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Application of multilocus sequence analysis for molecular characterization of enterococci with virulence factors recovered from a tropical recreational beach

Ahmad A, Dada AC, Usup G

Southeast Asian J. Trop. Med. Public Health, 2014 May;45(3):700-18.
PMID: 24974655

Partial gene sequences of phenylalanyl-tRNA synthase alpha subunit (pheS) and RNA polymerase alpha subunit (rpoA) were evaluated for species delineation and detection of recombination among enterococci populations recovered from a bathing beach impacted by low tide river flow. At inter-species level, a maximum similarity of 86.5% and 94.8% was observed among the enterococci pheS and rpoA sequence, respectively. A superimposed plot of delimited pairwise similarity values obtained for 266 pair-wise observations revealed that while there was a harmony between species identity obtained from both genes, pheS was more discriminatory than rpoA. The difference was more pronounced for inter-species comparison. A number of putative recombination events between indigenous and non-indigenous strains was detected based on a library of aligned sequences. Virulence genes cyl, esp, gelE and asa were detected in 7, 22, 100 and 63%, respectively among river isolates but at lower proportion of 0, 20, 67 and 42%, respectively among beach water isolates. Random amplified polymorphic DNA profiling presented evidence suggesting low tide river as a source of fecal enterococci entering the recreation beach water. Multilocus sequence typing analysis of a number of Enterococcus faecalis isolates presented four sequence types, ST59, 117, 181 and 474. The presence of genetically diverse fecal enterococci with associated virulence traits and a background of recombination events in surface recreational water could present a potential public health risk.
Occurrence of Enterococcus species with virulence markers in an urban flow-influenced tropical recreational beach

Ahmad A, Dada AC, Usup G, Heng LY

Mar Pollut Bull, 2014 May 15;82(1-2):26-38.
PMID: 24725825 DOI: 10.1016/j.marpolbul.2014.03.028

Median enterococci counts of beach water samples gradually increased at statistically significant levels (χ2: 26.53, df: 4; p<0.0001) with increasing proximity to river influx. The difference in proportion of antibiotic resistant enterococci in beach water and river water samples was statistically significant (p<0.05) for the tested antibiotics with river isolates generally presenting higher resistance frequencies. Virulence genes cyl, esp, gelE and asa were detected at varying frequencies (7.32%, 21.95%, 100% and 63.41% respectively) among river isolates. On the other hand, the prevalence of these genes was lower (0%, 20%, 67.27% and 41.82% respectively) among beach water isolates. Multi-Locus-Sequence-Typing analysis of Enterococcus faecalis presented four sequence types (ST) one of which shared six out of seven tested loci with ST6, a member of the clonal complex of multi-drug resistant strains associated with hospital outbreaks.
Pseudomonas putida Strain FStm2 Isolated from Shark Skin: A Potential Source of Bacteriocin

Ahmad A, Hamid R, Dada AC, Usup G

Probiotics Antimicrob Proteins, 2013 Sep;5(3):165-75.
PMID: 26782985 DOI: 10.1007/s12602-013-9140-4

Bacteriocin-producing Pseudomonas putida strain FStm2 isolated from shark showed broad range of antibacterial activity against all pathogens tested except Bacillus subtilis ATCC11774, MRSA N32064, Proteus mirabilis ATCC12453, Enterococcus faecalis ATCC14506, Salmonella typhimurium ATCC51312, Salmonella mutan ATCC25175, and Aeromonas hydrophila Wbf314. Of the three growth media tested in this study, TSB was observed to support the bacteriocin activity the most. While the highest bacteriocin activity was observed for media supplemented with 1 % NaCl, there was an observed reduction in bacteriocin activity with increasing salt concentration. Although the least bacteriocin activity was observed for marine broth, addition of increasing amounts of tryptone, glucose, or yeast extract increased bacteriocin activity. This was, however, contrary to the effect observed when MgSO4 and MnSO4 were added as supplements. In the presence of α-amylase, lipase, DNase, and RNase, a positive effect on bacteriocin production was observed. Proteinase K strongly inhibited bacteriocin production. Furthermore, the bacteriocins produced were heat stable within the temperature range of 30-70 °C. Bacteriocin activity also was not affected within a wide pH range of 3-9. Exposure to detergents did not inhibit the activity of the bacteriocin at the concentrations tested. Instead, a positive effect on the relative activity of produced bacteriocin was observed as sodium dodecyl sulfate (SDS), EDTA, and Tween 20 at 1 % concentration all improved bacteriocin activity when the cell-free supernatant was tested against Serratia marcescens ATCC 13880. The bacteriocin was purified by ammonium sulfate precipitation and gel filtration on a Superdex-200 column. SDS-PAGE analysis of the partially purified bacteriocin revealed an apparent molecular weight of ~32 kDa.
Fulltext Validation of the Enterococci indicator for bacteriological quality monitoring of beaches in Malaysia using a multivariate approach

Ahmad A, Dada AC, Usup G, Heng LY

Springerplus, 2013;2:425.
PMID: 24052928 DOI: 10.1186/2193-1801-2-425

There is currently no established bacteriological beach quality monitoring (BQM) program in place in Malaysia. To initiate cost-effective, sustainable bacteriological BQM schemes for the ultimate goal of protecting public health, policy decision makers need to be provided robust, indigenous empirical findings that validate appropriate water quality parameters for inclusion in such monitoring programs. This is the first study that assesses the validity of enterococci as an ideal indicator for bacteriological BQM in Malaysia using a multivariate approach. Beach water and sand samples from 7 beach locations were analyzed for a total of twenty-one microbial and non-microbial water quality parameters. A multivariate approach incorporating cluster analyses (CA), principal component analyses (PCA), and factor analysis (FA) was also adopted. Apart from the weak correlations of Staphylococcus aureus with concentrations of Vibro species (r = 0.302, p = 0.037) and total coliforms (r = 0.392, p = 0.006) in seawater, no correlation existed between S. aureus concentration and other parameters. Faecal coliforms failed to correlate with any of the tested parameters. Enterococci also correlated with more quality parameters than faecal coliforms or any other indicator. Multiple linear regressions highlighted a significant, best fit model that could predict enterococci concentrations in relation to other parameters with a maximum predictive success of 69.64%. PCA/FA clearly delineated enterococci and faecal coliforms as parameters that weighed strongly for BQM while Staphylococcus aureus, faecal coliforms and enterococci weighed strongly for beach sand quality monitoring. On the whole, higher correlations of enterococci levels with other parameters than was observed for faecal coliforms suggest that the former be considered a preferred parameter of choice for BQM in Malaysia. Our findings provide meaningful evidence particularly as it relates to the correlation of Enterococci with pathogens and other non-microbial parameters. It also provides empirical data to validate the applicability of the enterococci indicator paradigm for bacteriological beach quality monitoring in Malaysia. The current study thus provides policy decision makers evidenced based approach to parameter streamlining for optimized beach sampling and sustainable bacteriological quality monitoring.
Fulltext Insights into Alexandrium minutum Nutrient Acquisition, Metabolism and Saxitoxin Biosynthesis through Comprehensive Transcriptome Survey

Akbar MA, Yusof NYM, Sahrani FK, Usup G, Ahmad A, Baharum SN, et al.

Biology (Basel), 2021 Aug 25;10(9).
PMID: 34571703 DOI: 10.3390/biology10090826

The toxin-producing dinoflagellate Alexandrium minutum is responsible for the outbreaks of harmful algae bloom (HABs). It is a widely distributed species and is responsible for producing paralytic shellfish poisoning toxins. However, the information associated with the environmental adaptation pathway and toxin biosynthesis in this species is still lacking. Therefore, this study focuses on the functional characterization of A. minutum unigenes obtained from transcriptome sequencing using the Illumina Hiseq 4000 sequencing platform. A total of 58,802 (47.05%) unigenes were successfully annotated using public databases such as NCBI-Nr, UniprotKB, EggNOG, KEGG, InterPRO and Gene Ontology (GO). This study has successfully identified key features that enable A. minutum to adapt to the marine environment, including several carbon metabolic pathways, assimilation of various sources of nitrogen and phosphorus. A. minutum was found to encode homologues for several proteins involved in saxitoxin biosynthesis, including the first three proteins in the pathway of saxitoxin biosynthesis, namely sxtA, sxtG and sxtB. The comprehensive transcriptome analysis presented in this study represents a valuable resource for understanding the dinoflagellates molecular metabolic model regarding nutrient acquisition and biosynthesis of saxitoxin.
Fulltext Biosynthesis of Saxitoxin in Marine Dinoflagellates: An Omics Perspective

Akbar MA, Mohd Yusof NY, Tahir NI, Ahmad A, Usup G, Sahrani FK, et al.

Mar Drugs, 2020 Feb 05;18(2).
PMID: 32033403 DOI: 10.3390/md18020103

Saxitoxin is an alkaloid neurotoxin originally isolated from the clam Saxidomus giganteus in 1957. This group of neurotoxins is produced by several species of freshwater cyanobacteria and marine dinoflagellates. The saxitoxin biosynthesis pathway was described for the first time in the 1980s and, since then, it was studied in more than seven cyanobacterial genera, comprising 26 genes that form a cluster ranging from 25.7 kb to 35 kb in sequence length. Due to the complexity of the genomic landscape, saxitoxin biosynthesis in dinoflagellates remains unknown. In order to reveal and understand the dynamics of the activity in such impressive unicellular organisms with a complex genome, a strategy that can carefully engage them in a systems view is necessary. Advances in omics technology (the collective tools of biological sciences) facilitated high-throughput studies of the genome, transcriptome, proteome, and metabolome of dinoflagellates. The omics approach was utilized to address saxitoxin-producing dinoflagellates in response to environmental stresses to improve understanding of dinoflagellates gene-environment interactions. Therefore, in this review, the progress in understanding dinoflagellate saxitoxin biosynthesis using an omics approach is emphasized. Further potential applications of metabolomics and genomics to unravel novel insights into saxitoxin biosynthesis in dinoflagellates are also reviewed.
Fulltext Loktanella spp. Gb03 as an algicidal bacterium, isolated from the culture of Dinoflagellate Gambierdiscus belizeanus

Bloh AH, Usup G, Ahmad A

Vet World, 2016 Feb;9(2):142-6.
PMID: 27051199 DOI: 10.14202/vetworld.2016.142-146

AIM: Bacteria associated with harmful algal blooms can play a crucial role in regulating algal blooms in the environment. This study aimed at isolating and identifying algicidal bacteria in Dinoflagellate culture and to determine the optimum growth requirement of the algicidal bacteria, Loktanella sp. Gb-03.
MATERIALS AND METHODS: The Dinoflagellate culture used in this study was supplied by Professor Gires Usup's Laboratory, School of Environmental and Natural Resources Sciences, Faculty of Science and Technology, University Kebangsaan Malaysia, Malaysia. The culture was used for the isolation of Loktanella sp., using biochemical tests, API 20 ONE kits. The fatty acid content of the isolates and the algicidal activity were further evaluated, and the phenotype was determined through the phylogenetic tree.
RESULTS: Gram-negative, non-motile, non-spore-forming, short rod-shaped, aerobic bacteria (Gb01, Gb02, Gb03, Gb04, Gb05, and Gb06) were isolated from the Dinoflagellate culture. The colonies were pink in color, convex with a smooth surface and entire edge. The optimum growth temperature for the Loktanella sp. Gb03 isolate was determined to be 30°C, in 1% of NaCl and pH7. Phylogenetic analysis based on 16S rRNA gene sequences showed that the bacterium belonged to the genus Loktanella of the class Alphaproteobacteria and formed a tight cluster with the type strain of Loktanella pyoseonensis (97.0% sequence similarity).
CONCLUSION: On the basis of phenotypic, phylogenetic data and genetic distinctiveness, strain Gb-03, were placed in the genus Loktanella as the type strain of species. Moreover, it has algicidal activity against seven toxic Dinoflagellate. The algicidal property of the isolated Loktanella is vital, especially where biological control is needed to mitigate algal bloom or targeted Dinoflagellates.
Speciation and antimicrobial resistance of Enterococci isolated from recreational beaches in Malaysia

Dada AC, Ahmad A, Usup G, Heng LY

Environ Monit Assess, 2013 Feb;185(2):1583-99.
PMID: 22592782 DOI: 10.1007/s10661-012-2653-6

We report the first study on the occurrence of antibiotic-resistant enterococci in coastal bathing waters in Malaysia. One hundred and sixty-five enterococci isolates recovered from two popular recreational beaches in Malaysia were speciated and screened for antibiotic resistance to a total of eight antibiotics. Prevalence of Enterococcus faecalis and Enterococcus faecium was highest in both beaches. E. faecalis/E. faecium ratio was 0.384:1 and 0.375:1, respectively, for isolates from Port Dickson (PD) and Bagan Lalang (BL). Analysis of Fisher's exact test showed that association of prevalence of E. faecalis and E. faecium with considered locations was not statistically significant (p < 0.05). Chi-square test revealed significant differences (χ(2) = 82.630, df = 20, p < 0.001) in the frequency of occurrence of enterococci isolates from the considered sites. Resistance was highest to nalidixic acid (94.84 %) and least for chloramphenicol (8.38 %). One-way ANOVA using Tukey-Kramer multiple comparison test showed that resistance to ampicillin was higher in PD beach isolates than BL isolates and the difference was extremely statistically significant (p < 0.0001). Frequency of occurrence of multiple antibiotic resistance (MAR) isolates were higher for PD beach water (64.29 %) as compared to BL beach water (13.51 %), while MAR indices ranged between 0.198 and 0.48. The results suggest that samples from Port Dickson may contain MAR bacteria and that this could be due to high-risk faecal contamination from sewage discharge pipes that drain into the sea water.
High-level aminoglycoside resistance and virulence characteristics among Enterococci isolated from recreational beaches in Malaysia

Dada AC, Ahmad A, Usup G, Heng LY, Hamid R

Environ Monit Assess, 2013 Sep;185(9):7427-43.
PMID: 23417753 DOI: 10.1007/s10661-013-3110-x

We report the first study on the occurrence of high-level aminoglycoside-resistant (HLAR) Enterococci in coastal bathing waters and beach sand in Malaysia. None of the encountered isolates were resistant to high levels of gentamicin (500 μg/mL). However, high-level resistance to kanamycin (2,000 μg/mL) was observed in 14.2 % of tested isolates, the highest proportions observed being among beach sand isolates. High-level resistance to kanamycin was higher among Enterococcus faecalis and Enterococcus faecium than Enterococcus spp. Chi-square analysis showed no significant association between responses to tested antibiotics and the species allocation or source of isolation of all tested Enterococci. The species classification of encountered Enterococci resistance to vancomycin was highest among Enterococcus spp. (5.89 %) followed by E. faecium (4.785) and least among E. faecalis. A total of 160 isolates were also tested for virulence characteristics. On the whole, caseinase production was profoundly highest (15.01 %) while the least prevalent virulence characteristic observed among tested beach Enterococci was haemolysis of rabbit blood (3.65 %). A strong association was observed between the source of isolation and responses for each of caseinase (C = 0.47, V = 0.53) and slime (C = 0.50, V = 0.58) assays. Analysis of obtained spearman's coefficient showed a strong correlation between caseinase and each of the slime production (p = 0.04), gelatinase (p = 0.0035) and haemolytic activity on horse blood (p = 0.004), respectively. Suggestively, these are the main virulent characteristics of the studied beach Enterococci. Our findings suggest that recreational beaches may contribute to the dissemination of Enterococci with HLAR and virulence characteristics.
Fulltext Genome Sequence of Nitratireductor basaltis Strain UMTGB225, a Marine Bacterium Isolated from a Green Barrel Tunicate in Bidong Island, Malaysia

Danish-Daniel M, Gan HY, Gan HM, Saari NA, Usup G

Genome Announc, 2014;2(5).
PMID: 25301654 DOI: 10.1128/genomeA.01015-14

Nitratireductor basaltis strain UMTGB225 is a Gram-negative bacterium isolated from a marine tunicate found in Bidong Island, Terengganu, Malaysia. In this study, the genome of Nitratireductor basaltis UMTGB225 was sequenced to gain insight into the role of this bacterium and its association with tunicate hosts in a coral reef habitat.
Fulltext Draft genome sequence of Mameliella alba strain UMTAT08 isolated from clonal culture of toxic dinoflagellate Alexandrium tamiyavanichii

Danish-Daniel M, Han Ming G, Noor ME, Yeong YS, Usup G

Genom Data, 2016 Dec;10:12-4.
PMID: 27625991 DOI: 10.1016/j.gdata.2016.08.015

Mameliella alba strain UMTAT08 was isolated from clonal culture of paralytic shellfish toxin producing dinoflagellate, Alexandrium tamiyavanichii. Genome of the strain UMTAT08 was sequenced in order to gain insights into the dinoflagellate-bacteria interactions. The draft genome sequence of strain UMTAT08 contains 5.84Mbp with an estimated G + C content of 65%, 5717 open reading frames, 5 rRNAs and 49 tRNAs. It contains genes related to nutrients uptake, quorum sensing and environmental tolerance related genes. Gene clusters for the biosynthesis of type 1 polyketide synthase, bacteriocin, microcin, terpene and ectoine were also identified. This is suggesting that the bacterium possesses diverse adaptation strategy to survive within the dinoflagellate phycosphere. The draft genome sequence and annotation have been deposited at DDBJ/EMBL/GenBank under the accession number JSUQ00000000.
Fulltext Genome Sequence of Bacillus sp. Strain UMTAT18 Isolated from the Dinoflagellate Alexandrium tamiyavanichii Found in the Straits of Malacca

Danish-Daniel M, Ming GH, Mohd Noor ME, Sung YY, Usup G

Genome Announc, 2016 Oct 6;4(5).
PMID: 27795265 DOI: 10.1128/genomeA.01106-16

Bacillus sp. strain UMTAT18 was isolated from the harmful dinoflagellate Alexandrium tamiyavanichii Its genome consists of 5,479,367 bp with 5,546 open reading frames, 102 tRNAs, and 29 rRNAs. Gene clusters for biosynthesis of nonribosomal peptides, bacteriocin, and lantipeptide were identified. It also contains siderophore and genes related to stress tolerance.
Fulltext Genome Sequence of Vibrio campbellii Strain UMTGB204, a Marine Bacterium Isolated from a Green Barrel Tunicate

Gan HY, Noor ME, Saari NA, Musa N, Mustapha B, Usup G, et al.

Genome Announc, 2015;3(2).
PMID: 25814609 DOI: 10.1128/genomeA.00210-15

Vibrio campbellii strain UMTGB204 was isolated from a green barrel tunicate. The genome of this strain comprises 5,652,224 bp with 5,014 open reading frames, 9 rRNAs, and 116 tRNAs. It contains genes related to virulence and environmental tolerance. Gene clusters for the biosynthesis of nonribosomal peptides and bacteriocin were also identified.
Pathogenicity of Aeromonas hydrophila isolated from the Malaysian Sea against coral (Turbinaria sp.) and sea bass (Lates calcarifer)

Hamid R, Ahmad A, Usup G

Environ Sci Pollut Res Int, 2016 Sep;23(17):17269-76.
PMID: 27221587 DOI: 10.1007/s11356-016-6655-8

A study was carried out to determine the pathogenicity (hemolytic activity) on corals (Turbinaria sp.) and sea bass (Lates calcarifer) of Aeromonas hydrophila from water, sediment, and coral. Samples were collected from coastal water and coral reef areas. One hundred and sixty-two isolates were successfully isolated. Out of 162, 95 were from seawater, 49 from sediment, and 18 from coral. Sixteen isolates were picked and identified. Isolates were identified using a conventional biochemical test, the API 20NE kit, and 16S rRNA nucleotide sequences. Hemolytic activity was determined. Out of 16 isolates, 14 isolates were β-hemolytic and two isolates were non-hemolytic. Corals infected with A. hydrophila suffered bleaching. Similar effect was observed for both hemolytic and non-hemolytic isolates. Intramuscular injection of A. hydrophila into sea bass resulted in muscular bleeding and death. Higher infection rates were obtained from hemolytic compared to non-hemolytic strains of A. hydrophila isolates.
Physiological and transcriptional responses to inorganic nutrition in a tropical Pacific strain of Alexandrium minutum: Implications for the saxitoxin genes and toxin production

Hii KS, Lim PT, Kon NF, Takata Y, Usup G, Leaw CP

Harmful Algae, 2016 06;56:9-21.
PMID: 28073499 DOI: 10.1016/j.hal.2016.04.005

Saxitoxins (STXs) constitute a family of potent sodium channel blocking toxins, causative agents of paralytic shellfish poisoning (PSP), and are produced by several species of marine dinoflagellates and cyanobacteria. Two STX-core genes, sxtA and sxtG, have been well elucidated in Alexandrium but the expression of these genes under various nutritional modes in tropical species remains unclear. This study investigates the physiological responses of a tropical Pacific strain of Alexandrium minutum growing with nitrate or ammonium, and with various nitrogen to phosphorus (N:P) supply ratios. The transcriptional responses of the sxt genes were observed. Likewise, a putative sxtI encoding O-carbamoyltransferase (herein designated as AmsxtI) was recovered from the transcriptomic data, and its expression was investigated. The results revealed that the cellular toxin quota (Qt) was higher in P-depleted, nitrate-grown cultures. With cultures at similar N:P (<16), cells grown with excess ammonium showed a higher Qt than those grown with nitrate. sxtA1 was not expressed under any culture conditions, suggesting that this gene might not be involved in STX biosynthesis by this strain. Conversely, sxtA4 and sxtG showed positive correlations with Qt, and were up-regulated in P-depleted, nitrate-grown cultures and with excess ambient ammonium. On the other hand, AmsxtI was expressed only when induced by P-depletion, suggesting that this gene may play an important role in P-recycling metabolism, while simultaneously enhancing toxin production.
Transcriptional and physiological responses to inorganic nutrition in a tropical Pacific strain of Alexandrium minutum: Implications for nutrient uptakes and assimilation

Hii KS, Lim PT, Kon NF, Usup G, Gu H, Leaw CP

Gene, 2019 Aug 30;711:143950.
PMID: 31255736 DOI: 10.1016/j.gene.2019.143950

The marine dinoflagellate Alexandrium minutum is known to produce saxitoxins that cause paralytic shellfish poisoning in human worldwide through consumption of the contaminated shellfish mollusks. Despite numerous studies on the growth physiology and saxitoxin production of this species, the knowledge on the molecular basis of nutrient uptakes in relation to toxin production in this species is limited. In this study, relative expressions of the high-affinity transporter genes of nitrate, ammonium, and phosphate (AmNrt2, AmAmt1 and AmPiPT1) and the assimilation genes, nitrate reductase (AmNas), glutamine synthase (AmGSIII) and carbamoyl phosphate synthase (AmCPSII) from A. minutum were studied in batch clonal culture condition with two nitrogen sources (nitrate: NO3- or ammonium: NH4+) under different N:P ratios (high-P: N:P of 14 and 16, and low-P: N:P of 155). The expression of AmAmt1 was suppressed in excess NH4+-grown condition but was not observed in AmNrt2 and AmNas. Expressions of AmAmt1, AmNrt2, AmNas, AmGSIII, AmCPSII, and AmPiPT1 were high in P-deficient condition, showing that A. minutum is likely to take up nutrients for growth under P-stress condition. Conversely, relative expression of AmCPSII was incongruent with cell growth, but was well correlated with toxin quota, suggesting that the gene might involve in arginine metabolism and related toxin production pathway. The expression of AmGSIII is found coincided with higher toxin production and is believed to involve in mechanism to detoxify the cells from excess ammonium stress. The gene regulation observed in this study has provided better insights into the ecophysiology of A. minutum in relation to its adaptive strategies in unfavorable environments.
Influence of culture conditions and medium composition on the production of antibacterial compounds by marine Serratia sp. WPRA3

Jafarzade M, Yahya NA, Shayesteh F, Usup G, Ahmad A

J Microbiol, 2013 Jun;51(3):373-9.
PMID: 23812818 DOI: 10.1007/s12275-013-2440-2

This study was undertaken to investigate the influence of culture conditions and medium components on production of antibacterial compounds by Serratia sp. WPRA3 (JX020764) which was isolated from marine water of Port Dickson, Malaysia. Biochemical, morphological, and molecular characteristics suggested that the isolate is a new candidate of the Serratia sp. The isolate showed strong antimicrobial activity against fungi, Gram-negative and Gram-positive bacteria. This bacterium exhibited optimum antibacterial compounds production at 28°C, pH 7 and 200 rev/min aeration during 72 h of incubation period. Highest antibacterial activity was obtained when sodium chloride (2%), yeast extract (0.5%), and glucose concentration (0.75%) were used as salt, nitrogen, and carbon sources respectively. Different active fractions were obtained by Thin-Layer Chromatography (TLC) and Flash Column Chromatography (FCC) from ethyl acetate crude extracts namely OCE and RCE in different culture conditions, OCE (pH 5, 200 rev/min) and RCE (pH 7/without aeration). In conclusion, the results suggested different culture conditions have a significant impact on the types of secondary metabolites produced by the bacterium.
Life-history stages of natural bloom populations and the bloom dynamics of a tropical Asian ribotype of Alexandrium minutum

Lau WLS, Law IK, Liow GR, Hii KS, Usup G, Lim PT, et al.

Harmful Algae, 2017 12;70:52-63.
PMID: 29169568 DOI: 10.1016/j.hal.2017.10.006

In 2015, a remarkably high density bloom of Alexandrium minutum occurred in Sungai Geting, a semi-enclosed lagoon situated in the northeast of Peninsular Malaysia, causing severe discoloration and contaminated the benthic clams (Polymesoda). Plankton and water samples were collected to investigate the mechanisms of bloom development of this toxic species. Analysis of bloom samples using flow cytometry indicated that the bloom was initiated by the process of active excystment, as planomycetes (>4C cells) were observed in the early stage of the bloom. Increase in planozygotes (2C cells) was evident during the middle stage of the bloom, coinciding with an abrupt decrease in salinity and increase of temperature. The bloom was sustained through the combination of binary division of vegetative cells, division of planozygotes, and cyst germination through continuous excystment. Nutrient depletion followed by precipitation subsequently caused the bloom to terminate. This study provides the first continuous record of in situ life-cycle stages of a natural bloom population of A. minutum through a complete bloom cycle. The event has provided a fundamental understanding of the pelagic life-cycle stages of this tropical dinoflagellate, and demonstrated a unique bloom development characteristic shared among toxic Alexandrium species in coastal embayments.
New scenario for speciation in the benthic dinoflagellate genus Coolia (Dinophyceae)

Leaw CP, Tan TH, Lim HC, Teng ST, Yong HL, Smith KF, et al.

Harmful Algae, 2016 05;55:137-149.
PMID: 28073527 DOI: 10.1016/j.hal.2016.02.010

In this study, inter- and intraspecific genetic diversity within the marine harmful dinoflagellate genus Coolia Meunier was evaluated using isolates obtained from the tropics to subtropics in both Pacific and Atlantic Ocean basins. The aim was to assess the phylogeographic history of the genus and to clarify the validity of established species including Coolia malayensis. Phylogenetic analysis of the D1-D2 LSU rDNA sequences identified six major lineages (L1-L6) corresponding to the morphospecies Coolia malayensis (L1), C. monotis (L2), C. santacroce (L3), C. palmyrensis (L4), C. tropicalis (L5), and C. canariensis (L6). A median joining network (MJN) of C. malayensis ITS2 rDNA sequences revealed a total of 16 haplotypes; however, no spatial genetic differentiation among populations was observed. These MJN results in conjunction with CBC analysis, rDNA phylogenies and geographical distribution analyses confirm C. malayensis as a distinct species which is globally distributed in the tropical to warm-temperate regions. A molecular clock analysis using ITS2 rDNA revealed the evolutionary history of Coolia dated back to the Mesozoic, and supports the hypothesis that historical vicariant events in the early Cenozoic drove the allopatric differentiation of C. malayensis and C. monotis.
MORPHOLOGY AND MOLECULAR CHARACTERIZATION OF PSEUDO-NITZSCHIA (BACILLARIOPHYCEAE) FROM MALAYSIAN BORNEO, INCLUDING THE NEW SPECIES PSEUDO-NITZSCHIA CIRCUMPORA SP. NOV

Lim HC, Leaw CP, Su SN, Teng ST, Usup G, Mohammad-Noor N, et al.

J Phycol, 2012 Oct;48(5):1232-47.
PMID: 27011282 DOI: 10.1111/j.1529-8817.2012.01213.x

Field sampling was undertaken to investigate the occurrence of Pseudo-nitzschia Peragallo species in eight locations along the coast of Malaysian Borneo. A total of 108 strains of Pseudo-nitzschia species were isolated, and their morphology examined with SEM and TEM. Additionally, molecular data from nuclear-encoded partial LSU rDNA, and ITS regions, were characterized. A total of five species were confidently identified based on a combination of distinct morphological characteristics and supporting molecular evidence: P. brasiliana Lundholm, Hasle & Fryxell, P. cuspidata (Hasle) Hasle, P. dolorosa Lundholm & Moestrup, P. micropora Priisholm, Moestrup & Lundholm, and P. pungens (Grunow) Hasle var. pungens. However, one morphotype from Sarawak, while somewhat similar to P. caciantha, showed significant morphological distinction from this and any other of the currently described species. Most notably this morphotype possessed a characteristic pore arrangement in the poroids, with the fine pores in each perforation sector arranged in circles. Pair-wise sequence comparison of the LSU rDNA between this unidentified morphotype and P. caciantha Lundholm, Moestrup & Hasle, revealed 2.7% genetic divergence. Phylogenetic analyses strongly supported the monophyly of the morphotype. Based upon these supporting data it is here described as a new species, Pseudo-nitzschia circumpora sp. nov. A key to the six species of Pseudo-nitzschia from Malaysian Borneo is presented. Molecular signatures for all species were established based on structural comparisons of ITS2 rRNA transcripts.

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