CATEGORY 66 AUSTRALASIAN DENTIST As a progressive SEM sample in the same patient/same site, the healing bone lesion demonstrated a much thicker established biofilm and the identifiable bacteria are no longer pleomorphic rods but are coccoid. This suggests that there has been an evolution in the biofilm and its microbial inhabitants and that the biofilm might now be polymicrobial. There was no visual evidence of the original rod-shaped CLINICAL bacteria seen in the previous pathological persistence profile at 9 months postextraction. Longitudinal SEM suggests an apparent population shift in the resident biofilm requiring four surgical debridements in search of a regenerative health margin over 18 months. The healing bone lesion had recovered a large radiolucent ischaemic necrosis into a vascularised vertical cleft in the labial plate. Microbiome population shift after multiple surgical debridements Microbiome analysis revealed that change in the resident bacterial assemblages may correlate with a microbial community health/disease shift, corroborated by ‘corresponding same site SEM images. Persistent continuity of chronic pathology revealed by pyrosequencing at 6 months and 9 months post-extraction (Sample 41F) (Fig. 13a-b) corroborated SEM visually confirming deeply invading pathogenic rod-shaped bacteria (Fig. 12b). Health recovery population-shift with a turnover of 7 phyla between 9 and 12 months, following multiple debridements, at 12 months postextraction (Sample 57E) at revision (Fig. 13a-b). Repetitious surgical debridement drove a healing trajectory through habitat change and resident microbial population shift, according to habitat fitness. This allowed the placement of a revision implant into an ecologically healthy bone bed with a bony return to normal internal architecture. At a 9-year review, a normal bony architecture was fully returned (Fig. 14). Fig. 12: (a) SEM of a bone sample taken from (location A) side of the lesion in Fig. 11. 6 months after implant failure and 9 months post extraction. The definitive image shows White Blood Cells (WBC) in Biofilm (BF) with red blood cells (RBC), Fibrin (FIB), and Cretinated Erythrocytes (CE). This sample demonstrates extensive fibrous deposits with lace biofilm plus small areas of patchy biofilm clusters covering the bone surface; (b) SEM image of persistent pathologic sample (41F) taken from “location B” side of lesion invading deep within the bone with areas of bony destruction x7500. Deeply invading pathogenic rods persisting 9 months post extraction and multiple (3x) debridements in diffuse sclerotic bone (D1 Nelson and Viljoen bone quality index) (Viljoen, 2019); (c) SEM image of same site healed sample (57E) taken at 12 months post extraction from D side in Fig. 11 following further debridement and further 3 months post-surgical debridement healing. Rods are no longer present, now dominated by cocci. Fig. 13: (a) Microbial population shift achieved before implant revision. Diversity and stability are almost synonymous. Turnover of 5 Phyla between the pathologic sample at six months post-extraction, and the pathologic sample at nine months postextraction (41F). There was a 7 Phyla turnover between diseased bone at nine months postextraction, and healthy bone at twelve months postextraction (57E); (b) Example of population-shift, post-RSD. The shift of 7 phyla between 9 months (41F) post-extraction persistent pathology and 12 months (57E) healing recovery and population shift following further debridement and stipulated healing time. Resident bacterial diversity shifted from 13 genera to 37 genera with only 3 common genera after a major healing event of chronic resistant pathology. Ecologic diversity is recovered along with stability and bone health. Fig. 14: 9-year case review in 2021. Noted a full return to normal bony architecture. The invading pathogens appear to have completely replaced the resident community with a disease-orientated community. SEM visually confirmed biofilm phenotype pathogenicity and persistence as an ecological shift in a commensal (resident) population (Costerton, 2007). The bacterial biofilm health/ disease population shift has the primary aetiological role in disease and persistence which impedes and delays healing. Pathologic communities will produce direct toxin lysis and compression lysis of capillaries where ischaemic bone beds are subject to infection and disease by population shift. Persister cell populations will persist as substrate-bound subclinical biofilm niduses which will be resistant to non-surgical treatment (Cierny, 2011). Discussion SEM was a methodology critically pursued in 1985 by Gristina et al. to detect adhesive and coherent polymicrobial microcolonies in failed orthopedic prostheses and bone samples obtained during surgical debridement of long-bone chronic osteomyelitis (Gristina et al., 1985; 1987). This helped promote the definition of chronic osteomyelitis as a bacterial biofilm infection of the bone. SEM provided visual confirmation of bacteria and bacterial biofilm in the jawbone in health and disease. This included pathologically altered sclerotic sequestrum, a pathologically altered chronic periapical lesion, and an apparent same-site population-shift health recovery, corroborated by same-site longitudinal microbiome analysis. We may now suggest that the enhanced live bacterial culture (Tunney et al., 1998) that we had previously reported of 21% in a minimum three-month healed edentulous jawbone1, was a result of the surgical disturbance of live resident biofilms. A biofilm/planktonic phenotype A B
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