Bacterial strains and susceptibility testing
A clinical isolate of Salmonella enterica serovar Typhimurium was kindly provided by the Estonian Laboratory of Public Health Inspectorate.
For treatment of the experimental S. Typhimurium infection a fluoroquinolone ofloxacin (Hoechst, Germany) and a probiotic L. fermentum ME-3 were applied. The MIC values of ofloxacin to S. Typhimurium on Mueller-Hinton media (Oxoid, UK) were measured by the E-test following the manufacturer's instructions (AB Biodisk, Sweden) and estimated according to the CLSI guidelines [24]. Lactobacillus fermentum ME-3 originated from the Microbial strains collection of Department of Microbiology of University Tartu.
The following two tests were used to evaluate the combinative effect of OFX and ME-3 against S. Typhimurium. First, in the overlay test, 10 ml of the MRS agar (Oxoid, UK), containing 108 CFU/ml of ME-3, was poured onto agar plates and incubated in 10% CO2 at 37°C for 48 h. Then the plates were overlaid with 5 ml 1.0% (w/w) Isosensitest agar (Oxoid, UK) and 108 CFU/ml of S. Typhimurium was seeded into agar. Plates were incubated in microaerobic conditions at 37°C for 24 h and E-test was applied. Second, in the dilution test, serial two-fold dilutions of OFX in broth were prepared. S. Typhimurium and ME-3 solutions were adjusted to the 0.5 McFarland turbidity standards and 10 μl of the suspension was placed into the OFX broth (Nutrient broth No2 Oxoid, UK) and the MBC values were detected. All susceptibility tests were performed in duplicate.
Experimental murine model
The 4 to 6 week old NIH line mice (Kuopio, Finland) were inoculated orally by a single 0.5 ml dose of the S. Typhimurium suspension (105 CFU/ml) using a sterile syringe with the blunt-ended tube. After 48 hours animals were treated either with OFX or ME-3 alone or with their combination for 8 days. Control animals received PBS (phosphate buffered saline). OFX at doses of 20 mg/kg [25] was diluted in 0.5 ml of PBS and given by the sterile syringe with the blunt-ended tube once daily.
Lyophilised ME-3 (Probiotical s.r.l, Novara, Italy) was suspended in PBS to a final concentration of 5 × 107CFU/ml. During the experiments each mouse consumed daily approximately 5 millilitres of ME-3 containing PBS, receiving 2.5 × 108 CFU of lactobacilli.
The commercial diet R-70 (Lactamin, Sweden) with tap water was given ad libitum.
A total of 61 mice were infected with S. Typhimurium and divided into following groups: Gr1 (untreated; n = 22), Gr2 (treated with OFX; n = 13), Gr3 (treated with ME-3; n = 13) and Gr 4 (treated with OFX and ME-3; n = 13). In addition, 11 uninfected animals were treated with PBS and served as a control group for the biochemistry testing. On Day 10 all the surviving animals were sacrificed by cervical dislocation and an autopsy was performed. All experiments were approved by the Committee of Animal Experiments of Estonian Ministry of Agriculture.
Bacteriological assays
At autopsy 10 μl of the heart blood was cultured onto McConkey agar (Oxoid, UK) and on the de Man-Rogosa-Sharpe (MRS) medium (Oxoid, UK) for detection of S. Typhimurium and L. fermentum, respectively. The samples of the ileum and liver were homogenized with sterile glass powder and 10 μl of homogenate was cultured onto McConkey agar. All plates were incubated in the air at 37°C for 24 h. For quantification of lactobacilli the homogenised samples from liver and ileum were weighed and 10 μl of serial ten-fold dilutions in PBS (pH 7.2) were cultured on the MRS and incubated in the 10% CO2 at 37°C for 48 h. The total counts of lactobacilli were calculated as CFU/mg. The lower limit of detection was ≥ 3.0 log CFU/ml. For identification of Lactobacillus fermentum ME-3 the following criteria were applied: colony morphology on MRS, negative catalase reaction, growth on 15°C, lysozyme production and gas production from glucose [26].
Histology
Samples from the ileum, liver and spleen were fixed in 10% formaldehyde and processed further for paraffin embedding. Tissue sections were stained by haematoxylin and eosin. Two pathologists independently in a blinded manner using coded slides evaluated inflammatory and/or destructive lesions. The focal collections of inflammatory cells with various degrees of necrosis were defined as granulomas [27].
Biochemistry
The ileum mucosa collected at autopsy was stored at -80°C until further analysis for a maximum of three months. The indices of oxidative stress: LPO (lipid peroxides) and GSSG/GSH (glutathione redox ratio) were measured after homogenization in a 1.15% KCL solution (1:10).
The levels of LPO were detected using the commercial kit Bioxytech LPO-586 (Oxis International, USA). The value of the redox status of the ileum mucosa was expressed as the glutathione redox ratio GSSG/GSH. The total glutathione TSSG and the oxidized glutathione GSSG of the ileum mucosa were measured using the Griffith method [16, 28]. Glutathione content was determined on the basis of a standard curve generated with a known concentration of this substance. The amount of reduced glutathione GSH was calculated as the difference between TSSG and GSSG.
Statistics
The computer program Sigma Stat for Windows 2.0 (Jandel Corporation, USA) was applied. The tests were selected according to data distribution: the Fisher exact test in comparing categorical values, the Student t-test with Bonferroni correction for describing the continuous indices. The Mann-Whitney test was used for comparing unevenly distributed data.
