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Molecular diversity of HIV-1 and surveillance of transmitted drug resistance variants among treatment Naïve patients, 5 years after active introduction of HAART in Kuala Lumpur, Malaysia

Ong LY, Razak SN, Lee YM, Sri La Sri Ponnampalavanar S, Syed Omar SF, Azwa RI, et al.

J Med Virol, 2014 Jan;86(1):38-44.
PMID: 24127302 DOI: 10.1002/jmv.23772

Expansion of antiretroviral treatment programs have led to the growing concern for the development of antiretroviral drug resistance. The aims were to assess the prevalence of drug resistant HIV-1 variants and to identify circulating subtypes among HAART-naïve patients. Plasma specimens from N = 100 HIV+ HAART-naïve adult were collected between March 2008 and August 2010 and viral RNA were extracted for nested PCR and sequenced. PR-RT sequences were protein aligned and checked for transmitted drug resistance mutations. Phylogenetic reconstruction and recombination analysis were performed to determine the genotypes. Based on the WHO consensus guidelines, none of the recruited patients had any transmitted drug resistance mutations. When analyzed against the Stanford guidelines, 35% of patients had at least one reported mutation that may reduce drug susceptibility to PI (24%), NRTI (5%), and NNRTI (14%). The commonly detected mutation that may affect current first line therapy was V179D (3%), which may lead to reduced susceptibility to NNRTI. The predominant circulating HIV-1 genotypes were CRF01_AE (51%) and CRF33_01B (17%). The prevalence of unique recombinant forms (URF) was 7%; five distinct recombinant structures involving CRF01_AE and subtype B' were observed, among them a cluster of three isolates that could form a novel circulating recombinant form (CRF) candidate. Transmitted drug resistance prevalence among HAART-naïve patients was low in this cohort of patients in Kuala Lumpur despite introduction of HAART 5 years ago. Owing to the high genetic diversity, continued molecular surveillance can identify the persistent emergence of HIV-1 URF and novel CRF with significant epidemiological impact.
Fulltext Emerging and re-emerging viruses in Malaysia, 1997-2007

Tee KK, Takebe Y, Kamarulzaman A

Int J Infect Dis, 2009 May;13(3):307-18.
PMID: 19010076 DOI: 10.1016/j.ijid.2008.09.005

Over the past decade, a number of unique zoonotic and non-zoonotic viruses have emerged in Malaysia. Several of these viruses have resulted in significant morbidity and mortality to those affected and they have imposed a tremendous public health and economic burden on the state. Amongst the most devastating was the outbreak of Nipah virus encephalitis in 1998, which resulted in 109 deaths. The culling of more than a million pigs, identified as the amplifying host, ultimately brought the outbreak under control. A year prior to this, and subsequently again in 2000 and 2003, large outbreaks of hand-foot-and-mouth disease due to enterovirus 71, with rare cases of fatal neurological complications, were reported in young children. Three other new viruses - Tioman virus (1999), Pulau virus (1999), and Melaka virus (2006) - whose origins have all been linked to bats, have been added to the growing list of novel viruses being discovered in Malaysia. The highly pathogenic H5N1 avian influenza has also been detected in Malaysia with outbreaks in poultry in 2004, 2006, and 2007. Fortunately, no human infections were reported. Finally, the HIV/AIDS epidemic has seen the emergence of an HIV-1 recombinant form (CRF33_01B) in HIV-infected individuals from various risk groups, with evidence of ongoing and rapid expansion.
Fulltext Comparative global epidemiology of influenza, respiratory syncytial and parainfluenza viruses, 2010-2015

Lam TT, Tang JW, Lai FY, Zaraket H, Dbaibo G, Bialasiewicz S, et al.

J Infect, 2019 10;79(4):373-382.
PMID: 31323249 DOI: 10.1016/j.jinf.2019.07.008

OBJECTIVES: To improve our understanding of the global epidemiology of common respiratory viruses by analysing their contemporaneous incidence at multiple sites.
METHODS: 2010-2015 incidence data for influenza A (IAV), influenza B (IBV), respiratory syncytial (RSV) and parainfluenza (PIV) virus infections were collected from 18 sites (14 countries), consisting of local (n = 6), regional (n = 9) and national (n = 3) laboratories using molecular diagnostic methods. Each site submitted monthly virus incidence data, together with details of their patient populations tested and diagnostic assays used.
RESULTS: For the Northern Hemisphere temperate countries, the IAV, IBV and RSV incidence peaks were 2-6 months out of phase with those in the Southern Hemisphere, with IAV having a sharp out-of-phase difference at 6 months, whereas IBV and RSV showed more variable out-of-phase differences of 2-6 months. The tropical sites Singapore and Kuala Lumpur showed fluctuating incidence of these viruses throughout the year, whereas subtropical sites such as Hong Kong, Brisbane and Sydney showed distinctive biannual peaks for IAV but not for RSV and PIV.
CONCLUSIONS: There was a notable pattern of synchrony of IAV, IBV and RSV incidence peaks globally, and within countries with multiple sampling sites (Canada, UK, Australia), despite significant distances between these sites.
HIV-1 Tat biosensor: Current development and trends for early detection strategies

Fatin MF, Ruslinda AR, Arshad MK, Tee KK, Ayub RM, Hashim U, et al.

Biosens Bioelectron, 2016 Apr 15;78:358-66.
PMID: 26655174 DOI: 10.1016/j.bios.2015.11.067

Human immunodeficiency virus (HIV) has infected almost 35 million people worldwide. Various tests have been developed to detect the presence of HIV during the early stages of the disease in order to reduce the risk of transmission to other humans. The HIV-1 Tat protein is one of the proteins present in HIV that are released abundantly approximately 2-4 weeks after infection. In this review, we have outlined various strategies for detecting the Tat protein, which helps transcribe the virus and enhances replication. Detection strategies presented include immunoassays, biosensors and gene expression, which utilize antibodies or aptamers as common probes to sense the presence of Tat. Alternatively, measuring the levels of gene transcription is a direct method of analysing the HIV gene to confirm the presence of Tat. By detection of the Tat protein, virus transmission can be detected in high-risk individuals in the early stages of the disease to reduce the risk of an HIV pandemic.
Fulltext Whole-Genome Analysis of Quorum-Sensing Burkholderia sp. Strain A9

Chan KG, Chen JW, Tee KK, Chang CY, Yin WF, Chan XY

Genome Announc, 2015;3(2).
PMID: 25745000 DOI: 10.1128/genomeA.00063-15

Burkholderia spp. rely on N-acyl homoserine lactone as quorum-sensing signal molecules which coordinate their phenotype at the population level. In this work, we present the whole genome of Burkholderia sp. strain A9, which enables the discovery of its N-acyl homoserine lactone synthase gene.
Fulltext N-acyl homoserine lactone-producing Pseudomonas putida strain T2-2 from human tongue surface

Chen JW, Chin S, Tee KK, Yin WF, Choo YM, Chan KG

Sensors (Basel), 2013;13(10):13192-203.
PMID: 24084113 DOI: 10.3390/s131013192

Bacterial cell-to-cell communication (quorum sensing) refers to the regulation of bacterial gene expression in response to changes in microbial population density. Quorum sensing bacteria produce, release and respond to chemical signal molecules called autoinducers. Bacteria use two types of autoinducers, namely autoinducer-1 (AI-1) and autoinducer-2 (AI-2) where the former are N-acylhomoserine lactones and the latter is a product of the luxS gene. Most of the reported literatures show that the majority of oral bacteria use AI-2 for quorum sensing but rarely the AI-1 system. Here we report the isolation of Pseudomonas putida strain T2-2 from the oral cavity. Using high resolution mass spectrometry, it is shown that this isolate produced N-octanoylhomoserine lactone (C8-HSL) and N-dodecanoylhomoserine lactone (C12-HSL) molecules. This is the first report of the finding of quorum sensing of P. putida strain T2-2 isolated from the human tongue surface and their quorum sensing molecules were identified.
Fulltext Complete genome sequencing of Pandoraea pnomenusa RB38 and Molecular Characterization of Its N-acyl homoserine lactone synthase gene ppnI

Lim YL, Ee R, How KY, Lee SK, Yong D, Tee KK, et al.

PeerJ, 2015;3:e1225.
PMID: 26336650 DOI: 10.7717/peerj.1225

In this study, we sequenced the genome of Pandoraea pnomenusa RB38 using Pacific Biosciences RSII (PacBio) Single Molecule Real Time (SMRT) sequencing technology. A pair of cognate luxI/R homologs was identified where the luxI homolog, ppnI, was found adjacent to a luxR homolog, ppnR1. An additional orphan luxR homolog, ppnR2, was also discovered. Multiple sequence alignment and phylogenetic analysis revealed that ppnI is an N-acyl homoserine lactone (AHL) synthase gene that is distinct from those of the nearest phylogenetic neighbor viz. Burkholderia spp. High resolution tandem mass spectrometry (LC-MS/MS) analysis showed that Escherichia coli BL21 harboring ppnI produced a similar AHL profile (N-octanoylhomoserine lactone, C8-HSL) as P. pnomenusa RB38, the wild-type donor strain, confirming that PpnI directed the synthesis of AHL in P. pnomenusa RB38. To our knowledge, this is the first documentation of the luxI/R homologs of the genus Pandoraea.
Streptomyces humi sp. nov., an actinobacterium isolated from soil of a mangrove forest

Zainal N, Ser HL, Yin WF, Tee KK, Lee LH, Chan KG

Antonie Van Leeuwenhoek, 2016 Mar;109(3):467-74.
PMID: 26786500 DOI: 10.1007/s10482-016-0653-1

A novel Streptomyces strain, MUSC 119(T), was isolated from a soil collected from a mangrove forest. Cells of MUSC 119(T) stained Gram-positive and formed light brownish grey aerial mycelium and grayish yellowish brown substrate mycelium on ISP 2 medium. A polyphasic approach was used to determine the taxonomic status of strain MUSC 119(T), which shows a range of phylogenetic and chemotaxonomic properties consistent with those of the genus Streptomyces. The cell wall peptidoglycan consisted of LL-diaminopimelic acid. The predominant menaquinones were identified as MK-9(H8), MK-9(H6) and MK-9(H4). The polar lipid profile consisted of phosphatidylinositol, phosphatidylethanolamine, glycolipids, diphosphatidylglycerol and four phospholipids. The predominant cellular fatty acids were anteiso-C15:0, iso-C16:0, and anteiso-C17:0. The cell wall sugars were glucose, mannose, ribose and rhamnose. The phylogenetic analysis based on 16S rRNA gene sequence similarity showed that strain MUSC119(T) to be closely related to Streptomyces rhizophilus JR-41(T) (99.0 % sequence similarity), S. panaciradicis 1MR-8(T) (98.9 %), S. gramineus JR-43(T) (98.8 %) and S. graminisoli JR-19(T) (98.7 %). These results suggest that MUSC 119(T) should be placed within the genus Streptomyces. DNA-DNA relatedness values between MUSC 119(T) to closely related strains ranged from 14.5 ± 1.3 to 27.5 ± 0.7 %. The G+C content was determined to be 72.6 mol %. The polyphasic study of MUSC 119(T) showed that this strain represents a novel species, for which the name Streptomyces humi sp. nov. is proposed. The type strain of S. humi is MUSC 119(T) (=DSM 42174(T) = MCCC 1K00505(T)).
Fulltext Complete Genome Sequence Analysis of Pandoraea pnomenusa Type Strain DSM 16536(T) Isolated from a Cystic Fibrosis Patient

Lim YL, Ee R, Yong D, Yu CY, Ang GY, Tee KK, et al.

Front Microbiol, 2016;7:109.
PMID: 26903988 DOI: 10.3389/fmicb.2016.00109
Fulltext Quorum sensing activity of Citrobacter amalonaticus L8A, a bacterium isolated from dental plaque

Goh SY, Khan SA, Tee KK, Abu Kasim NH, Yin WF, Chan KG

Sci Rep, 2016;6:20702.
PMID: 26860259 DOI: 10.1038/srep20702

Cell-cell communication is also known as quorum sensing (QS) that happens in the bacterial cells with the aim to regulate their genes expression in response to increased cell density. In this study, a bacterium (L8A) isolated from dental plaque biofilm was identified as Citrobacter amalonaticus by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). Its N-acylhomoserine-lactone (AHL) production was screened by using two types of AHL biosensors namely Chromobacterium violaceum CV026 and Escherichia coli [pSB401]. Citrobacter amalonaticus strain L8A was identified and confirmed producing numerous types of AHL namely N-butyryl-L-homoserine lactone (C4-HSL), N-hexanoyl-L-homoserine lactone (C6-HSL), N-octanoyl-L-homoserine lactone (C8-HSL) and N-hexadecanoyl-L-homoserine lactone (C16-HSL). We performed the whole genome sequence analysis of this oral isolate where its genome sequence reveals the presence of QS signal synthase gene and our work will pave the ways to study the function of the related QS genes in this bacterium.
Fulltext Insights of biosurfactant producing Serratia marcescens strain W2.3 isolated from diseased tilapia fish: a draft genome analysis

Chan XY, Chang CY, Hong KW, Tee KK, Yin WF, Chan KG

Gut Pathog, 2013;5(1):29.
PMID: 24148830 DOI: 10.1186/1757-4749-5-29

Serratia marcescens is an opportunistic bacterial pathogen with broad range of host ranging from vertebrates, invertebrates and plants. S. marcescens strain W2.3 was isolated from a diseased tilapia fish and it was suspected to be the causal agent for the fish disease as virulence genes were found within its genome. In this study, for the first time, the genome sequences of S. marcescens strain W2.3 were sequenced using the Illumina MiSeq platform.
Fulltext Characterization and Transcriptome Studies of Autoinducer Synthase Gene from Multidrug Resistant Acinetobacter baumannii Strain 863

Ng CK, How KY, Tee KK, Chan KG

Genes (Basel), 2019 04 08;10(4).
PMID: 30965610 DOI: 10.3390/genes10040282

Quorum sensing (QS) is a cell-to-cell communication system that uses autoinducers as signaling molecules to enable inter-species and intra-species interactions in response to external stimuli according to the population density. QS allows bacteria such as Acinetobacter baumannii to react rapidly in response to environmental changes and hence, increase the chances of survival. A. baumannii is one of the causative agents in hospital-acquired infections and the number of cases has increased remarkably in the past decade. In this study, A. baumannii strain 863, a multidrug-resistant pathogen, was found to exhibit QS activity by producing N-acyl homoserine lactone. We identified the autoinducer synthase gene, which we named abaI, by performing whole genome sequencing analysis of A. baumannii strain 863. Using high resolution tandem triple quadrupole mass spectrometry, we reported that abaI of A. baumannii strain 863 produced 3-hydroxy-dodecanoyl-homoserine lactone. A gene deletion mutant was constructed, which confirmed the functionality of abaI. A growth defect was observed in the QS-deficient mutant strain. Transcriptome profiling was performed to determine the possible genes regulated by QS. Four groups of genes that showed differential expression were discovered, namely those involved in carbon source metabolism, energy production, stress response and the translation process.
Fulltext Methylome Characterization of Burkholderia pseudomallei Strain 982 at Single-Base Resolution

Hong KW, Tee KK, Yin WF, Roberts RJ, Chan KG

Microbiol Resour Announc, 2019 Oct 24;8(43).
PMID: 31649075 DOI: 10.1128/MRA.00898-19

Burkholderia pseudomallei is the etiological agent of melioidosis, which has been studied by transcriptome and secretome analyses. However, little is known about the methylome of this pathogen. Here, we present the complete genome and methylome of melioidosis-causing B. pseudomallei strain 982.
Complete genome sequence of Pandoraea thiooxydans DSM 25325(T), a thiosulfate-oxidizing bacterium

Yong D, Ee R, Lim YL, Yu CY, Ang GY, How KY, et al.

J Biotechnol, 2016 Jan 10;217:51-2.
PMID: 26603120 DOI: 10.1016/j.jbiotec.2015.11.009

Pandoraea thiooxydans DSM 25325(T) is a thiosulfate-oxidizing bacterium isolated from rhizosphere soils of a sesame plant. Here, we present the first complete genome of P. thiooxydans DSM 25325(T). Several genes involved in thiosulfate oxidation and biodegradation of aromatic compounds were identified.
Complete genome of Pandoraea pnomenusa RB-38, an oxalotrophic bacterium isolated from municipal solid waste landfill site

Lim YL, Ee R, Yong D, Tee KK, Yin WF, Chan KG

J Biotechnol, 2015 Nov 20;214:83-4.
PMID: 26393955 DOI: 10.1016/j.jbiotec.2015.09.018

Pandoraea pnomenusa RB-38 is a bacterium isolated from a former sanitary landfill site. Here, we present the complete genome of P. pnomenusa RB38 in which an oxalate utilization pathway was identified. The genome analysis suggested the potential of this strain as an effective biocontrol agent against oxalate-producing phytopathogens.
Complete genome sequence of Serratia fonticola DSM 4576 T, a potential plant growth promoting bacterium

Lim YL, Yong D, Ee R, Krishnan T, Tee KK, Yin WF, et al.

J Biotechnol, 2015 Nov 20;214:43-4.
PMID: 26376471 DOI: 10.1016/j.jbiotec.2015.09.005

Here, we present the first complete genome sequence of Serratia fonticola DSM 4576(T), a potential plant growth promoting (PGP) bacterium which confers solubilization of inorganic phosphate, indole-3-acetic acid production, hydrogen cyanideproduction, siderophore production and assimilation of ammonia through the glutamate synthase (GS/GOGAT) pathway. This genome sequence is valuable for functional genomics and ecological studies which are related to PGP and biocontrol activities.
Complete genome sequence of Serratia multitudinisentens RB-25(T), a novel chitinolytic bacterium

Lim YL, Yong D, Ee R, Tee KK, Yin WF, Chan KG

J Biotechnol, 2015 Aug 10;207:32-3.
PMID: 25975625 DOI: 10.1016/j.jbiotec.2015.04.027

Serratia multitudinisentens RB-25(T) (=DSM 28811(T) =LMG 28304(T)) is a newly proposed type strain in the genus of Serratia isolated from a municipal landfill site. Here, we present the complete genome of S. multitudinisentens RB-25(T) which contains a complete chitinase operon and other chitin and N-acetylglucosamine utilisation enzymes. To our knowledge, this is the first report of the complete genome sequence of this novel isolate and its chitinase gene discovery.
Comparative genomic and phylogenetic analysis of a toxigenic clinical isolate of Corynebacterium diphtheriae strain B-D-16-78 from Malaysia

Hong KW, Asmah Hani AW, Nurul Aina Murni CA, Pusparani RR, Chong CK, Verasahib K, et al.

Infect Genet Evol, 2017 Oct;54:263-270.
PMID: 28711373 DOI: 10.1016/j.meegid.2017.07.015

In this study, we report the comparative genomics and phylogenetic analysis of Corynebacterium diphtheriae strain B-D-16-78 that was isolated from a clinical specimen in 2016. The complete genome of C. diphtheriae strain B-D-16-78 was sequenced using PacBio Single Molecule, Real-Time sequencing technology and consists of a 2,474,151-bp circular chromosome with an average GC content of 53.56%. The core genome of C. diphtheriae was also deduced from a total of 74 strains with complete or draft genome sequences and the core genome-based phylogenetic analysis revealed close genetic relationship among strains that shared the same MLST allelic profile. In the context of CRISPR-Cas system, which confers adaptive immunity against re-invading DNA, 73 out of 86 spacer sequences were found to be unique to Malaysian strains which harboured only type-II-C and/or type-I-E-a systems. A total of 48 tox genes which code for the diphtheria toxin were retrieved from the 74 genomes and with the exception of one truncated gene, only nucleotide substitutions were detected when compared to the tox gene sequence of PW8. More than half were synonymous substitution and only two were nonsynonymous substitutions whereby H24Y was predicted to have a damaging effect on the protein function whilst T262V was predicted to be tolerated. Both toxigenic and non-toxigenic toxin-gene bearing strains have been isolated in Malaysia but the repeated isolation of toxigenic strains with the same MLST profile suggests the possibility of some of these strains may be circulating in the population. Hence, efforts to increase herd immunity should be continued and supported by an effective monitoring and surveillance system to track, manage and control outbreak of cases.
Characterization and Comparative Overview of Complete Sequences of the First Plasmids of Pandoraea across Clinical and Non-clinical Strains

Yong D, Tee KK, Yin WF, Chan KG

Front Microbiol, 2016;7:1606.
PMID: 27790203

To date, information on plasmid analysis in Pandoraea spp. is scarce. To address the gap of knowledge on this, the complete sequences of eight plasmids from Pandoraea spp. namely Pandoraea faecigallinarum DSM 23572(T) (pPF72-1, pPF72-2), Pandoraea oxalativorans DSM 23570(T) (pPO70-1, pPO70-2, pPO70-3, pPO70-4), Pandoraea vervacti NS15 (pPV15) and Pandoraea apista DSM 16535(T) (pPA35) were studied for the first time in this study. The information on plasmid sequences in Pandoraea spp. is useful as the sequences did not match any known plasmid sequence deposited in public databases. Replication genes were not identified in some plasmids, a situation that has led to the possibility of host interaction involvement. Some plasmids were also void of par genes and intriguingly, repA gene was also not discovered in these plasmids. This further leads to the hypothesis of host-plasmid interaction. Plasmid stabilization/stability protein-encoding genes were observed in some plasmids but were not established for participating in plasmid segregation. Toxin-antitoxin systems MazEF, VapBC, RelBE, YgiT-MqsR, HigBA, and ParDE were identified across the plasmids and their presence would improve plasmid maintenance. Conjugation genes were identified portraying the conjugation ability amongst Pandoraea plasmids. Additionally, we found a shared region amongst some of the plasmids that consists of conjugation genes. The identification of genes involved in replication, segregation, toxin-antitoxin systems and conjugation, would aid the design of drugs to prevent the survival or transmission of plasmids carrying pathogenic properties. Additionally, genes conferring virulence and antibiotic resistance were identified amongst the plasmids. The observed features in the plasmids shed light on the Pandoraea spp. as opportunistic pathogens.
Characterization of quorum sensing genes and N-acyl homoserine lactones in Citrobacter amalonaticus strain YG6

Kher HL, Krishnan T, Letchumanan V, Hong KW, How KY, Lee LH, et al.

Gene, 2019 Feb 05;684:58-69.
PMID: 30321658 DOI: 10.1016/j.gene.2018.10.031

In the phylum of Proteobacteria, quorum sensing (QS) system is widely driven by synthesis and response of N-acyl homoserine lactone (AHL) signalling molecules. AHL is synthesized by LuxI homologue and sensed by LuxR homologue. Once the AHL concentration achieves a threshold level, it triggers the regulation of target genes. In this study, QS activity of Citrobacter amalonaticus strain YG6 which was isolated from clams was investigated. In order to characterise luxI/R homologues, the genome of C. amalonaticus strain YG6 (4.95 Mbp in size) was sequenced using Illumina MiSeq sequencer. Through in silico analysis, a pair of canonical luxI/R homologues and an orphan luxR homologue were identified and designated as camI, camR, and camR2, respectively. A putative lux box was identified at the upstream of camI. The camI gene was cloned and overexpressed in E. coli BL21 (DE3)pLysS. High-resolution triple quadrupole liquid chromatography mass spectrometry (LC-MS/MS) analysis verified that the CamI is a functional AHL synthase which produced multiple AHL species, namely N‑butyryl‑l‑homoserine lactone (C4-HSL), N‑hexanoyl‑l‑homoserine lactone (C6-HSL), N‑octanoyl‑l‑homoserine lactone (C8-HSL), N‑tetradecanoyl‑l‑homoserine lactone (C14-HSL) and N‑hexadecanoyl‑l‑homoserine lactone (C16-HSL) in C. amalonaticus strain YG6 and camI gene in recombinant E. coli BL21(DE3)pLysS. To our best knowledge, this is the first functional study report of camI as well as the first report describing the production of C14-HSL by C. amalonaticus.

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