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Fulltext Characterization of Escherichia coli isolated from cultured catfish by antibiotic resistance and RAPD analysis

Samuel, L., Marian, M.M.,, Apun, K., Lesley, M.B., Son, R.

International Food Research Journal, 2011;18(3):-.
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Antibiotic susceptibility and genetic diversity of E. coli isolated from cultured catfish and their surrounding environment were determined. The levels of resistance of the E. coli isolates towards six different antibiotics tested differed considerably. Though the isolates displayed resistance towards some of the antibiotics tested, none of the isolates showed resistant towards norfloxacin, sulphametoxazole/trimethoprim and chloramphenicol. RAPD-PCR analysis using single primer and primers combination clustered the E. coli isolates into 3 and 5 groups, respectively. The results of this study suggest that the E. coli isolates from the catfish and their surrounding environment derived from a mixture of sensitive and resistant strains with diverse genetic contents. The use of the RAPD analysis is sufficiently discriminatory for the typing of the E. coli isolates.
Fulltext Molecular confirmation and characterization of Vibrio parahaemolyticus from retailed fish

Vincent, M., Chan, C. S. W., Apun, K.

International Food Research Journal, 2015;22(4):1705-1710.
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The present study was conducted to assess the rapid molecular identification and characterization of 45 Vibrio parahaemolyticus isolates from 15 samples of 3 different types of fish (Kembung, Bawal and Sangeh) in the Kuching-Samarahan district. Polymerase chain reaction (PCR) based confirmation was done targeting the 450 bp fragment of the thermolabile (tl) gene, while DNA fingerprinting was performed using Randomly Amplified Polymorphic DNA (RAPD) PCR with the primer GEN15008. All the 45 V. parahaemolyticus isolates were positive for the tl gene, however, only 34 were typable via RAPD-PCR with bands sizes ranging from slightly over 250 bp to 2.5 kbp. The degree of diversity was then determined via the Simpson Index which showed a value of 0.891, indicating high diversity among the isolates. Data from the RAPD-PCR fingerprints were later used to construct a dendrogram for clustal analysis. From the dendrogram, the 34 isolates were grouped into 2 major clusters containing 26 and 8 isolates, respectively. Further analyses of the dendrogram also indicated that the 34 isolated were clustered according to the period of sampling. This is an interesting observation as it shows the high discriminatory capability of RAPD-PCR to be used as molecular epidemiological tool to study the temporal distribution of V. parahaemolyticus.
Screening and isolation of a cellulolytic and amylolytic Bacillus from sago pith waste

Apun K, Jong BC, Salleh MA

J Gen Appl Microbiol, 2000 Oct;46(5):263-267.
PMID: 12483578
Spatially varying correlation between environmental conditions and human leptospirosis in Sarawak, Malaysia

Kira R, Bilung LM, Ngui R, Apun K, Su'ut L

Trop Biomed, 2021 Jun 01;38(2):31-39.
PMID: 33973570 DOI: 10.47665/tb.38.2.034

The spatial distribution of environmental conditions may influence the dynamics of vectorborne diseases like leptospirosis. This study aims to investigate the global and localised relationships between leptospirosis with selected environmental variables. The association between environmental variables and the spatial density of geocoded leptospirosis cases was determined using global Poisson regression (GPR) and geographically weighted Poisson regression (GWPR). A higher prevalence of leptospirosis was detected in areas with higher water vapour pressure (exp(â): 1.12; 95% CI: 1.02 - 1.25) and annual precipitation (exp(â): 1.15; 95% CI: 1.02 - 1.31), with lower precipitation in the driest month (exp(â): 0.85; 95% CI: 0.75 - 0.96) and the wettest quarter (exp(â): 0.88; 95% CI: 0.77 - 1.00). Water vapor pressure (WVP) varied the most in the hotspot regions with a standard deviation of 0.62 (LQ: 0.15; UQ; 0.99) while the least variation was observed in annual precipitation (ANNP) with a standard deviation of 0.14 (LQ: 0.11; UQ; 0.30). The reduction in AICc value from 519.73 to 443.49 indicates that the GWPR model is able to identify the spatially varying correlation between leptospirosis and selected environmental variables. The results of the localised relationships in this study could be used to formulate spatially targeted interventions. This would be particularly useful in localities with a strong environmental or socio-demographical determinants for the transmission of leptospirosis.
Investigation of the antibacterial activity of Ag-NPs conjugated with a specific antibody against Staphylococcus aureus after photoactivation

Al-Sharqi A, Apun K, Vincent M, Kanakaraju D, Bilung LM, Sum MSH

J Appl Microbiol, 2020 Jan;128(1):102-115.
PMID: 31596989 DOI: 10.1111/jam.14471

AIM: This work reports a new method for the use of lasers for the selective killing of bacteria targeted using light-absorbing Silver nanoparticles (Ag-NPs) conjugated with a specific antibody against the Gram-positive bacterium Staphylococcus aureus (S. aureus).
METHODS AND RESULTS: Ag-NPs were synthesized using a chemical reduction method and characterized with respect to their surface plasmon resonance, surface morphology via transmission electron microscopy (TEM) and dynamic light scattering (DLS). The bacterial surface was targeted using 20 nm Ag-NPs conjugated with an anti-protein A antibody. Labelled bacteria were irradiated with blue visible laser at 2·04 W/cm2 . The antibacterial activity of functionalized Ag-NPs was investigated by fluorescence microscopy after irradiation, and morphological changes in S. aureus after laser treatment were assessed using scanning electron microscopy (SEM). The laser-irradiated, functionalized Ag-NPs exhibited significant bactericidal activity, and laser-induced bacterial damage was observed after 10 min of laser irradiation against S. aureus. The fluorescence microscopic analysis results supported that bacterial cell death occurred in the presence of the functionalized Ag-NPs.
CONCLUSIONS: The results of this study suggest that a novel method for the preparation of functionalized nanoparticles has potential as a potent antibacterial agent for the selective killing of resistant disease-causing bacteria.
SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that Ag-NPs functionalized with a specific antibody, could be used in combination with laser radiation as a novel treatment to target resistant bacterial and fungal pathogens with minimal impact on normal microflora.
Fulltext Evaluation of BOX-PCR and ERIC-PCR as Molecular Typing Tools for Pathogenic Leptospira

Bilung LM, Pui CF, Su'ut L, Apun K

Dis Markers, 2018;2018:1351634.
PMID: 30154937 DOI: 10.1155/2018/1351634

In the last decades, leptospirosis had gained public health concern due to morbidity and mortality rates caused by pathogenic Leptospira. The need for rapid and robust molecular typing methods to differentiate this zoonotic pathogen is of utmost importance. Various studies had been conducted to determine the genetic relatedness of Leptospira isolates using molecular typing methods. In this study, 29 pathogenic Leptospira isolates from rat, soil, and water samples in Sarawak, Malaysia, were characterized using BOX-PCR and ERIC-PCR. The effectiveness of these two methods with regard to the ease of interpretation, reproducibility, typeability, and discriminatory power was also being evaluated. Using BOX-PCR, six clusters and 3 single isolates were defined at a genetic distance percentage of 11.2%. ERIC-PCR clustered the isolates into 6 clusters and 2 single isolates at a genetic distance percentage of 6.8%. Both BOX-PCR and ERIC-PCR produced comparable results though the discriminatory index for ERIC-PCR (0.826) was higher than that for BOX-PCR (0.809). From the constructed dendrogram, it could be summarized that the isolates in this study were highly heterogeneous and genetically diverse. The findings from this study indicated that there is no genetic relatedness among the pathogenic Leptospira isolates in relation to the locality, source, and identity, with some exceptions. Out of the 29 pathogenic Leptospira isolates studied, BOX-PCR and ERIC-PCR successfully discriminated 4 isolates (2 isolates each) into the same cluster in relation to sample sources, as well as 2 isolates into the same cluster in association with the sample locality. Future studies shall incorporate the use of other molecular typing methods to make a more thorough comparison on the genetic relatedness of pathogenic Leptospira.
Fulltext Diversity of Leptospira spp. in Rats and Environment from Urban Areas of Sarawak, Malaysia

Pui CF, Bilung LM, Apun K, Su'ut L

J Trop Med, 2017;2017:3760674.
PMID: 28348601 DOI: 10.1155/2017/3760674

Various prevalence studies on Leptospira in animals and humans, as well as environmental samples, had been conducted worldwide, including Malaysia. However, limited studies have been documented on the presence of pathogenic, intermediate, and saprophytic Leptospira in selected animals and environments. This study was therefore conducted to detect Leptospira spp. in rats, soil, and water from urban areas of Sarawak using the polymerase chain reaction (PCR) method. A total of 107 rats, 292 soil samples, and 324 water samples were collected from April 2014 to February 2015. Pathogenic Leptospira was present in 5.6% (6/107) of rats, 11.6% (34/292) of soil samples, and 1.9% (6/324) of water samples. Intermediate Leptospira was present in 2.7% (8/292) of soil samples and 1.9% (6/324) of water samples. Saprophytic Leptospira was present in 10.3% (11/107) of rats, 1.4% (4/292) of soil samples, and 0.3% (1/324) of water samples. From this study, 76 Leptospira spp. were isolated. Based on DNA sequencing, the dominant Leptospira spp. circulating in urban areas of Sarawak are pathogenic Leptospira noguchii, intermediate Leptospira wolffii serovar Khorat, and saprophytic Leptospira meyeri, respectively. Overall, this study provided important surveillance data on the prevalence of Leptospira spp. from rats and the environment, with dominant local serovars in urban areas of Sarawak.
Molecular characterization of Vibrio cholerae O1 outbreak strains in Miri, Sarawak (Malaysia)

Radu S, Vincent M, Apun K, Abdul-Rahim R, Benjamin PG, Yuherman, et al.

Acta Trop, 2002 Aug;83(2):169-76.
PMID: 12088858

Bacterial resistance to various antimicrobial agents is common in area with high usage of antibiotics. In this study, the data on antimicrobial susceptibility patterns of Vibrio cholerae O1 from patients during an outbreak period was found to be high but variable rates of multidrug resistance. Thirty-two of 33 V. cholerae isolates harboured the tcp, ctx, zot and ace genes, suggesting their possible roles in the outbreak cases. We analyzed the molecular diversity of a total of 33 strains of V. cholerae O1 isolated from 33 patients between November 1997 and April 1998 using random amplified polymorphic DNA (RAPD) analysis. The 30 typable isolates could be separated into four major clusters containing 5, 17, 2 and 6 isolates, respectively. However, no particular RAPD pattern was predictive of a particular pattern of antibiotic susceptibility. The findings of this study showed that multiple clones seemed to be responsible for cases in the outbreaks in the study area.
Epidemiological study of human intestinal parasites in Sarawak, East Malaysia: A review

Tahar AS, Bilung LM, Apun K, Richard RL, Lim YAL

Trop Biomed, 2021 Sep 01;38(3):377-386.
PMID: 34508347 DOI: 10.47665/tb.38.3.083

Intestinal parasitic infections are endemic in rural settings and may account for asymptomatic infections to various health complications. These infections are a cause of concern for communities of lower economic status, especially in developing countries. In Sarawak, indigenous populations residing in geographically inaccessible areas are socially and economically disadvantaged. Through close association with nature, these populations are prone to intestinal parasitism. Currently, scattered information has led to a continual state of neglect at each level of parasitic infection control. This urges for a review of their distribution and transmission based on previous reports to understand the pattern of the diseases in the state which can further address the improvement of mass controlling programs. A literature search was conducted to collect previous reports on human intestinal parasites in Sarawak, East Malaysia from PubMed (Medline), SCOPUS, ScienceDirect and Web of Science from January 2019 to March 2021. Extrapolating the current data in Sarawak which is still considered limited, further interdisciplinary strategies are demanded to give insights in the epidemiology and true prevalence of intestinal parasites in Sarawak. This review addresses for redirection of attitude towards intestinal parasitic infections where it should be given with ample attention by rural populations. In tandem to that, improvement of rural livelihood such as standard of living and sanitation in Sarawak should be accredited as part of the efforts to reduce the number of intestinal parasitic infections in the state. As a control measure, mass deworming should be reconsidered especially to the rural populations.
Fulltext Prevalence, Genetic Heterogeneity, and Antibiotic Resistance Profile of Listeria spp. and Listeria monocytogenes at Farm Level: A Highlight of ERIC- and BOX-PCR to Reveal Genetic Diversity

Maurice Bilung L, Sin Chai L, Tahar AS, Ted CK, Apun K

Biomed Res Int, 2018;2018:3067494.
PMID: 30065935 DOI: 10.1155/2018/3067494

This study aimed to identify Listeria spp. and L. monocytogenes, characterize the isolates, and determine the antibiotic resistance profiles of the isolates Listeria spp. and L. monocytogenes in fresh produce, fertilizer, and environmental samples from vegetable farms (organic and conventional farms). A total of 386 samples (vegetables, soil, water, and fertilizer with manure) were examined. The identification of bacterial isolates was performed using PCR and characterized using ERIC-PCR and BOX-PCR. The discriminating power of the typing method was analyzed using Simpson's Index of Diversity. Thirty-four (n=34) Listeria isolates were subjected to antimicrobial susceptibility test using the disc-diffusion technique. The PCR analysis revealed that Listeria spp. were present in 7.51% (29/386) of all the samples (vegetable, soil, fertilizer, and water). None of the samples examined were positive for the presence of L. monocytogenes. Percentages of 100% (15/15) and 73.30% (11/15) of the Listeria spp. isolated from vegetables, fertilizer, and soil from organic farm B had indistinguishable DNA fingerprints by using ERIC-PCR and BOX-PCR, respectively. Listeria spp. isolated from 86 samples of vegetable, fertilizer, and environment of organic farm A and conventional farm C had distinct DNA fingerprints. Simpson's Index of Diversity, D, of ERIC-PCR and BOX-PCR is 0.604 and 0.888, respectively. Antibiotic susceptibility test revealed that most of the Listeria spp. in this study were found to be resistant to ampicillin, rifampin, penicillin G, tetracycline, clindamycin, cephalothin, and ceftriaxone. The isolates had MAR index ranging between 0.31 and 0.85. In conclusion, hygienic measures at farm level are crucial to the reduction of Listeria transmission along the food chain.
Fulltext High Occurrence of Staphylococcus aureus Isolated from Fitness Equipment from Selected Gymnasiums

Maurice Bilung L, Tahar AS, Kira R, Mohd Rozali AA, Apun K

J Environ Public Health, 2018;2018:4592830.
PMID: 30245728 DOI: 10.1155/2018/4592830

Introduction: Staphylococcus aureus is a leading cause of cutaneous bacterial infection involving community.
Methods: In this study, a total of 42 swab samples were collected from the surface of various fitness equipment such as back machines, exercise mats, dip stations, dumbbells, and treadmills. Identification of the bacterial isolates was conducted using biochemical tests and further analysed molecularly using the PCR method targeting nuc gene (270 bp). The nuc gene encodes for the thermonuclease enzyme, a virulent factor of S. aureus.
Results: The findings showed 31 out of 42 swab samples (73.81%) were positive with S. aureus.
Conclusion: This study showed that gymnasium equipment is a potential reservoir for S. aureus and might play an important role in transmitting the pathogen to humans.
Objective: This study was undertaken to assess the presence of S. aureus on the surface of fitness equipment from selected gymnasiums in Kuching and Kota Samarahan, Sarawak (Malaysia).
Fulltext Detection of Cryptosporidium and Cyclospora Oocysts from Environmental Water for Drinking and Recreational Activities in Sarawak, Malaysia

Bilung LM, Tahar AS, Yunos NE, Apun K, Lim YA, Nillian E, et al.

Biomed Res Int, 2017;2017:4636420.
PMID: 29234679 DOI: 10.1155/2017/4636420

Cryptosporidiosis and cyclosporiasis are caused by waterborne coccidian protozoan parasites of the genera Cryptosporidium and Cyclospora, respectively. This study was conducted to detect Cryptosporidium and Cyclospora oocysts from environmental water abstracted by drinking water treatment plants and recreational activities in Sarawak, Malaysia. Water samples (12 each) were collected from Sungai Sarawak Kanan in Bau and Sungai Sarawak Kiri in Batu Kitang, respectively. In addition, 6 water samples each were collected from Ranchan Recreational Park and UNIMAS Lake at Universiti Malaysia Sarawak, Kota Samarahan, respectively. Water physicochemical parameters were also recorded. All samples were concentrated by the iron sulfate flocculation method followed by the sucrose floatation technique. Cryptosporidium and Cyclospora were detected by modified Ziehl-Neelsen technique. Correlation of the parasites distribution with water physicochemical parameters was analysed using bivariate Pearson correlation. Based on the 24 total samples of environmental water abstracted by drinking water treatment plants, all the samples (24/24; 100%) were positive with Cryptosporidium, and only 2 samples (2/24; 8.33%) were positive with Cyclospora. Based on the 12 total samples of water for recreational activities, 4 samples (4/12; 33%) were positive with Cryptosporidium, while 2 samples (2/12; 17%) were positive with Cyclospora. Cryptosporidium oocysts were negatively correlated with dissolved oxygen (DO).

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