Isolation and characterization of anaerobic bacteria for symbiotic recycling of uric acid nitrogen in the gut of various termites.

Recycling of the nitrogenous waste uric acid (UA) of wood-feeding termites by their gut bacteria is one of the significant aspects of symbiosis for the conservation of nitrogen sources. Diverse anaerobic UA-degrading bacteria comprising 16 species were isolated from the gut of eight termite species, and were assigned to Clostridia, Enterobacteriaceae, and low G+C Gram-positive cocci. UA-degrading Clostridia had never been isolated from termite guts. UA-degrading ability was sporadically distributed among phylogenetically various culturable anaerobic bacteria from termite guts. A strain of Clostridium sp., which was commonly isolated from three termite species and represented a probable new species in cluster XIVa of clostridia, utilized UA as a nitrogen source but not as a sole carbon and energy source. This feature is in clear contrast to that of well-studied purinolytic clostridia or previously isolated UA degraders from termite guts, which also utilize UA as a sole carbon and energy source. Ammonia is the major nitrogenous product of UA degradation. Various purines stimulated the growth of this strain when added to an otherwise growth-limiting, nitrogen poor medium. The bacterial species involved the recycling of UA nitrogen in the gut microbial community of termites are more diverse in terms of both taxonomy and nutritional physiology than previously recognized.

Variable-number tandem repeats typing of Mycobacterium tuberculosis isolates with low copy numbers of IS6110 in Thailand.

Spoligotyping and variable-number tandem repeats (VNTR) typing have been increasingly used for differentiating Mycobacterium tuberculosis strains with low copy numbers of IS6110. However, there are few studies comparing their potential to type the strains originating from South and Southeast Asia where many of the isolates have only a few copies, or even single copy, of IS6110. Here, we evaluated the genotyping of 187M. tuberculosis isolates harboring 1-6 copies of IS6110, available from a population-based study in Chiangrai, northern Thailand during 1998-2000, using spoligotyping and VNTR typing. The low-copy-number isolates constituted about 34% of all M. tuberculosis isolated in the province. Discriminating capacities and cluster identification by the two methods were compared with each other and to those obtained by the standard IS6110-restriction fragment length polymorphism (RFLP) method. We found that VNTR typing based on the studied 10-loci set generated more distinct patterns (151 patterns) than spoligotyping (54 patterns) and IS6110-RFLP (65 patterns). Most of the RFLP- or spoligotyping-defined clusters were subdivided by VNTR typing. Combining IS6110-RFLP with VNTR typing produced 164 distinct patterns and 21.9% of clustered isolates whereas the combination of IS6110-RFLP and spoligotyping gave 103 different patterns and 59.4% of clustered isolates. Our results confirm the utility of VNTR typing as the secondary method of choice for investigating the epidemiology of M. tuberculosis with low copy numbers of IS6110.

Evolution of some variable-number tandem repeat loci among a group of Beijing strains of Mycobacterium tuberculosis.

The patterns of variable-number tandem repeats (VNTR) genotypes of a clonal group of Mycobacterium tuberculosis Beijing isolates with very similar IS6110-restriction fragment length polymorphism (RFLP) patterns were studied. Differences between VNTR were mostly by a single repeat unit. However, a multiple-unit change also occurred. This suggests that a mechanism other than the slipped-strand mispairing might be responsible for the instability of VNTR in M. tuberculosis as well. This finding is useful for inferring phylogenetics of M. tuberculosis based on the VNTR genotypes.

Polymorphism of variable-number tandem repeats at multiple loci in Mycobacterium tuberculosis.

Genotyping based on variable-number tandem repeats (VNTR) is currently a very promising tool for studying the molecular epidemiology and phylogeny of Mycobacterium tuberculosis. Here we investigate the polymorphisms of 48 loci of direct or tandem repeats in M. tuberculosis previously identified by our group. Thirty-nine loci, including nine novel ones, were polymorphic. Ten VNTR loci had high allelic diversity (Nei's diversity indices >or= 0.6) and subsequently were used as the representative VNTR typing set for comparison to IS 6110-based restriction fragment length polymorphism (RFLP) typing. The 10-locus VNTR set, potentially providing >2 x 10(9) allele combinations, obviously showed discriminating capacity over the IS 6110 RFLP method for M. tuberculosis isolates with fewer than six IS 6110-hybridized bands, whereas it had a slightly better resolution than IS 6110 RFLP for the isolates having more than five IS 6110-hybridized bands. Allelic diversity of many VNTR loci varied in each IS 6110 RFLP type. Genetic relationships inferred from the 10-VNTR set supported the notion that M. tuberculosis may have evolved from two different lineages (high and low IS 6110 copy number). In addition, we found that the lengths of many VNTR loci had statistically significant relationships to each other. These relationships could cause a restriction of the VNTR typing discriminating capability to some extent. Our results suggest that VNTR-PCR typing is practically useful for application to molecular epidemiological and phylogenetic studies of M. tuberculosis. The discriminating power of the VNTR typing system can still be enhanced by the supplementation of more VNTR loci.