Asmat Ahmad

Alexandrium(Dinophyceae) species in Malaysian waters

A study was carried out to determine the presence of paralytic shellfish poisoning (PSP) toxin-producing dinoflagellates in the coastalwaters of Peninsula Malaysia. This followed first ever occurrences of PSP in the Straits of Malacca and the northeast coast of the peninsula. The toxic tropical dinoflagellate Pyrodinium bahamense var. compressum was never encountered in any of the plankton samples. On the other hand, five species of Alexandrium were found. They were Alexandrium affine, Alexandrium leei, Alexandrium minutum, Alexandrium tamarense and Alexandrium tamiyavanichii. Not all species were present at all sites. A. tamiyavanichii was present only in the central to southern parts of the Straits of Malacca. A. tamarense was found in the northern part of the straits, while A. minutum was only found in samples from the northeast coast of the peninsula. A. leei and A. affine were found in both the north and south of the straits. Cultured isolates of A. minutum and A. tamiyavanichii were proven toxic by the receptor binding assay for PSP toxins but A. tamarense clones were not toxic. Mean toxin content for the A. tamiyavanichii and A. minutum clones were 26 and 15 fmol per cell STX equivalent, respectively. This study has provided evidence on the presence of PSP toxin-producing Alexandrium species in Malaysian waters which suggests that PSP could increase in importance in the future.

Genetic Diversity of Ostreopsis ovata (Dinophyceae) from Malaysia

Abstract: The genus Ostreopsis is an important component of benthic and epiphytic dinoflagellate assemblages in coral reefs and seaweed beds of Malaysia. Members of the species may produce toxins that contribute to ciguatera fish poisoning. In this study, two species have been isolated and cultured, Ostreopsis ovata and Ostreopsis lenticularis. Analyses of the 5.8S subunit and internal transcribed spacer regions ITS1 and ITS2 of the ribosomal RNA gene sequences of these two species showed that they are separate species, consistent with morphological designations. The nucleotide sequences of the 5.8S subunit and ITS1 and ITS2 regions of the rRNA gene were also used to evaluate the interpopulation and intrapopulation genetic diversity of O. ovata found in Malaysian waters. Results showed a low level of sequence divergence within populations. At the interpopulation level, the rRNA gene sequence distinguished two groups of genetically distinct strains, representative of a Malacca Straits group (isolates from Port Dickson) and a South China Sea group (isolates from Pulau Redang and Kota Kinabalu). Part of the sequences in the ITS regions may be useful in the design of oligonucleotide probes specific for each group. Results from this study show that the ITS regions can be used as genetic markers for taxonomic, biogeographic, and fine-scale population studies of this species.

Phylogenetic analysis of Alexandrium species and Pyrodinium bahamense (Dinophyceae) based on theca morphology and nuclear ribosomal gene sequence

C.P. Leaw, P.T. Lim, B.K. Ng, M.Y. Cheah, A. Ahmad and G. Usup. 2005. Phylogenetic analysis of Alexandrium species and Pyrodinium bahamense (Dinophyceae) based on theca morphology and nuclear ribosomal gene sequence. Phycologia 44: 550–565. A phylogenetic analysis of Alexandrium species and Pyrodinium bahamense was carried out. The analysis was based on nucleotide sequences of the large subunit ribosomal RNA gene and 16 morphological characters considered taxonomically informative. Maximum likelihood, maximum parsimony and Bayesian approaches were used. Molecular and morphological data were analysed independently and in combination. The outcomes of all the analyses were the same. Pyrodinium was consistently grouped in the same clade with Alexandrium, specifically with the subgenus Gessnerium, A. pseudogoniaulax and A. taylori. Two monophyletic clades were resolved. The first comprised A. tamarense, A. fundyense, A. catenella, A. tamiyavanichii, A. affine and A. concavum, with the base formed by A. pseudogoniaulax, A. taylori and P. bahamense. The second clade comprised the species A. minutum, A. insuetum, A. tamutum, A. andersoni, A. ostenfeldii and A. leei, with A. margalefi forming the base. Mapping of morphological characters onto the phylogenetic trees indicated that posterior sulcal plate probably has the highest value in the taxonomy of Alexandrium. Some other characters considered taxonomically important, such as the ventral pore and position of the anterior attachment pore, are most probably homoplastic.

Phylogenetic relationship of Alexandrium tamiyavanichii (Dinophyceae) to other Alexandrium species based on ribosomal RNA gene sequences

The phylogenetic relationship of the thecate PSP-toxin producing dinoflagellate Alexandrium tamiyavanichii Balech to other species of Alexandrium was studied based on nucleotide sequences of the ITS1, ITS2, 5.8S, 18S and 28S subunits of the ribosomal RNA gene. These are the first such sequences available for A. tamiyavanichii, which is one of the producers of paralytic shellfish poisoning toxins in tropical waters. Based on the nucleotide sequences of the 28S, 18S and 5.8S subunits of the rRNA gene, A. tamiyavanichii grouped together with A. tamarense, A. catenella and A. fundyense. More interestingly, A. tamiyavanichii was most closely affiliated to A. tamarense isolates from Thailand. This result reaffirmed conclusions from previous studies that, for the A. tamarense/fundyense/catenella species complex, geographical origin rather than morphology seems to determine genetic relatedness. Results of this study also suggest that A. tamiyavanichii most probably belongs to the same species complex. Ribosomal RNA gene sequences do not separate the PSP toxin producing from the non-producing species of Alexandrium.

Marked Differences in Fatty Acid Profiles of Some Planktonic and Benthic Marine Dinoflagellates from Malaysian Waters

G. Usup, S.Z. Hamid, P.K. Chiet, C.K. Wah and A. Ahmad. 2007. Marked differences in fatty acid profiles of some planktonic and benthic marine dinoflagellates from Malaysian waters. Phycologia 47: 105–111. DOI: 10.2216/07-55.1 This study was carried out to characterize the fatty acid profiles of some planktonic and benthic marine dinoflagellates from Malaysian waters. Clonal batch cultures of Alexandrium affine, A. leei, A. minutm, A. tamarense, A. tamiyavanichii, Coolia monotis, Prorocentrum emarginatum, P. mexicanum, Ostreopsis ovata and Amphidinium sp. were harvested at late exponential phase, and total lipid was extracted. Samples were derivatized to produce fatty acid methyl esters (FAMEs). FAMEs were analyzed on a gas chromatograph with flame ionization detection. The total number of fatty acids detected in the clones ranged from 10 in the A. tamarense AtPA04 clone to 22 in the C. monotis CmPL01 clone. Fatty acids found in all clones were myristic acid (14 : 0), palmitic acid (16 : 0), stearic acid (18 : 0), linoleic acid (18 : 2ω6c) and oleic acid (18 : 1ω9c). In all clones only a few fatty acids were dominant. In the Alexandrium clones the dominant fatty acids were 16 : 0, 18 : 0, cis-13,16-docosadienoic acid (22 : 2), 18 : 2ω6c and 18 : 1ω9c. There was almost complete absence of omega-3 polyunsaturated fatty acids (PUFA) in the Alexandrium clones. In the benthic species the major fatty acids were 16 : 0, eicosapentaenoic acid (EPA, 20 : 5ω3), docosahexaenoic acid (22 : 6ω3), 18 : 2ω6c and 18 : 1ω9c. In the Prorocentrum clones the major fatty acids were 14 : 0, 16 : 0, palmitoleic acid (16 : 1) and EPA. Total PUFA content in the benthic species were 37%–56%, while in the planktonic species the content was 19%–44%. The fatty acid profiles could not differentiate between species. However, cluster analysis and principal components analysis were able to clearly discriminate between the Alexandrium group, Prorocentrum group and benthic species group.

Secondary metabolites produced by marine streptomyces as antibiofilm and quorum-sensing inhibitor of uropathogen Proteus mirabilis

Quorum-sensing regulates bacterial biofilm formation and virulence factors, thereby making it an interesting target for attenuating pathogens. In this study, we investigated anti-biofilm and anti-quorum-sensing compounds from secondary metabolites of halophiles marine streptomyces against urinary catheter biofilm forming Proteus mirabilis without effect on growth viability. A total of 40 actinomycetes were isolated from samples collected from different places in Iraq including marine sediments and soil samples. Fifteen isolates identified as streptomyces and their supernatant screened as anti-quorum-sensing by inhibiting quorum-sensing regulated prodigiosin biosynthesis of Serratia marcescens strain Smj-11 as a reporter strain. Isolate Sediment Lake Iraq (sdLi) showed potential anti-quorum-sensing activity. Out of 35 clinical isolates obtained from Urinary catheter used by patient at the Universiti Kebangsaan Malaysia Medical Center, 22 isolates were characterized and identified as Proteus mirabilis. Isolate Urinary Catheter B4 (UCB4) showed the highest biofilm formation with highest resistance to used antibiotic and was chosen for further studies. Ethyl acetate secondary metabolites extract was produced from sdLi isolate. First, we determined the Minimum Inhibitory Concentration (MIC) of sdLi crude extract against UCB4 isolate, and all further experiments used concentrations below the MIC. Tests of subinhibitory concentrations of sdLi crude extract showed good inhibition against UCB4 isolate biofilm formation on urinary catheter and cover glass using Scanning electron microscopy and light microscopy respectively. The influence of sub-MIC of sdLi crude extract was also found to attenuate the quorum sensing (QS)—dependent factors such as hemolysin activity, urease activity, pH value, and motility of UCB4 isolate. Evidence is presented that these nontoxic secondary metabolites may act as antagonists of bacterial quorum sensing by competing with quorum-sensing signals for receptor binding.

Microencapsulated Aliivibrio fischeri in Alginate Microspheres for Monitoring Heavy Metal Toxicity in Environmental Waters

In this article a luminescence fiber optic biosensor for the microdetection of heavy metal toxicity in waters based on the marine bacterium Aliivibrio fischeri (A. fischeri) encapsulated in alginate microspheres is described. Cu(II), Cd(II), Pb(II), Zn(II), Cr(VI), Co(II), Ni(II), Ag(I) and Fe(II) were selected as sample toxic heavy metal ions for evaluation of the performance of this toxicity microbiosensor. The loss of bioluminescence response from immobilized A. fischeri bacterial cells corresponds to changes in the toxicity levels. The inhibition of the luminescent biosensor response collected at excitation and emission wavelengths of 287 ± 2 nm and 487 ± 2 nm, respectively, was found to be reproducible and repeatable within the relative standard deviation (RSD) range of 2.4–5.7% (n = 8). The toxicity biosensor based on alginate micropsheres exhibited a lower limit of detection (LOD) for Cu(II) (6.40 μg/L), Cd(II) (1.56 μg/L), Pb(II) (47 μg/L), Ag(I) (18 μg/L) than Zn(II) (320 μg/L), Cr(VI) (1,000 μg/L), Co(II) (1700 μg/L), Ni(II) (2800 μg/L), and Fe(III) (3100 μg/L). Such LOD values are lower when compared with other previous reported whole cell toxicity biosensors using agar gel, agarose gel and cellulose membrane biomatrices used for the immobilization of bacterial cells. The A. fischeri bacteria microencapsulated in alginate biopolymer could maintain their metabolic activity for a prolonged period of up to six weeks without any noticeable changes in the bioluminescence response. The bioluminescent biosensor could also be used for the determination of antagonistic toxicity levels for toxicant mixtures. A comparison of the results obtained by atomic absorption spectroscopy (AAS) and using the proposed luminescent A. fischeri-based biosensor suggests that the optical toxicity biosensor can be used for quantitative microdetermination of heavy metal toxicity in environmental water samples.

Occurrence of Enterococcus species with virulence markers in an urban flow-influenced tropical recreational beach.

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.

Antibiotic resistance profiling and phenotyping of Aeromonas species isolated from aquatic sources

This study aimed to investigate antibiotics resistance pattern and phenotyping of Aeromonas species isolated from different aquatic sources in Melaka, Malaysia. A total of 53 Aeromonas species were isolated from the following sources: sediment (n = 13), bivalve (n = 10), sea cucumber (n = 16) and sea water (n = 14) and resistance to 12 antibiotics – Tetracycline (30 μg), Kanamycin (30 μg), Oxytetracycline (30 μg), Ampicillin (10 μg), Streptomycin (10 μg), Gentamicin (10 μg), Sulphamethoxazole (25 μg), Nalixidic acid (30 μg), Trimethoprim (1.25 μg), Novobiocin (5 μg), Penicilin (10 μg) and Chloramphenicol (10 μg) was tested. The results obtained from this study reveal multi drug resistance pattern among the isolates. All the isolates were completely resistant to Ampicillin, Novobiocin, Sulphamethoxazole and Trimethoprim, respectively but susceptible to Tetracycline (100%), Kanamycin (5.7%), Gentamicin (5.7%) and Oxytetracycline (24.5%). Antibiotics phenotyping of the bacteria revealed 21 different phenotypes among the isolates.

Influence of culture conditions and medium composition on the production of antibacterial compounds by marine Serratia sp. WPRA3

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.