Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.516826
Title: Microbial and immunological influences on the composition of human oral microbiota
Author: Madhwani, Tejal
Awarding Body: The University of Manchester
Current Institution: University of Manchester
Date of Award: 2010
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Abstract:
Whilst considerable temporal microbial stability has been demonstrated within the oral cavity, the mechanisms responsible for the development, maintenance and stability of individualspecific human oral microbiotas are not fully understood. This doctoral thesis presents a series of in-vitro and ex-vivo investigations that focused on four factors believed to be involved; the microbial factors i) colonisation resistance (CR; a consortial-intrinsic homeostatic mechanism); ii) probiosis (the introduction of bacteria claimed to positively influence the microbial balance of human microbiotas); and the host-derived factors iii) antimicrobial peptides (innate immunity); and iv) secretory antibodies (homoral immunity). To investigate microbial homeostasis in the absence of immunological influences (i.e CR), long-term multiple sorbarod oral microcosms were established using fresh saliva from three volunteers (designated A, B and C). Following the establishment of dynamic steady-states, a reciprocal exchange of pcrfusates (analogous to dispersed plaque bacteria) was performed between MSDs A and B and the resistance of microbial profiles to immigration was tested by differential culture and PCR-DGGE. Fermenter C was maintained to investigate long-term stability of modelled plaque in the absence of host immune factors. 1fSD-C maintained short-term dynamic bacterial stability but streptococci and lactobacilli decreased to undetectable levels after 20d. Whilst increases in streptococci and reductions in lactobacilli occurred in MSDs A and B following mixing, CR was apparent as both communities maint.ained their original DGGE-profiles. CR together with transient stability in unmixed in-vitro plaques indicates the importance of intrinsic homeostatic mechanisms in maintaining short-term stability but that additional factors must be responsible for long-term stability in-vivo, such as the innate and adaptive immune systems and bacterial immigration. The potential influence of the oral probiotic bacterium Lactobacillus reuteri was studied in constant depth film ferrnenter plaques. Dosing in-vitro, plaques with L. reuteri formulations resulted in decreases in Gram-negative anaerobes (6 to 4.5 10glOCFU/mm2) and significant decreases in streptococci (7 to 4 loglOCFU/mm2). Morphological and q-PCR tracking of the probiotic indicated its persistence three weeks after treatment was ceased. Addition of L. reuteri to invitro oral microbiotas therefore markedly influenced microbial composition reaffirming the role of external factors in maintenance of homeostasis. The effect of host defence peptides (HDPs; innate immune factors) on maintenance of stability of in-vitro plaques was therefore tested. The peptides (defensins, histatins and a cathelicidin) caused differential decreases in bacterial viability and the ~ defensins and His-5 additionally inhibited coaggregation. Peptide-exposed plaques exhibited closest similarity to salivary incocula following exposure to the peptides in combination, according to DGGE analyses. These observations indicate a role for physiological levels of host defence peptides in influencing the composition of oral microbiotas. In order to investigate the role of salivary immunoglobulins (adaptive immunity), saliva from three volunteers was fract.ionated by centrifugation into antibody (supernatant) and bacterial (pellet) fractions and streptavidin-coated magnetic bead separation was used to identify and retain bacteria according to their recognition by host-specific Igs. Bead-selected bacteria were then profiled using eubacterial PCR-DGGE and cluster analyses and identified by DNA sequencing. Analyses indicated that whilst a large proportion of oral bacteria (c. 50% of DGGE bands) were recognised by salivary IgA and IgG, the specificity of host antibodies for exogenous oral bacteria and putatively transient species was great.er in all cases. These investigations indicate that the composition and stability of oral microbiotas are variously influenced (individually and probably combinatorially) by the adaptive and innate immune systems, by CR and by bacterial immigration (as occurs naturally and as applied in probiosis).
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.516826  DOI: Not available
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