UNIVERSITY OF MALAYA 1000 GENOME CONSORTIUM PROJECT ON MULTIDRUG RESISTANT BACTERIA (UM 1K GENOME PROJECT)

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research project conducted at University of Malaya.

Signing of MoU

Signing of MoU between RIKEN, JAPAN and University of Malaya

Multidrug resistant (MDR) bacteria is a major public health problem in the Asia Pacific region as well as globally. Although the regional disease burden is difficult to quantify, it can be assumed to be very substantial. The abuse of antibiotics usage in the region, both in humans and livestock, has led to the evolution of resistant pathogens in a wide array of bacteria, thus rendering these drugs ineffective in the treatment of the infection. In some cases, “superbugs” are being created such as the methicillin-resistant Staphylococcus aureus (MRSA), which are resistant to practically all available drugs.

There is a compelling need to obtain information on the extent of bacterial drug resistance and its evolution from the genomic point of view. Next Generation Sequencing (NGS) technology is a powerful tool that makes it possible to scan the entire genome of drug resistant bacterial strains at a low cost, in a matter of hours to days.

The University of Malaya-Ministry of Higher Education High Impact Research (UM-MoHE HIR) Nature Microbiome programme is proposing to set up the UM 1000 Genome Consortium Project (UM 1K GENOME PROJECT) for the study of MDR bacteria from human and animal origin in Malaysia, using the Next Generation Sequencing (NGS) technology. In the last three months, the use of NGS on several Malaysian bacteria strains has already resulted in >30 publications. In January 2013, UM HIR shortlisted the following for further studies using two main platforms, Illumina (HiSeq & MiSeq) and PacBio (Single Molecule Real Time Sequencer, SMRT).

1) Mycobacteria as follows:

1.1) MDR-TB, XDR-TB and rifampicin/isoniazid mono-resistant strains of M. tuberculosis

1.2) Non-tuberculous mycobacteria (NTM) that are resistant to anti-NTM drugs e.g. clarithromycin, amikacin, ciprofloxacin, linezolid, doxycycline (clinical or environmental strains; from humans or animals)

2) MRSA (nosocomial or community strains; pathogens or commensals; particularly those resistant to new antibiotics such as streptogramins, daptomycin and linezolid)

3) Penicillin-resistant and MDR Streptococcus pneumoniae (from putum/nasopharyngeal source, superficial or invasive infections) as follows:

3.1) Acinetobacter spp. (imipenem-resistant, as well as MDR strains; clinical or environmental source)

3.2) Aeromonas spp. (or Pseudomonas with plasmidmediated resistance)

OBJECTIVES OF THE PROJECT

1) To perform large-scale parallel sequencing of multidrug resistant pathogenic bacteria from Malaysia and other Asia Pacific countries;

2) To identify the origin of multidrug resistance in the region and to understand the dynamics of the development of resistance;

3) To create a database for MDR pathogenic bacteria genomes sequences for the Asia Pacific region;

4) To collaborate with researchers outside the Asia Pacific region.

PROJECTED OUTPUT
Using the Illumina HiSeq and PacBio, the genomic sequencing and in-depth genomic study output in UM-HIR will be 140 de novo bacterial genomes in 20 days which is equivalent to 250-300 bacterial genomes per month or 600 bacterial genomes per year.

Signing of MoU

Signing of MoU between RIKEN, JAPAN and University of Malaya

OPTION TO EXPAND THE SCOPE OF THE PROJECT:
PHASE 2

This project can be extended to the next phase on pathogen transcriptome analysis including the use of RNAseq. RNAseq is one of the newest transcriptome analyses that investigate the global gene expression at a particular time which will provide a very precise and clear picture on genes expression in the cell. RNAseq per se is an excellent tool for gene expression work, and also it can be explored for drug testing that is used to study the effects of drugs on the pathogen genes expression, and also environmental factors on the pathogen genes expression such as heat shock, pH shock, chemical or UV treatments, etc.

Comparative genomes can be done on pathogens of the same group. This will provide insights into the various sources and origins of resistance (e.g. transposon, chromosomal mutation, or even lateral genes transfer). To illustrate, the Vibrio cholerae strain that caused a lethal diarrhoeal outbreak recently in Haiti was studied using the NGS method and its resistance origin was confirmed.

Single cell genome will be the next niche area in NGS since >90% of the current scientific literature is based on population DNA extraction instead of DNA extraction from single bacterial cells. It is assumed that all bacterial cells are clonal but this is due to limitation of instrumentation to study single cells. The next phase of NGS work will venture into this niche research area that will help UM-HIR to establish high impact, informative and competitive advantage on single cell genome NGS involving micromanipulation of cells and microfluidic devices. This will provide
individual genome NGS datasets and provide comparative genomes at the single cell level.

EXPERIMENTAL DETAILS
DNA extractions will be done on various multidrug resistant bacteria from clinical and environmental settings, using robotic workflow for speedy, standardized and unbiased nucleic acids extraction. Purified DNA will be subjected to NGS.

DATA ANALYSIS
Using bioinformatics software, the NGS data will be analysed to:

1) Determine the spectrum of mutations by screening for mutations known to be associated with resistance to antibiotics (in all selected isolates)

2) Identify new mutations (by comparing pre- and post-treatment isolates) to provide a genetic basis for new antibiotic resistance mechanisms

3) Study genetic features associated with rapid acquisition (emergence) of resistance

4) Correlate strain genotypic features with host/environmental features (to determine what drives the development of resistance)

5) Describe the origin of drug resistance and the circulation of resistant strains (clades) over geographical regions

DELIVERABLES
1. Publication of ISI-indexed journals [target: New England Journal of Medicine (Impact factor or IF: 53.29), Lancet (IF: 38.28), Nature (IF: 36.28), Science(IF: 31.20), Nature Medicine (IF: 22.46), PLoS Medicine (IF: 13.05), PLoS Biology (IF: 11.452), PLoS Pathogens (IF: 9.172), PNAS (IF: 9.681)].

2. The setting up of a bacterial genome database hosted by UM-HIR with free access for the global scientific community. Find out more by visiting: http://www.hir.um.edu.my/

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