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Long term effects of H.pylori eradication on Gastro intestinal tract microbiome and development of screening system for detection of extended-spectrum beta-lactamase coding genes within feces samples

 

 

Project Title:Long term effects of H.pylori eradication on Gastro intestinal tract microbiome and development of screening system for detection of extended-spectrum beta-lactamase coding genes within feces samples.

Funding: European Regional Development Fund (ERDF), Measure 1.1.1.1 “Industry-Driven Research”

Project Nr.: 1.1.1.1/16/A/272

Period: 1st March 2017 – 29 st February 2020

Project costs: 648 648, 00 EUR

Principle Investigator: Dr. biol. D. Fridmanis

Collaboration:

 

The current international guidelines are encouraging “search-and-treat” strategy for H.pylori to prevent gastric cancer. The highest yield of this approach is expected in countries with high incidence of gastric cancer and high prevalence of H.pylori infection. In Latvia this would require antibiotic treatment to 79% of population. The potential adverse events caused by such therapies to microbiome are insufficiently studied; the expert opinion is generally limited to the consideration that single-time antibiotic treatment would be a minor intervention upon microbiome. However, limited data suggests, that even one-week treatment with macrolides is increasing the resistance of macrolide-resistant S.pneumoniae in pharynx; this difference was statistically significant within a period of 180 days.

The aim of this Project is, within the scope of industrial research, to evaluate the long term effects of H.pylori eradication on GIT microbiome, asses its effects on abundance and prevalence of extended-spectrum b-lactamases coding genes and develop cost effective ESBL screening test prototype. The results acquired through the implementation of this Project shall provide us with enhanced understanding on long term effects of antimicrobial agents on GIT microbiome and development of antibiotic resistance. They shall also contribute to development of National eradication treatment strategy.

Information published: 01.03.2017.

Summary of results

from 1 march to 31 may

Project scientific management

During initial stages of the project the application was submitted to the Riga Eastern Clinical University Hospital’s support fund’s Medical and Biomedical ethics committee for evaluation of project “Long term effects of H.pylori eradication on Gastro intestinal tract microbiome and development of screening system for detection of extended-spectrum beta-lactamase coding genes within feces samples”. Following the receipt of the application the committee provided positive response, thus allowing us to proceed with the implementation of the project.

Acquisition of samples

Acquisition of initial sample set – within the scope of this activity using various types of sample containers 20 fecal samples were collected from five healthy subjects. Since the purpose of this sample set is to evaluate the applicability of standardized fecal occult blood test containers for microbiome studies, following the acquisition they were subjected to various temperature regimes to simulate different environmental condition that could occur before the samples reach the laboratory. During the following project stages these samples shall be used for DNA extraction using extraction method that is developed within the scope of “Development of experimental procedures” activity.

Genetic analysis

During the first stages of this activity detailed analysis of available literature was carried out, with the goal to identify and develop the most suitable primers for amplification of V3 region of 16S rRNA gene. Following their development and acquisition, DNA samples that were acquired during “Development of experimental procedures” activity were used to identify the most suitable conditions for the amplification of the region of interest, which was followed sequencing of acquired products using IonTorrent PGM sequencing system. Next stages of the project shall involve sequencing of 16S rRNA V3 for all other samples thus identifying the most suitable DNA extraction method.

Development of experimental procedures

During the initial stages of this activity in-depth literature analysis and compilation of both project partners knowledge was carried out to identify the most effective sample acquisition and storage method. As the result of these actions the sample acquisition methodology was developed that in a form of detailed instruction shall be issued to all enrolled patients. In parallel development of effective gastrointestinal tract microbiome DNA extraction method was also carried out. During this latest action fresh – in-house acquired samples were used to evaluate the DNA extraction efficiency for various extraction reagent and material kits. During the next project stages within the scope of “Genetic analysis” activity all acquired DNA samples shall be analyzed through 16S rRNA V3 sequencing and as the result we shall identify the most suitable DNA extraction method.

 

Information published: 31.05.2017.

Summary of results

from 1 June to 31 August

Acquisition of samples

During the period from May to July the sample collection of experimental sample set was started. Within the scope of this activity we enrolled patients that underwent H.pyori eradication with standard 10 day antibiotic therapy. In parallel the DNA extraction from preliminary sample set was performed employing method that was developed during “Development of experimental procedures” activity.

Genetic analysis

Within the scope of this activity the genetic analysis of fresh – in-house acquired samples was performed to evaluate the DNA extraction efficiency for various extraction reagent and material kits. During this analysis relative abundances of microorganisms species and its internal diversity was calculated. Further the data was compared to that described in scientific literature and further evaluated within the scope of “Development of experimental procedures” to determine the most suitable DNA extraction method.

Following the completion of these procedure the genetic analysis of preliminary sample set was started.

Development of experimental procedures

Within the scope of this activity the analysis of results that were gained during sequencing of fresh – in-house acquired samples was performed to evaluate the DNA extraction efficiency for various extraction reagent and material kits. As the result the most suitable DNA extraction method was identified.

Information published: 31.08.2017.

 

Summary of results

from 1 September to 30 November

Acquisition of samples

In accordance with the project implementation plans, within the scope of experimental sample set creation, we continued acquisition of samples from patients that were undergoing the standard 10 day H.pylori eradication. In addition we also carried out the extraction of genetic material from already acquired samples, using method that was previously developed.

Genetic analysis

Within scope of this activity the quality of extracted DNA samples from experimental sample set was evaluated. Following this assessment for number of samples 16S rRNA gene V3 region sequencing library was also prepared, which involved amplification the region using specific primers, purification of acquired amplification products and evaluation of their quality.

Development of experimental procedures

During this project period within the scope of this activity we initiated the development of ESBL gene amplification panel. The first stage of this process involved downloading of all known ESBL gene sequences and their manual curation to extract usable information.

Information published: 30.11.2017.

 

Summary of results

01.12.2017.-28.02.2018.

Acquisition of samples

During this period the collection of experimental sample set, that comprised samples from patients that underwent standard 10 day H.pylori eradication therapy, was finalized. In addition the DNA extraction, employing previously developed extraction method, was performed for 50 samples. In summary at the present moment DNA extraction has been performed for 110 fecal samples. In parallel to all these activities the manuscript on applicability of fecal occult blood test tubes for microbiome studies was prepared.

Genetic analysis

Within the scope of this activity we performed quality assessment for 50 DNA samples from experimental sample set, as well as prepared 50 16S rRNA gene V3 region sequencing libraries and carried out sequencing analysis for 20 libraries that were prepared during previous periods.

Development of experimental procedures

Within the scope of this activity we continued our work on development of ESBL amplification panel, it involved manual curation of downloaded information and clustering analysis of all known ESBL genes, with the goal to divide them in allele groups, identify single nucleotide variations and conservative regions.

Information published: 28.02.2018.

 

Summary of results

01.03.2018.-31.05.2018.

Acquisition of samples

During this period the extraction of genetic material, using previously developed extraction method, was performed on last samples of experimental sample set. Thus in total extraction procedure has been carried out on 120 fecal samples. Further, to continue this activity, the collection of samples for validation sample set was initiated. The purpose of this set shall be validation of micro-bead technology based ESB L test. Within the scope of these actions first two samples were collected. In parallel the manuscript on sample stability within collection tubes was sent to journal “Gut” for publication and upon receiving of response from journal editors the required changes were implemented.

Genetic analysis

Within the scope of this activity quality assessment was performed for next 50 experimental sample set DNA samples.in addition preparation of 16S rRNA gene V3 region sequencing libraries was carried out for 50 samples and sequencing analysis was performed on libraries that were generated during previous project stages.

Development of experimental procedures

Within the scope of this activity creation of ESBL gene amplification panel was finished and during the further project stages it shall be used for targeted ESBL gene amplification and preparation of

Information published: 31.05.2018.

Summary of results

01.06.2018.-31.08.2018.

Acquisition of samples

During this project period the work on creation of a validation sample set, that shall be further used to validate the micro-bead technology based ESBL gene test was continued. During these activities, 3 samples were collected. In parallel, a revised publication manuscript on the applicability of FIT test tubes for microbiome studies was re-submitted to the magazine "Gut".

Genetic analysis

Within the framework of the activity, a sequencing analysis as well as analysis of all obtained sequencing data was carried out for 50 previously created libraries.

Development of experimental procedures

The preparation of 4 ESBL gene sequencing libraries was carried out in order to access the effectiveness of the panel. 16SrRNA gene primers were added to two of the libraries to evaluate whether this amplicon can be used for the normalization between the samples.

Information published: 31.08.2018.

 

Summary of results

01.09.2018.-30.11.2018.

Acquisition of samples

During this project period, the collection of samples for validation sample set, which shall be used for validation of the ESBL gene test, was continued. Within the framework of these activities 4 additional samples were collected. In parallel confirmation was received from editors of journal “Gut” that the submitted manuscript on the subject of FIT test tube applicability in microbiome studies has been accepted for publication.

Genetic analysis

Within the scope of this activity the work on previously acquired sequencing data analysis was continued. In parallel sequencing of first 4 ESBL gene libraries was performed and, since acquired results confirmed, that developed amplification panel is working with sufficient efficiency and it can be applied for identification of presence of specific ESBL genes, then the work on preparation of ESBL sequencing libraries was continued and at the moment 56 have been prepared. These shall be sequenced during the next project stages.

Development of experimental procedures

During this project period analysis of sequencing data that was acquired from previously prepared ESBL gene libraries was carried out. Acquired results confirmed, that as the result of amplification the library was enriched with ESBL coding sequences and that frequencies of sequences from various types of ESBL genes differed significantly, while the differences in frequencies between technical replicates were not significant. Thus it was concluded that developed amplification panel is sufficiently effective and is applicable for detections of ESBL gene presence.

Information published: 30.11.2018. 




Mājas lapas izstrādi finansēja ERAF 2.1.1.2. aktivitātes projekts Nr. 2010/0196/2DP/2.1.1.2.0/10/APIA/VIAA/004 "Latvijas biomedicīnas pētījumu integrācija Eiropas zinātnes telpā".