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A single-gene approach for the subspecies classification of Mycobacteroides abscessus

Ng HF, Ngeow YF

Pathog Dis, 2020 11 11;78(8).
PMID: 32945880 DOI: 10.1093/femspd/ftaa055

The subspecies classification of Mycobacteroides abscessus complex into M. abscessus, M. massiliense and M. bolletii requires the amplification and sequencing of multiple genes. The objective of this study was to evaluate the possibility of subspecies classification using a single PCR target. An in silico study was performed to classify 1613 strains deposited in a public database using 9 genes (partial gene sequences of hsp65, rpoB, sodA, argH, cya, glpK, gnd, and murC, and the full gene sequence of MAB_3542c). We found the housekeeping gene gnd to be able to classify the M. abscessus subspecies with high accuracy (99.94%). A single-gene PCR approach based on gnd would be a suitable replacement for the more expensive, labor-intensive and time-consuming multi-gene PCR analysis currently in use for the subspecies identification of M. abscessus.
A simple spreadsheet-based method for relative quantification using quantitative real-time PCR

Ng HF, Ngeow YF

Biochem Mol Biol Educ, 2022 Jan;50(1):99-103.
PMID: 34854201 DOI: 10.1002/bmb.21596

Relative quantification is a popular analysis in gene expression studies using quantitative real-time PCR (qPCR). However, the calculation steps using the major algorithms for this analysis are rather complicated. In this study, we developed an easy-to-use spreadsheet-based method for relative quantification. The inputs from end-users are the efficiencies of both target and reference genes and the Cq values of those genes from cases and controls. This method performed normalization (with one or more reference genes), calculation of fold change of gene expression, and statistical analysis to analyze the difference between the groups in a step-by-step manner, which would allow the end-users to understand how the analysis arrived at the conclusion. Four previously published data sets with different experimental designs were used as examples. The calculated results were concordant with the results computed by the Relative Expression Software Tool (REST) 2009, a popular tool for relative quantification. Altogether, our method, which offers easy-to-understand calculation steps and does not require specialized instruments, software, or expertise to operate, would be a useful tool for students, educators, and scientists in the field of molecular biology.
Mutations in Genes Encoding 23S rRNA and FadD32 May be Associated with Linezolid Resistance in Mycobacteroides abscessus

Ng HF, Ngeow YF

Microb Drug Resist, 2023 Feb;29(2):41-46.
PMID: 36802272 DOI: 10.1089/mdr.2022.0068

Linezolid is one of the antibiotics used to treat the Mycobacteroides abscessus infection. However, linezolid-resistance mechanisms of this organism are not well understood. The objective of this study was to identify possible linezolid-resistance determinants in M. abscessus through characterization of step-wise mutants selected from a linezolid-susceptible strain, M61 (minimum inhibitory concentration [MIC]: 0.25 mg/L). Whole-genome sequencing and subsequent PCR verification of the resistant second-step mutant, A2a(1) (MIC: >256 mg/L), revealed three mutations in its genome, two of which were found in the 23S rDNA (g2244t and g2788t) and another one was found in a gene encoding the fatty-acid-CoA ligase FadD32 (c880t→H294Y). The 23S rRNA is the molecular target of linezolid and mutations in this gene are likely to contribute to resistance. Furthermore, PCR analysis revealed that the c880t mutation in the fadD32 gene first appeared in the first-step mutant, A2 (MIC: 1 mg/L). Complementation of the wild-type M61 with the pMV261 plasmid carrying the mutant fadD32 gene caused the previously sensitive M61 to develop a reduced susceptibility to linezolid (MIC: 1 mg/L). The findings of this study uncovered hitherto undescribed mechanisms of linezolid resistance in M. abscessus that may be useful for the development of novel anti-infective agents against this multidrug-resistant pathogen.
The mRNA expression of soluble urokinase plasminogen activator surface receptor in human adipose tissue is positively correlated with body mass index

Ng HF, Chin KF, Chan KG, Ngeow YF

Genome, 2015 Jun;58(6):315-21.
PMID: 26284904 DOI: 10.1139/gen-2015-0028

suPLAUR is the transcript variant that encodes the soluble form of the urokinase plasminogen activator surface receptor (suPLAUR). This soluble protein has been shown to enhance leukocyte migration and adhesion, and its circulatory level is increased in inflammatory states. In this pilot study, we used RNA-Seq to examine the splicing pattern of PLAUR in omental adipose tissues from obese and lean individuals. Of the three transcript variants of the PLAUR gene, only the proportion of suPLAUR (transcript variant 2) increases in obesity. After removing the effects of gender and age, the expression of suPLAUR is positively correlated with body mass index. This observation was validated using RT-qPCR with an independent cohort of samples. Additionally, in our RNA-Seq differential expression analysis, we also observed, in obese adipose tissues, an up-regulation of genes encoding other proteins involved in the process of chemotaxis and leukocyte adhesion; of particular interest is the integrin beta 2 (ITGB2) that is known to interact with suPLAUR in leukocyte adhesion. These findings suggest an important role for suPLAUR in the recruitment of immune cells to obese adipose tissue, in the pathogenesis of obesity.
25S rDNA genotyping and multi-locus sequence typing of Candida albicans of pathogenic and commensal origins in the Klang Valley, Malaysia

Illyaaseen Z, Ngeow YF, Yap SF, Ng HF

Malays J Pathol, 2021 Apr;43(1):55-61.
PMID: 33903306

Candida albicans is an important opportunistic fungal pathogen capable of causing fatal systemic infections in humans. Presently in Malaysia, there is little information available on the genetic diversity of this organism and trends in behavioural characteristics. In this project, three genotyping methods: 25S rDNA genotyping, Alternative Lengthening of Telomerase (ALT) sequence typing and Multi-Locus Sequence Typing (MLST) were applied to study the genetic diversity of strains from infected hospital in-patients and asymptomatic individuals in the community. The results showed that, with the 25S rDNA genotyping, as in other parts of the world, the most common genotype was type A which accounted for approximately 70% of the 111 isolates tested. Further typing with the ALT sequence showed type 3 to be the most common in the isolates tested. MLST analysis revealed many possibly novel sequence types, as well as a statistically significant association between pathogenicity and a group of closely related isolates, most of which were from hospital samples. Further work on genotypes associated with enhanced virulence will help to clarify the value of genotyping for clinical and epidemiological investigations.
A stop-gain mutation in sigma factor SigH (MAB_3543c) may be associated with tigecycline resistance in Mycobacteroides abscessus

Lee CL, Ng HF, Ngeow YF, Thaw Z

J Med Microbiol, 2021 Jul;70(7).
PMID: 34236301 DOI: 10.1099/jmm.0.001378

Introduction. Tigecycline is currently acknowledged to be one of the most effective antibiotics against infections caused by Mycobacteroides abscessus.Gap statement. The genetic determinants of tigecycline resistance in M. abscessus are not well understood.Aim. In this study, we characterized a tigecycline-resistant M. abscessus mutant, designated CL7, to identify the potential resistance mechanism.Methodology. CL7 was characterized using antimicrobial susceptibility testing, whole-genome sequencing, PCR and RT-qPCR. For biological verification, gene overexpression assays were carried out.Results. Whole-genome sequencing and the subsequent gene overexpression assays showed that CL7 harboured a stop-gain mutation in MAB_3543 c, which may be responsible for the tigecycline resistance phenotype. This gene encodes an orthologue of SigH, which is involved in the positive regulation of physiological stress response and is negatively regulated by the RshA anti-sigma factor in Mycobacterium tuberculosis. We hypothesized that the MAB_3543 c mutation may disrupt the interaction between SigH and RshA (MAB_3542 c). RT-qPCR analyses revealed the upregulation of MAB_3543 c and other key stress response genes, which has previously been shown to be a hallmark of SigH-RshA bond disruption and tigecycline resistance.Conclusion. The MAB_3543c mutation may represent a novel determinant of tigecycline resistance in M. abscessus. The findings of this study will hopefully contribute to our knowledge of potential tigecycline resistance mechanisms in M. abscessus, which may lead to better diagnostics and treatment modalities in the future.
Fulltext Hierarchical Image Transformation and Multi-Level Features for Anomaly Defect Detection

Farady I, Kuo CC, Ng HF, Lin CY

Sensors (Basel), 2023 Jan 15;23(2).
PMID: 36679785 DOI: 10.3390/s23020988

Anomalies are a set of samples that do not follow the normal behavior of the majority of data. In an industrial dataset, anomalies appear in a very small number of samples. Currently, deep learning-based models have achieved important advances in image anomaly detection. However, with general models, real-world application data consisting of non-ideal images, also known as poison images, become a challenge. When the work environment is not conducive to consistently acquiring a good or ideal sample, an additional adaptive learning model is needed. In this work, we design a potential methodology to tackle poison or non-ideal images that commonly appear in industrial production lines by enhancing the existing training data. We propose Hierarchical Image Transformation and Multi-level Features (HIT-MiLF) modules for an anomaly detection network to adapt to perturbances from novelties in testing images. This approach provides a hierarchical process for image transformation during pre-processing and explores the most efficient layer of extracted features from a CNN backbone. The model generates new transformations of training samples that simulate the non-ideal condition and learn the normality in high-dimensional features before applying a Gaussian mixture model to detect the anomalies from new data that it has never seen before. Our experimental results show that hierarchical transformation and multi-level feature exploration improve the baseline performance on industrial metal datasets.
Fulltext Comparative genomic analysis of Mycobacterium iranicum UM_TJL against representative mycobacterial species suggests its environmental origin

Tan JL, Ngeow YF, Wee WY, Wong GJ, Ng HF, Choo SW

Sci Rep, 2014;4:7169.
PMID: 25417557 DOI: 10.1038/srep07169

Mycobacterium iranicum is a newly reported mycobacterial species. We present the first comparative study of M. iranicum UM_TJL and other mycobacteria. We found M. iranicum to have a close genetic association with environmental mycobacteria infrequently associated with human infections. Nonetheless, UM_TJL is also equipped with many virulence genes (some of which appear to be the consequence of transduction-related gene transfer) that have been identified in established human pathogens. Taken all together, our data suggest that M. iranicum is an environmental bacterium adapted for pathogenicity in the human host. This comparative study provides important clues and forms the basis for future functional studies on this mycobacterium.
Fulltext First Whole-Genome Sequence of Mycobacterium iranicum, a Newly Reported Mycobacterial Species

Tan JL, Ng HF, Wee WY, Ang MY, Wong GJ, Ngeow YF, et al.

Genome Announc, 2013;1(5).
PMID: 24072861 DOI: 10.1128/genomeA.00732-13

Mycobacterium iranicum is a new species of nontuberculous mycobacterium reported in 2013. Here, we describe the first whole-genome sequence of this species, that of M. iranicum strain UM_TJL, isolated from a patient in Malaysia.
Fulltext Identification of new genomospecies in the Mycobacterium terrae complex

Ngeow YF, Wong YL, Tan JL, Hong KW, Ng HF, Ong BL, et al.

PLoS One, 2015;10(4):e0120789.
PMID: 25830768 DOI: 10.1371/journal.pone.0120789

Members of the Mycobacterium terrae complex are slow-growing, non-chromogenic acid-fast bacilli found in the natural environment and occasionally in clinical material. These genetically closely-related members are difficult to differentiate by conventional phenotypic and molecular tests. In this paper we describe the use of whole genome data for the identification of four strains genetically similar to Mycobacterium sp. JDM601, a newly identified member of the M. terrae complex. Phylogenetic information from the alignment of genome-wide orthologous genes and single nucleotide polymorphisms show consistent clustering of the four strains together with M. sp. JDM601 into a distinct clade separate from other rapid and slow growing mycobacterial species. More detailed inter-strain comparisons using average nucleotide identity, tetra-nucleotide frequencies and analysis of synteny indicate that our strains are closely related to but not of the same species as M. sp. JDM601. Besides the 16S rRNA signature described previously for the M. terrae complex, five more hypothetical proteins were found that are potentially useful for the rapid identification of mycobacterial species belonging to the M. terrae complex. This paper illustrates the versatile utilization of whole genome data for the delineation of new bacterial species and introduces four new genomospecies to add to current members in the M. terrae complex.
A mutation in anti-sigma factor MAB_3542c may be responsible for tigecycline resistance in Mycobacterium abscessus

Ng HF, Tan JL, Zin T, Yap SF, Ngeow YF

J Med Microbiol, 2018 Dec;67(12):1676-1681.
PMID: 30351265 DOI: 10.1099/jmm.0.000857

In this study, we characterized 7C, a spontaneous mutant selected from tigecycline-susceptible Mycobacterium abscessus ATCC 19977. Whole-genome sequencing (WGS) was used to identify possible resistance determinants in this mutant. Compared to the wild-type, 7C demonstrated resistance to tigecycline as well as cross-resistance to imipenem, and had a slightly retarded growth rate. WGS and subsequent biological verifications showed that these phenotypes were caused by a point mutation in MAB_3542c, which encodes an RshA-like protein. In Mycobacterium tuberculosis, RshA is an anti-sigma factor that negatively regulates the heat/oxidative stress response mechanisms. The MAB_3542c mutation may represent a novel determinant of tigecycline resistance. We hypothesize that this mutation may dysregulate the stress-response pathways which have been shown to be linked to antibiotic resistance in previous studies.
Influence of job demands and job control on work-life balance among Taiwanese nurses

Ng LP, Chen IC, Ng HF, Lin BY, Kuar LS

J Nurs Manag, 2017 Sep;25(6):438-448.
PMID: 28419626 DOI: 10.1111/jonm.12482

AIM: This study investigated the extent to which the job demands and job control of nurses were related to their work-life balance.
BACKGROUND: The inability to achieve work-life balance is one of the major reasons for the declining retention rate among nurses. Job demands and job control are two major work domain factors that can have a significant influence on the work-life balance of nurses.
METHOD: The study measured the job demands, job control and work-life balance of 2040 nurses in eight private hospitals in Taiwan in 2013.
RESULTS: Job demands and job control significantly predicted all the dimensions of work-life balance. Job demands increased the level of work-life imbalance among nurses. While job control showed positive effects on work/personal life enhancement, it was found to increase both work interference with personal life and personal life interference with work.
CONCLUSION: Reducing the level of job demands (particularly for psychological demands) between family and career development and maintaining a proper level of job control are essential to the work-life balance of nurses.
IMPLICATIONS FOR NURSING MANAGEMENT: Flexible work practices and team-based management could be considered by nursing management to lessen job demand pressure and to facilitate job engagement and participation among nurses, thus promoting a better balance between work and personal life.
Fulltext QuaBingo: A Prediction System for Protein Quaternary Structure Attributes Using Block Composition

Tung CH, Chen CW, Guo RC, Ng HF, Chu YW

Biomed Res Int, 2016;2016:9480276.
PMID: 27610389 DOI: 10.1155/2016/9480276

Background. Quaternary structures of proteins are closely relevant to gene regulation, signal transduction, and many other biological functions of proteins. In the current study, a new method based on protein-conserved motif composition in block format for feature extraction is proposed, which is termed block composition. Results. The protein quaternary assembly states prediction system which combines blocks with functional domain composition, called QuaBingo, is constructed by three layers of classifiers that can categorize quaternary structural attributes of monomer, homooligomer, and heterooligomer. The building of the first layer classifier uses support vector machines (SVM) based on blocks and functional domains of proteins, and the second layer SVM was utilized to process the outputs of the first layer. Finally, the result is determined by the Random Forest of the third layer. We compared the effectiveness of the combination of block composition, functional domain composition, and pseudoamino acid composition of the model. In the 11 kinds of functional protein families, QuaBingo is 23% of Matthews Correlation Coefficient (MCC) higher than the existing prediction system. The results also revealed the biological characterization of the top five block compositions. Conclusions. QuaBingo provides better predictive ability for predicting the quaternary structural attributes of proteins.
Tigecycline resistance may be associated with dysregulated response to stress in Mycobacterium abscessus

Ng HF, Ngeow YF, Yap SF, Zin T, Tan JL

Int J Med Microbiol, 2019 Nov 18.
PMID: 31784213 DOI: 10.1016/j.ijmm.2019.151380

Previously, we characterized 7C, a laboratory-derived tigecycline-resistant mutant of Mycobacterium abscessus ATCC 19977, and found that the resistance was conferred by a mutation in MAB_3542c, which encodes an RshA-like protein. In M. tuberculosis, RshA is an anti-sigma factor that negatively regulates the SigH-dependent heat/oxidative stress response. We hypothesized that this mutation in 7C might dysregulate the stress response which has been generally linked to antibiotic resistance. In this study, we tested this hypothesis by subjecting 7C to transcriptomic dissection using RNA sequencing. We found an over-expression of genes encoding the SigH ortholog, chaperones and oxidoreductases. In line with these findings, 7C demonstrated better survival against heat shock when compared to the wild-type ATCC 19977. Another interesting observation from the RNA-Seq analysis was the down-regulation of ribosomal protein-encoding genes. This highlights the possibility of ribosomal conformation changes which could negatively affect the binding of tigecycline to its target, leading to phenotypic resistance. We also demonstrated that transient resistance to tigecycline could be induced in the ATCC 19977 by elevated temperature. Taken together, these findings suggest that dysregulated stress response may be associated with tigecycline resistance in M. abscessus.
Fulltext Global-and-Local Context Network for Semantic Segmentation of Street View Images

Lin CY, Chiu YC, Ng HF, Shih TK, Lin KH

Sensors (Basel), 2020 May 21;20(10).
PMID: 32455537 DOI: 10.3390/s20102907

Semantic segmentation of street view images is an important step in scene understanding for autonomous vehicle systems. Recent works have made significant progress in pixel-level labeling using Fully Convolutional Network (FCN) framework and local multi-scale context information. Rich global context information is also essential in the segmentation process. However, a systematic way to utilize both global and local contextual information in a single network has not been fully investigated. In this paper, we propose a global-and-local network architecture (GLNet) which incorporates global spatial information and dense local multi-scale context information to model the relationship between objects in a scene, thus reducing segmentation errors. A channel attention module is designed to further refine the segmentation results using low-level features from the feature map. Experimental results demonstrate that our proposed GLNet achieves 80.8% test accuracy on the Cityscapes test dataset, comparing favorably with existing state-of-the-art methods.
Concentrated specimen smear microscopy utilising a polymer membrane sandwich filtration vessel for the detection of acid-fast bacilli in health facilities in Sabah, East Malaysia

Paul DC, Ngeow YF, Yap SF, Dony JF, Avoi R, Mohammad R, et al.

Tuberculosis (Edinb), 2022 Mar;133:102183.
PMID: 35180496 DOI: 10.1016/j.tube.2022.102183

A simple, ready-to-use concentrated specimen smear microscopy method employing a nanometer silicon polyvinylidene fluoride (PVDF) polymer membrane sandwich filtration vessel to concentrate acid-fast bacilli (AFB) in samples (SFV-CSSM, Hunan-Tech New Medical System Co. Ltd. China) was compared with direct sputum smear microscopy (DSSM) to determine its performance using culture on modified Ogawa agar as reference. The results for 4114 clinical samples collected from health facilities in Sabah were interpreted with reference to culture results, sample collection-transportation conditions and clinical data including responses to anti-TB drug treatment. The SFV-CSSM showed higher sensitivity than DSSM (79.4% versus 60.5%) and less background interference. Its ability to detect low levels of AFB at an affordable cost makes it an excellent tool for the screening of pauci-bacillary samples as well as for active case finding in TB control programs.
Mutations in atpG2 may confer resistance to gentamicin in Listeria monocytogenes

Ng JML, Ngeow YF, Saw SH, Ng HF, Zin T

J Med Microbiol, 2022 Dec;71(12).
PMID: 36748567 DOI: 10.1099/jmm.0.001618

Introduction Listeriosis, a foodborne infection caused by Listeria monocytogenes, could lead to febrile listerial gastroenteritis and a more invasive form which is often associated with a high mortality and hospitalisation rate. Gentamicin, used as an adjunct therapy with ampicillin, remains the treatment of choice for this life-threatening and invasive infection.Gap statement Nevertheless, there is little data on gentamicin resistance determinants in L. monocytogenes.Aim In this study, we selected and characterised B2b, a gentamicin-resistant mutant derived from L. monocytogenes ATCC 19115 to determine the target(s) of resistance in L. monocytogenes after exposure to gentamicin.Methodology Whole-genome sequencing was carried out to identify the mutation site(s) and possible mechanism(s) of resistance. The mutant was characterised using antimicrobial susceptibility testing and PCR. For biological verifications, complementation and allelic exchange mutagenesis were carried out.Results We found that the gentamicin resistance in B2b was caused by a 10 bp deletion in atpG2 which encodes a gamma subunit of the ATP synthase in L. monocytogenes. Using atpG2 PCR, various other mutations were identified in other gentamicin resistant mutants derived from ATCC 19115. In addition, the mutation from B2b, when introduced into L. ivanovii, also caused gentamicin resistance in this Listeria species.Conclusion Hence, atpG2 mutations appear to be important determinants of gentamicin resistance not only in L. monocytogenes but possibly also in other Listeria species.
Navigating the intricacies of RT-qPCR data analysis in gene expression studies

Wetthasinghe L, Ng HF, Ngeow YF, Chew KS, Lee WS

Funct Integr Genomics, 2024 Jun 24;24(4):115.
PMID: 38910215 DOI: 10.1007/s10142-024-01393-0
Tuberculosis in captive Asian elephants (Elephas maximus) in Peninsular Malaysia

Ong BL, Ngeow YF, Razak MF, Yakubu Y, Zakaria Z, Mutalib AR, et al.

Epidemiol Infect, 2013 Jul;141(7):1481-7.
PMID: 23414617 DOI: 10.1017/S0950268813000265

A cross-sectional study was conducted from 10 January to 9 April 2012, to determine the seroprevalence of tuberculosis (TB) of all captive Asian elephants and their handlers in six locations in Peninsular Malaysia. In addition, trunk-wash samples were examined for tubercle bacillus by culture and polymerase chain reaction (PCR). For 63 elephants and 149 elephant handlers, TB seroprevalence was estimated at 20.4% and 24.8%, respectively. From 151 trunkwash samples, 24 acid-fast isolates were obtained, 23 of which were identified by hsp65-based sequencing as non-tuberculous mycobacteria. The Mycobacterium tuberculosis-specific PCR was positive in the trunk-wash samples from three elephants which were also seropositive. Conversely, the trunk wash from seven seropositive elephants were PCR negative. Hence, there was evidence of active and latent TB in the elephants and the high seroprevalence in the elephants and their handlers suggests frequent, close contact, two-way transmission between animals and humans within confined workplaces.
Fulltext Multiphasic strain differentiation of atypical mycobacteria from elephant trunk wash

Chan KG, Loke MF, Ong BL, Wong YL, Hong KW, Tan KH, et al.

PeerJ, 2015;3:e1367.
PMID: 26587340 DOI: 10.7717/peerj.1367

Background. Two non-tuberculous mycobacterial strains, UM_3 and UM_11, were isolated from the trunk wash of captive elephants in Malaysia. As they appeared to be identical phenotypes, they were investigated further by conventional and whole genome sequence-based methods of strain differentiation. Methods. Multiphasic investigations on the isolates included species identification with hsp65 PCR-sequencing, conventional biochemical tests, rapid biochemical profiling using API strips and the Biolog Phenotype Microarray analysis, protein profiling with liquid chromatography-mass spectrometry, repetitive sequence-based PCR typing and whole genome sequencing followed by phylogenomic analyses. Results. The isolates were shown to be possibly novel slow-growing schotochromogens with highly similar biological and genotypic characteristics. Both strains have a genome size of 5.2 Mbp, G+C content of 68.8%, one rRNA operon and 52 tRNAs each. They qualified for classification into the same species with their average nucleotide identity of 99.98% and tetranucleotide correlation coefficient of 0.99999. At the subspecies level, both strains showed 98.8% band similarity in the Diversilab automated repetitive sequence-based PCR typing system, 96.2% similarity in protein profiles obtained by liquid chromatography mass spectrometry, and a genomic distance that is close to zero in the phylogenomic tree constructed with conserved orthologs. Detailed epidemiological tracking revealed that the elephants shared a common habitat eight years apart, thus, strengthening the possibility of a clonal relationship between the two strains.

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