Potential impact of gut Lactobacillus acidophilus and Bifidobacterium bifidum on hepatic histopathological changes in non-cirrhotic hepatitis C virus patients with different viral load

Patients population

This case control study was conducted on 40 non-cirrhotic chronically infected patients with hepatitis C virus (group I), and 10 healthy control subjects well matched with respect to age and sex (group II) after obtaining their informed consent. This study was carried out in Hepatology outpatient clinic, Internal Medicine department, Ain Shams University Hospital after taking the approval of the Research Ethical Committee of the institute. Our exclusion criteria included all patients complaining of chronic hepatitis other than HCV, bilharziasis, splenomegaly, ascites, portal hypertension, recent diarrhea or constipation, liver cirrhosis, collagen disease, diabetes mellitus, or other medical illness, and any patient on alcohol intake, antibiotic, interferon or any antiviral medication. Detailed history taking, clinical examination, abdominal ultrasound (U/S), and laboratory investigations including complete blood count (CBC), liver function tests, serum transaminases, Hepatitis B surface antigen (HBs Ag), and Human immune deficiency antibody (HIV Ab) were done for both groups.

Quantitative assessment of the RNA viral load

HCV-RNA level was detected using real-time polymerase chain reaction (RT-PCR) (Bioline International, UK) with a lower limit of detection 15 IU/ml. Mild viremia considered when the detected copy number of the virus < 200,00 IU/ml. Moderate viremia ranged from 200,000 to 2000,000 IU/ml. High viremia considered when the viral load more than 2,000,000 IU/ml [17].

Liver biopsy and histopathology

Percutaneous liver biopsy was exclusively performed for the group I under U/S guidance using 16 gauge needles. The fixation of tissue samples were done in buffered formalin for 2–4 hrs then embedded in paraffin with melting point 55–57 °C. Sections of 3–4 um were cut followed by staining with haematoxylin-eosin and Masson's trichrome stains to identify collagen fibers. Specimens of 2.5 cm in length, including at least of 12 portal tracts were considered efficient for adequate grading and staging. They were scored for overall necro-inflammatory activity (grades 0–3) and fibrosis (stages 0–4) according to the Metavir scoring system. Examination of liver biopsies was done by only one pathologist who was blind to the clinical data.

Detection of total lactic acid bacteria and Lactobacillus acidophilus using stool analysis and culture

De Man Rogosa Sharp (MRS) agar (CONDA, Spain) was used for the culture of lactic acid bacteria from fecal samples that were collected in the early morning. For quantitative culture of Lactobacillus acidophilus, fecal samples were diluted in sterile saline (weighing 1 gm in 2 ml saline). Sequential dilutions in the sterile saline solution were prepared followed by inoculation of 1 ml of each dilution into MRS agar plates using a standard loop. Then, the plates were incubated anaerobically at 37 °C for 2–3 days. For each stool specimen, colony forming unit per gram was calculated using the following formula (CFU/gm) = (D × N × 2)/W, where D: the dilution; N: the number of colonies on the plate; 2: the original dilution of fecal specimen; and W: the weight of fecal specimen in gm). Normal bacterial count reaches up to 100,000 CFU/ml.

Lactobacillus acidophilus was identified using the morphology of colonies. Typical colonies of Lactobacillus acidophilus on MRS agar appear as small, round, rough, white, translucent, raised colonies and catalase negative. Under microscope Lactobacillus acidophilus appears as gram positive bacilli or rods arranged in short chains. For distinguishing Lactobacillus acidophilus from other lactobacilli, biochemical reactions were performed. As, Lactobacillus acidophilus shows inability to ferment lactose, mannitol and sorbitol [18]. The confirmatory polymerase chain reaction (PCR) assay, which was subjected on every single colony, was used as a specific identifier to the lactic acid bacteria species; (Lactobacillus acidophilus ATCC 4356 and Bifidobacterium bifidum ATCC 11863 microbiota).

PCR detection of the lactic acid bacteria in fecal samplesDNA extraction

QIA amp DNA stool mini kit (QIAGEN, Hilden, Germany) was used for genomic isolation of bacterial DNA from fecal samples according to the manufacturing protocol.

PCR primer

Genus specific primers for identification of Lactobacillus and Bifidobacteria species of lactic acid producing bacteria were designed according to 16S/23S ribosomal RNA intergenic spacer region (16SrRNA) sequence of each species (Sigma, USA) as described by Dubernet et al., and Matsuki et al. [19, 20], respectively. LBA–F (5′-CTT GTA CAC ACC GCC CGT CA-3′), and LBA–R (5-CTC AAA ACT AAA CAA AGT TTC-3) were designed for the amplification of Lactobacillus with a PCR product at 123 pb. BIF-R (GGT GTT CTT CCC GAT ATC A), and BIF-F (CTC CTG GAA ACG GGT GG) were designed for the amplification Bifidobacteria with a PCR product between 549 and 563 bp (Fig. 1).

Figure 1figure 1

Gel Electrophoresis of Lactobacillus acidophilus and Bifidobacteria bifidum gene amplification. Lanes: M, 100 bp DNA ladder marker (Bioron HmbH, Ludwigshafen, Germany). R; reference strains as positive control (Lactobacillus acidophilus ATCC 4356, and Bifidobacterium bifidum ATCC 11863). Well (1, 3, 5, 7, 9) showed Lactobacillus acidophilus gene amplification fragment at 123 bp. Well (4, 6, 10) showed Bifidobacterium bifidum gene amplification fragment between 549-563 bp.

PCR conditions

The PCR reaction mixture (25 ul) was composed of 1×PCR buffer (50 mM Tris-HCL,PH 8.8, 2.5 mM MgCl2 ,15 Mm (NH4)2SO4 0.45% Triton X-100), 0.2 mM of each dNTP, 25 pmol of each primer, 10 ng of bacterial DNA and 1 U of Taq DNA Polymerase (Biotech International, Australia). The PCR was done in a Touch down Thermal Cycler (Hybaid Middlesex, UK). The PCR amplification program included 1st initial denaturation period for 5 min at 94 °C, followed by 30 cycles involving denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 30 s. Finally, the program ended with a final extension step at 72 °C for 7 min. The amplification products were detected using 1% agarose gels (Electrophoresis grade, Invitrogen) electrophoresis. The strain Bifidobacterium bifidum ATCC 11863 and Lactobacillus acidophilus ATCC 4356 were used as positive controls for the PCR runs.

Statistical analysis

Version 12 of the Statistical Program for Social Sciences (SPSS) software were used for data analysis. Mean and standard deviation (SD) or interquartile ranges were used to represent the data. For the comparison between two or three groups, Student’s t-test and one-way analysis of variance (ANOVA) with post Hoc analysis (Tukey’s test) were used respectively. For comparing cateviral load and fecalgorical data, Chi-square test was used. Pearson correlation was used for measuring the correlation between the different variables. A P-value < 0.05 was considered significant.

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