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Featured researches published by Richard Yuen Chong Kong.


Water Research | 2002

Rapid detection of six types of bacterial pathogens in marine waters by multiplex PCR

Richard Yuen Chong Kong; Simon Kwok-Ying Lee; T.W.F. Law; Sheran H.W. Law; Rudolf S.S. Wu

A rapid multiplex PCR (m-PCR) method that allows the simultaneous detection, in a single tube, of six commonly encountered waterborne pathogens is developed. The target genes used were: the aerolysin (aero) gene of Aeromonas hydrophila, the invasion plasmid antigen H (ipaH) gene of Shigella flexneri, the attachment invasion locus (ail) gene of Yersinia enterocolitca, the invasion plasmid antigen B (ipaB) gene of Salmonella typhimurium, the enterotoxin extracellular secretion protein (epsM) gene of Vibrio cholerae and a species-specific region of the 16S-23S rDNA (Vpara) gene of Vibrio parahaemolyticus were used as the gene targets. Multiplex PCR using the six pairs of primers produced specific amplicons of the expected sizes from mixed populations of reference bacterial strains in seawater and from pure cultures. The m-PCR assay was specific and rapid, with a turnaround time of < 12 h. The detection limit of the assay for the bacterial targets was estimated at 10(0)-10(2) cfu. Multiplex PCR analysis was performed on 19 seawater samples collected around Hong Kong and the results indicated significant levels of four bacterial pathogens at several sites where primary sewage wastes are discharged, and the levels of which showed no correlation with E. coli counts. Overall, both laboratory and field validation results demonstrated that the m-PCR assay developed in this study could provide a cost-effective and informative supplement to conventional microbiological methods for routine monitoring and risk assessment of water quality.


Emerging Infectious Diseases | 2008

Vibrio cholerae O1 Hybrid El Tor Strains, Asia and Africa

Ashrafus Safa; Jinath Sultana; Phung Dac Cam; James Mwansa; Richard Yuen Chong Kong

To the Editor: Vibrio cholerae is a water-borne pathogen that causes a severe watery diarrhea disease known as cholera. On the basis of variable somatic O antigen composition, >200 serogroups of V. cholerae have been recognized. Classical and El Tor are 2 well-established biotypes within the V. cholerae O1 serogroup, and they can be distinguished by differences in their biochemical reactions or phenotypic traits (1). In addition to phenotypic traits, genetic markers have recently been used in the identification of the biotypes of V. cholerae. For example, the major toxin-coregulated pilus (TCP) gene, tcpA, of the TCP cluster possesses classical- and El Tor–specific alleles that encode identical functions but differ in their DNA sequence composition; however, the rtxC gene of the repeat in toxin (RTX) cluster is present in El Tor strains only and absent in classical strains (2,3). The cholera toxin, encoded by the ctxA and ctxB genes, is the principal toxin produced by V. cholerae O1 and O139 and is responsible for the disease cholera. Heterogeneity within the ctxB gene and protein was first reported in the early 1990s, and, on this basis, 3 ctxB genotypes of the V. cholerae O1 strains have been identified. Based on amino acid residue substitutions at positions 39, 46, and 68, all classical and US Gulf Coast El Tor strains have been categorized as genotype 1, the Australian El Tor strains as genotype 2, and the El Tor strains of the seventh pandemic and the Latin American epidemic as genotype 3. Genotyping of ctxB has indicated that the classical strains harbor a unique cholera toxin gene that is not in the El Tor strains except for the US Gulf Coast El Tor clone (4). The US Gulf Coast hybrid El Tor strains that harbor the classical cholera toxin have been associated with sporadic outbreaks in the United States (5) and, until recently, had not been reported anywhere else in the world. Then in 2004, hybrid El Tor strains that encode the classical cholera toxin were isolated from cholera patients in Matlab, Bangladesh (6), and in Beira, Mozambique (7). In 2006, Nair et al. reported that the current seventh pandemic prototype El Tor strains had been replaced by hybrid El Tor strains in Bangladesh (8). We now report how far the hybrid El Tor strains have spread in Asia and Africa. We examined 41 clinical V. cholerae strains from Asia and Africa that were isolated from 1991 through 2004 (Table) and confirmed as serogroup O1 by O-antigen biosynthesis gene (rfbO1)-specific PCR. Biotyping was performed by using standard procedures, and all strains were confirmed as El Tor (Table). All strains were PCR-positive for the El Tor–specific 451-bp tcpA and 263-bp rtxC amplicons but negative for the classical-specific 620-bp tcpA amplicon. All 41 strains were PCR-positive for ctxAB (1,037 bp) and produced cholera toxin, as demonstrated by the VET-RPLA Toxin Detection Kit (Oxoid, Basingstoke, UK). Sequence comparison of the PCR-amplified ctxB gene (460 bp) of each strain with the reference strains (569B and N16961) showed that 30 strains harbored classical cholera toxin (with histidine at position 39, phenylalanine at position 46, and threonine at position 68), whereas the remaining 11 strains carried the El Tor cholera toxin gene (with tyrosine at position 39, phenylalanine at position 46, and isoleucine at position 68) (Table). The overall analysis showed that all test strains are El Tor biotype but that most harbor the classical cholera toxin gene. Table Phenotypic and genotypic traits of Vibrio cholerae O1 clinical strains isolated from Asia and Africa, 1991–2004* The major finding of this study is that El Tor strains that harbor the classical cholera toxin gene are not limited to the US Gulf Coast, Bangladesh, and Mozambique; they have spread to several other countries in Asia and Africa. Since 1817, 7 cholera pandemics have occurred around the world. Firm evidence indicates that the fifth and sixth cholera pandemics were caused by the classical biotype whereas the most extensive and ongoing seventh pandemic is caused by the El Tor biotype. Since the onset of El Tor dominance in 1961, the classical strains have been gradually replaced by the El Tor strains and are now believed to be extinct. However, reports from Bangladesh (6), Mozambique (7), and this study have provided sufficient evidence to indicate that the classical cholera toxin gene has reappeared but that for these cases its carrier has been El Tor. Although how the classical cholera toxin in El Tor strains would affect V. cholerae pathogenicity is unclear, cholera caused by the classical biotype is more severe, whereas the El Tor biotype is considered to be better able to survive in the environment (1,9). Given that cholera toxin is directly responsible for the major clinical sign of the disease, such a genetic change could result in substantial alteration in the clinical manifestation of cholera. Additionally, this subtle genetic change may also influence the effectiveness of current cholera vaccines, which could stimulate both antitoxic and antibacterial immunity.


Aquatic Toxicology | 2008

Real-time PCR array to study effects of chemicals on the Hypothalamic-Pituitary-Gonadal axis of the Japanese medaka

Xiaowei Zhang; Markus Hecker; June-Woo Park; Amber R. Tompsett; John L. Newsted; Kei Nakayama; Paul D. Jones; Doris Wai-Ting Au; Richard Yuen Chong Kong; Rudolf S.S. Wu; John P. Giesy

This paper describes the development and validation of a PCR array for studying chemical-induced effects on gene expression of selected endocrine pathways along the hypothalamic-pituitary-gonadal (HPG) axis of the small, oviparous fish, the Japanese medaka (Oryzias latipes). The Japanese medaka HPG-PCR array combines the quantitative performance of SYBR Green-based real-time PCR with the multiple gene profiling capabilities of a microarray to examine expression profiles of 36 genes associated with endocrine pathways in brain, liver and gonad. The performance of the Japanese medaka HPG-PCR array was evaluated by examining effects of two model compounds, the synthetic estrogen, 17alpha-ethinylestradiol (EE2) and the anabolic androgen, 17beta-trenbolone (TRB) on the HPG axis of the Japanese medaka. Four-month-old medaka was exposed to three concentrations of EE2 (5, 50, 500 ng/L) or TRB (50, 500, 5000 ng/L) for 7d in a static renewal exposure system. A pathway-based approach was implemented to analyze and visualize concentration-dependent mRNA expression in the HPG axis of Japanese medaka. The compensatory response to EE2 exposure included the down-regulation of male brain GnRH RI and testicular CYP17. The down-regulation of AR-alpha expression in brain of EE2-exposed males was associated with suppression of male sexual behavior. Compensatory responses to TRB in the female HPG axis included up-regulation of brain GnRH RII and ovary steroidogenic CYP19A. Overall, the results suggested that the Japanese medaka HPG-PCR array has potential not only as a screening tool of potential endocrine-disrupting chemicals but also in elucidating mechanisms of action.


Aquatic Toxicology | 2002

Biodegradation and enzymatic responses in the marine diatom Skeletonema costatum upon exposure to 2,4-dichlorophenol

Shao Yang; Rudolf S.S. Wu; Richard Yuen Chong Kong

The biodegradation and responses of selected detoxification and antioxidant enzymes in the marine diatom, Skeletonema costatum, upon exposure to sublethal concentrations of 2,4-dichlorophenol (2,4-DCP) were investigated. Results show that 2,4-DCP was readily metabolised, but bioaccumulation and adsorption were negligible. Glutathione S-transferase, ascorbate peroxidase and superoxide dismutase activities were increased markedly after exposure to 2,4-DCP for 96 h, while no appreciable change in peroxidase activity was observed. The addition of exogeneous glutathione to diatom culture enhanced the degradation of 2,4-DCP, and promoted diatom growth. The inhibition of glutathione synthesis enhanced the toxicity of 2,4-DCP. These results suggest that glutathione conjugation was one of the principal mechanisms involved in the degradation of 2,4-DCP in this diatom.


Fungal Biology | 1999

Studies on Amphisphaeriales : the Amphisphaeriaceae (sensu stricto)

Ji C. Kang; Kevin D. Hyde; Richard Yuen Chong Kong

The Amphisphaeriaceae ( sensu lato ) presently includes 36 genera and 23 synonyms and is a heterogeneous assemblage of ascomycetes. In this paper molecular and morphological data and teleomorph—anamorph connections are examined and confirm that the Amphisphaeriaceae ( sensu stricto ) should be confined to ten teleomorphic genera and their Pestalotia -like anamorphs. These include Amphisphaeria, Broomella, Discostroma, Ellurema, Griphosphaerioma and Neobroomella which are described and illustrated; and Blogiascospora, Lepteutypa, Paracainiella and Pestalosphaeria which are discussed.


Marine Pollution Bulletin | 1995

Co-detection of three species of water-borne bacteria by multiplex PCR

Richard Yuen Chong Kong; W.F. Dung; Lilian L.P. Vrijmoed; Rudolf S.S. Wu

Abstract Monitoring of water-borne pathogens is important to safeguard public health. In view of various limitations inherent in the traditional culture methods, the feasibility of using the polymerase chain reaction (PCR) to monitor water-borne pathogens was investigated. The STN enterotoxin gene of Salmonella typhimurium , the STO enterotoxin gene of Vibrio cholerae , the LTI and LTII enterotoxin genes of Escherichia coli , and the house-keeping genes, ARO-A and PHO-A of S. typhimurium and E. coli , respectively, were used as gene targets for PCR detection of toxigenic and general strains of these organisms. Six pairs of oligonucleotide primers were chosen to amplify internal fragments of the respective genes, and the identity of the PCR products was confirmed by restriction endonuclease digestion. The specificity of individual primer pairs in PCR was evaluated on DNA templates of 54 different bacterial isolates. The results showed that the LTI, LTII and STO primer sets were highly specific for toxigenic strains of E. coli H10407 (LTI+), E. coli SA53 (LTII+) and V. cholerae NRT (STO+), respectively. The PHO-A primers showed species-specific amplification products for all nine E. coli isolates examined, while the STN and ARO-A primer sets yielded species-specific amplification products for the 10 S. typhimurium isolates tested. Detection sensitivity of the ARO-A and PHO-A primer sets for S. typhimurium and E. coli , respectively, was estimated at 10 3 CFU. Using three different combinations of the above primer sets, multiplex PCR was performed to detect toxigenic and non-toxigenic strains of V. cholerae, S. typhimurium and E. coli in seawater samples artificially spiked with the organisms. The technique, which showed positive co-detection of the respective target genes in each case, only required a turnaround time of 5 h. Results of the present study indicate that the multiplex PCR is a potentially powerful technique for the rapid co-detection of enteropathogenic bacteria in routine water quality monitoring.


Environmental Toxicology and Chemistry | 2008

Time‐Dependent transcriptional profiles of genes of the hypothalamic‐pituitary‐gonadal axis in medaka (Oryzias latipes) exposed to fadrozole and 17β‐trenbolone

Xiaowei Zhang; Markus Hecker; June-Woo Park; Amber R. Tompsett; Paul D. Jones; John L. Newsted; Doris W.T. Au; Richard Yuen Chong Kong; Rudolf S.S. Wu; John P. Giesy

Both the anabolic androgen 17beta-trenbolone (TRB) and the aromatase inhibitor fadrozole (FAD) can cause decreased plasma concentrations of estrogen (E2) and reduce fecundity of fish. However, the underlying mechanisms and the molecular pathways involved are largely unknown. The present study was designed to assess time-dependent effects of FAD and TRB on the transcriptional responses of the hypothalamic-pituitary-gonadal (HPG) axis of Japanese medaka (Oryzias latipes). Fourteen-week-old Japanese medaka were exposed to 50 microg FAD/L or 2 microg TRB/L in a 7-d static renewal test, and the expression profiles of 36 HPG axis genes were measured by means of a medaka HPG real-time reverse-transcription polymerase chain reaction array after 8 h, 32 h, or 7 d of exposure. Exposure to TRB or FAD caused lesser fecundity of Japanese medaka and down-regulated transcription of vitellogenin and choriogenin (CHG) gene expression in the liver of females. Exposure to FAD for 8 h resulted in an 8-fold and 71-fold down-regulation of expression of estrogen receptor alpha and choriogenin L (CHG L), respectively, in female liver. 17beta-Trenbolone caused similar down-regulation of these genes, but the effects were not observed until 32 h of exposure. These results support the hypothesis that FAD reduces plasma E2 more quickly by inhibiting aromatase enzyme activity than does TRB, which inhibits the production of the E2 precursor testosterone. Exposure to FAD and TRB resulted in rapid (after 8 h) down-regulation of luteinizing hormone receptor and low-density-lipoprotein receptor in the testis to compensate for excessive androgen levels. Overall, the molecular responses observed in the present study differentiate the mechanisms of the reduced fecundity by TRB and FAD.


Marine Pollution Bulletin | 2002

Analysis of the 16S-23S rDNA intergenic spacers (IGSs) of marine vibrios for species-specific signature DNA sequences.

Simon Kwok-Ying Lee; Huizhi Wang; Sheran H.W. Law; Rudolf S.S. Wu; Richard Yuen Chong Kong

Vibrios are widespread in the marine environment and a few pathogenic species are known to be commonly associated with outbreaks of diarrheal diseases in humans due to the consumption of raw or improperly cooked seafood. However, there are also many Vibrio species which are potentially pathogenic to vertebrate and invertebrate aquatic animals, and of which little is known. In an attempt to develop rapid PCR detection methods for these latter class of vibrios, we have examined the 16S-23S intergenic spacers (IGSs) of 10 lesser-known Vibrio species and successfully developed species-specific primers for eight of them--Vibrio costicola, V. diazotrophicus, V. fluvialis, V. nigripulchritudo, V. proteolyticus, V. salmonicida, V. splendidus and V. tubiashii. The IGS amplicons were amplified using primers complementary to conserved regions of the 16S and 23S rRNA genes, and cloned into plasmid vectors and sequenced. Analysis of the IGS sequences showed that 37 ribosomal RNA (rrn) operons representing seven different IGS types have been cloned from the 10 vibrios. The three IGS types--IGS(0), IGS(IA) and IGS(Glu)--were the most prevalent forms detected. Multiple alignment of representative sequences of these three IGS types from different Vibrio species revealed several domains of high sequence variability, which were used to design species-specific primers for PCR. The specificity of the primers were evaluated using total DNA prepared from different Vibrio species and bacterial genera. The results showed that the PCR method can be used to reliably detect eight of the 10 Vibrio species in marine waters in this study.


Marine Pollution Bulletin | 1999

A Sensitive and Versatile Multiplex PCR System for the Rapid Detection of Enterotoxigenic (ETEC), Enterohaemorrhagic (EHEC) and Enteropathogenic (EPEC) Strains of Escherichia coli

Richard Yuen Chong Kong; C.L So; W.F Law; Rudolf S.S. Wu

Although Escherichia coli is widely distributed in the marine environment, only a small percentage are pathogenic to humans. Nonetheless, the widespread occurrence of waterborne infections of E. coli origin in humans has become one of the major health problems worldwide. To date, several types of enterovirulent E. coli have been recognized as the aetiologic agents of various gastrointestinal infections in humans. The most commonly encountered are those belonging to the enterotoxigenic (ETEC), enteroinvasive (EIEC), enterohaemorrhagic (EHEC) and enteropathogenic (EPEC) subtypes. In order to better determine the health risks that are associated with exposure to some of these specific subtypes, we have developed a very sensitive multiplex PCR system for the rapid detection and typing of ETEC, EHEC and possibly EPEC strains of E. coli in the aquatic environment. The target genes chosen for this investigation included: the PHO-A housekeeping gene (present in all E. coli); the LT1, LT2 and ST1 genes of ETEC; the VT1 and VT2 verotoxin, and EAE virulence genes of EHEC and EPEC, respectively. Six pairs of oligonucleotide primers were designed to simultaneously amplify internal fragments of these genes by multiplex PCR to generate PCR products that could be analysed and confirmed with relative ease by gel electrophoresis and HincII enzyme digestion. The results showed that the six sets of PCR primers were highly specific for their target genes and produced specific amplimers of the expected size from several control strains of E. coli – ATCC 35401 (LT1+/ST1+); SA53 (LT2+/VT2+); and O157 (VT1+/VT2+/EAE+). The detection sensitivity of the multiplex PCR system for the six target genes in an E. coli cell mixture was optimized and enhanced by preincubating serially diluted cells in Luria-Bertani broth for 6 h prior to PCR analysis. The results obtained indicated a detection sensitivity of 10° CFU (of each strain) per 100 μl reaction. Multiplex PCR analysis of seawater samples collected from four sewage-polluted sites in Hong Kong indicated the presence in all four samples of E. coli bacteria that were positive for LT1, ST1, VT1 and EAE virulence genes. Overall, the data indicated that the multiplex PCR system described in this study is a potentially very useful and powerful method for routine monitoring and risk assessment of water quality.


PLOS ONE | 2014

Identification and Expression Profiling of MicroRNAs in the Brain, Liver and Gonads of Marine Medaka (Oryzias melastigma) and in Response to Hypoxia

Karen Lau; Keng Po Lai; Jessie Y.J. Bao; Na Zhang; Anna Tse; Amy Hin Yan Tong; Jing-Woei Li; Si Lok; Richard Yuen Chong Kong; Wing-Yee Lui; Alice Wong; Rudolf S.S. Wu

The marine medaka (Oryzias melastigma) has been increasingly used as a fish model for detecting environmental stresses and chemical contaminants in the marine environment. Recent mammalian studies have shown that environmental stresses can alter the expression profiles of microRNAs (miRNAs), leading to transgenerational effects. Here, we use high-throughput Illumina RNA sequencing (RNA-Seq) for miRNA transcriptome analysis of brain, liver, and gonads from sexually mature male and female marine medaka. A total of 128,883,806 filtered sequence reads were generated from six small RNA libraries, identifying a total of 2,125,663 non-redundant sequences. These sequences were aligned and annotated to known animal miRNAs (miRBase) using the BLAST method. A total of 223 distinct miRNA types were identified, with the greatest number expressed in brain tissue. Our data suggested that 55 miRNA types from 34 families are common to all tested tissues, while some of the miRNAs are tissue-enriched or sex-enriched. Quantitative real-time PCR analysis further demonstrated that let-7a, miR-122, and miR-9-3p were downregulated in hypoxic female medaka, while miR-2184 was specifically upregulated in the testis of hypoxic male fish. This is the first study to identify miRNAs in O. melastigma using small RNA deep sequencing technology. Because miRNA expression is highly conserved between marine medaka and other vertebrates, marine medaka may serve as a good model for studies on the functional roles of miRNAs in hypoxia stress response and signaling in marine fish.

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Rudolf S.S. Wu

City University of Hong Kong

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Richard Man Kit Yu

City University of Hong Kong

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John P. Giesy

University of Saskatchewan

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Jing-Woei Li

The Chinese University of Hong Kong

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Doris W.T. Au

City University of Hong Kong

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Helen O. L. Mok

City University of Hong Kong

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Keng Po Lai

City University of Hong Kong

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Minnie Man Lai Wong

City University of Hong Kong

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S. W. Leung

City University of Hong Kong

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