Nutrient Deficiencies Impact on the Cellular and Metabolic Responses of Saxitoxin Producing Alexandrium minutum: A Transcriptomic Perspective.

Dinoflagellate Alexandrium minutum Halim is commonly associated with harmful algal blooms (HABs) in tropical marine waters due to its saxitoxin production. However, limited information is available regarding the cellular and metabolic changes of A. minutum in nutrient-deficient environments. To fill this gap, our study aimed to investigate the transcriptomic responses of A. minutum under nitrogen and phosphorus deficiency. The induction of nitrogen and phosphorus deficiency resulted in the identification of 1049 and 763 differently expressed genes (DEGs), respectively. Further analysis using gene set enrichment analysis (GSEA) revealed 702 and 1251 enriched gene ontology (GO) terms associated with nitrogen and phosphorus deficiency, respectively. Our results indicate that in laboratory cultures, nitrogen deficiency primarily affects meiosis, carbohydrate catabolism, ammonium assimilation, ion homeostasis, and protein kinase activity. On the other hand, phosphorus deficiency primarily affects the carbon metabolic response, cellular ion transfer, actin-dependent cell movement, signalling pathways, and protein recycling. Our study provides valuable insights into biological processes and genes regulating A. minutum's response to nutrient deficiencies, furthering our understanding of the ecophysiological response of HABs to environmental change.

Serinicoccus kebangsaanensis sp. nov: A new bacterium isolated from the toxic diatom, Nitzschia navis-varingica.

Serinicoccus kebangsaanensis sp. nov strain P2D13-UKM is a new species of Gram-positive bacteria isolated from a toxic diatom, Nitzschia navis-varingica. It is a halophilic aerobic, oxidase-negative, catalase-positive, circular, and colonies with white colour. Based on the 16S rRNA gene, the closest species were Serinicoccus profundi MCCC 1A05965 strain 0714S6-1, 97.41%, and Serinicoccus hydrothermalis strain JLT9, 97.35%. This bacteria's predominant cellular fatty acids were iso-C15: 0 (30.1 %) and iso-C16: 0 (16.2 %). The polar lipids identified in this bacterium were phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol, and an unknown glycolipid. The whole-genome sequence analysis of strain P2D13-UKM showed less than 85% similarity from other Serinicoccus species. The genomic DNA G + C content is 72.2 %. Here, we report the main characteristic of strain P2D13-UKM as a new species of bacteria according to its draft genome sequence, which was deposited in Gene Bank and is publicly available under the accession number VSLG00000000.

Harmful Microalgae Detection: Biosensors versus Some Conventional Methods.

In the last decade, there has been a steady stream of information on the methods and techniques available for detecting harmful algae species. The conventional approaches to identify harmful algal bloom (HAB), such as microscopy and molecular biological methods are mainly laboratory-based and require long assay times, skilled manpower, and pre-enrichment of samples involving various pre-experimental preparations. As an alternative, biosensors with a simple and rapid detection strategy could be an improvement over conventional methods for the detection of toxic algae species. Moreover, recent biosensors that involve the use of nanomaterials to detect HAB are showing further enhanced detection limits with a broader linear range. The improvement is attributed to nanomaterials' high surface area to volume ratio, excellent biological compatibility with biomolecules, and being capable of amplifying the electrochemical signal. Hence, this review presents the potential usage of biosensors over conventional methods to detect HABs. The methods reported for the detection of harmful algae species, ranging from conventional detection methods to current biosensor approaches will be discussed, along with their respective advantages and drawbacks to indicate the future prospects of biosensor technology for HAB event management.

Insights into Alexandrium minutum Nutrient Acquisition, Metabolism and Saxitoxin Biosynthesis through Comprehensive Transcriptome Survey.

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.

Pathogenic hitchhiker diversity on international ships' ballast water at West Malaysia port.

This study investigates bacterial diversity and potential pathogens in the international ships' ballast water at Tanjung Pelepas Port, Malaysia, using 16S rRNA amplicon sequencing. Thirty-four bacterial phylum, 305 families, 577 genera, and 941 species were detected in eight ballast water samples of different origins. The similarity of the bacterial composition between samples was found to be random and not tied to geographical locations. The bacterial abundance did not seem to be affected by related physicochemical except for temperature. Ballast water samples with a temperature lower than 25 °C showed a relatively lower bacterial abundance. A total of 33 potential pathogens were detected from all ballast water samples. Pseudomonas spp., Tenacibaculum spp., Flavobacteriaceae spp., Halomonas spp., and Acinetobacter junii are the potential pathogens with more than 10% OTU prevalence. This study would provide beneficial information for further enhancing ballast water microorganism guidelines in Malaysia.

Characterization of putative pathogenic Shewanella algae isolated from ballast water.

BACKGROUND AND AIM: Shewanella algae is ubiquitous in marine-associated environments and has been increasingly recognized as a significant human pathogen that can cause serious infections mainly associated with exposure to seawater and ingestion of raw seafood. This study aimed to isolate and characterize S. algae from ballast water of ships berthed at Port Klang, Malaysia. MATERIALS AND METHODS: Ballast water was sampled from nine ships docked at Port Klang, Malaysia. The isolates were identified and characterized based on biochemical and enzymatic properties, 16S rRNA and gyrB sequencing, biofilm formation capability, and antibiotic susceptibility. RESULTS: A total of four S. algae isolates were isolated from four ballast water samples tentatively name Sa-BW1, Sa-BW2, Sa-BW7, and Sa-BW8. All isolates showed positive reaction for cytochrome oxidase, catalase, high tolerance to NaCl (6% and 8%), ability to grow at 42°C, and on Salmonella-Shigella agar. The strains also exhibited b-hemolytic activity on sheep blood and human blood agar, positive reaction for lipase, protease, DNase and gelatinase, strong biofilm adherence capabilities and multiple antibiotic resistances against ampicillin, carbenicillin, cephalothin, colistin, novobiocin, oxacillin, penicillin, rifampicin, and tobramycin which suggested their potential pathogenicity. CONCLUSION: This study demonstrated the occurrence of putative pathogen S. algae in ballast water of ships docked at Malaysian port.

In vitro anti-biofilm activity of bacteriocin from a marine Bacillus sp. strain Sh10 against Proteus mirabilis.

BACKGROUND AND OBJECTIVES: Biofilm formed by Proteus mirabilis strains is one of the most important medical problems especially in the case of device-related urinary tract infections. This study was conducted to evaluate the bacteriocin produced by a marine isolate of Bacillus sp. Sh10, for it's in vitro inhibitory activity against pre-formed biofilm and in interference with the biofilm-forming of two biofilm-producing bacteria (P. mirabilis UCa4 and P. mirabilis UCe1). MATERIALS AND METHODS: Sensitivity of two biofilm-producing bacteria (P. mirabilis UCa4 and P. mirabilis UCe1) to bacteriocin, was investigated in planktonic and biofilm states by cell viability and crystal violet assay, respectively. Scanning electron microscopy (SEM) was also performed to determine the effect of bacteriocin on the morphology of the cells associated with biofilm. RESULTS: It was found that bacteriocin possessed bactericidal activity to biofilm-forming isolates in the planktonic state. However, bacteriocin interferes with the formation of biofilms and disrupts established biofilms. Bacteriocin reduced biofilm formation in the isolates of P. mirabilis UCa4 and P. mirabilis UCe1 with SMIC50 of 32 and 128 µg/mL, desirable SMIC50 of bacteriocin for biofilm disruption were 128 and 256 µg/mL, respectively. The SEM results indicated that bacteriocin affected the cell morphology of biofilm-associated cells. CONCLUSION: The present findings indicated that bacteriocin from Bacillus sp. Sh10 has bactericidal properties against biofilm-forming isolates of P. mirabilis UCa4 and P. mirabilis UCe1 and has the ability to inhibit the formation of biofilm and disrupt established biofilm.

Biosynthesis of Saxitoxin in Marine Dinoflagellates: An Omics Perspective.

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.

Isolation, detection of virulence genes, antibiotic resistance genes, plasmid profile, and molecular typing among Vibrio parahaemolyticus isolated in Malaysian seawater from recreational beaches and fish.

BACKGROUND AND AIM: Despite the importance of the global emergence of Vibrio parahaemolyticus infections worldwide, there has been scanty information on its occurrence in Malaysian seawaters and fish. This study aimed to determine the occurrence of V. parahaemolyticus isolates using polymerase chain reaction targeted at toxin operon gene, thermostable direct hemolysin (tdh), and tdh-related hemolysin genes and to determine antibiotic resistance pattern, genes, and plasmid profile of V. parahaemolyticus from Malaysian seawaters and fish. MATERIALS AND METHODS: Samples were collected from four recreational beaches in Malaysia (Port Klang; Bachok; Port Dickson; and Mersing). Thiosulfate-citrate-bile salts-sucrose (TCBS) agar and chromogenic Vibrio agar were used for isolation and identification. Colonies with yellow color on TCBS and green color on chromogenic vibrio (CV) agar were considered to be V. parahaemolyticus and they were subjected to biochemical tests. All V. parahaemolyticus isolates were further subjected to identification using seven specific gene markers. RESULTS: Seventy-three Vibrio isolates were recovered. Only one gene thermostable direct hemolysin (tdh) from seawater isolates of Vibrio has high virulence gene percentage (95.23%). Two genes alkaline serine protease (asp) and (tdh) had high percentage of virulence (83.87% and 80.64%, respectively) from fish. Comparatively, fish isolates have a higher virulence percentage compared to seawater isolates. Only gene streptomycin resistance B (strB) from seawater had 100% of the resistance genes. All isolates were multi-antibiotic resistant. Seventeen antibiotic resistance patterns were observed. The isolates had plasmids of varying sizes ranging from 2.7 kb to 42.4 kb. Dendrogram based on antibiotic resistance patterns of V. parahaemolyticus isolates discriminated the isolates into three clusters. CONCLUSION: This study demonstrated the occurrence of pathogenic, multi-antibiotic-resistant V. parahaemolyticus strains in Malaysian coastal waters and fish, and this could constitute potential public health risks.

Rapid determination of kappa-carrageenan using a biosensor from immobilized Pseudomonas carrageenovora cells.

A potentiometric whole cell biosensor based on immobilized marine bacterium, Pseudomonas carrageenovora producing κ-carrageenase and glycosulfatase enzymes for specific and direct determination of κ-carrageenan, is described. The bacterial cells were immobilized on the self-plasticized hydrogen ion (H+)-selective acrylic membrane electrode surface to form a catalytic layer. Hydrogen ionophore I was incorporated in the poly(n-butyl acrylate) [poly(nBA)] as a pH ionophore. Catalytic decomposition of κ-carrageenan by the bienzymatic cascade reaction produced neoagarobiose, an inorganic sulfate ion and a proton. The latter was detectable by H+ ion transducer for indirect potentiometric quantification of κ-carrageenan concentration. The use of a disposable screen-printed Ag/AgCl electrode (SPE) provided no cleaning requirement and enabled κ-carrageenan detection to be carried out conveniently without cross contamination in a complex food sample. The SPE-based microbial biosensor response was found to be reproducible with high reproducibility and relative standard deviation (RSD) at 2.6% (n = 3). The whole cell biosensor demonstrated a broad dynamic linear response range to κ-carrageenan from 0.2-100 ppm in 20 mM phosphate buffer saline (PBS) at pH 7.5 with a detection limit at 0.05 ppm and a Nernstian sensitivity of 58.78±0.87 mV/decade (R2 = 0.995). The biosensor showed excellent selectivity towards κ-carrageenan compared to other types of carrageenans tested e.g. ι-carrageenan and λ-carrageenan. No pretreatment to the food sample was necessary when the developed whole cell biosensor was employed for direct assay of κ-carrageenan in dairy product.

An ultrasensitive hollow-silica-based biosensor for pathogenic Escherichia coli DNA detection.

A novel electrochemical DNA biosensor for ultrasensitive and selective quantitation of Escherichia coli DNA based on aminated hollow silica spheres (HSiSs) has been successfully developed. The HSiSs were synthesized with facile sonication and heating techniques. The HSiSs have an inner and an outer surface for DNA immobilization sites after they have been functionalized with 3-aminopropyltriethoxysilane. From field emission scanning electron microscopy images, the presence of pores was confirmed in the functionalized HSiSs. Furthermore, Brunauer-Emmett-Teller (BET) analysis indicated that the HSiSs have four times more surface area than silica spheres that have no pores. These aminated HSiSs were deposited onto a screen-printed carbon paste electrode containing a layer of gold nanoparticles (AuNPs) to form a AuNP/HSiS hybrid sensor membrane matrix. Aminated DNA probes were grafted onto the AuNP/HSiS-modified screen-printed electrode via imine covalent bonds with use of glutaraldehyde cross-linker. The DNA hybridization reaction was studied by differential pulse voltammetry using an anthraquinone redox intercalator as the electroactive DNA hybridization label. The DNA biosensor demonstrated a linear response over a wide target sequence concentration range of 1.0×10-12-1.0×10-2 µM, with a low detection limit of 8.17×10-14 µM (R2 = 0.99). The improved performance of the DNA biosensor appeared to be due to the hollow structure and rough surface morphology of the hollow silica particles, which greatly increased the total binding surface area for high DNA loading capacity. The HSiSs also facilitated molecule diffusion through the silica hollow structure, and substantially improved the overall DNA hybridization assay. Graphical abstract Step-by-step DNA biosensor fabrication based on aminated hollow silica spheres.

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.

Population dynamics, antibiotics resistance and biofilm formation of Aeromonas and Vibrio species isolated from aquatic sources in Northern Malaysia.

This study aimed to compare population dynamics, antibiotic resistance and biofilm formation of Aeromonas and Vibrio species from seawater and sediment collected from Northern Malaysia. Isolates with different colony morphology were characterized using both biochemical and molecular methods before testing for antibiotic resistance and biofilm formation. Results obtained from this study showed that in Kedah, the population of Aeromonas isolated from sediment was highest in Pantai Merdeka (8.22 log CFU/ml), Pulau Bunting recorded the highest population of Aeromonas from sediment (8.43 log CFU/g). It was observed that Vibrio species isolated from seawater and sediment were highest in Kuala Sanglang (9.21 log CFU/ml). In Kuala Perlis, the population of Aeromonas isolated from seawater was highest in Jeti (7.94 log CFU/ml). Highest population of Aeromonas from sediment was recorded in Kampong Tanah Baru (7.99 log CFU/g). It was observed that Vibrio species isolated from seawater was highest in Padang Benta (8.42 log CFU/g) while Jeti Kuala Perlis had highest population of Vibrio isolated from sediment. It was observed that location does not influence population of Aeromonas. The results of the independent t - test revealed that there was no significant relationship between location and population of Vibrio (df = 10, t = 1.144, p > 0.05). The occurrence of biofilm formation and prevalence of antibiotic resistant Aeromonas and Vibrio species in seawater and sediment pose danger to human and aquatic animals' health.

Pathogenicity of Aeromonas hydrophila isolated from the Malaysian Sea against coral (Turbinaria sp.) and sea bass (Lates calcarifer).

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 ß-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.

Loktanella spp. Gb03 as an algicidal bacterium, isolated from the culture of Dinoflagellate Gambierdiscus belizeanus.

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.

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.

An optical biosensor from green fluorescent Escherichia coli for the evaluation of single and combined heavy metal toxicities.

A fluorescence-based fiber optic toxicity biosensor based on genetically modified Escherichia coli (E. coli) with green fluorescent protein (GFP) was developed for the evaluation of the toxicity of several hazardous heavy metal ions. The toxic metals include Cu(II), Cd(II), Pb(II), Zn(II), Cr(VI), Co(II), Ni(II), Ag(I) and Fe(III). The optimum fluorescence excitation and emission wavelengths of the optical biosensor were 400 ± 2 nm and 485 ± 2 nm, respectively. Based on the toxicity observed under optimal conditions, the detection limits of Cu(II), Cd(II), Pb(II), Zn(II), Cr(VI), Co(II), Ni(II), Ag(I) and Fe(III) that can be detected using the toxicity biosensor were at 0.04, 0.32, 0.46, 2.80, 100, 250, 400, 720 and 2600 µg/L, respectively. The repeatability and reproducibility of the proposed biosensor were 3.5%-4.8% RSD (relative standard deviation) and 3.6%-5.1% RSD (n = 8), respectively. The biosensor response was stable for at least five weeks, and demonstrated higher sensitivity towards metal toxicity evaluation when compared to a conventional Microtox assay.

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.

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

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.

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.