Geik Yong Ang

Molecular Evidence of Cholera Outbreak Caused by a Toxigenic Vibrio cholerae O1 El Tor Variant Strain in Kelantan, Malaysia

ABSTRACT A total of 20 Vibrio cholerae isolates were recovered for investigation from a cholera outbreak in Kelantan, Malaysia, that occurred between November and December 2009. All isolates were biochemically characterized as V. cholerae serogroup O1 Ogawa of the El Tor biotype. They were found to be resistant to multiple antibiotics, including tetracycline, erythromycin, sulfamethoxazole-trimethoprim, streptomycin, penicillin G, and polymyxin B, with 35% of the isolates being resistant to ampicillin. All isolates were sensitive to ciprofloxacin, norfloxacin, chloramphenicol, gentamicin, and kanamycin. Multiplex PCR analysis confirmed the biochemical identification and revealed the presence of virulence genes, viz., ace, zot, and ctxA, in all of the isolates. Interestingly, the sequencing of the ctxB gene showed that the outbreak strain harbored the classical cholera toxin gene and therefore belongs to the newly assigned El Tor variant biotype. Clonal analysis by pulsed-field gel electrophoresis demonstrated that a single clone of a V. cholerae strain was responsible for this outbreak. Thus, we present the first molecular evidence that the toxigenic V. cholerae O1 El Tor variant has invaded Malaysia, highlighting the need for continuous monitoring to facilitate early interventions against any potential epidemic by this biotype.

Ambient temperature detection of PCR amplicons with a novel sequence-specific nucleic acid lateral flow biosensor

In the field of diagnostics, molecular amplification targeting unique genetic signature sequences has been widely used for rapid identification of infectious agents, which significantly aids physicians in determining the choice of treatment as well as providing important epidemiological data for surveillance and disease control assessment. We report the development of a rapid nucleic acid lateral flow biosensor (NALFB) in a dry-reagent strip format for the sequence-specific detection of single-stranded polymerase chain reaction (PCR) amplicons at ambient temperature (22-25°C). The NALFB was developed in combination with a linear-after-the-exponential PCR assay and the applicability of this biosensor was demonstrated through detection of the cholera toxin gene from diarrheal-causing toxigenic Vibrio cholerae. Amplification using the advanced asymmetric PCR boosts the production of fluorescein-labeled single-stranded amplicons, allowing capture probes immobilized on the NALFB to hybridize specifically with complementary targets in situ on the strip. Subsequent visual formation of red lines is achieved through the binding of conjugated gold nanoparticles to the fluorescein label of the captured amplicons. The visual detection limit observed with synthetic target DNA was 0.3 ng and 1 pg with pure genomic DNA. Evaluation of the NALFB with 164 strains of V. cholerae and non-V. cholerae bacteria recorded 100% for both sensitivity and specificity. The whole procedure of the low-cost NALFB, which is performed at ambient temperature, eliminates the need for preheated buffers or additional equipment, greatly simplifying the protocol for sequence-specific PCR amplicon analysis.

Dry-reagent gold nanoparticle-based lateral flow biosensor for the simultaneous detection of Vibrio cholerae serogroups O1 and O139

Cholera is a communicable disease caused by consumption of contaminated food and water. This potentially fatal intestinal infection is characterised by profuse secretion of rice watery stool that can rapidly lead to severe dehydration and shock, thus requiring treatment to be given immediately. Epidemic and pandemic cholera are exclusively associated with Vibrio cholerae serogroups O1 and O139. In light of the need for rapid diagnosis of cholera and to prevent spread of outbreaks, we have developed and evaluated a direct one-step lateral flow biosensor for the simultaneous detection of both V. cholerae O1 and O139 serogroups using alkaline peptone water culture. Serogroup specific monoclonal antibodies raised against lipopolysaccharides (LPS) were used to functionalize the colloidal gold nanoparticles for dual detection in the biosensor. The assay is based on immunochromatographic principle where antigen-antibody reaction would result in the accumulation of gold nanoparticles and thus, the appearance of a red line on the strip. The dry-reagent dipstick format of the biosensor ensure user-friendly application, rapid result that can be read with the naked eyes and cold-chain free storage that is well-suited to be performed at resource-limited settings.

Complete genome sequencing revealed novel genetic contexts of the mcr-1 gene in Escherichia coli strains.

Sir, Polymyxin-based treatment is often considered as the last-line therapy for MDR Gram-negative bacterial infections. Resistance to polymyxins, in particular colistin, was previously linked to adaptive or mutational mechanisms involving chromosomally encoded genes before the discovery in late 2015 of a plasmid-mediated mobile colistin-resistance determinant (mcr-1). The widespread dissemination of mcr-1 was subsequently discovered as this resistance determinant was found in at least 20 countries from various continents within a span of 3 months. A retrospective study has shown that the mcr-1 gene can be traced back to the early 1980s, which coincides with the introduction of colistin into animal husbandry in China, and hence the current distribution of MCR-1-producing Enterobacteriaceae may only represent the tip of the iceberg. The mcr-1 gene has attracted significant interest due to its potential for horizontal transfer and impact on clinical treatment options. In order to gain insights into the localization and genetic organization of the mcr-1 gene, we sequenced six different STs of mcr-1-positive Escherichia coli strains (Table S1, available as Supplementary data at JAC Online) that were previously isolated from various sources in Malaysia. Genomic DNAs were extracted using a MasterPure DNA Purification Kit (Epicenter, Madison, WI, USA) and long-read sequencing was performed on a PacBio RS II instrument (Pacific Biosciences, Menlo Park, CA, USA). The singlemolecule sequencing reads were de novo assembled using Hierarchical Genome Assembly Process (HGAP) v. 3.0, and overlapping regions were assessed with Gepard followed by circularization using Minimus2 pipeline in the AMOS software package. The genomes were annotated via RAST and replicon sequences in the plasmids were analysed using PlasmidFinder (https://cge.cbs.dtu.dk/ services/). The genetic contexts of the mcr-1-harbouring plasmids and chromosome are summarized in Figure 1. pEC5-1, pEC13-1 and pS2.14-2 were IncI2 plasmids that shared over 96% of their sequences (at 99% nucleotide identity) with pmcr1_IncI2 (KU761326)—an mcr-1-harbouring plasmid that was isolated from a clinical ESBL-producing E. coli strain in China (Figure S1). The genetic context of mcr-1 in pEC5-1, pEC13-1 and pS2.14-2 was identical to that of pmcr1_IncI2 (nikB–mcr-1–pap), whereas, in pHNSHP45 (KP347127), an ISApl1 mobile element was inserted between nikB and mcr-1. The upstream ISApl1 insertion in mcr-1-bearing precursor plasmids such as pEC5-1, pEC13-1, pS2. 14-2 and pmcr1_IncI2 was hypothesized to be the key genetic event underlying the rapid mobilization and acquisition of this colistin-resistance gene. In IncHl1 megaplasmids pEC2-4 and pEC2_1-4 the mcr-1-associated cassettes interrupted a gene that encodes a putative DNA repair protein and the pap elements were also found to be truncated. The ISApl1–mcr-1–Dpap–ISApl1 in pEC2-4 was bracketed by a 2 bp (TG) target site duplication, while, in pEC2_1-4, a 2961 bp deletion surrounding the downstream ISApl1 element resulted in a different genetic arrangement of ISApl1–mcr-1–Dpap– Dorfunknown–int. Both plasmids also harboured other clinically relevant antibiotic resistance genes, including aadA1 (aminoglycoside), qnrS1 (fluoroquinolone), floR (florfenicol/chloramphenicol) and sul3 (sulphonamide), whereas only pEC2-4 carried an additional fosfomycin resistance determinant (fosA). pEC2-4 and pEC2_1-4 bear the closest resemblance to pB71 (KP899806; 63% query coverage; 99% nucleotide identity) and p109/9 (KP899805; 70% query coverage; 99% nucleotide identity), respectively, from Salmonella Typhimurium (Figure S1). The finding of mcr-1carrying IncHl1 plasmids in addition to previously reported classes of mcr-1-positive plasmids that include IncI2, IncF, IncX4, IncHl2 and IncP incompatibility groups provided further evidence that transmission of this resistance gene occurred via distinct plasmids. An ISApl1-mediated translocation of mcr-1 into the chromosome of E. coli was recently proposed by Veldman et al., but the site of integration and the mcr-1-associated genetic structures were not reported. In the present study, multiple copies of ISApl1–mcr-1–Dpap that were arranged in tandem were found to be inserted between nhaA and sokC genes in the chromosome of clinical isolate EC590 resulting in a unique genetic arrangement of nhaA–ISApl1–mcr-1–Dpap–ISApl1–mcr-1–Dpap–ISApl1–mcr1–Dpap–ISApl1–sokC, which was further confirmed by long-range

Enzymatic electrochemical detection of epidemic-causing Vibrio cholerae with a disposable oligonucleotide-modified screen-printed bisensor coupled to a dry-reagent-based nucleic acid amplification assay.

In this study, we developed a nucleic acid-sensing platform in which a simple, dry-reagent-based nucleic acid amplification assay is combined with a portable multiplex electrochemical genosensor. Preparation of an amplification reaction mix targeting multiple DNA regions of interest is greatly simplified because the lyophilized reagents need only be reconstituted with ultrapure water before the DNA sample is added. The presence of single or multiple target DNAs causes the corresponding single-stranded DNA (ssDNA) amplicons to be generated and tagged with a fluorescein label. The fluorescein-labeled ssDNA amplicons are then analyzed using capture probe-modified screen-printed gold electrode bisensors. Enzymatic amplification of the hybridization event is achieved through the catalytic production of electroactive α-naphthol by anti-fluorescein-conjugated alkaline phosphatase. The applicability of this platform as a diagnostic tool is demonstrated with the detection of toxigenic Vibrio cholerae serogroups O1 and O139, which are associated with cholera epidemics and pandemics. The platform showed excellent diagnostic sensitivity and specificity (100%) when challenged with 168 spiked stool samples. The limit of detection was low (10 colony-forming units/ml) for both toxigenic V. cholerae serogroups. A heat stability assay revealed that the dry-reagent amplification reaction mix was stable at temperatures of 4-56 °C, with an estimated shelf life of seven months. The findings of this study highlight the potential of combining a dry-reagent-based nucleic acid amplification assay with an electrochemical genosensor in a more convenient, sensitive, and sequence-specific detection strategy for multiple target nucleic acids.

Development of a dry-reagent-based nucleic acid-sensing platform by coupling thermostabilised LATE-PCR assay to an oligonucleotide-modified lateral flow biosensor

In this study, we report for the first time the development of a dry-reagent-based nucleic acid-sensing platform by combining a thermostabilised linear-after-the-exponential (LATE)-PCR assay with a one-step, hybridisation-based nucleic acid lateral flow biosensor. The nucleic acid-sensing platform was designed to overcome the need for stringent temperature control during transportation or storage of reagents and reduces the dependency on skilled personnel by decreasing the overall assay complexity and hands-on time. The platform was developed using toxigenic Vibrio cholerae as the model organism due to the bacteriums propensity to cause epidemic and pandemic cholera. The biosensor generates result which can be visualised with the naked eyes and the limit of detection was found to be 1pg of pure genomic DNA and 10CFU/ml of toxigenic V. cholerae. The dry-reagent-based nucleic acid-sensing platform was challenged with 95 toxigenic V. cholerae, 7 non-toxigenic V. cholerae and 66 other bacterial strains in spiked stool sample and complete agreement was observed when the results were compared to that of monosialoganglioside (GM1)-ELISA. Heat-stability of the thermostabilised LATE-PCR reaction mixes at different storage temperatures (4-56°C) was investigated for up to 90days. The dry-reagent-based genosensing platform with ready-to-use assay components provides an alternative method for sequence-specific detection of nucleic acid without any cold chain restriction that is associated with conventional molecular amplification techniques.

A colloidal gold-based lateral flow immunoassay for direct determination of haemoglobin A1c in whole blood

We developed an immunosensor that operates based on the lateral flow principle designed to detect haemoglobin A1c (HbA1c), a biomarker for type 2 diabetes mellitus in human blood samples. Two different clones of antibodies were used to form a “sandwich” when HbA1c was present. Functionalization of colloidal gold with antibodies was carefully optimized to generate stable gold conjugates to amplify the signal from the formed “sandwich” for the immunoassay. The ideal blocking reagent to minimize background noises, the test line format on the strip, the selectivity of the assay towards HbA1c against HbA0 and glycated species of HbA0, and the potential interference contributed by the colour of the blood sample were investigated. Captured HbA1c on the lateral flow immunosensor can be distinguished based on the number and intensity of the test lines shown; visual detection of the lines shown then indicated the distinctive groups at normal, under control, and elevated levels of HbA1c. Also, a calibration curve that covered the detection range of 4% (20 mmol mol−1) and 12% (108 mmol mol−1) HbA1c was reported, indicating that the prototype can be used for future quantification utilizing a lateral flow reader. The resultant immunosensor was found to report results that were easier to be interpreted and relatively inexpensive compared to electrochemical biosensors developed for the detection of HbA1c.

Comparative genomic and phylogenetic analysis of a toxigenic clinical isolate of Corynebacterium diphtheriae strain B-D-16-78 from Malaysia

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.

Draft genome sequence of multidrug-resistant Salmonella enterica serovar Brancaster strain PS01 isolated from chicken meat, Malaysia

OBJECTIVES Salmonella spp. represent one of the main diarrhoeal pathogens that are transmitted via the food supply chain. Here we report the draft genome sequence of a multidrug-resistant Salmonella enterica serovar Brancaster (PS01) that was isolated from poultry meat in Malaysia. METHODS Genomic DNA was extracted from Salmonella strain PS01 and was sequenced using an Illumina HiSeq 2000 platform. The generated reads were de novo assembled using CLC Genomics Workbench. The draft genome was annotated and the presence of antimicrobial resistance genes was identified. RESULTS The 5 036 442bp genome contains various antimicrobial resistance genes conferring resistance to aminoglycosides, fluoroquinolones, fosfomycin, macrolides, phenicols, sulphonamides, tetracyclines and trimethoprim. The β-lactamase gene blaTEM-176 encoding TEM-176 was also found in this strain. CONCLUSIONS The genome sequence will aid in the understanding of drug resistance mechanisms in foodborne Salmonella Brancaster and highlights the need to ensure the judicious use of antibiotics in animal husbandry as well as the importance of implementing proper food handling and preparation practices.

Detection of CYP2C19 Genetic Variants in Malaysian Orang Asli from Massively Parallel Sequencing Data.

The human cytochrome P450 (CYP) is a superfamily of enzymes that have been a focus in research for decades due to their prominent role in drug metabolism. CYP2C is one of the major subfamilies which metabolize more than 10% of all clinically used drugs. In the context of CYP2C19, several key genetic variations that alter the enzyme’s activity have been identified and catalogued in the CYP allele nomenclature database. In this study, we investigated the presence of well-established variants as well as novel polymorphisms in the CYP2C19 gene of 62 Orang Asli from the Peninsular Malaysia. A total of 449 genetic variants were detected including 70 novel polymorphisms; 417 SNPs were located in introns, 23 in upstream, 7 in exons, and 2 in downstream regions. Five alleles and seven genotypes were inferred based on the polymorphisms that were found. Null alleles that were observed include CYP2C19*3 (6.5%), *2 (5.7%) and *35 (2.4%) whereas allele with increased function *17 was detected at a frequency of 4.8%. The normal metabolizer genotype was the most predominant (66.1%), followed by intermediate metabolizer (19.4%), rapid metabolizer (9.7%) and poor metabolizer (4.8%) genotypes. Findings from this study provide further insights into the CYP2C19 genetic profile of the Orang Asli as previously unreported variant alleles were detected through the use of massively parallel sequencing technology platform. The systematic and comprehensive analysis of CYP2C19 will allow uncharacterized variants that are present in the Orang Asli to be included in the genotyping panel in the future.