose of gut sterilization facilitated physiological studies of the nutritionally important relationship between the intestinal microflora and the host. However, when carrying such studies one must consider the extreme variability of such gut flora and thus expect considerable variations of the efficacy of antibiotics in gut sterilization between and within species. Therefore, it is necessary to test effectiveness of any antibiotic cocktail before its implementation . Since the microflora of laboratory specific pathogen-free (SPF) mice is partially controlled and these animals do not come into contact with antimicrobial substances, they are the most suitable model for decontamination . Due to the frequent testing, these animals do not serve as a reservoir of multiresistant or nosocomial micro-organisms . By using antibiotics for decontamination of these animals, one can reduce considerably the number and species diversity of their microbiota.
Our study focused on obtaining an animal model with reduced and controlled microflora ensuring at the same time good health of these model animals.
2. Material and method
2.1. Isolator technology
The experiment was carried out in three germ free isolators (Velaz s.r.o., Prague, Czech Republic) using a gnototechnology described previously by Gancarčíková et al. . A routine microbiological control of isolators was performed throughout the experimental study. Microbiological swabs were taken from gnotobiotic isolator walls, surface of animals and from their rectum. They were inoculated onto TSA agar (tryptic soy agar) with 5% ram’s blood (BBL, Microbiology Systems, Cockeysville, USA).
2.2. Animals, housing and diet
The experiment was carried out on 66 specific pathogen-free (SPF) BALB/c female mice, (4 weeks old), obtained from Velaz s.r.o. (Prague, Czech Republic). All experimental procedures were approved by the Ethics Commission of the University of Veterinary Medicine and Pharmacy in Košice, Slovakia. The experimental protocol No. 1177/14–221 was approved by the State Veterinary and Food Administration of the Slovak Republic and the animals were handled and sacrificed in humane manner in compliance with the guidelines established by the relevant commission. All applicable institutional, national and international regulations for the care and use of experimental animals were observed. The conventional SPF mice were transported by air in special transport containers to the experimental facilities of the Laboratory of Gnotobiology, University of Veterinary Medicine and Pharmacy (UVMP) in Košice. After a thorough surface disinfection of the containers with peracetic acid, these were transferred to gnotobiotic isolators (Velaz s.r.o., Prague, Czech Republic). After subsequent venting of peracetic acid vapors, the mice were transferred to three breeding polypropylene cages, 7–9 mice per cage. The following groups were formed: negative control C (n = 7); decontaminated/antibiotic- treated group DC (n = 9); decontaminated/antibiotic- treated and convalesced group DC + R (n = 8). All animals were fed
2.3. Antibiotic treatment of SPF mice
The experimental mice were administered amoxicillin and clavulanate potassium (Amoksiklav 2 × 457 mg/5 mL, Sandoz Pharmaceuticals, Ljubljana, Slovenia) perorally at a dose of 387.11 mg/kg body weight (0.2 mL of dilution) every 12 h during the first 5 days of the experiment.
Ciprofloxacin (Ciloxan 1 × 5 mL/15 mg, Alcon Cusí S.A., Barcelona, Spain) was administered subcutaneously at a dose of 18.87 mg/kg body weight (0.1 mL of dilution) every 12 h during the first 5 days of the experiment.
2.4. Sampling procedures
Health of the animals and consistency of feces were observed and recorded daily. Fresh fecal samples were collected on days 0, 1, 2, 3, 5 and 15 of the study. Blood samples for hematological and biochemical analysis were collected from anesthetized animals using retro-orbital technique. Anesthesia was induced with sodium pentobarbital at a dose of 86 mg/kg body weight. The mice were euthanized by cervical dislocation at the end of the study for the purpose of sample collection. During dissection, weight of internal organs (heart, liver, spleen, kidneys and lungs) was recorded and samples of feces, caecum and
2.5. Microbiological analysis
2.5.1. Bacterial enumeration
For microbiological analysis, samples of feces and caecum were collected individually from each animal. The samples (1 g) were homogenized Stomacher Lab Blender 80 (Seward Medical Limited, London, UK) with 9 mL of a sterile anaerobic diluent (0.4 g NaHCO3, 0.05 g), L-cysteine-HCl, 1 mL resazurin (0.1%), 7.5 mL mineral solution I (0.6% K2HPO4), 7.5 mL mineral solution II (1.2% NaCl, 1.2% (NH4)2SO4, 0.6% KH2PO4, 0.12% CaCl2, 0.25% MgSO4) and 84 mL distilled water (pH 6.8). A series of 10-fold dilutions (10−1 to 10−9) were made under a CO2 atmosphere. From appropriate dilutions, 0.1 mL aliquots were spread onto Trypticase soy blood agar (Oxoid Unipath, Ltd., Basingstoke, UK) with 10% sheep blood for total aerobes, Schaedler agar (BBL Microbiology systems, Cockeysville, USA) with 1% vitamin K1 - hemin solution for total anaerobes, and Man-Rogosa-Sharpe agar (MRS, Merck, Darmstadt, Germany) for lactic acid bacteria. Incubation of the inoculated media for anaerobic and lactic acid bacteria was carried out at 37°C for 3 days under anaerobic conditions (Gas Pak Plus, BBL). Plates for the enumeration of aerobic bacteria were incubated for 24 h at 37°C. Numbers of colony-forming units (CFU) were expressed as log CFU per gram of sample. The results were presented as arithmetical means ± standard deviation (SD).
2.5.2. Viability of microorganisms on fluorescence-activated cell sorting visualized with viability fluorescent quick test on a polycarbonate filter (VFQTOPF)
The samples of feces and cecal contents were diluted 1:100 in PBS (37°C; MP Biomedicals, France) and filtered through 70 μm and subsequently through 45 μm cell strainers (BD Falcon, NJ, USA). The prepared suspensions were stained with carboxyfluorescein diacetate (cFDA; Sigma) in final concentration of 25 μM and with propidium iodide (PI; Sigma) in final concentration of 45 μM at 37°C for 20 min. Flow cytometric analysis was performed employing a BD FACSCanto™ flow cytometer (Becton Dickinson Biosciences, USA) and BD FACS Diva™ software. The percentages of live and dead bacteria were evaluated based on presence of carboxyfluorescein (cF) (metabolized form of cFDA) detectable only in live bacteria, measured in FL-1 channel (530/30 nm) and the intensity of fluorescence was measured in FL-3 channel (695/40 nm) for propidium iodide (PI) which enters only damaged or dead bacteria . Simultaneously, samples stained with cFDA and PI were analyzed by epifluorescence microscopy. Vacuum filtered samples were fixed on polycarbonate filters (Merck Millipore, Billerica, USA) and stained also with DAPI solution (1 mg of 4′,6-diamidino-2-phenylindole/mL). The filters were placed on microscopic slides and mounted with Vectashield Medium (Vector Laboratories, Peterborough, UK). The slides were examined under a Carl Zeiss Axio Observer Z1 epifluorescence microscope using filter sets 38HE, 64HE and Set 49 for detection of cF, PI and DAPI, respectively. Microphotography analysis was performed using Axio Vision Rel 4.8 software
2.5.3. Determination of the minimum inhibitory concentration of antibiotics
The minimum inhibitory concentrations (MICs) of antibiotics against the tested strains were determined by Etest® strips for ciprofloxacin (AB bioMérieux, Marcy l’Étoile, France) and M.I.C. evaluator strips for amoxicillin and clavulanate potassium (Thermo Fisher Scientific, Basingstoke, UK). The results were read in accordance with the manufacturers’ protocol, which is essentially identical for both strip products.
2.5.4. Phenotypical identification
Phenotypical identification of
2.5.5. DNA identification
After microbiological cultivation on blood agar, DNAzol direct (Molecular Research Center Inc., Cincinnati, USA) was used to isolate DNA from bacterial colonies. The PCR reaction was performed with the help of primers 27F (5-AGAGTTTGATCMTGGCTCAG-3 and 1492R (5-CGGYTACCTTGTTACGACTT-3). The amplification protocol for PCR reaction was: 5-min at 94°C, 1 min at 94°C, 1 min at 55°C and 3 min at 72°C and a final at 72°C 10-min (TProfesional Basic, Biometra GmbH, Göttingen, Germany). PCR products were separated by electrophoresis on 0.7% agarose gel with the help of TAE buffer. The PCR amplicons were stained with GelRed™ (Biotium Inc., Hayward, USA) and visualized after the separation under UV light. Purification of PCR products was carried out by means of a kit NucleoSpin® Gel and PCR Clean-Up Kit (Mancherey-Nagel GmbH & Co. KG, Düren, Germany). The amplicons were submitted for sequence analysis to
2.6. Blood and serum analysis
Hematological analysis was carried out using a BC-2008 VET automatic analyzer (Mindray, Shenzhen, China). An automated biochemical analyzer Ellipse (AMS, Rome, Italy) and standard kits (Dialab, Prague, Czech Republic) were used to determine concentrations of the following biochemical parameters: glucose; triglycerides; cholesterol; HDL-cholesterol; LDL-cholesterol; total protein; urea; albumin; creatinine; activities of enzymes aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP). Total activity of lactate dehydrogenase (CLDH) was determined spectrophotometrically (Alizé, Lisabio, France) and its isoenzymes (LDH-1: LDH-5) were determined by an electrophoretic method (Hydrasys, Lisses, France).
2.7. Short chain fatty acids (SCFAs) analysis
The produced organic acids were determined by isotachophoresis as described by Gancarčíková et al. . After the collection, 0.5 g of feces and caecum contents were dissolved in 25 mL deionized water and 30 μL aliquots were used for analysis of short-chain fatty acids (SCFAs). The measurements were done on an Isotachophoretic analyzer ZKI 01 (Radioecological Institute, Košice, Slovakia). A leading electrolyte of the following composition was used in the pre-separatory capillary: 10−2 mol/L HCl + 2.2. 10−2 mol/L ε-aminocaproic acid + 0.1% methylhydroxyethylcellulosic acid, pH = 4.3. A solution of 5. 10−3 mol/L caproic acid + 2. 10−2 mol/L histidine was used as a finishing electrolyte. This electrolytic system worked at 150 μA in the pre-separatory and at 40 μA in the analytic capillary.
2.8. Histology of the liver and kidneys
Liver samples from
Statistical evaluation of the results was performed using Statistic software GraphPad Prism 3.0 for Windows (GraphPad Software, San Diego, USA). One-way analysis of variance (ANOVA) was used, followed by a multiple comparison Tukey’s test. Significance of differences between the groups of mice was tested using analysis of variance and unpaired Student’s
3.1. Clinical examination of animals
Laboratory SPF BALB/c mice were subjected to complex clinical examination during quarantine and at the end of the experiment. During experiment, all changes in clinical status were observed and recorded twice daily (8.00 and 15.00 h). The regular observation of overall health manifested by uptake of food, agility of animals and consistency of feces allowed us to detect changes in consistency of feces from solid to pasty on day 3 of the experiment in 10 out of 17 animals treated with antibiotics. All SPF BALB/c mice were agile and their intake of food was unchanged.
3.2. Total body weight and relative weight of internal organs
On day 5 of the experiment, the total body weight of animals from experimental group DC (Table 1) was insignificantly lower by 0.23 g in comparison to negative control (C). Examination of internal organs showed a significant decrease in relative weight of the liver (
|Group||The organ dimensions (g/kg)||Body weight (g)|
|Heart||Liver||Spleen||Right kidney||Left kidney||Lungs|
|C||5.49 ± 0.28||53.87 ± 2.6||4.40 ± 0.30||7.19 ± 0.34||7.16 ± 0.24||8.27 ± 0.67||16.13 ± 0.34|
|DC||5.07 ± 0.21||47.41 ± 0.68 *C||2.82 ± 0.13 **C||7.31 ± 0.30||7.33 ± 0.33||7.40 ± 0.33||15.90 ± 0.36|
|DC + R||5.96 ± 0.21||5.70 ± 1.49||4.71 ± 0.31||7.29 ± 0.29||7.16 ± 0.31||8.23 ± 0.27||17.33 ± 0.65|
3.3. Hematology parameters
Total counts of leukocytes (WBC) and lymphocytes (Ly) in all investigated groups (Table 2) were in physiological ranges. However, the decontaminated group (DC) showed insignificantly lower counts of WBC (by 1.73 G/L) and lymphocytes (by 1.65 G/L) in comparison with control group C.
|Group||C||DC||DC + R||Ref BALB/c|
|WBC (G/L)||7.76 + 1.55||8.80 + 1.92||6.03 + 0.98||5.69–9.87|
|Ly (G/L)||6.08 + 1.31||5.18 + 1.09||4.43 + 0.65||3.60–7.29|
|Mo (G/L)||0.14 + 0.04||0.74 + 0.30*C||0.15 + 0.04*DC||0.34–0.70|
|Gran (G/L)||1.54 + 0.35||2.88 + 0.70||1.45 + 0.34||0.74 – 1.78|
|Ly (%)||77.64 ± 2.83||60.10 ± 5.06**C||74.67 ± 1.98**DC||55.06–73.44|
|Mo (%)||1.94 ± 0.20||7.62 ± 2.13**C||2.75 ± 0.30**DC||3.75–7.26|
|Gran (%)||20.42 ± 2.70||32.28 ± 3.34*C||22.58 ± 1.77*DC||10.46–18.94|
|RBC (T/L)||9.06 ± 1.17||10.77 ± 0.39||9.44 ± 1.16||8.16–9.98|
|HGB (g/L)||156.4 ± 19.85||189.00 ± 7.96||145.90 ± 13.52||124–154|
|HCT (%)||51.50 ± 7.01||61.00 ± 2.42||47.37 ± 4.20||43.50 – 55.4|
|MCV (fL)||56.60 ± 0.69||56.66 ± 0.45||55.88 ± 0.42||50.80 – 55.60|
|MCH (pg)||17.26 ± 0.28||17.46 ± 0.15||15.85 ± 0.96||13–15.5|
|MCHC (g/L)||306.4 ± 6.74||309.4 ± 1.03||297.5 ± 12.19||239–280|
|RDW (%)||14.78 ± 0.50||13.62 ± 0.43||13.76 ± 0.24||16.9–19.1|
The changes in red blood components recorded in group DC after decontaminated with antibiotics (ATB) resembled those observed in white blood components in this group (Table 2). Increased counts exceeding the physiological range, although insignificantly different, were observed for erythrocyte counts (RBC), level of hemoglobin (HGB) and hematocrit (HCT) in comparison with control (C).
3.4. Biochemical parameters
3.4.1. Nitrogen profile
Nitrogen profile (Table 3) represented by concentration of total proteins (TP) and albumin showed significant differences between groups DC and C on day 5 of the experiment. Despite decreased exogenous intake of feed by animals of group DC, this group exhibited significantly higher concentration of both TP and albumin (
|Group||C||DC||DC + R||Ref BALB/c|
|Total protein (g/L)||70.20 ± 1.65||89.98 ± 0.90**C||66.60 + 5.78**DC||60.8–73.0|
|Urea (mmol/L)||6.66 ± 0.63||6.68 ± 0.08||5.87 ± 0.16||5.70–7.14|
|Albumin (g/L)||33.98 ± 0.48||37.48 ± 0.57**C||31.57 ± 0.82***DC||31.0–37.0|
|Creatinine (μmol/L)||27.50 ± 0.96||24.50 ± 0.96||30.00 ± 0.58**DC||up to 33.59|
|Glucose (mmol/L)||8.03 ± 0.17||6.35 ± 0.06**C||8.03 ± 0.47**DC||4.72–10.71|
|Triglyceride (mmol/L)||2.59 ± 0.05||2.26 ± 2.09*C||2.96 ± 0.06*C, ***DC||up to 3.42|
|Cholesterol (mmol/L)||2.62 ± 0.09||3.41 ± 0.03***C||3.22 ± 0.16*C||2.09–3.65|
|HDL cholesterol (mmol/L)||1.77 ± 0.06||1.76 ± 0.02||1.79 ± 0.02||up to 1.78|
|LDL cholesterol (mmol/L)||0.38 ± 0.01||0.75 ± 0.02***C||0.57 ± 0.01***C,DC||up to 0.38|
|DC||up to 0.38|
|AST (μkat/L)||3.27 ± 0.18||3.64 ± 0.10||3.13 ± 1.57||2.67–3.05|
|ALT (μkat/L)||2.50 ± 0.24||3.26 ± 0.55||8.20 ± 1.63**C,DC||0.68–2.89|
|ALP (μkat/L)||6.47 ± 0.30||5.48 ± 0.48||5.96 ± 0.45||1.83–6.23|
|LDH-Total (μkat/L)||58.4 ± 2.9||78.98 ± 9.81||64.83 ± 12.3|
|% z LDH-T||2.9 ± 1.1||1.55 ± 0.13||2.0 ± 0.15|
|(μkat/L)||1.73 ± 0.73||1.22 ± 0.16||1.27 ± 0.18|
|% z LDH-T||2.6 ± 0.1||2.38 ± 0.15||3.0 ± 0.15|
|(μkat/L)||1.53 ± 0.14||1.85 ± 0.16||1.98 ± 0.43|
|% z LDH-T||16.75 ± 3.15||14.35 ± 2.54||21.2 ± 1.25|
|(μkat/L)||9.88 ± 2.33||10.98 ± 1.53||13.57 ± 2.16|
|% z LDH-T||9.25 ± 0.55||8.53 ± 0.24||10.97 ± 1.52|
|(μkat/L)||5.39 ± 0.06||6.7 ± 0.74||7.44 ± 2.38|
|% z LDH-T||68.5 ± 3.8||73.2 ± 2.7||62.83 ± 0.8|
|(μkat/L)||39.9 ± 0.24||58.24 ± 8.7||40.58 ± 7.4|
3.4.2. Energy and lipid profile
3.4.3. Enzymatic profile
While on days 5 and 15 of the experiment none of the investigated groups showed increased activity of enzyme ALP (Table 3), activities of enzymes AST and ALT were insignificantly increased in group DC in comparison with group C. ALT is a liver-specific enzyme and its increased activity indicates irritation or damage to the liver. Its increase is associated with damage to membrane of liver cells, even at the absence of their necrosis, and the enzyme is excreted at both reversible and irreversible damage to liver parenchyma. Increased activity up to 3-fold the reference level is considered a moderate increase. After
3.4.4. Activity of LDH-total and isoenzymes
LDH-T is a multi-organ cytosol enzyme that exists as 5 isoenzymes. It is released to circulation already at slight tissue damage. Observation of specific activity of total LDH (Table 3) and its isoenzymes in the serum of mice of the investigated groups (C, DC a DC + R) showed no significant differences.
An insignificant decrease in total LDH was observed again in group DC + R after convalescence period. The activity of this enzyme was lower by 14.15 μkat/L in comparison with group DC.
3.5. Microbiological parameters
3.5.1. Determination of counts of cultivable microorganisms in mice feces
Before the application of antibiotics (ATB), the plate counts (Figure 1) of microorganisms in feces in all groups of SPF mice (C, DC, DC + R) ranged between 8.15 and 9.19 log10 CFU/mL. Determination of plate counts 24 h after the antibiotic treatment showed a significant decrease by 4 logs (4.58 ± 0.31 log10 CFU/mL) after aerobic cultivation and by 3–4 logs (4.89 ± 0.46 log10 CFU/mL) after anaerobic cultivation when compared with the initial counts determined before antibiotic treatment (8.51 ± 0.31 log10 CFU/mL). Cultivation at 48 h from the beginning of antibiotic treatment revealed less pronounced decrease in plate counts of cultivable microorganisms. The counts were reduced by 1 log under aerobic conditions (3.54 ± 0.47 log10 CFU/mL) and by 1–2 logs when cultivated anaerobically (3.38 ± 0.48 log10 CFU/mL), in comparison with the counts determined at 24 h after the antibiotic treatment. The following investigations on days 3 and 5 of cultivation revealed absence of cultivable microorganisms in the feces (Figure 1). Determination of plate counts on day 10 after termination of antibiotic treatment showed recurrence of cultivable microorganisms in feces after both aerobic cultivation (8.36 ± 0.08 log10 CFU/mL) and anaerobic cultivation (8.36 ± 0.29 log10 CFU/mL).
3.5.2. Survivability of microorganisms in samples of feces and caecum content determined by FACS, visualized by means of VFQTOPF
Survivability of microorganisms in mice feces (BD FACS Canto flow cytometer, BD, USA) decreased significantly (
3.5.3. Cultivable bacteria detected in the study
At day 10 after termination of antibiotic treatment, the microbiota was reduced to two cultivable species. They were differentiated and identified on the basis of morphological, biochemical and genetic differences. The first species isolated from DC + R group was a Gram-negative rod-shaped bacterium. Determination of biochemical properties of this bacterium by means commercial ENTEROtest 24 N (Erba Lachema s.r.o., Brno, Czech Republic) showed that this involved species
The second species isolated from DC + R group was a Gram-positive coccus. By analyzing the DNA section corresponding to 16S rRNA of bacteria by BLAST analysis and comparing it with DNA templates, the best match obtained indicated
3.6. Production of SCFAs in feces and caecum
Production of organic acids (Figure 6) in the
Within the 5-day decontamination period, examination of
Despite the fact that control mice (C) were not treated with ATB, they showed a significant decrease in concentrations of acetic and lactic acid (
More pronounced although insignificant decrease in
Although the concentration of
While the concentrations of acetic, lactic, succinic and propionic acids in groups DC and DC + R showed a decreasing tendency in the decontamination period (days 2–5) the concentrations of
The concentration of
3.7. Histological examinations of livers and kidneys
The liver and kidney cells are highly sensitive to harmful effects of xenobiotics including antibiotics; therefore, we examined histomorphology of the livers and kidneys from control mice without treatment (C), from treated mice (DC) and from treated group of mice after a period of recovery (DC + R). Light microscopy revealed that liver sections from control mice showed normal liver architecture and hepatocytes were arranged in rows radiating out from central veins (Figure 10). In the livers from DC group, we observed sporadic occurrence of lesions (arrows) with advanced vacuolization containing a few, usually necrotic, hepatocytes and disrupted sinusoids (arrowheads). Such altered or loose liver parenchyma indicated an early metabolic injury to the cells. In this group, fluorescent staining specific for neutral lipids demonstrated the presence of lipids droplets in some hepatocytes (arrowheads) and in these lesions (arrows). However, we did not find the fragmented nuclei of hepatocytes and other cells indicating that treatment did not elicit apoptosis. The representative microphotograph of the liver from DC + R group showed normal tissue morphology without any histopathological changes.
The representative Figure 11 (C) of paraffin section after hematoxylin/eosin staining of cortex from untreated group demonstrates multiple renal corpuscles consisting of the glomerulus and the capsule around it (arrows). In DC group, the overall morphology of cortex and appearance of these Bowman’s capsules did not show any pathological alterations or damage to cells. Central medullar part of kidneys from DC group had the same morphology as was observed on sections from control mice (not shown). A representative image of kidney’s cortex from DC + R group (Figure 11) showed normal morphology. Using the fluorescent double staining methods we demonstrated the positive signal for neutral lipids droplets in the cortex (left, arrows) and in some of renal cells in medulla of kidneys (right, arrowheads) in all groups. No apoptotic process in kidney cells was seen in either of examined groups.
Animal gut microbiota is a complex community of trillions of microbes colonizing the digestive tract of animals. This extensive community, comprising as many as 1012 colony-forming units/mL in the colon, affects physiology of the gastrointestinal tract, the function of distant organs and susceptibility of animals to diseases . Despite the enormous bacterial load carried by the gastrointestinal tract and the sheer variety of species present, an exquisite balance is maintained at almost all times. The combination of an efficient, self-repairing barrier, abundant mucus secretion, continuous luminal flow of contents and a vigorous yet finely regulated immune system is capable of keeping a massive foreign population contained within the limits of the mucosa . This delicate equilibrium represents a well-balanced opposition of considerable forces. Alteration of this equilibrium is pivotal in the development of diseases of gastrointestinal tract.
Laboratory animals such as germ-free (GF) rodents have proved important for studying the effects of microbial mono- and poly-colonizations on host phenotype [38, 39, 40] and in the search for a mechanistic understanding of microbe-mediated changes in several disease models [41, 42, 43, 44, 45]. An alternative is temporary gut sterilization, which may involve absolute or selective elimination of microflora [27, 28]. The first studies devoted to decontamination of the digestive tract by ATB investigated successfulness of such decontamination and removal of microorganisms from the animal digestive tract. Results indicate that decontamination of mice , monkeys , dogs , Syrian hamsters  and pigs  with oral antibiotics is feasible. However, these studies did not investigate the effect of ATB on animal health. In human medicine, the beginnings of decontamination of digestive tract were related to prevention of septicemia in patients with granulopenia , in studies of burns therapy , acute pancreatitis , and later in acute stroke , critically ill patients  or esophageal resection  and prevention of acute graft-versus-host disease following allogeneic bone marrow transplantation . Selective antibiotic treatment resulting in decontamination of the digestive tract was capable of preventing severe infections and reducing mortality rate in patients in the critical stages of diseases. Concern about development of bacterial resistance associated with the use of such decontamination and the absence of its influence on mortality, have not been confirmed . The aim of SDD (Selective Digestive Decontamination) is to prevent or eradicate, if present, the oropharyngeal and intestinal abnormal carriage of potentially pathogenic microorganisms, such as Gram-negative aerobic microorganisms, methicillin-sensitive
Various antibiotic cocktails have been shown to completely or selectively sterilize the gastrointestinal tracts of mice and rats [31, 32, 60]. Our study was aimed at decontamination of BALB/c SPF mice in a way that would not have adverse effect on their health. Similar to Johnson et al. , we strived to develop a non-invasive, relatively simple and inexpensive method of decontamination of the gut, testing for the sterility and maintaining controlled microbiota in model animals suitable for further experiments. In the study by Johnson et al.  animals were decontaminated and sterile environment in their gastrointestinal tract was maintained by enrofloxacin in Baytril 10% (Bayer, Germany) without barrier maintenance or using a laminar box. In other studies, the decontamination of gastrointestinal tract was carried out using ampicillin [61, 62, 63], bacitracin and neomycin , meropenem [64, 65] and vancomycin  added to the drinking water. On the basis of our previous results , mice in our study were decontaminated with amoxicillin administered
Some research studies were conducted dealing with the comparison of antibiotic decontamination carried out on the basis of cultivation and studies based on commonly used antibiotic combinations. They included the clinical study E.O.R.T.C. , which investigated the effect of ATB selected on the basis of cultivation and compared it with the effect of combination of neomycin, cephaloridine, polymyxin (B or E) and nystatin or amphotericin B in granulocytopenic patients. Comparisons indicated good effectiveness of both methods and the differences were insignificant. However, it is worth mentioning that only non-absorbable ATB were used in the E.O.R.T.C.  study. In our study, we used the ATB selected on the basis of cultivation, as recommended by Johnson et al.  with the aim to eliminate the ATB with marked adverse impacts on animal health.
The length of antibiotic administration in the available studies differed. In our study, we administered ATB for 5 days. This was based on preliminary examinations and procedures carried out at our institution that showed null cultivation recovery of bacteria from feces on day 3 of antibiotic administration. Van der Waaij et al.  arrived to similar conclusions while the length of administration of ATB in other studies varied as follows: 4 days [27, 76], 7 days , 14 days , 21 days  or 28 days [73, 78, 79, 80]. While in our study the DC + R group of animals was kept under gnotobiotic conditions for 10 days following the antibiotic administration, in other studies, the mice convalescence period lasted from 14 days [71, 77] up to 5 weeks .
Following the 10-day convalescence period, the cultivable colonies obtained from feces and caecum content of SPF mice were tested biochemically and subjected to 16S ribosomal DNA (rDNA) sequencing that allowed us to identify
The ciprofloxacin MIC against
Flow cytometry results obtained in our study showed a decrease in viability of microorganisms in feces. The differences were significant (
Short chain fatty acids are the principal metabolites of intestinal fermentation and their concentrations in the digestive tract reflect the level of this fermentation. The most pronounced decrease in production of organic acids, particularly acetic, lactic and propionic acids in feces of both decontaminated groups (DC, DC + R), was recorded as soon as 24 h after starting with administration of ATB, which correlates with decreased plate counts of microorganisms in these groups by 4 logs after aerobic cultivation and 3–4 logs after anaerobic cultivation. Also during the following days of administration of ATB (days 2–5), low level of intestinal fermentation was detected in the feces of decontaminated mice. Eleven days lasting antibiotic treatment (ampicillin, bacitracin, meropenem, neomycin and vancomycin) caused marked changes in colon microbiota and gut dysbiosis was reflected in changed concentrations of several metabolites in the colon luminal contents . The depletion of the SCFAs acetate, n-butyrate and propionate, the products of microbial fermentation of dietary fiber, agreed with results presented in other studies [88, 89, 90]. In our study, we observed high concentrations of acetoacetic and butyric acids, the products of biodegradation of lipid tissue. Keto compounds that formed at physiological state of passive degradation of lipid stores became a substitute source of energy for normal functioning of the organism at the time of energy starvation. However, by day 15 of the study, the intestinal fermentation activity was restored in group DC + R and production of organic acids returned to the level before the treatment with ATB. An interesting observation was that concentration of organic acids decreased also in the feces of control group of mice C that was not treated with ATB. This decrease could be explained by the fact that these animals were fed sterile commercial feed, supplied sterile water in bottles and sterile bedding was replaced every day.
The macroscopic picture of all decontaminated mice was typical of germ-free animals, such as megacaecum condition and a significant decrease in relative weight of the liver (
The potential effect of antibiotic decontamination of mice on overall health of treated animals was investigated only in small number of relevant studies. Our study showed only a slight change in the blood picture of mice from DC group in comparison with group C. Moreover, after the convalescence, all parameters determined in group DC + R returned to the physiological range . The mice from DC group showed increased levels of Mo (
Animals obtained under this protocol can be used in our further studies such as nutritionally important relationship between the intestinal microflora and the host, interactions between microorganisms in the gut or modulation of metabolic and physiological parameters of host with selected probiotics.
In conclusion, decontamination of SPF BALB/c mice with combination of per oral administration of amoxicillin and clavulanate potassium and subcutaneous administration of ciprofloxacin every 12 h during 5 days reduced viability of microorganisms in feces and caecum content and resulted in absence of cultivable microorganisms in feces. After 10-day convalescence of antibiotic-treated SPF mice under gnotobiotic conditions the diversity of gut microbiota of mice was not recovered as it was reduced to only two detectable cultivable species, specifically to
Results presented in this publication were obtained with the support of the Slovak Research and Development Agency under the contracts APVV-16-0176 and APVV-15-0377 and the projects VEGA No. 1/0009/15 and No.1/0081/17.