An earlier electrochemical mechanism of DNA detection was adapted and specified for the detection of Vibrio parahaemolyticus in real samples. The reader, based on a screen printed carbon electrode, was modified with polylactide-stabilized gold nanoparticles and methylene blue was employed as the redox indicator. Detection was assessed using a microprocessor to measure current response under controlled potential. The fabricated sensor was able to specifically distinguish complementary, non-complementary and mismatched oligonucleotides. DNA was measured in the range of 2.0 × 10(-8)-2.0 × 10(-13) M with a detection limit of 2.16 pM. The relative standard deviation for 6 replications of differential pulse voltammetry (DPV) measurement of 0.2 µM complementary DNA was 4.33%. Additionally, cross-reactivity studies against various other food-borne pathogens showed a reliably sensitive detection of the target pathogen. Successful identification of Vibrio parahaemolyticus (spiked and unspiked) in fresh cockles, combined with its simplicity and portability demonstrate the potential of the device as a practical screening tool.
The demands for applicable tissue-engineered scaffolds that can be used to repair load-bearing segmental bone defects (SBDs) is vital and in increasing demand. In this study, seven different combinations of 3 dimensional (3D) novel nanocomposite porous structured scaffolds were fabricated to rebuild SBDs using an extraordinary blend of cockle shells (CaCo3) nanoparticles (CCN), gelatin, dextran and dextrin to structure an ideal bone scaffold with adequate degradation rate using the Freeze Drying Method (FDM) and labeled as 5211, 5400, 6211, 6300, 7101, 7200 and 8100. The micron sized cockle shells powder obtained (75 µm) was made into nanoparticles using mechano-chemical, top-down method of nanoparticles synthesis with the presence of the surfactant BS-12 (dodecyl dimethyl bataine). The phase purity and crystallographic structures, the chemical functionality and the thermal characterization of the scaffolds' powder were recognized using X-Ray Diffractometer (XRD), Fourier transform infrared (FTIR) spectrophotometer and Differential Scanning Calorimetry (DSC) respectively. Characterizations of the scaffolds were assessed by Scanning Electron Microscopy (SEM), Degradation Manner, Water Absorption Test, Swelling Test, Mechanical Test and Porosity Test. Top-down method produced cockle shell nanoparticles having averagely range 37.8±3-55.2±9 nm in size, which were determined using Transmission Electron Microscope (TEM). A mainly aragonite form of calcium carbonate was identified in both XRD and FTIR for all scaffolds, while the melting (Tm) and transition (Tg) temperatures were identified using DSC with the range of Tm 62.4-75.5 °C and of Tg 230.6-232.5 °C. The newly prepared scaffolds were with the following characteristics: (i) good biocompatibility and biodegradability, (ii) appropriate surface chemistry and (iii) highly porous, with interconnected pore network. Engineering analyses showed that scaffold 5211 possessed 3D interconnected homogenous porous structure with a porosity of about 49%, pore sizes ranging from 8.97 to 337 µm, mechanical strength 20.3 MPa, Young's Modulus 271±63 MPa and enzymatic degradation rate 22.7 within 14 days.
This article contains data on the bacterial communities and its diversity associated with Anadara granosa. The A. granosa samples were obtained from two major estuaries in Penang, Malaysia using a culture dependent and 16S rRNA Illumina sequencing approaches. A. granosa, a commercial blood cockles and popular seafoods, is fragile to the surrounding environments. Thus, our research focused to better understand the bacterial communities and it diversity in the A. granosa, as well as on the generation of a metagenomic library from A. granosa to further understanding on it diversity. The bacteria Vibrionaceae (34.1%) was predominant in the A. granosa from both environments followed by Enterobacteriaceae (33.3%) and Bacillaceae (16.75%). Vibrio sp., Klebsiella sp., and Bacillus subtilis were the most abundant species present. The data generated in this research is the first metagenomic examination of A. granosa and will provide as a baseline to understand the bacterial communities associated with A. granosa and its surrounding natural environments.
This paper presents the results of mercury analysis on 786 abiotic (surface sediments) and biotic (plant and animal tissues) samples collected from 10 sites at Matang Mangrove Forest Reserve in Peninsular Malaysia. Sediment samples were collected at the surface level from both river bank and forest understory. Whereas plant tissues obtained from Rhizophora apiculata Blume and Rhizophora mucronata L. consisted of leaves (in four stages namely young, mature, senescent and decomposing), bark and roots (divided into xylem, cortex and epidermis), the animal samples were represented by muscle tissue of the gastropod Cassidula aurisfelis Bruguière and the cockle Tegillarca granosa L. The mercury concentration measurements were obtained through a cold vapor atomic absorption spectrometer. The core data have been analysed and interpreted in the paper "Distribution of mercury in sediments, plant and animal tissues in Matang Mangrove Forest Reserve, Malaysia" [1].
The occurrence of tributyltin (TBT) is reported in the coastal waters of a few selected sites in Peninsular Malaysia. Water, bivalves and sediment samples collected were analysed specifically for TBT using sensitive analytical methods which involved a solvent extraction procedure with appropriate clean-up followed by graphite furnace atomic absorption spectrometric measurements. The levels of TBT in the seawater in unexposed areas were found in the range from <3.4 to 20 ng litre(-1) as compared to coastal areas with high boat and ship activities where TBT levels in seawater were generally above 30 ng litre(-1), with the highest level found at 281.8 ng litre(-1). TBT levels in the tissues of random cockle and soft-shell clam samples from local markets were found in the range from <0.5 to 3.7 ng g(-1) wet weight. The levels of TBT found in green mussel samples both from the market (23.5 ng g(-1) wet weight) and those from a mussel farm (14.2 ng g(-1) wet weight) indicate slight accumulation of TBT. In sediments, TBT levels were found ranging from <0.7 ng g(-1) dry weight in unexposed coastal sites to as high as 216.5 ng g(-1) dry weight for a site within a port area.
The paper demonstrates the capability of using cockle shells as an adsorbent for phosphorus removal from simulated petrochemical wastewater, focusing on the actual condition of the petrochemical facultative pond. In this study, the physicochemical properties of shell powder were determined, such as the functional groups, surface morphology, crystalline structure, and surface area using FTIR, SEM, EDX, XRD, and BET. It was observed that the optimum conditions for effective phosphorus removal are under the presence of rotational speed (125 rpm), higher dosage (7 g/L), and larger surface area (smaller particle size) of the shell powder. Fine powder achieved up to 52.27% of phosphorus removal after 40 min compared to coarse powder which could only give 16.67% removal. Additionally, calcined shell powder demonstrated a higher phosphorus removal rate, i.e., up to 62.37%, compared to raw shell powders. The adsorption isotherm was studied using Langmuir and Freundlich models, but the isothermal data fit better for the Freundlich model (R2 = 0.9836). Overall, this study has successfully generated a greener and low-cost adsorbent.
The CO2 capture capacity and cyclic stability of calcium oxide (CaO) prepared from cockle shells (CS) were enhanced by incorporating rice husk (RH) and binder through wet-mixing method. The cyclic reaction of calcination and carbonation was demonstrated using thermal gravimetric analyzer (TGA) which the calcination was performed in a pure N2 environment at 850 °C for 20 min and carbonation at 650 °C for 30 min in 20 vol% of CO2 in N2. The analysis using x-ray fluorescence (XRF) identified silica (Si) as the major elements in the sorbents. The RH-added sorbents also contained several types of metal elements such as which was a key factor to minimize the sintering of the sorbent during the cyclic reaction and contributed to higher CO2 capture capacity. The presence of various morphologies also associated with the improvement of the synthesized sorbents performance. The highest initial CO2 capture capacity was exhibited by CS+10%RH sorbent, which was 12% higher than the RH-free sorbent (CS). However, sorbents with the higher RH loading amount such as 40 and 50 wt% were preferred to maintain high capture capacity when the sorbents were regenerated and extended to the cyclic reaction. The sorbents also demonstrated the lowest average sorption decay, which suggested the most stable sorbent for cyclic-reaction. Once regenerated, the capture capacity of the RH-added sorbent was further increased by 12% when clay was added into the sorbent. Overall, the metal elements in RH and clay were possibly the key factor that enhances the performance of CaO prepared from CS, particularly for cyclic CO2 capture. Graphical abstract Cyclic calcination and carbonation reaction.
A total of 24 strains of Vibrio alginolyticus were isolated from cockles (Anadara granosa) and identified for VibA and gyrB genes. All V. alginolyticus isolates were then tested against nine different antibiotics. In this study, the highest percentage of antibiotic resistance was obtained against penicillin (37.50%), followed by ampicillin, vancomycin (12.50%) and erythromycin (8.33%). All of V. alginolyticus isolates were susceptible against streptomycin, kanamycin, tetracycline, chloramphenicol and sulfamethoxazole. Polymerase chain reaction (PCR) assay has confirmed the presence of four antibiotic resistance genes of penicillin (pbp2a), ampicillin (blaOXA), erythromycin (ermB) and vancomycin (vanB). Out of 24 V. alginolyticus isolates, 2 isolates possessed the tdh-related hemolysin (trh) (strains VA15 and VA16) and none for the thermostable direct hemolysin (tdh) gene. Both strains of the tdh-related hemolysin (trh) were susceptible to all antibiotics tested. The multiple antibiotic resistance (MAR) index ranging between 0.2 and 0.3 with 5 antibiograms (A1-A5) was observed. Combination of enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) and antibiotic resistance indicated 18 genome types which showed genetic heterogeneity of those V. alginolyticus isolates. The results demonstrated the presence of V. alginolyticus strain found in cockles can be a potential risk to consumers and can contribute to the deterioration of human health in the study area. Thus, it is essential for local authority to provide the preventive measures in ensuring the cockles are safe for consumption.
The development and utilization of nano-antibiotics is currently gaining attention as a possible solution to antibiotic resistance. The aim of this study was therefore to determine the pharmacokinetics of free oxytetracycline (OTC) and oxytetracycline loaded cockle shell calcium carbonate-based nanoparticle (OTC-CNP) after a single dose of intraperitoneal (IP) administration in BALB/c mice. A total of 100 female BALB/c mice divided into two groups of equal number (n = 50) were administered with 10 mg/kg OTC and OTC-CNP, respectively. Blood samples were collected before and post-administration from both groups at time 0, 5, 10, 15, and 30 min and 1, 2, 6, 24, and 48 h, and OTC plasma concentration was quantified using a validated HPLC-UV method. The pharmacokinetic parameters were analyzed using a non-compartment model. The Cmax values of OTC in OTC-CNP and free OTC treated group were 64.99 and 23.53 μg/ml, respectively. OTC was detected up to 24 h in the OTC-CNP group as against 1 h in the free OTC group following intraperitoneal administration. In the OTC-CNP group, the plasma elimination rate of OTC was slower while the half-life, the area under the curve, and the volume of the distribution were increased. In conclusion, the pharmacokinetic profile of OTC in the OTC-CNP group differs significantly from that of free OTC. However, further studies are necessary to determine the antibacterial efficacy of OTC-CNP for the treatment of bacterial diseases.
Blood cockles are among the most economically important brackish water invertebrates found in Malaysia. However, our knowledge of blood cockle phylogeny and systematics is rudimentary, especially for the species Tegillarca granosa. It is unclear, for instance, whether the cockles occurring on the west coast of peninsular Malaysia constitute a single species, or multiple, phylogenetically distinct species. We performed the first DNA molecular phylogenetic analysis of T. granosa to distinguish it from other related species found in other parts of the world and to create a DNA database for the species. An approximately 585-nucleotide fragment of the mitochondrial DNA (cytochrome oxidase I, COI) was sequenced for 150 individual cockles, representing 10 populations: three from the north, four from the central part and three from the southern part of peninsular Malaysia. Phylogenetic analyses of the resulting dataset yielded tree topologies that not only showed the relationship between T. granosa and its closest relatives but its position in the evolutionary tree. Three mitochondrial clades were evident, each containing an individual genus. Using the mutation rate of the COI gene, the divergence time between T. granosa and its closest related species was estimated to be 460 thousand years ago. This study provides a phylogenetic framework for this ecologically prominent and commercially important cockle species.
We examined genetic variation in blood cockles in an effort to obtain information useful for the sustainability, management, and the stability of this species as a major commodity in the fisheries sector. Ten populations of cockles were sampled from the north to the south of the west coast of peninsular Malaysia. The cockles were collected in collaboration with the Fisheries Research Institute, Penang. The population genetic analysis of the cockles were studied via RAPD-PCR and mtDNA sequencing. Three hundred individuals were analyzed with RAPD-PCR experiments. High gene diversity over all loci was observed (Shannon index = 0.549 ± 0.056 and Nei's gene diversity = 0.4852 ± 0.0430 among 35 loci). The second method, mtDNA sequencing, was employed to complement the information obtained from RAPD-PCR. The gene selected for mtDNA sequencing was cytochrome c oxidase I (COI). One hundred and fifty individuals were sequenced, yielding a partial gene of 585 bp. Statistical analysis showed homogeneity in general but did reveal some degree of variability between the populations in Johor and the rest of the populations. The Mantel test showed a positive but nonsignificant correlation between geographic and genetic distances (r = 0.2710, P = 0.622), as in the RAPD analysis. We propose that the homogeneity between distant populations is caused by two factors: 1) the translocation of the spats; 2) larvae are carried by current movement from the north of the peninsula to the south. The different genetic composition found in Johor could be due to pollution, mutagenic substances or physical factors such as the depth of the water column. This population genetic study is the first for this species in peninsular Malaysia. The data from this study have important implications for fishery management, conservation of blood cockles and translocation policies for aquaculture and stock enhancement programs.
Vibrio parahaemolyticus is a gram negative bacterium and causes gastrointestinal illness in humans. In this study, twenty five out of fifty cockle samples from Padang, Indonesia produced purple colonies when they were grown on selective medium, CHROMagarTM Vibrio. Specific–PCR for toxR gene detection gave positive results in which a band with 368 base pairs size appeared on the gel for all the isolates that confirmed the presence of V. parahaemolyticus. In the virulence properties test, all the isolates showed negative results for tdh and trh genes detection. The results indicate that the isolates under this study do not contain virulence properties that correlate to the ability of infection and diseases, which means that they are nonpathogenic.
A study was carried out to determine the effect of enzyme concentration, temperature and incubation time of bromelain on nitrogen content (NC) and degree of hydrolysis (DH) of hydrolysate from cockle (Anadara granosa) meat wash water. Protein precipitation of cockle meat wash water was conducted at pH 4. The precipitate was then hydrolyzed using bromelain at concentrations of 0.5, 1.5 and 2.5% (enzyme/substrate). The best enzyme concentration was subsequently used to study the effect of incubation temperature at 30, 45 and 60°C. The best temperature was then used to determine the effect of incubation time at 0, 24 and 48 hours. Increasing bromelain concentration from 0 to 2.5% produced an increase in NC and DH. Similarly, increasing the incubation time from 0 to 48 hours also increased the value of NC and DH. However, while the increasing of incubation temperature from 30 to 60°C produced an increase in NC, no significant difference was observed for DH.
Cockle (Anadara granosa) meat wash water precipitate was hydrolyzed using bromelain. Experiments were carried out to determine optimum conditions for temperature, enzyme concentration and hydrolysis time using response surface methodology (RSM) based on a central composite rotatable design (CCRD) to obtain the highest value of nitrogen content (NC) and degree of hydrolysis (DH). Results revealed that the optimum conditions for temperature, enzyme concentration and hydrolysis time were 33.7°C, 1.45% (E/S) and 28.42 hrs, respectively. At the optimum condition, hydrolysis of cockle meat wash water precipitate using bromelain resulted in a NC of 0.6% and DH of 48%. The NC and DH were significantly influenced by temperature, enzyme concentration and hydrolysis time. When the bromelain concentration, hydrolysis time and temperature were increased, the values of NC and DH also increased. The hydrolysate produced contained flavor compounds found in clam and oyster which were 3-methylbutanol and 1-pentanol. The compound 3-MCPD was not found in the hydrolysate.
A total of sixty V. parahaemolyticus strains isolated from local cockles (Anadara granosa) were investigated by their antibiotic resistance patterns and plasmid profiles. The isolates showed multiple resistances towards most of the antibiotics tested. All strains of V. parahaemolyticus isolated harbored 1-3 plasmids, with sizes ranging from 2.7 to 54 kb. All V. parahaemolyticus strains showed high multiple antibiotics in frequencies of 0.58 – 0.94 indicating that the strains were derived from high-risk sources. In addition, no particular plasmid profile was predictive of a particular pattern of antibiotic susceptibility. These findings are essential because of the suggested involvement of seafood especially shellfish and environment in transmission of this pathogen to human. Thus, indicating that seafood may be a source of food- acquired antibiotic resistant bacteria to consumer.
Pathogenic Vibrio parahaemolyticus is one of the leading causes of bacterial gastroenteritis in many countries. Among the strains examined, 36 RAPD-types were found when amplified with primers OPA8 and OPA10. The analysis shows the majority of V. parahaemolyticus isolates originated from seafood were branched into four major clusters at 18.2%, 20.7% 34% and 3.4% similarity levels. This suggests that there is potential for a single strain to be distributed widely within a population and there also potential for multiple contaminating strains of different clonal lineages to be present within the same population. Optimum temperature (37ºC) was the highest and stable formation of biofilm. The total percentage of biofilm formation at 37ºC was 33.33% for each of weak, moderate and strong biofilm producers. Room temperature produces 61.1% of weak biofilm producer, while 13. 89% for moderate biofilm producers and produce 25% of strong biofilm. While a total of 91.67% weak biofilm producers at 4ºC and 8:33% for room temperature and no growth of strong biofilm. Upon analysis, strong biofilm was tracked from the largest group at 37°C and room temperature which produce 27.27% of strong biofilm producer respectively. Interestingly, they are derived from cockles.
Hepatitis A is a liver infection caused by the hepatitis A virus (HAV). Outbreaks of hepatitis A have been linked to the consumption of both raw and cooked shellfish. These outbreaks could induce a public confidence problem over shellfish safety and may result in important economic losses for the seafood industry. The work presented in this study investigated the presence of HAV in shellfish from Peninsular Malaysia. A total of 365 of cultured and wild shellfish from 36 sampling locations located throughout Peninsular Malaysia were examined using a commercial nucleic acid extraction and reverse transcription -polymerase chain reaction (RT-PCR) kit. HAV was not detected in almost all of the shellfish samples xamined. Only one cockle sample from Changkat, Seberang Perai was positive for HAV. The results suggest the absence of HAV or very low amount of HAV viral particles in most of the shellfish examined.
In this study, RAPD-PCR and ERIC-PCR were used to study the epidemiology of V. parahaemolyticus isolated from cockles in Padang, Indonesia. The Gold Oligo OPAR3 primer produced bands ranged from 1-8 with sizes from 0.2 – 5.0 kb and the Gold Oligo OPAR8 primer produced 1-7 bands with sizes 0.7 – 1.5 kb. Both primers produced twenty five RAPD patterns with a few isolates failed to produce any products. Based on phylogenetic dendrogram, all the isolates can be divided into 6 major clusters with similarity between 0 to 52%. For the ERIC primer, it produced bands ranged from 3-15 with sizes from 0.1 – 5.0 kb and twenty seven different ERIC patterns. Construction of the phylogenetic dendogram showed the isolates can be divided into 4 major clusters with similarity between 56 to 86%. The high diversity of both processes may be due to the multiple contamination sources of V. parahaemolyticus.
Aimed of this study was to determine the presence of Vibrio cholerae in cockles (Anadara granosa)
from different coasts in Malaysia and to measure the biosafety of V. cholerae in raw cockles at wet market in Malaysia using the polymerase chain reaction (PCR) in combination with the most probable number (MPN) method. A total of 100 samples from 4 different wet markets in the West and East were examined for the presence of V. cholerae. The prevalence of V. cholerae between the two coasts was not significant different. In fact, the 74% of samples from West coast area was found positive while the 69% for samples collected in the East coast. West coast samples showed a prevalence of 60% for the wet market A=, 64% for B=, 88% for C= and 84% for the market D); East coast samples showed the same percentage with 72% for the wet markets E, F and H, followed by wet market G with 60%.With the MPN-PCR method, using 80 samples of raw cockles obtained from 4 wet markets, the occurrence of V. cholerae detected was of 95%. The frequency of V. cholerae in raw cockles obtained from wet market I and L was higher (100%) compared to other wet market (Wet market B=, 90%; Wet market C=, 95%).The density of V. cholerae detected in all samples ranged from 24000 MPN/g, but most of the samples (24 samples) were in category >24000 MPN/g concentration. V. cholerae was present in raw cockles in higher number. Hence, these results demonstrate the presence of pathogenic V.cholerae in cockles harvested and reveal the potential risk of illness associated with their consumption. This study will be the first biosafety assessment of V. choleare in raw cockles in Malaysia and it will provide significant insights about Malaysian scenario.
This study aimed to investigate the prevalence and antibiogram of Vibrio parahaemolyticus in processed bivalve molluscs in Kuala Terengganu. A total of 80 seafood samples, namely mussels (n=20), carpet clams (n=20), cockles (n=20) and scallops (n=20), were subjected to PCR and conventional plating method for the detection of V. parahaemolyticus. V. parahaemolyticus was found in green mussels (55%), carpet clam (80%), cockles (40%) and scallops (55%). Fifty-five V. parahaemolyticus isolates were subjected to 9 types antibiotic sensitivity test using discs diffusion method. All isolates were susceptible to Tetracycline and Gentamycin. Isolates showed high resistance towards Vancomycin (52.73%), Penicillin (45.45%) and Amplicillin (32.73%). Resistance towards Amikacin, Ciprofloxacin and Norfloxacin were found to be 1.82%. It can be concluded that local bivalve molluscs were contaminated with V. parahaemolyticus and isolates showed resistance towards certain antibiotics. Therefore, consumption of raw or semi-cooked bivalve molluscs is not advisable.