Genotypic determination of resistance and heteroresistance to clarithromycin in Helicobacter pylori isolates from antrum and corpus of Colombian symptomatic patients.

BACKGROUND: The effectiveness of Helicobacter pylori first-line treatment has decreased drastically with the rise of strains resistant to clarithromycin. Therapy failure has also been described in patients with infections by strains with dissimilar antimicrobial susceptibilities. The present study aims to estimate the prevalence of resistance and heteroresistance to clarithromycin in H. pylori isolates from antrum and corpus of Colombian patients. METHODS: The study material included 126 isolates from antrum and corpus biopsies from 63 symptomatic patients over 18 years old who had a gastric endoscopy performed on them between June 2014 to August 2016. PCR amplification and sequencing of the H. pylori 23S rDNA gene was performed to determine the presence of mutations associated with clarithromycin resistance. Random amplified polymorphic DNA analysis was implemented in cases of resistance and heteroresistance. RESULTS: The overall frequency of resistance to clarithromycin was 38.1% (24/63 patients), of which 19 patients had resistant isolates in both stomach segments (14 with A2143G mutation and 5 with A2142G mutation), and 5 patients had a heteroresistant status. The remaining 61.9% (39/63 patients) presented only susceptible isolates. DNA fingerprinting analysis showed different patterns in 4/22 paired isolates. CONCLUSIONS: The high prevalence of H. pylori clarithromycin-resistance obtained (> 15%) constitutes an alert for gastroenterologists and suggests the need for reconsideration of the current eradication regimen for H. pylori in the studied population. The data show that heteroresistance status is an additional factor to be considered in the assessment of resistance. In consequence, it is advisable to examine at least two biopsies from different gastric segments.

Tryptophan usage by Helicobacter pylori differs among strains.

Because of its association with severe gastric pathologies, including gastric cancer, Helicobacter pylori has been subject of research for more than 30 years. Its capacity to adapt and survive in the human stomach can be attributed to its genetic flexibility. Its natural competence and its capacity to turn genes on and off allows H. pylori to adapt rapidly to the changing conditions of its host. Because of its genetic variability, it is difficult to establish the uniqueness of each strain obtained from a human host. The methods considered to-date to deliver the best result for differentiation of strains are Rapid Amplification of Polymorphic DNA (RAPD), Multilocus Sequence Typing (MLST) and Whole Genome Sequencing (WGS) analysis. While RAPD analysis is cost-effective, it requires a stable genome for its reliability. MLST and WGS are optimal for strain identification, however, they require analysis of data at the bioinformatics level. Using the StainFree method, which modifies tryptophan residues on proteins using 2, 2, 2, - trichloroethanol (TCE), we observed a strain specific pattern of tryptophan in 1D acrylamide gels. In order to establish the effectiveness of tryptophan fingerprinting for strain identification, we compared the graphic analysis of tryptophan-labelled bands in the gel images with MLST results. Based on this, we find that tryptophan banding patterns can be used as an alternative method for the differentiation of H. pylori strains. Furthermore, investigating the origin for these differences, we found that H. pylori strains alters the number and/or position of tryptophan present in several proteins at the genetic code level, with most exchanges taking place in membrane- and cation-binding proteins, which could be part of a novel response of H. pylori to host adaptation.

Intramolecular energies of the cytotoxic protein CagA of Helicobacter pylori as a possible descriptor of strains' pathogenicity level.

The Helicobacter pylori cytotoxin-associated gene A (CagA) is known for causing gastroduodenal diseases, such as atrophic gastritis and peptic ulcerations. Furthermore Helicobacter pylori CagA positive strains has been reported as one of the main risk factors for gastric cancer (Parsonnet et al., 1997). Structural variations in the CagA structure can alter its affinity with the host proteins, inducing differences in the pathogenicity of H. pylori. CagA N-terminal region is characterized for be conserved among all H. pylori strains since the C-terminal region is characterized by an intrinsically disorder behavior. We generated complete structural models of CagA using different conformations of the C-terminal region for two H. pylori strains. These models contain the same EPIYA (ABC1C2) motifs but different level of pathogenicity: gastric cancer and duodenal ulcer. Using these structural models we evaluated the pathogenicity level of the H. pylori strain, based on the affinity of the interaction with SHP-2 and Grb2 receptors and on the number of interactions with the EPIYA motif. We found that the main differences in the interaction was due to the contributions of certain types of energies from each strain and not from the total energy of the molecule. Specifically, the electrostatic energy, helix dipole energy, Wander Waals clashes, torsional clash, backbone clash and cis bond energy allowed a separation between severe and mild pathology for the interaction of only CagA with SHP2.

Computational approaches for evaluating the effect of sequence variations and the intrinsically disordered C-terminal region of the Helicobacter pylori CagA protein on the interaction with tyrosine kinase Src.

The Helicobacter pylori CagA protein was the first bacterial oncoprotein to be identified as important in the development of human malignancies such as gastric cancer. It is not clear how it is able to deregulate a set of cell control mechanisms to induce carcinogenesis following translocation into human gastric epithelial cells. It is likely, however, that structural variations in the CagA sequence alter its affinity with the host proteins inducing differences in the pathogenicity of different H. pylori strains. Using the recently elucidated N-terminal 3D structure of H. pylori CagA, information on the full cagA gene sequence, and intrinsically disordered protein structure predictions methods we evaluated the interaction of different CagA variants with the kinase Src. An automated docking followed by molecular dynamics simulations were performed to explore CagA interaction modes with Src, one of its cellular partners. The computational approach let us establish that even in the presence of the same number and type of EPIYA motifs, CagA protein can reveal different spatial distributions. Based on the lowest affinity energy and higher number of interactions it was established that the principal forces governing the CagA-Src interaction are electrostatic. Results showed that EPIYA-D models presents higher affinity with some host proteins than EPIYA-C. Thus, we highlight the importance and advantage of the use of computational tools in combining chemical and biological data with bioinformatics for modeling and prediction purposes in some cases where experimental techniques present limitations.

Genetic structure of sylvatic, peridomestic and domestic populations of Triatoma dimidiata (Hemiptera: Reduviidae) from an endemic zone of Boyaca, Colombia.

Randomly amplified polymorphic DNA (RAPD) technique was used to study the genetic structure of sylvatic, peridomestic and domestic populations of Triatoma dimidiata. The genetic flow among them was calculated to establish the epidemiological risk of non-domiciliated populations in the transmission of Chagas disease in an endemic area of Boyaca, Colombia. A total of 83 adult specimens were studied: 26 sylvatic, 27 peridomestic and 30 domestic insects. Wright's Fst was 0.071 and the effective migration rate (Nm) 3.3, suggestive of low genetic differentiation and a movement of at least three insects per generation. The calculated percentage of polymorphic loci was 99%, confirming a large average heterozygosity due to a permanent contact between insects of the three populations. These results imply that non-domiciliated populations of T. dimidiata represent an epidemiological risk in the transmission of Chagas disease owing to the fact that they can colonize human dwellings. Close surveillance of non-strictly domiciliated species of triatomines such as T. dimidiata should entail not only the domicile but also the peridomicile and should include control programs of animal reservoirs. Houses enhancement, educational programs, surveillance of reinfestation and of individuals at risk of infection should be priorities in the control policies in endemic regions such as Boavita, Boyaca.