Canine Oral Biofilms: Cultural, Molecular, and in Vitro Studies
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Canine oral biofilms: cultural, molecular, and in vitro studies David Richard Elliott September 2005 Eastman Dental Institute (University College London) Thesis submitted for the degree of Doctor of Philosophy in Microbiology (clinical dentistry) 1 Abstract The canine oral microbiota is poorly understood compared to that of humans. The aim of this work was to improve understanding of the canine oral microbiota. This was achieved by surveying the canine oral microbiota, determining coaggregation interactions between its mem- bers, and developing a laboratory microcosm. Bacteria were isolated from the dental plaque and saliva of dogs, and isolates were identified by comparative 16S rRNA gene sequenc- ing. From 339 isolates, 84 phylotypes belonging to 37 genera were identified. Approximately half were identified to species level, and 28 % of these were also members of the human oral microbiota. Thirty eight phylotypes were tentatively identified as candidate new species. The genera most frequently isolated from saliva were Actinomyces, Streptococcus, and Granulicatella. Porphyromonas, Actinomyces, and Neisseria were most frequently isolated from plaque. On average, se- quences from this study differed by almost 7 % in the 16S rRNA gene compared to similar organisms from humans. Targeted PCR was used to detect culture resistant bacteria from ca- nine plaque. Successful amplification indicated that Spirochaetes and candidate division TM7 bacteria were present, however the identities of the originating organisms were not determined. The entire cultivable plaque microbiota from a single dog was as- sessed for coaggregation reactions. Eight (6.7 %) unique interactions were detected from 120 crosses, indicating that the prevalence of coag- gregation is similar in the canine and human oral microbiotas. Genera common to both hosts generally exhibited similar coaggregation reac- tions, however autoaggregation was more common among bacteria iso- lated from dogs. The constant depth film fermenter was used to grow microcosms from canine plaque and saliva using a mucin containing artificial saliva supplemented with horse serum as the growth medium. The model produced biofilms similar to natural dental plaque, which could be used to investigate the canine oral microbiota further. 2 Acknowledgements Many people have contributed to this work by encouraging and sup- porting me both academically and personally. I dedicate this thesis to my wife Pamela, whose continual love and support has been invaluable. Thank you for tolerating my indulgence so happily, and for the sacrifices you have made on my behalf. I thank my parents and extended family for providing a loving en- vironment in which my childhood interest in science and nature was encouraged. I also wish to thank my parents for their generous finan- cial assitance throughout my education. Thanks to my colleagues and friends at the Eastman Dental Institute for your technical assistance, discussion, and fun times. I also acknowledge assistance with DNA sequencing given by Prof William Wade at Guys and St Thomas’s, and thank Dr Marilou Ciantar and Lisa Milella for their kind assistance with sampling. Financial sup- port for this project was generously provided by the BBSRC and the Waltham Centre for Pet Nutrition. Finally, special thanks go to my academic and industrial supervi- sors. Dr Neil Culham, Dr Catherine Buckley, and Dr Marie-Louise Baillon were all involved in my supervision at the Waltham Centre. At the Eastman, Prof Mike Wilson and Dr Dave Spratt were my academic supervisors. Between them they inspired and motivated me, enabled me to work productively, and allowed me to develop as an independent researcher. 3 Abbreviations General abbreviations AA anaerobe agar AS artificial saliva BHI brain heart infusion bp basepairs BPA black pigmented anaerobic bacteria CAS canine artificial saliva CBA Columbia agar base CFAT cadmium fluoride acriflavin tellurite cfu colonyformingunits CP canine plaque CPP canine pooled plaque CPS canine pooled saliva DGGE denaturing gradient gel electrophoresis DNA deoxyribonucleicacid FISH fluorescent in situ hybridisation GCF gingival crevicular fluid h hour PBS phosphatebuffered saline PCR polymerase chain reaction ptype phylotype RCF relative centrifugal force (multiples of g) RNA ribonucleic acid rRNA ribosomal RNA S Svedberg unit (measure of density) sp. species (singular) spp. species (plural) Tm melting temperature tvc totalviablecount UPGMA unweighted pair group method with arithmetic means V volt(electricpotentialdifference) Units m metre min minute M moleslitre−1 (molarity) MY millionyear MYA million years ago ym yearmonth Unit prefixes m milli-(×10−3) µ micro- (×10−6) n nano-(×10−9) 4 Contents Abstract................................... 2 Acknowledgements ............................. 3 Abbreviations ................................ 4 List of Figures 10 List of Tables 12 1 Introduction 14 1.1 Microbially-mediatedoraldiseases . .. 15 1.1.1 Periodontaldiseases . 16 1.1.2 Diseasesofthetooth . 16 1.2 Oraldiseaseinthedog . .. .. .. .. .. .. .. 18 1.3 Oralmicrobiotaofhumansanddogs . 19 1.3.1 Firmicutes .......................... 20 1.3.2 Fusobacteria ......................... 26 1.3.3 Actinobacteria ........................ 27 1.3.4 Spirochaetes ......................... 30 1.3.5 Proteobacteria ........................ 30 1.3.6 Bacteroidetes ......................... 35 1.3.7 Deferribacteres ........................ 37 1.3.8 CandidatedivisionsTM7andOP11 . 37 1.4 Modellingtheoralmicrobiota . 38 1.4.1 In vivo systems........................ 39 1.4.2 Planktonic in vitro systems.................. 40 1.4.3 Biofilm in vitro systems ................... 40 5 CONTENTS 1.5 Methodsofcommunityanalysis . 43 1.5.1 Microscopic.......................... 44 1.5.2 Cultural............................ 46 1.5.3 Molecular........................... 47 1.5.4 Metabolic........................... 49 1.6 Aimsandobjectives. .. .. .. .. .. .. .. 49 2 Materials and methods 50 2.1 Cohorts ................................ 50 2.2 Clinicalscoring ............................ 51 2.3 Sampling ............................... 51 2.3.1 Saliva............................. 51 2.3.2 Plaque............................. 51 2.4 Suppliersofchemicalsandreagents . 52 2.5 GrowthMedia............................. 53 2.5.1 Agars ............................. 53 2.5.2 Canineartificialsaliva . 54 2.6 Microscopy .............................. 55 2.6.1 Lightmicroscopy. 55 2.6.2 Scanningelectronmicroscopy . 55 2.6.3 Transmissionelectronmicroscopy . 55 2.6.4 Confocallaserscanningmicroscopy . 56 2.7 DNAextraction ............................ 56 2.8 DNApurification ........................... 56 2.9 Polymerasechainreaction . 57 2.10 DNAfragmentanalysis . 57 2.10.1 Primers ............................ 57 2.10.2 StandardPCRconditions. 57 2.11 DNAsequencing ........................... 58 2.12 DNAsequenceassemblyandcomparison . 60 2.13 PCR-Cloning ............................. 61 6 CONTENTS 3 Culture-based analyses 62 3.1 Introduction.............................. 62 3.2 Materialsandmethods . 63 3.2.1 Samples............................ 63 3.2.2 Viablecounting. .. .. .. .. .. .. .. 63 3.2.3 Isolateidentificationandstorage . 63 3.2.4 ComparativeDNAsequenceanalyses . 64 3.2.5 Phylogeneticanalyses . 65 3.2.6 Literaturesearch . 66 3.3 Results................................. 66 3.3.1 Phylogeneticdiversity . 66 3.3.2 ViableCounts......................... 72 3.3.3 Comparisontohumanoralmicrobiota . 74 3.4 Discussion............................... 74 3.4.1 Overview ........................... 74 3.4.2 Comparisonwithhumanand animaloral microbiota . 76 3.4.3 Evolution,taxonomy,andecology . 80 3.5 Conclusions.............................. 84 4 Culture-independent analyses 86 4.1 Introduction.............................. 86 4.2 MaterialsandMethods . 87 4.2.1 TemplateDNA ........................ 87 4.2.2 Denaturing gradient gel electrophoresis (DGGE) . .. 88 4.2.3 PCR detectionofculture-resistantbacteria . ... 90 4.3 Results................................. 91 4.3.1 Communityprofiling(DGGE) . 91 4.3.2 Culture-resistantbacteria . 93 4.4 Discussion............................... 96 4.4.1 Communityprofiling . 96 4.4.2 Culture-resistantbacteria . 97 4.5 Conclusions.............................. 98 7 CONTENTS 5 Coaggregation 100 5.1 Introduction..............................100 5.2 MaterialsandMethods . .101 5.2.1 Bacterialisolates . .101 5.2.2 Culturepreparation . 101 5.2.3 Standardcoaggregationassay . 103 5.2.4 Buffertimecourseassessment . 104 5.2.5 Canine-adaptedcoaggregationassay . 104 5.2.6 Transmissionelectronmicroscopy(TEM) . 105 5.3 Results.................................106 5.3.1 Standardcoaggregationassay . 106 5.3.2 Buffertimecourseassessment . 106 5.3.3 Canine-adaptedcoaggregationassay . 106 5.3.4 Transmission electron microscopy of coaggregates . 108 5.4 Discussion...............................113 5.5 Conclusions..............................116 6 In vitro microcosm development 117 6.1 Introduction..............................117 6.2 MaterialsandMethods . .118 6.2.1 Constantdepthfilmfermenter(CDFF). 118 6.2.2 Serumadditionexperiment. 121 6.3 Results.................................122 6.3.1 Culturalanalyses . .122 6.3.2 Serumaddition . .122 6.4 Discussion...............................124 6.4.1 Overview ...........................124 6.4.2 Modeldesign .........................124 6.5 Conclusions..............................126 7 Validation of in vitro microcosm 128 7.1 Introduction..............................128 7.2 MaterialsandMethods