Prevalent bacterial species and novel phylotypes in advanced noma lesions

Abstract

The purpose of this study was to determine the bacterial diversity in advanced noma lesions using culture-independent molecular methods. 16S ribosomal DNA bacterial genes from DNA isolated from advanced noma lesions of four Nigerian children were PCR amplified with universally conserved primers and spirochetal selective primers and cloned into Escherichia coli. Partial 16S rRNA sequences of approximately 500 bases from 212 cloned inserts were used initially to determine species identity or closest relatives by comparison with sequences of known species or phylotypes. Nearly complete sequences of approximately 1,500 bases were obtained for most of the potentially novel species. A total of 67 bacterial species or phylotypes were detected, 25 of which have not yet been grown in vitro. Nineteen of the species or phylotypes, including Propionibacterium acnes, Staphylococcus spp., and the opportunistic pathogens Stenotrophomonas maltophilia and Ochrobactrum anthropi were detected in more than one subject. Other known species that were detected included Achromobacter spp., Afipia spp., Brevundimonas diminuta, Capnocytophaga spp., Cardiobacterium sp., Eikenella corrodens, Fusobacterium spp., Gemella haemoylsans, and Neisseria spp. Phylotypes that were unique to noma infections included those in the genera Eubacterium, Flavobacterium, Kocuria, Microbacterium, and Porphyromonas and the related Streptococcus salivarius and genera Sphingomonas and TREPONEMA: Since advanced noma lesions are infections open to the environment, it was not surprising to detect species not commonly associated with the oral cavity, e.g., from soil. Several species previously implicated as putative pathogens of noma, such as spirochetes and Fusobacterium spp., were detected in at least one subject. However, due to the limited number of available noma subjects, it was not possible at this time to associate specific species with the disease.

FIG. 1.

Phylogenetic tree of full 16S rDNA sequences of clonal libraries derived from advanced noma lesions. Only those species or phylotypes detected in noma lesions are shown. The information presented includes bacterial species or phylotype and GenBank accession number. Species or phylotypes detected in more than one subject are highlighted in red boldface lettering and are also listed in Table 1. Color-coordinated symbols are used to identify those species or phylotypes that were also detected in oral sites other than noma lesions, e.g., in subgingival plaque of healthy and diseased sites (26), in supragingival plaque on tooth surfaces (2), on or in subgingival crevicular epithelial cells (5), and on epithelial cells of the tongue dorsum (18). The superscripted “1” indicates a partial sequence of about 500 bp. The bar at top represents a 5% difference in nucleotide sequences. The superscripted “2” indicates a newly described bacterial phylum. Two hundred bootstrap trees were generated, and bootstrap confidence levels as percentages (only values over 50%) are shown at tree nodes. Symbols: red lightning bolt, refractory periodontitis subgingival plaque; black diamond, periodontitis subgingival plaque; blue cross, acute necrotizing ulcerative gingivitis subgingival plaque; golden triangle, necrotizing ulcerative periodontitis subgingival plaque; green heart, healthy subgingival plaque; yellow star, dental supragingival plaque; fuchsia cube, on or in crevicular epithelial cells; orange flag, on or in tongue dorsum epithelial cells.