Background: There is an urgent need for an extensive evaluation of benzimidazole efficacy in humans. In veterinary science, benzimidazole resistance has been mainly associated with three single... Show moreBackground: There is an urgent need for an extensive evaluation of benzimidazole efficacy in humans. In veterinary science, benzimidazole resistance has been mainly associated with three single-nucleotide polymorphisms (SNPs) in the isotype-1 beta-tubulin gene. In this study, we optimized the stool sample processing methodology and resistance allele frequency assessment in Trichuris trichiura and Necator americanus anthelmintic-related SNPs by pyrosequencing, and standardized it for large-scale benzimidazole efficacy screening use.Methods: Three different protocols for stool sample processing were compared in 19 T. trichiura-positive samples: fresh stool, egg concentration using metallic sieves with decreasing pore size, and egg concentration followed by flotation with saturated salt solution. Yield of each protocol was assessed by estimating the load of parasite DNA by real-time PCR. Then, we sequenced a DNA fragment of the beta-tubulin gene containing the putative benzimidazole resistance SNPs in T. trichiura and N. americanus. Afterwards, resistant and susceptible-type plasmids were produced and mixed at different proportions, simulating different resistance levels. These mixtures were used to compare previously described pyrosequencing assays with processes newly designed by our own group. Once the stool sample processing and the pyrosequencing methodology was defined, the utility of the protocols was assessed by measuring the frequencies of putative resistance SNPs in 15 T. trichiura- and 15 N. americanus-positive stool samples.Results: The highest DNA load was provided by egg concentration using metallic sieves with decreasing pore size. Sequencing information of the beta-tubulin gene in Mozambican specimens was highly similar to the sequences previously reported, for T. trichiura and N. americanus, despite the origin of the sample. When we compared pyrosequencing assays using plasmids constructs, primers designed in this study provided the most accurate SNP frequencies. When pooled egg samples were analysed, none of resistant SNPs were observed in T. trichiura, whereas 17% of the resistant SNPs at codon 198 were found in one N. americanus sample.Conclusions: We optimized the sample processing methodology and standardized pyrosequencing in soil-transmitted helminth (STH) pooled eggs. These protocols could be used in STH large-scale screenings or anthelmintic efficacy trials. Show less
Chitin is the second most abundant polymer in nature. In aquatic ecosystems, chitin is mainly present as part of the exoskeleton of arthropods; in soil, chitin is mainly present as component of... Show moreChitin is the second most abundant polymer in nature. In aquatic ecosystems, chitin is mainly present as part of the exoskeleton of arthropods; in soil, chitin is mainly present as component of fungal cell walls. The main enzymes involved in the breakdown of chitin are chitinases. Chitinases are produced by both fungi and bacteria. Chitinases of soil-borne bacteria can decompose chitin of dead fungal hyphae and other resources, but they may also play a role in antagonistic activities against fungi by destroying the chitin in the fungal cell walls. In the research described in this thesis, I tested the hypothesis that bacterial chitinases may perform different functions in different environments and under different circumstances, while the genetic composition and function of bacterial chitinases vary between different habitats. The results obtained in this study have contributed to a better understanding of the ecological functions of bacterial chitinases. New insights in the composition of the bacterial chitinolytic system and the importance of its components were obtained. The potential ecological functions of the bacterial chitinase complex were explored and the role of chitinases in bacteria-fungi interactions, which are vital to the functioning of terrestrial ecosystems, was revealed further. Show less
