AbstractInflammatory bowel disease (IBD) is a series of disorders characterised by chronic intestinal inflammation, with the principal examples being Crohn’s Disease (CD) and ulcerative colitis (UC). A paradigm of these disorders is that the composition of the colon microbiota changes, with increases in bacterial numbers and a reduction in diversity, particularly within the Firmicutes. Sulfate reducing bacteria (SRB) are believed to be involved in the etiology of these disorders, because they produce hydrogen sulphide which may be a causative agent of epithelial inflammation, although little supportive evidence exists for this possibility. The purpose of this study was (1) to detect and compare the relative levels of gut bacterial populations among patients suffering from ulcerative colitis and healthy individuals using PCR-DGGE, sequence analysis and biochip technology; (2) develop a rapid detection method for SRBs and (3) determine the susceptibility of Desulfovibrio indonesiensis in biofilms to Manuka honey with and without antibiotic treatment.
Mucosal biopsy DNA from 4 colitis patients and one healthy individual was used to amplify 16S rRNA fragments, which were separated either by DGGE or by molecular cloning prior to sequencing, and dissimilatory sulphite reductase (dsr) gene fragments, which were cloned and sequenced. The number of bands separated by DGGE varied from 3 to 12 for an individual. The profiles from the UC patients had a greater similarity than the one from the healthy individual. In total, 25 bands were excised for sequence analysis but only 4 produced usable sequences. The 16S rRNA gene library comprised250 clones, the sequences of which showed great changes in diversity from the healthy individual to the UC patients. The sequences from the healthy individual represented members of the Bacteroidetes, Clostridiaceae, Ruminococcaceae, Enterobacteriaceae,Coriobacteriaceae and Lactobacillaceae, whereas those form the UC patients comprise only members of the Enterobacteriaceae. The library for the dsr gene comprised 30 clones, with sequences from the healthy individual representing members of the Desulfobacteraceae (45%), and the Desulfovibrionaceae (33%), with minor components from the Desulfohalobiaceae, Desulfomicrobiaceae and Desulfonatronaceae. Sequences from the UC patients were less diverse, being composed primarily of Desulfovibrionaceae sequences.
Oligonucleotide probes targeting SRB and other bacterial species found to be involved in UC were developed or selected from published sources. Cassettes with multiple probes were used to evaluate the hybridisation probes for inclusion in a biochip. Validatedprobes (23), with similar binding profiles, were then used to assess the levels of SRB and other bacteria associated with UC in healthy control and UC patient’s samples. The DNA from the healthy individual gave a strong hybridisation signal for Desulfovibrio piger, and weaker signals for Escherichia coli, Bacteroides fragilis, and a Clostridium species, whereas the signals Desulfobacter and Desulfovibrio gigas were present in all of the samples from the UC patients. Signals were also recorded for species of Fuscobacterium, which have been implicated in colitis.
In this investigation, procedure for rapid detection and quantification of SRB was developed. The SRB growth was monitored in the presence and absence of Escherichiacoli BP, the copy number of the dsrA and adenosine-5′-phosphosulfate reductase (aps) genes was detected in DNA and RNA purified from different ages of SRB culture using TaqMan qPCR. The result showed that in the presence of E.coli BP enhanced the growth of SRB and allowed the detection of SRB in low dilutions comparing with the growth of SRB only.
E. coli and SRB are both implicated in colonic infections. They pose several mechanisms for immune evasion and antibiotic resistance, one of these being their ability to grow in a biofilm. The present investigation has highlighted that Manuka honey and antibiotic treatment might have a protective role against SRBs and enteric bacteria. However, mixed bacterial population in biofilms exhibit greater resistance to both treatments, and this needs to be taken into account when devising a treatment based on ingestion of honey.
The present investigation were conducted as a baseline study for determination the role of SRB in the pathogenesis of colitis through the use of novel methodology allowing for rapid detection and screening investigations into species and genera of interest present in colonic mucosa; that may allowed in further investigation to evaluate the bacterial role in UC through extensive studies in broad multinational cohort of patients suffering from this UC.
|Date of Award||Oct 2017|
|Supervisor||Vitaly Zinkevich (Supervisor) & Julian Mitchell (Supervisor)|