Real-time analysis of gut flora in Entamoeba histolytica infected patients of Northern India - PubMed (original) (raw)
Real-time analysis of gut flora in Entamoeba histolytica infected patients of Northern India
Anil Kumar Verma et al. BMC Microbiol. 2012.
Abstract
Background: Amebic dysentery is caused by the protozoan parasite Entamoeba histolytica and the ingestion of quadrinucleate cyst of E. histolytica from fecally contaminated food or water initiates infection. Excystation occurs in the lumen of small intestine, where motile and potentially invasive trophozoites germinate from cysts. The ability of trophozoites to interact and digest gut bacteria is apparently important for multiplication of the parasite and its pathogenicity; however the contribution of resident bacterial flora is not well understood. We quantified the population of Bacteroides, Bifidobacterium, Ruminococcus, Lactobacillus, Clostridium leptum subgroup, Clostridium coccoides subgroup, Eubacterium, Campylobacter, Methanobrevibacter smithii and Sulphur reducing bacteria using genus specific primers in healthy (N = 22) vs amebic patients (E. histolytica positive, N = 17) stool samples by Real-time PCR.
Results: Absolute quantification of Bacteroides (p = .001), Closrtridium coccoides subgroup (p = 0.002), Clostridium leptum subgroup (p = 0.0001), Lactobacillus (p = 0.037), Campylobacter (p = 0.0014) and Eubacterium (p = 0.038) show significant drop in their population however, significant increase in Bifdobacterium (p = 0.009) was observed where as the population of Ruminococcus (p = 0.33) remained unaltered in healthy vs amebic patients (E. histolytica positive). We also report high prevalence of nimE gene in stool samples of both healthy volunteers and amebic patients. No significant decrease in nimE gene copy number was observed before and after the treatment with antiamebic drug.
Conclusions: Our results show significant alteration in predominant gut bacteria in E. histolytica infected individuals. The frequent episodes of intestinal amoebic dysentery thus result in depletion of few predominant genera in gut that may lead to poor digestion and absorption of food in intestine. It further disturbs the homeostasis between gut epithelium and bacterial flora. The decrease in beneficial bacterial population gives way to dysbiosis of gut bacteria which may contribute to final outcome of the disease. Increase in the copy number of nimE gene harboring bacteria in our population reflects possible decrease in the availability of metronidazole drug during treatment of amoebiasis.
Figures
Figure 1
Screening of stool samples by Dot-Blot method. (A) Linear map of EhRI episome (24.5 kb) showing the position of HMe probe (4.5 kb in size) common for both E. histolytica and E. dispar), E - EcoR1 site and H- Hind III site; rDNA I and rDNA II represent two inverted repeats of transcription units with various restriction sites and repeats (B) Representative figure of Dot-blot analysis of stool sample using HMe probe. Rows 1 to 6 (column A-D) represent spots of DNA from stool samples. About 20 ng of DNA was loaded on each spot in triplicate on nylon membrane. Row 7 was blank. Row 8 (column A) E. histolytica HM1: IMSS genomic DNA as positive control; (column B) E. dispar SAW760 genomic DNA as positive control; (column C) E.Coli DH5α as negative control; (column D) Plasmid with cloned HMe as positive control. All samples were loaded in triplicate. Experimental details are provided in material and methods.
Figure 2
Screening of Stool samples by PCR. (A) Schematic representation of location of Entamoeba histolytica specific primer. BH16197 is Genbank accession number of Entamoeba histolytica SINE-2 (EhSINE2) element; (B) Schematic representation of location of Entamoeba dispar specific primer from rDNA molecule. 18 S, 5.8 S and 28 S are corresponding ribosomal gene sequences and ITS-1 and ITS-2 refers to internal transcribed spacer 1 and 2; (C) Detection of E. histolytica in stool DNA sample using E. histolytica specific primers, Lane 1 = Marker 100 bp, Lane 2 = EhHM1 genomic DNA as positive control, Lane 3 & 4 stool sample DNA, Lane 5 = Genomic DNA of E. dispar SAW760 as negative control_._ Sample in lane 4 is E. histolytica positive; (D) Detection of E. dispar in Stool sample using E. dispar specific primers. Lane 1 = Marker 1 kb, Lane 2,3,4 and 5 stool sample DNA, Lane 6 = Genomic DNA of E. dispar as positive control. Sample in Lane 3 and 5 are E. dispar positive. Lane 4 stool sample is E. histolytica positive and was used as negative control.
Figure 3
Real-time analysis for quantification of different bacterial genera in Healthy vs E. histolytica positive (Eh + ve) samples. (A) Bacteroides (B) Clostridium coccoides subgroup (C) Clostridium leptum subgroup (D) Lactobacillus (E) Campylobacter (F) Eubacterium. P value = .05 or below was considered significant. CI stands for confidence interval.
Figure 4
Real-time analysis of population of (A) Rumminococcus in Healthy vs E. histolytica positive (Eh + ve) samples (B) Bifidobacterium in Healthy vs E. histolytica positive (Eh + ve) samples. P value = .05 or below was considered significant. CI stands for confidence interval.
Figure 5
Detection and identification of nim gene in stool samples. (A) Detection of nim gene using nim gene specific primers. Lane 1 = Marker 100 bp, Lane 2 = clone of nim gene as positive control, Lane 3–5 = DNA from stool samples from healthy volunteer, Lane 6–8 = DNA from stool samples from E. histolytica positive patients and Lane 9 = No template control PCR (B) Restriction map of TaqI restriction sites in 458 bp nimE gene fragment. (C) HpaII does not digest nimE,where as digestion of nimE by TaqI generates four fragment of 274 bp,155 bp,6 bp and 25 bp. Lane 1 = Marker 100 bp, Lane H1, H2, E1 and E2 show RFLP profile of PCR product digested with HpaII; Lane H3, H4, E3 and E4 show RFLP profile of PCR product digested with TaqI. H1-H4, DNA from stool samples of Healthy volunteers and E1-E4 are DNA from stool samples of E. histolytica positive patients.
Figure 6
Detemination of copy number of nimE gene by Real-time PCR. (A) Standard curve, slope = −3.6 and R2 = 0.998 showing good efficiency. (B) Dissociation curve showing specific amplification of target (nimE gene) and NTC = No template control. (C) Absolute quantification of copy no. of nimE gene in Healthy vs E. histolytica positive samples. (D) Absolute quantification of copy no. of nimE gene in stool sample DNA of Healthy volunteers before and after satronidazole treatment. P value = .05 or below was considered significant. CI stands for confidence interval.
References
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