Minh Duy Phan

The University of Queensland, Australia, School of Chemistry and Molecular Biosciences, Post-Doc

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Papers by Minh Duy Phan

Differential Afa/Dr Fimbriae Expression in the Multidrug-Resistant Escherichia coli ST131 Clone

mBio

E. coli ST131 is the most common antibiotic resistant UPEC clone associated with human urinary tr... more

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Ultra-Deep Sequencing of Plasma-Circulating DNA for the Detection of Tumor- Derived Mutations in Patients with Nonmetastatic Colorectal Cancer

Cancer Investigation

Identification of tumor-derived mutation (TDM) in liquid biopsies (LB), especially in early-stage... more Identification of tumor-derived mutation (TDM) in liquid biopsies (LB), especially in early-stage patients, faces several challenges, including low variant-allele frequencies, interference by white blood cell (WBC)-derived mutations (WDM), benign somatic mutations and tumor heterogeneity. Here, we addressed the above-mentioned challenges in a cohort of 50 nonmetastatic colorectal cancer patients, via a workflow involving parallel sequencing of paired WBC- and tumor-gDNA. After excluding potential false positive mutations, we detected at least one TDM in LB of 56% (28/50) of patients, with the majority showing low-patient coverage, except for one TDM mapped to KMT2D that recurred in 30% (15/30) of patients.

Genetic analysis of vietnamese patients with early-onset alzheimer's disease

The International journal of neuroscience, 2021

Alzheimer's disease (AD) is the most common type of dementia and its prevalence is rapidly in... more Alzheimer's disease (AD) is the most common type of dementia and its prevalence is rapidly increasing worldwide. Early-onset Alzheimer's disease (EOAD) constitutes of patients with age of onset earlier than 65 year-old and is known to be associated with genetic mutations. In this study, we reported the first genetic analysis of Vietnamese patients with EOAD. We analyzed targeted sequencing data obtained from a cohort of 51 Vietnamese EOAD patients to identify pathogenic variants in twenty nine well-characterized neurodengerative genes. We identified four missense mutations in APP/PSEN1 genes from six individuals, which accounts for 11.8% of all tested cases. Three of these mutations were previously reported as pathogenic and one mutation in the APP gene was newly identified and might be specific for Vietnamese patients. Our study also found eight individuals carrying homozygous APOE ε4 allele, the main risk factor gene for late-onset AD. Our findings showed that mutation rat...

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Genomic characterisation and context of the blaNDM-1 carbapenemase in Escherichia coli ST101

Carbapenems are last-resort antibiotics; however, the spread of plasmid-encoded carbapenemases su... more Carbapenems are last-resort antibiotics; however, the spread of plasmid-encoded carbapenemases such as the New Delhi metallo-β-lactamase 1 (NDM-1) challenges their effectiveness. The rise of NDM-1 has coincided with the emergence of extensively multidrug resistant (MDR) lineages such as Escherichia coli ST101. Here we present a comprehensive genomic analysis of seven E. coli ST101 isolates that carry the blaNDM-1 gene. We determined the complete genomes of two isolates and the draft genomes of five isolates, enabling complete resolution of the plasmid context of blaNDM-1. Comparisons with thirteen previously published ST101 genomes revealed a monophyletic lineage within the B1 phylogroup forming two clades (designated Clade 1 and Clade 2). Most Clade 1 strains are MDR, encoding resistance to at least 9 different antimicrobial classes, including extended spectrum cephalosporins. Additionally, we characterised different pathways for blaNDM-1 carriage and persistence in the ST101 linea...

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Genetic landscape of recessive diseases in the Vietnamese population from large-scale clinical exome sequencing

PurposeAccurate profiling of population-specific recessive diseases is essential for the design o... more PurposeAccurate profiling of population-specific recessive diseases is essential for the design of cost-effective carrier screening programs. However, minority populations and ethnic groups, including Vietnamese, are still under-represented in existing genetic studies. Here we reported the first comprehensive study of recessive diseases in the Vietnamese population.MethodsClinical exome sequencing (CES) data of 4,503 disease-associated genes obtained from a cohort of 985 Vietnamese individuals was analyzed to identify pathogenic variants, associated diseases and their carrier frequencies in the population.ResultsEighty-five recessive diseases were identified in the Vietnamese population, among which seventeen diseases had carrier frequencies of at least 1% (1 in 100 individuals). Three diseases were especially prevalent in the Vietnamese population with carrier frequencies of 2-12 times higher than in other East Asia or the world populations, including Beta-thalassemia (1 in 25), ci...

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Uncovering novel susceptibility targets to enhance the efficacy of third-generation cephalosporins against ESBL-producing uropathogenic Escherichia coli

Journal of Antimicrobial Chemotherapy

Background Uropathogenic Escherichia coli (UPEC) are a major cause of urinary tract infection (UT... more Background Uropathogenic Escherichia coli (UPEC) are a major cause of urinary tract infection (UTI), one of the most common infectious diseases in humans. UPEC are increasingly associated with resistance to multiple antibiotics. This includes resistance to third-generation cephalosporins, a common class of antibiotics frequently used to treat UTI. Methods We employed a high-throughput genome-wide screen using saturated transposon mutagenesis and transposon directed insertion-site sequencing (TraDIS) together with phenotypic resistance assessment to identify key genes required for survival of the MDR UPEC ST131 strain EC958 in the presence of the third-generation cephalosporin cefotaxime. Results We showed that blaCMY-23 is the major ESBL gene in EC958 responsible for mediating resistance to cefotaxime. Our screen also revealed that mutation of genes involved in cell division and the twin-arginine translocation pathway sensitized EC958 to cefotaxime. The role of these cell-division a...

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TraDIS-Xpress: a high-resolution whole-genome assay identifies novel mechanisms of triclosan action and resistance

Genome Research

Understanding the genetic basis for a phenotype is a central goal in biological research. Much ha... more Understanding the genetic basis for a phenotype is a central goal in biological research. Much has been learnt about bacterial genomes by creating large mutant libraries and looking for conditionally important genes. However, current genome-wide methods are largely unable to assay essential genes which are not amenable to disruption. To overcome this limitation, we developed a new version of “TraDIS” (transposon directed insertion-site sequencing) that we term “TraDIS-Xpress” that combines an inducible promoter into the transposon cassette. This allows controlled overexpression and repression of all genes owing to saturation of inserts adjacent to all open reading frames as well as conventional inactivation. We applied TraDIS-Xpress to identify responses to the biocide triclosan across a range of concentrations. Triclosan is endemic in modern life, but there is uncertainty about its mode of action with a concentration-dependent switch from bacteriostatic to bactericidal action unexp...

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Actionable Mutation Profiles of Non-Small Cell Lung Cancer patients from Vietnamese population

Scientific Reports

Comprehensive profiling of actionable mutations in non-small cell lung cancer (NSCLC) is vital to... more Comprehensive profiling of actionable mutations in non-small cell lung cancer (NSCLC) is vital to guide targeted therapy, thereby improving the survival rate of patients. Despite the high incidence and mortality rate of NSCLC in Vietnam, the actionable mutation profiles of Vietnamese patients have not been thoroughly examined. Here, we employed massively parallel sequencing to identify alterations in major driver genes (EGFR, KRAS, NRAS, BRAF, ALK and ROS1) in 350 Vietnamese NSCLC patients. We showed that the Vietnamese NSCLC patients exhibited mutations most frequently in EGFR (35.4%) and KRAS (22.6%), followed by ALK (6.6%), ROS1 (3.1%), BRAF (2.3%) and NRAS (0.6%). Interestingly, the cohort of Vietnamese patients with advanced adenocarcinoma had higher prevalence of EGFR mutations than the Caucasian MSK-IMPACT cohort. Compared to the East Asian cohort, it had lower EGFR but higher KRAS mutation prevalence. We found that KRAS mutations were more commonly detected in male patients ...

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Integrating multiple genomic technologies to investigate an outbreak of carbapenemase-producing Enterobacter hormaechei

Nature Communications

Carbapenem-resistant Enterobacteriaceae (CRE) represent an urgent threat to human health. Here we... more Carbapenem-resistant Enterobacteriaceae (CRE) represent an urgent threat to human health. Here we report the application of several complementary whole-genome sequencing (WGS) technologies to characterise a hospital outbreak of blaIMP-4 carbapenemase-producing E. hormaechei. Using Illumina sequencing, we determined that all outbreak strains were sequence type 90 (ST90) and near-identical. Comparison to publicly available data linked all outbreak isolates to a 2013 isolate from the same ward, suggesting an environmental source in the hospital. Using Pacific Biosciences sequencing, we resolved the complete context of the blaIMP-4 gene on a large IncHI2 plasmid carried by all IMP-4-producing strains across different hospitals. Shotgun metagenomic sequencing of environmental samples also found evidence of ST90 E. hormaechei and the IncHI2 plasmid within the hospital plumbing. Finally, Oxford Nanopore sequencing rapidly resolved the true relationship of subsequent isolates to the initial...

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Genetic profiling of 2,683 Vietnamese genomes from non-invasive prenatal testing data

Background: The under-representation of Vietnamese ethnic groups in existing genetic databases an... more Background: The under-representation of Vietnamese ethnic groups in existing genetic databases and studies have undermined our understanding of the genetic variations and associated traits or diseases in the population. Cost and technology limitations remain the challenges in performing large-scale genome sequencing projects in Vietnam and many developing countries. Non-invasive prenatal testing (NIPT) data offers an alternative untapped resource to study genetic variations in the Vietnamese population. Results: We analyzed the low-coverage genomes of 2,683 pregnant Vietnamese women using their NIPT data and identified a comprehensive set of 8,054,515 single-nucleotide polymorphisms, among which 8.2% were new to the Vietnamese population. Our study also revealed 24,487 disease-associated genetic variants and their allele frequency distribution, especially five pathogenic variants for prevalent genetic disorders in Vietnam. We also observed major discrepancies in the allele frequency...

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Plasma circulating tumor DNA-based genetic profiling of lung cancer patients in Vietnam using ultra-deep massive parallel sequencing with unique identifier tagging

Journal of Global Oncology

58 Background: Lung cancer is by far the leading cause of cancer death worldwide, with non-small ... more 58 Background: Lung cancer is by far the leading cause of cancer death worldwide, with non-small cell lung cancer (NSCLC) accounting for the majority of cases. Genotype-directed therapy becomes a promising method for cancer treatment beside surgery and chemo-radiotherapy. Liquid biopsy using massive parallel sequencing has emerged as a non-invasive alternative procedure in profiling cancer driver mutations. In this study, we report the spectrum of clinically actionable mutations in plasma circulating tumor DNA of 299 non-small cell lung cancer patients using ultra-deep massive parallel sequencing with unique identifier tagging. Methods: Plasma circulating tumor DNA was extracted, ligated with unique identifier (Swift Bioscience), enriched of the target coding regions of EGFR, KRAS, NRAS, BRAF and the breakpoints of ALK, ROS1 (IDT) and sequenced using NextSeq 550 (Illumina) at mean coverage depth of 20,000X. Results: Out of 299 patients tested, 128 (42,8%) carried driver mutations. G...

Bioinformatic and Molecular Analysis of Inverse Autotransporters from Escherichia coli

mSphere

Proteins secreted by the type V secretion system possess multiple functions, including the capaci... more Proteins secreted by the type V secretion system possess multiple functions, including the capacity to mediate adhesion, aggregation, and biolfilm formation. The type V secretion system can be divided into five subclasses, one of which is the type Ve system. Proteins of the type Ve secretion system are also referred to as inverse autotransporters (IATs). In this study, we performed an in silico analysis of 126 completely sequenced Escherichia coli genomes available in the NCBI database and identified several distinct IAT-encoding gene families whose distribution varied throughout the E. coli phylogeny. The genes included three characterized IATs (intimin, fdeC, and yeeJ) and four uncharacterized IATs (here named iatA, iatB, iatC, and iatD). The four iat genes were cloned from the completely sequenced environmental E. coli strain SMS-3-5 and characterized. Three of these IAT proteins (IatB, IatC, and IatD) were expressed at the cell surface and possessed the capacity to mediate biofi...

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Global gene expression profiling of a virulent Klebsiella pneumoniae strain during pulmonary infection

Access Microbiology

Background Klebsiella pneumoniae (Kpn) is an important respiratory pathogen associated with signi... more Background Klebsiella pneumoniae (Kpn) is an important respiratory pathogen associated with significant mortality, fierce inflammatory responses and high rates of antimicrobial resistance. The increasing incidence of multidrug resistant Kpn has significantly narrowed the therapeutic options available; as such, there is an urgent need to better understand Kpn pathophysiology to identify novel therapeutic targets. Here we performed an in vivo transcriptomic analysis of Kpn isolated from a mammalian host with pulmonary infection. Methods C57BL/6 mice were intranasally inoculated with the virulent Kp52.145 strain (serotype O1:K2); with lungs extracted, homogenised and pooled (n=3; in duplicate) at 32 h post-infection for bacterial RNA purification and RNA-Seq (Illumina). Differential gene expression (analysed using Degust [Voom/Limma]; FDR cut-off =0.01, abs log-FC=2) was assessed in comparison with mid-log phase growth in Lennox broth. Results Overall, we identified >900 differentially expressed genes (DEGs), comprising ∼17 % of the combined chromosomal and plasmid coding sequence repertoire. 52 % of the DEGs were upregulated during infection, including several siderophore-independent iron-, manganese- and zinc-uptake systems (e.g. hmuRSTUV, sitABCD, mntH and znuACB). We also observed a marked in vivo oxidative stress signal, with several Kpn oxidative stress response genes (e.g. oxyR, katE, katG) upregulated during infection. In contrast, expression of mgrB, a negative-regulator of the PhoPQ two-component system associated with resistance to host antimicrobial peptides was downregulated. Conclusion This study provides a novel insight into Kpn gene expression during pulmonary infection. Overall, our data suggest that adaptation to metal starvation, oxidative stress and innate immune defenses are critical for the success of Kpn lung infection.

A new massively-parallel transposon mutagenesis approach comparing multiple datasets identifies novel mechanisms of action and resistance to triclosan

The mechanisms by which antimicrobials exert inhibitory effects against bacterial cells and by wh... more The mechanisms by which antimicrobials exert inhibitory effects against bacterial cells and by which bacteria display resistance vary under different conditions. Our understanding of the full complement of genes which can influence sensitivity to many antimicrobials is limited and often informed by experiments completed in a small set of exposure conditions. Capturing a broader suite of genes which contribute to survival under antimicrobial stress will improve our understanding of how antimicrobials work and how resistance can evolve. Here, we apply a new version of ‘TraDIS’ (Transposon Directed Insert Sequencing); a massively parallel transposon mutagenesis approach to identify different responses to the common biocide triclosan across a 125-fold range of concentrations. We have developed a new bioinformatic tool ‘AlbaTraDIS’ allowing both predictions of the impacts of individual transposon inserts on gene function to be made and comparisons across multiple TraDIS data sets. This n...

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Variation in hemolysin A expression between uropathogenic Escherichia coli isolates determines NLRP3-dependent vs. -independent macrophage cell death and host colonization

The FASEB Journal

Uropathogenic Escherichia coli (UPEC) is the major cause of urinary tract infections (UTIs). The ... more Uropathogenic Escherichia coli (UPEC) is the major cause of urinary tract infections (UTIs). The multidrug‐resistant E. coli sequence type 131 (ST131) clone is a serious threat to human health, yet its effects on immune responses are not well understood. Here we screened a panel of ST131 isolates, finding that only strains expressing the toxin hemolysin A (HlyA) killed primary human macrophages and triggered maturation of the inflammasome‐dependent cytokine IL‐1β. Using a representative strain, the requirement for the hlyA gene in these responses was confirmed. We also observed considerable heterogeneity in levels of cell death initiated by different HlyA+ve ST131 isolates, and this correlated with secreted HlyA levels. Investigation into the biological significance of this variation revealed that an ST131 strain producing low levels of HlyA initiated cell death that was partly dependent on the nod‐like receptor family pyrin domain–containing 3 (NLRP3) inflammasome, with this response being associated with a host‐protective role in a mouse UTI model. When the same ST131 strain was engineered to overexpress high HlyA levels, macrophage cell death occurred even when NLRP3 function was abrogated, and bladder colonization was significantly increased. Thus, variation in HlyA expression in UPEC affects mechanisms by which macrophages die, as well as host susceptibility vs. resistance to colonization.—Murthy, A. M. V., Sullivan, M. J., Nhu, N. T. K., Lo, A. W., Phan, M.‐D., Peters, K. M., Boucher, D., Schroder, K., Beatson, S. A., Ulett, G. C., Schembri, M. A., Sweet, M. J. Variation in hemolysin A expression between uropathogenic Escherichia coli isolates determines NLRP3‐dependent vs. ‐independent macrophage cell death and host colonization. FASEB J. 33, 7437–7450 (2019). www.fasebj.org

Detection of a heterozygous germline APC mutation in a three-generation family with familial adenomatous polyposis using targeted massive parallel sequencing in Vietnam

BMC medical genetics, Jan 19, 2018

Familial adenomatous polyposis (FAP) is an autosomal dominant hereditary syndrome characterised b... more Familial adenomatous polyposis (FAP) is an autosomal dominant hereditary syndrome characterised by the development of hundreds to thousands of adenomatous colonic polyps during the second decade of life. FAP is caused by germ line mutations in the adenomatous polyposis coli (APC) gene located on chromosome 5q21-22. A 36-year-old female was presented with 100-1000 adenomatous colonic polyps, typical of classic FAP symptoms. Genetic testing using massively parallel sequencing identified a 5-bp deletion (c.3927_3931delAAAGA) which causes frameshift (p.Glu1309Aspfs) and creates a premature stop codon, resulting in the replacement of the last 1535 amino acids of APC by five incorrect amino acids. Two of the proband's four siblings also exhibited classic FAP symptoms and carried the same 5-bp heterozygous deletion in the APC gene. One of the proband's two nephews also tested positive for this mutation but has not been examined by endoscopy due to his young age. We reported here fo...

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Discovery of New Genes Involved in Curli Production by a Uropathogenic Escherichia coli Strain from the Highly Virulent O45:K1:H7 Lineage

mBio, Aug 21, 2018

Curli are bacterial surface-associated amyloid fibers that bind to the dye Congo red (CR) and fac... more Curli are bacterial surface-associated amyloid fibers that bind to the dye Congo red (CR) and facilitate uropathogenic (UPEC) biofilm formation and protection against host innate defenses. Here we sequenced the genome of the curli-producing UPEC pyelonephritis strain MS7163 and showed it belongs to the highly virulent O45:K1:H7 neonatal meningitis-associated clone. MS7163 produced curli at human physiological temperature, and this correlated with biofilm growth, resistance of sessile cells to the human cationic peptide cathelicidin, and enhanced colonization of the mouse bladder. We devised a forward genetic screen using CR staining as a proxy for curli production and identified 41 genes that were required for optimal CR binding, of which 19 genes were essential for curli synthesis. Ten of these genes were novel or poorly characterized with respect to curli synthesis and included genes involved in purine biosynthesis, a regulator that controls the Rcs phosphorelay system, and a nove...

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Establishing and validating noninvasive prenatal testing procedure for fetal aneuploidies in Vietnam

The journal of maternal-fetal & neonatal medicine : the official journal of the European Association of Perinatal Medicine, the Federation of Asia and Oceania Perinatal Societies, the International Society of Perinatal Obstetricians, Jan 4, 2018

Noninvasive prenatal testing (NIPT) for fetal aneuploidies has been widely adopted in developed c... more Noninvasive prenatal testing (NIPT) for fetal aneuploidies has been widely adopted in developed countries. Despite the sharp decrease in the cost of massively parallel sequencing, the technical know-how and skilled personnel are still one of the major limiting factors for applying this technology to NIPT in low-income settings. Here, we present the establishment and validation of our NIPT procedure called triSure for detection of fetal aneuploidies. We established the triSure algorithm based on the difference in proportion of fetal and maternal fragments from the target chromosome to all chromosomes. Our algorithm was validated using a published data set and an in-house data set obtained from high-risk pregnant women in Vietnam who have undergone amniotic testing. Several other aneuploidy calling methods were also applied to the same data set to benchmark triSure performance. The triSure algorithm showed similar accuracy to size-based method when comparing them using published data ...

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Regulation of hemolysin in uropathogenic Escherichia coli fine-tunes killing of human macrophages

Virulence, Dec 23, 2018

Uropathogenic E. coli (UPEC) causes the majority of urinary tract infections (UTIs), which are a ... more Uropathogenic E. coli (UPEC) causes the majority of urinary tract infections (UTIs), which are a major global public health concern. UPEC uses numerous mechanisms to subvert the innate immune system, including targeting macrophage functions. We recently showed that some UPEC strains rapidly kill human macrophages via an NLRP3-independent pathway, and also trigger NLRP3-dependent IL-1β processing. In this study, we used random transposon mutagenesis in the reference strain CFT073 to identify UPEC genes that mediate human macrophage cell death. Our approach revealed that the hemolysin A (HlyA) toxin is essential for triggering both cell death and NLRP3 inflammasome-mediated IL-1β release in human macrophages. Random transposon mutagenesis also identified the cof gene, which encodes a poorly characterized phosphatase, as a novel hemolysin regulator; a CFT073 mutant deleted for the cof gene secreted significantly reduced levels of HlyA, had diminished hemolytic activity, and was impaire...

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Genome-Wide Discovery of Genes Required for Capsule Production by Uropathogenic Escherichia coli

mBio, Oct 24, 2017

Uropathogenic Escherichia coli (UPEC) is a major cause of urinary tract and bloodstream infection... more Uropathogenic Escherichia coli (UPEC) is a major cause of urinary tract and bloodstream infections and possesses an array of virulence factors for colonization, survival, and persistence. One such factor is the polysaccharide K capsule. Among the different K capsule types, the K1 serotype is strongly associated with UPEC infection. In this study, we completely sequenced the K1 UPEC urosepsis strain PA45B and employed a novel combination of a lytic K1 capsule-specific phage, saturated Tn5 transposon mutagenesis, and high-throughput transposon-directed insertion site sequencing (TraDIS) to identify the complement of genes required for capsule production. Our analysis identified known genes involved in capsule biosynthesis, as well as two additional regulatory genes (mprA and lrhA) that we characterized at the molecular level. Mutation of mprA resulted in protection against K1 phage-mediated killing, a phenotype restored by complementation. We also identified a significantly increased ...

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Differential Afa/Dr Fimbriae Expression in the Multidrug-Resistant Escherichia coli ST131 Clone

mBio

E. coli ST131 is the most common antibiotic resistant UPEC clone associated with human urinary tr... more

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Ultra-Deep Sequencing of Plasma-Circulating DNA for the Detection of Tumor- Derived Mutations in Patients with Nonmetastatic Colorectal Cancer

Cancer Investigation