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Fulltext Methicillin-Resistant Staphylococcus aureus Biofilms and Their Influence on Bacterial Adhesion and Cohesion

Dakheel KH, Abdul Rahim R, Neela VK, Al-Obaidi JR, Hun TG, Yusoff K

Biomed Res Int, 2016;2016:4708425.
PMID: 28078291 DOI: 10.1155/2016/4708425

Twenty-five methicillin-resistant Staphylococcus aureus (MRSA) isolates were characterized by staphylococcal protein A gene typing and the ability to form biofilms. The presence of exopolysaccharides, proteins, and extracellular DNA and RNA in biofilms was assessed by a dispersal assay. In addition, cell adhesion to surfaces and cell cohesion were evaluated using the packed-bead method and mechanical disruption, respectively. The predominant genotype was spa type t127 (22 out of 25 isolates); the majority of isolates were categorized as moderate biofilm producers. Twelve isolates displayed PIA-independent biofilm formation, while the remaining 13 isolates were PIA-dependent. Both groups showed strong dispersal in response to RNase and DNase digestion followed by proteinase K treatment. PIA-dependent biofilms showed variable dispersal after sodium metaperiodate treatment, whereas PIA-independent biofilms showed enhanced biofilm formation. There was no correlation between the extent of biofilm formation or biofilm components and the adhesion or cohesion abilities of the bacteria, but the efficiency of adherence to glass beads increased after biofilm depletion. In conclusion, nucleic acids and proteins formed the main components of the MRSA clone t127 biofilm matrix, and there seems to be an association between adhesion and cohesion in the biofilms tested.
Fulltext Genomic analyses of two novel biofilm-degrading methicillin-resistant Staphylococcus aureus phages

Dakheel KH, Rahim RA, Neela VK, Al-Obaidi JR, Hun TG, Isa MNM, et al.

BMC Microbiol, 2019 05 28;19(1):114.
PMID: 31138130 DOI: 10.1186/s12866-019-1484-9

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) biofilm producers represent an important etiological agent of many chronic human infections. Antibiotics and host immune responses are largely ineffective against bacteria within biofilms. Alternative actions and novel antimicrobials should be considered. In this context, the use of phages to destroy MRSA biofilms presents an innovative alternative mechanism.
RESULTS: Twenty-five MRSA biofilm producers were used as substrates to isolate MRSA-specific phages. Despite the difficulties in obtaining an isolate of this phage, two phages (UPMK_1 and UPMK_2) were isolated. Both phages varied in their ability to produce halos around their plaques, host infectivity, one-step growth curves, and electron microscopy features. Furthermore, both phages demonstrated antagonistic infectivity on planktonic cultures. This was validated in an in vitro static biofilm assay (in microtiter-plates), followed by the visualization of the biofilm architecture in situ via confocal laser scanning microscopy before and after phage infection, and further supported by phages genome analysis. The UPMK_1 genome comprised 152,788 bp coding for 155 putative open reading frames (ORFs), and its genome characteristics were between the Myoviridae and Siphoviridae family, though the morphological features confined it more to the Siphoviridae family. The UPMK_2 has 40,955 bp with 62 putative ORFs; morphologically, it presented the features of the Podoviridae though its genome did not show similarity with any of the S. aureus in the Podoviridae family. Both phages possess lytic enzymes that were associated with a high ability to degrade biofilms as shown in the microtiter plate and CLSM analyses.
CONCLUSIONS: The present work addressed the possibility of using phages as potential biocontrol agents for biofilm-producing MRSA.
Proteomic analysis revealed the biofilm-degradation abilities of the bacteriophage UPMK_1 and UPMK_2 against Methicillin-resistant Staphylococcus aureus

Dakheel KH, Abdul Rahim R, Al-Obaidi JR, Neela VK, Hun TG, Mat Isa MN, et al.

Biotechnol Lett, 2022 Feb 05.
PMID: 35122191 DOI: 10.1007/s10529-022-03229-y

OBJECTIVE: The degradation activity of two bacteriophages UPMK_1 and UPMK_2 against methicillin-resistant Staphylococcus aureus phages were examined using gel zymography.
METHODS: The analysis was done using BLASTP to detect peptides catalytic domains. Many peptides that are related to several phage proteins were revealed.
RESULTS: UPMK_1 and UPMK_2 custom sequence database were used for peptide identification. The biofilm-degrading proteins in the bacteriophage UPMK_2 revealed the same lytic activity towards polysaccharide intercellular adhesin-dependent and independent of Methicillin-resistant Staphylococcus aureus (MRSA) biofilm producers in comparison to UPMK_1, which had lytic activity restricted solely to its host.
CONCLUSION: Both bacteriophage enzymes were involved in MRSA biofilm degradation during phage infection and they have promising enzybiotics properties against MRSA biofilm formation.
Survival of beneficial microbes in liquid bioformulation and optimization of different carrier materials using RSM technique

Chompa SS, Zuan ATK, Amin AM, Hun TG, Ghazali AHA, Sadeq BM, et al.

Int Microbiol, 2023 Aug 31.
PMID: 37651053 DOI: 10.1007/s10123-023-00423-4

Soil salinity in rice cultivation areas is considered a severely limiting factor that adversely affects the quantity and quality of rice production in wetlands. Recently, the alternative use of salt-tolerant plant growth-promoting rhizobacteria (PGPR) inhabiting extreme saline conditions has gained remarkable attention and had positive effects on soil and crops. Therefore, a study has been initiated to develop a liquid biofertilizer formulation from locally isolated multi-strain salt-tolerant PGPR strains such as Bacillus tequilensis and Bacillus aryabhattai, using glycerol (5 mM), trehalose (10 mM), and polyvinylpyrrolidone (PVP) at 1% as additives to prolong the shelf-life of the bacteria. After 3 months of incubation, the bacterial population in the trehalose-supplemented mixed strain was highest at 9.73×107 CFU/mL, followed by UPMRE6 and UPMRB9 at 9.40×107 CFU/mL and 8.50×107 CFU/mL respectively. The results showed that the optimal trehalose concentration successfully prolonged the shelf-life of bacteria with minimal cell loss. Validation of quadratic optimization by response surface methodology revealed that the cell density of the mixed strain was 4.278×107 log CFU/mL after 24 h. The precision ratio was 99.7% higher than the predicted value in the minimized medium formulation: 0.267 g/mL trehalose, 1% glycerol, at 120 rpm agitation using the data analysis tools of Design Expert software. The population study confirmed the better and longer survival of salt-tolerant PGPR fortified with 10 mM trehalose, which was considered the best liquid biofertilizer formulation. Moreover, the optimized trehalose-glycerol liquid formulation can be used commercially as it is cost-effective.
Growth and protein response of rice plant with plant growth-promoting rhizobacteria inoculations under salt stress conditions

Chompa SS, Zuan ATK, Amin AM, Hun TG, Ghazali AHA, Sadeq BM, et al.

Int Microbiol, 2024 Jan 03.
PMID: 38172302 DOI: 10.1007/s10123-023-00469-4

Soil salinity has been one of the significant barriers to improving rice production and quality. According to reports, Bacillus spp. can be utilized to boost plant development in saline soil, although the molecular mechanisms behind the interaction of microbes towards salt stress are not fully known. Variations in rice plant protein expression in response to salt stress and plant growth-promoting rhizobacteria (PGPR) inoculations were investigated using a proteomic method and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Findings revealed that 54 salt-responsive proteins were identified by mass spectrometry analysis (LC-MS/MS) with the Bacillus spp. interaction, and the proteins were functionally classified as gene ontology. The initial study showed that all proteins were labeled by mass spectrometry analysis (LC-MS/MS) with Bacillus spp. interaction; the proteins were functionally classified into six groups. Approximately 18 identified proteins (up-regulated, 13; down-regulated, 5) were involved in the photosynthetic process. An increase in the expression of eight up-regulated and two down-regulated proteins in protein synthesis known as chaperones, such as the 60 kDa chaperonin, the 70 kDa heat shock protein BIP, and calreticulin, was involved in rice plant stress tolerance. Several proteins involved in protein metabolism and signaling pathways also experienced significant changes in their expression. The results revealed that phytohormones regulated the manifestation of various chaperones and protein abundance and that protein synthesis played a significant role in regulating salt stress. This study also described how chaperones regulate rice salt stress, their different subcellular localizations, and the activity of chaperones.
Fulltext First Report of Fusarium wilt disease on Watermelon Caused by Fusarium oxysporum f. sp. niveum (FON) in Malaysia

Rahman MZ, Ahmad K, Siddiqui Y, Saad N, Hun TG, Mohd Hata E, et al.

Plant Dis, 2021 May 27.
PMID: 34042494 DOI: 10.1094/PDIS-04-21-0780-PDN

Fusarium wilt disease incited by Fusarium oxysporum f. sp. niveum (FON) is the utmost devastating soil-inhabiting fungal pathogen limiting watermelon (Citrullus lanatus) production in Malaysia and globally. The field disease survey of fusarium wilt was carried out during December 2019 and November 2020, in three major production areas (3 farmer fields per location) in Peninsular Malaysia namely, Mersing, Serdang and Kuantan and disease incidence of 30 and 45%, was recorded for each year, respectively. Infected watermelon plants showed symptoms such as vascular discoloration, brown necrotic lesions to the soil line or the crown, one-sided wilt of a plant, or a runner or the whole plant. Infected root and stem tissues, 1-2 cm pieces were surface sterilized with 0.6% NaOCl for 1 minute followed by double washing with sterile water. The disinfected tissues were air-dried and transferred onto semi-selective Komada's medium (Komada 1975) and incubated for 5 days. The fungal colonies produced were placed on potato dextrose agar (PDA) to attain a pure culture and incubated at 25±2℃ for 15 days. The pure fungal colony was flat, round and light purple in color. Macroconidia were straight to slightly curved, 18.56-42.22 µm in length, 2.69-4.08 µm width, predominantly 3 septate and formed in sporodochia. Microconidia measured 6.16-10.86 µm in length and 2.49-3.83 µm in width, kidney-shaped, aseptate and were formed on short monophialides in false-heads. Chlamydospores were single or in pairs with smooth or rough walls, found both terminally or intercalary. To confirm their pathogenicity, two-week-old watermelon seedlings (cv. NEW BEAUTY) were dipped into spore suspension (1 ˟ 106 spores/ml) of representative isolates of JO20 (Mersing), UPM4 (Serdang) and KU41 (Kuantan) for 30 second and then moved into 10 cm diameter plastic pots containing 300 g sterilized soil mix. Disease symptoms were assessed weekly for one month. Control seedlings were immersed in sterile distilled water before transplanting. The inoculated seedlings showed typical Fusarium wilt symptoms like yellowing, stunted growth, and wilting, which is similar to the farmer field infected plants. However, the seedlings inoculated by sterile distilled water remained asymptomatic. The pathogen was successfully re-isolated from the infected seedlings onto Komada's medium, fulfilling the Koch's postulate. For the PCR amplification, primers EF-1 and EF-2 were used to amplify the tef1-α region. A Blastn analysis of the tef1-α sequences of the isolates JO20 (accession nos. MW315902), UPM4 (MW839560) and KU41 (MW839562) showed 100% similarity; with e-value of zero, to the reference sequences of F. oxysporum isolate FJAT-31690 (MN507110) and F. oxysporum f. sp. niveum isolate FON2 790-2 (MN057702). In Fusarium MLST database, isolates JO20, UPM4 and KU41 revealed 100% identity with the reference isolate of NRRL 22518 (accession no. FJ985265). Though isolate FJ985265 belongs to the f. sp. melonis, earlier findings had revealed Fusarium oxysporum f. sp. are naturally polyphyletic and making clusters with diverse groups of the Fusarium oxysporum species complex (O'Donnell et al. 2015). The isolates JO20, UPM4 and KU41 were identified as F. oxysporum f. sp. niveum based on the aligned sequences of tef1-α and molecular phylogenetic exploration by the maximum likelihood method. To the best of our knowledge, this is the first report of F. oxysporum f. sp. niveum as a causative pathogen of Fusarium wilt disease of watermelon in Malaysia. Malaysia enables to export watermelon all-year-round in different countries like Singapore, Hong-Kong, The United Arab Emirates (UAE), and Netherlands. The outburst of this destructive soil-borne fungal pathogen could cause hindrance to watermelon cultivation in Malaysia. Thus, growers need to choice multiple management tactics such as resistant varieties, cultural practices (soil amendments and solarization), grafting, cover crops and fungicide application to control this new pathogen.
Fulltext First Report of Fusarium equiseti, Causing Fruit Rot Disease of Watermelon in Malaysia

Rahman MZ, Ahmad K, Siddiqui Y, Saad N, Hun TG, Mohd Hata E, et al.

Plant Dis, 2021 Aug 02.
PMID: 34340562 DOI: 10.1094/PDIS-05-21-1027-PDN

Watermelon (Citrullus lanatus) accounts for almost 13% of all tropical fresh fruit production in Malaysia. They are grown, mostly in Johor, Kedah, Kelantan, Pahang, and Terengganu areas of Malaysia on 10,406 ha and yielding 172,722 Mt. In 2019, a new fruit rot disease was observed in two major production areas in Peninsular Malaysia. Disease symptoms included water-soaked brown lesions on the fruit surface in contact with the soil. The lesions enlarged gradually and ultimately covered the whole fruit with white mycelium leading to internal fruit decay. Disease surveys were conducted in December 2019 and November 2020 in fields at Kuantan, Pahang and Serdang, Selangor. Disease incidence was 10% in 2019 and 15% in 2020. Infected fruits were collected and washed under running tap water to wash off adhering soil and debris. Fruit tissue sections 1 to 2 cm in length were surface sanitized with 0.6% sodium hypochlorite (NaOCl) for 3 min. and washed twice with sterile distilled water. The disinfected air-dried tissues were then transferred onto potato dextrose agar (PDA) media and incubated at 25±2℃ for 3 days. Fungal colonies with whitish mycelium and pink pigment isolated using single spore culture. The pure cultures were placed onto carnation leaf agar (CLA), and the culture plates were incubated at 25±2℃ for 15 days for morphological characterization. On CLA, macroconidia were produced from monophialides on branched conidiophores in orange sporodochia. Macroconindia were thick-walled, strong dorsiventral curvature, 5 to 7 septate with a tapered whip-liked pointed apical cell and characteristic foot-shaped basal cell, 21.9 to 50.98 μm long and 2.3 to 3.60 μm wide. Typical verrucose thick chlamydospores with rough walls were profuse in chains or clumps, sub-globose or ellipsoidal. Based on morphological characteristics they were identified as Fusarium equiseti (Leslie and Summerell 2006). Molecular identification of both U4-1 and N9-1 pure culture isolates were carried out using two primer pair sets; internal transcribed spacer (ITS) ITS-1/ ITS-4 and translation elongation factor 1 alpha (TEF1-α) (EF-1/EF-2). A Blastn analysis of the ITS gene sequence of U4-1(MW362286) and N9-1 (MW362287) showed >99% similarity index to the reference gene sequence of F. equiseti isolate 19MSr-B3-4 (LC514690). The TEF1-α sequences of U4-1 (accession no. MW839563) and N9-1 (accession no. MW839564) showed 100% identity; with an e-value of zero, to the reference gene sequence of F. equiseti isolate URM: 7561 (accession no. LS398490). Each isolate also had a >99% identity with isolate NRRL 34070 (accession no. GQ505642) in Fusarium MLST database that belongs to the F. incarnatum-equiseti species complex (O'Donnell et al. 2015). Based on phylogenetic analysis of the aligned sequences (TEF1-α) by the maximum likelihood method, the U4-1 and N9-1 isolates were confirmed to be F. equiseti as was reported in Georgia, USA (Li and Ji 2015) and in Harbin, Heilongjiang Province, China (Li et al. 2018). Finally, the two pure culture isolates of U4-1 and N9-1 were used to fulfill Koch's postulates. Stab inoculations of five healthy watermelon fruits (cv. 345-F1 hybrid seedless round watermelon) were performed with a microconidial suspension of individual isolates (4x106 spores/mL). Five control fruits were stabbed with double distilled water. The inoculated fruits were incubated under 95% relative humidity at a temperature of 25±2℃ for 48 h followed by additional incubation inside an incubator at 25±2℃ for 8 days. Ten days post-inoculation, the control fruits showed no disease symptoms. However, inoculated fruits exhibited typical symptoms of fruit rot disease like water-soaked brown lesions, white mycelium on the fruit surface and internal fruit decay, which is similar to the farmer's field infected fruits. The suspected pathogen was successfully re-isolated from the symptomatic portion of inoculated fruit and morphologically identified for verification. To our knowledge, this is the first report of F. equiseti causing fruit rot of watermelon in Malaysia. Malaysia exports watermelon year-round to many countries around the world. The outbreak of this new fruit rot disease could potentially pose a concern to watermelon cultivation in Malaysia.