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.
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.
Vibrio parahaemolyticus is one of the most widely recognized pathogenic Vibrio species due to numerous outbreaks and its’ wide occurrence in marine environment. In this study, 32 isolates of V. parahaemolyticus isolated from cockles were tested for sensitivity to 16 antibiotics and the presence of plasmids. All the isolates were multi-resistance, defined as resistant to atleast three different antibiotics with multiple antibiotic resistance indexes ranging from 0.31 to 0.69, indicating the isolates originate from high risk sources of contamination where antibiotics are often used. In the plasmid profiling test, only 15 isolates (47%) harbored plasmid DNA, which ranged in size from 2.7 to 56.2 kb, separating the isolates into 14 plasmid profiles. Hence, food contaminated with antibiotic resistant V. parahaemolyticus could be a major threat to public health due to the distinct possibility that they can be a significant reservoir of genes encoding antibiotic resistance determinants that can be transferred intra or interspecies. As in many developing countries, raw food hygiene and antimicrobial resistance epidemiology is still in the infancy stage in the locality of the study and thus our data provide a current baseline profile of antimicrobial resistance and plasmid of V. parahaemolyticusfrom cockles in Padang, Indonesia.
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.
The cockle, Anadara granosa, was experimentally exposed to low (0.1 mg/L) and sublethal (1.0 mg/L) doses of copper (Cu) for a period of 24 hrs. Significant increase in Cu concentrations in whole tissues and hepatopancreas compared to control animals were observed. In order to study the effect of copper exposure at molecular levels, a subtractive cDNA library was constructed from the hepatopancreas of cockles exposed to 1.0 mg/L Cu. Screening of the subtractive cDNA library using reverse northern analysis resulted in several differentially expressed genes, including one that codes for metallothionein (MT). The complete coding sequence of the MT gene (designated as AnaMT2) reveals an open reading frame of 234 bp in length that encodes a 77 amino acid polypeptide as revealed by the deduced amino acid composition. Although showing similarities with other molluscan MTs, AnaMT2 can be distinguished by its lower glycine and higher asparagine and proline content. Expression analysis of the AnaMT2 by northern analysis indicated higher mRNA level in cockle exposed to 1.0 mg/L Cu and was undetectable in those treated with 0.1 mg/L. This suggests that AnaMT2 represents a primarily inducible MT not highly expressed under basal conditions.
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].
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.
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 concentration of carcinogenic poly aromatic hydrocarbons (c-PAHs) present in water and sediment of Klang Strait as well as in the edible tissue of blood cockle (Anadara granosa) was investigated. The human health risk of c-PAHs was assessed in accordance with the standards of the United States Environmental Protection Agency (US EPA). The cancer risks of c-PAHs to human are expected to occur through the consumption of blood cockles or via gastrointestinal exposure to polluted sediments and water in Kalng Strait. The non-carcinogenic risks that are associated with multiple pathways based on ingestion rate and contact rates with water were higher than the US EPA safe level at almost all stations, but the non-carcinogenic risks for eating blood cockle was below the level of US EPA concern. A high correlation between concentrations of c-PAHs in different matrices showed that the bioaccumulation of c-PAHs by blood cockles could be regarded as a potential health hazard for the consumers.
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.
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.
Genomic DNA of Vibrio parahaemolyticus were characterized by antibiotic resistance, enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) and random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) analysis. These isolates originated from 3 distantly locations of Selangor, Negeri Sembilan and Melaka (East coastal areas), Malaysia. A total of 44 (n = 44) of tentatively V. parahaemolyticus were also examined for the presence of toxR, tdh and trh gene. Of 44 isolates, 37 were positive towards toxR gene; while, none were positive to tdh and trh gene. Antibiotic resistance analysis showed the V. parahaemolyticus isolates were highly resistant to bacitracin (92%, 34/37) and penicillin (89%, 33/37) followed by resistance towards ampicillin (68%, 25/37), cefuroxime (38%, 14/37), amikacin (6%, 2/37) and ceftazidime (14%, 5/37). None of the V. parahaemolyticus isolates were resistant towards chloramphenicol, ciprofloxacin, ceftriaxone, enrofloxacin, norfloxacin, streptomycin and vancomycin. Antibiogram patterns exhibited, 9 patterns and phenotypically less heterogenous when compared to PCR-based techniques using ERIC- and RAPD-PCR. The results of the ERIC- and RAPD-PCR were analyzed using GelCompare software. ERIC-PCR with primers ERIC1R and ERIC2 discriminated the V. parahaemolyticus isolates into 6 clusters and 21 single isolates at a similarity level of 80%. While, RAPD-PCR with primer Gen8 discriminated the V. parahaemolyticus isolates into 11 clusters and 10 single isolates and Gen9 into 8 clusters and 16 single isolates at the same similarity level examined. Results in the presence study demonstrated combination of phenotypically and genotypically methods show a wide heterogeneity among cockle isolates of V. parahaemolyticus.
This study evaluated microplastic (MP) abundances and physico-chemical characteristics in sediments and Anadara granosa along the East Java coast and their health implications. Fibers (74 %) dominated sediment MPs at south coast, while fragments (49-61 %) dominated north coast. Fiber (43-52 %) is the predominant MP in cockle tissues in all locations. Most MP in sediments (31-47 %) and cockle tissues (41-49 %) is black. The majority of microplastics (100-1500 μm) are found in sediment (73-90 %), and cockles (77-79 %). Very weak correlations found between the amount of MP and the length of the cockle shell. However, Spearman correlation shows that as the amount of MP in sediment increases, so does the amount of MP in cockle tissue. Each year, individuals of varying ages consume an average of 20,800 to 156,000 MP items. Cockles contain plasticizer components and microplastic polymers which are classified from II to V regarding of hazard levels, with V being the most hazardous.
This study was conducted to determine the proximate composition and four micronutrients (Cd, Cu, Mn and Zn) of Indian Mackerel (Rastrelliger kanagurta), Eel (Monopterus albus) and Cockle (Anadara granosa). All fish and shellfish were purchased from local fish market in Kuantan city. All samples of each species were mixed and divided into two groups based on random selection. Each group were again divided into 3 sub-groups which were considered as replications. The first group were kept uncooked. The second group were fried in a beaker of 400 mL palm cooking oil capacity at a temperature approximately of 180 degrees C for a 15 min period. Both raw and fried samples were analysed following standard methods to determine protein, lipid, ash, moisture, carbohydrate, Cd, Cu, Mn and Zn contents. Results showed that protein content was higher in Indian mackerel and eel than cockle while overall Cd, Cu, Mn and Zn contents were higher in cockle than Indian mackerel and eel. Therefore, fish is better than shellfish in the nutritional point of view. Fried fish and shellfish had very high fat content. Therefore, frying cannot be recommended to prepare a healthy diet. More research is needed including all cooking methods of fish to know the nutritional changes by each cooking method. Fish contains many important fatty acids and amino acids which might be lost during frying. Therefore, future study should include the effects of different cooking methods on amino acids and fatty acids compositions of fish and shellfish.
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 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.
Examination of the impact of Diuron contamination on blood cockles (Tegillarca granosa) was conducted by combining field screening at three sampling events and a toxicity test. Diuron was extracted using the liquid-liquid extraction (LLE) technique and analyzed using HPLC-UV. The median lethal concentration (LC50) of Diuron on T. granosa was tested under a 72-h exposure. Diuron in water samples ranged from not detected (ND) to 3910 ppb, which was the highest concentration detected in samples after the irrigation water was discharged from the paddy plantation. Diuron was not detected in sediment samples. Mortality of T. granosa ranged from 4.74 to 38.33% with the highest percentages recorded after the release of the irrigation water. The LC50 value of Diuron was 1.84 ppm. This study suggests that irrigation water from paddy plantation that drifts to coastal areas containing Diuron harms T. granosa at the study area.
The concentration of carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) present in the sediment and water of Peninsular Malaysia as well as in the cockle Anadara granosa was investigated. Samples were extracted and analysed with gas chromatography-mass spectrometry. The concentrations of total carcinogenic polycyclic aromatic hydrocarbons (t-PAHs) were measured between 0.80 +/- 0.04 to 162.96 +/- 14.74 ng/g wet weight (ww) in sediment, between 21.85 +/- 2.18 to 76.2 +/- 10.82 ng/L in water samples and between 3.34 +/- 0.77 to 46.85 +/- 5.50 ng/g ww in the cockle tissue. The risk assessment of probable human carcinogens in the Group B2 PAHs was calculated and assessed in accordance with the standards of the United States Environmental Protection Agency (US EPA). Case I in the toxicity assessment analysed the cancer risk to consumers of Malaysian blood cockle. Case II assessed the risk of cancer from exposure to PAHs from multiple pathways. The average cancer risk of case I and case II were found to be classifiable as unsafe according to the US EPA standard. The cancer risk due to c-PAHs acquired by the ingestion of blood cockle was (8.82 +/- 0.54) x 10-6 to (2.67 +/- 0.06) x 10(-2), higher than the US EPA risk management criterion. The non-cancer risks associated with multiple pathways in Kuala Gula, Kuala Juru and Kuala Perlis were higher than the U.S. EPA safe level, but the non-cancer risk for eating blood cockle was below the level of U.S. EPA concern.
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.