Vibrio parahaemolyticus is a foodborne pathogen that is frequently isolated from a variety of seafood. To control this pathogenic Vibrio spp., the implementation of bacteriophages in aquaculture and food industries have shown a promising alternative to antibiotics. In this study, six bacteriophages isolated from the seafood samples demonstrated a narrow host range specificity that infecting only the V. parahaemolyticus strains. Morphological analysis revealed that bacteriophages Vp33, Vp22, Vp21, and Vp02 belong to the Podoviridae family, while bacteriophages Vp08 and Vp11 were categorized into the Siphoviridae family. All bacteriophages were composed of DNA genome and showed distinctive restriction fragment length polymorphism. The optimal MOI for bacteriophage propagation was determined to be 0.001 to 1. One-step growth curve revealed that the latent period ranged from 10 to 20 min, and the burst size of bacteriophage was approximately 17 to 51 PFU/cell. The influence of temperature and pH levels on the stability of bacteriophages showed that all bacteriophages were optimally stable over a wide range of temperatures and pH levels. In vitro lytic activity of all bacteriophages demonstrated to have a significant effect against V. parahaemolyticus. Besides, the application of a bacteriophage cocktail instead of a single bacteriophage suspension was observed to have a better efficiency to control the growth of V. parahaemolyticus. Results from this study provided a basic understanding of the physiological and biological properties of the isolated bacteriophages before it can be readily used as a biocontrol agent against the growth of V. parahaemolyticus.
Industrial enzymes are important for various biotechnological applications. Currently, the diversity of industrial enzymes-producing marine bacteria from Malaysia remains mostly unknown. This study investigated the diversity of industrial enzyme-producing marine bacteria from culture collections at the Institute of Marine Biotechnology, Universiti Malaysia Terengganu. Out of 200 bacterial isolates revived, 163 bacteria isolate were successfully growth. Marine bacteria produced enzymes with total scoring higher than four were selected for molecular identification using 16S rDNA. About 161 bacteria isolate secreted amylase (68.7 %), lipase (88.3 %) and protease (68.7 %). The phylogenetic analysis led to the identification of three major phyla, namely Proteobacteria, Firmicutes and Bacteroidetes. These phyla were differentiated into nine genera consisted of Bacillus, Chryseomicrobium, Photobacterium, Pseudoalteromonas, Ruegeria, Shewanella, Solibacillus, Tenacibaculum and Vibrio. Genetic variation was more likely to occur within similar marine bacteria species. The microbial community was found to affect the production of industrial enzymes and the diversity of marine bacteria.
Serving raw oysters with lemon juice is a delicacy in many restaurants in
Malaysia. Oysters (Crassostrea virginica) live in the seacoast and they share the same
environment as Vibrio parahaemolyticus. Consumption of raw oysters contaminated with V.
parahaemolyticus can lead to severe gastroenteritis. A study was performed to determine
whether lemon (Citrus limon) juice is able to inhibit the growth of V. parahaemolyticus after
being inoculated in raw oysters. Methods: Frozen oysters bought from a local supplier
weighing 6 g each were minced and placed in two bottles using sterile technique.
Approximately 1 ml of 107 CFU of V. parahaemolyticus (ATCC strain 17802) was added and
mixed in both bottles. The mixture was treated with 1 ml of lemon juice in only one of the
bottles and the other bottle served as a control. At every 30 s intervals for 2 min, 1 g of the
sample was taken for enumeration of viable cells onto thiosulphate citrate bile salt sucrose
(TCBS). Results: After 30 s of treatment with the lemon juice, it was observed that the
number of colonies in the treated samples reduced from 7 Log to 3 Log. Subsequently, no
viable V. parahaemolyticus was seen. It was also observed that there were 3 Log reductions
of V. parahaemolyticus after 30 s in untreated samples, however the number of colonies
remained stable until the end of the experiment. Conclusion: This study therefore shows
that lemon juice has some antimicrobial effect on V. parahaemolyticus in raw oysters.
Biohydrogen is one of the most suitable clean energy sources for sustaining a fossil fuel independent society. The use of both land and ocean bioresources as feedstocks show great potential in maximizing biohydrogen production, but sodium ion is one of the main obstacles in efficient bacterial biohydrogen production. Vibrio tritonius strain AM2 can perform efficient hydrogen production with a molar yield of 1.7 mol H2/mol mannitol, which corresponds to 85% theoretical molar yield of H2 production, under saline conditions. With a view to maximizing the hydrogen production using marine biomass, it is important to accumulate knowledge on the effects of salts on the hydrogen production kinetics. Here, we show the kinetics in batch hydrogen production of V. tritonius strain AM2 to investigate the response to various NaCl concentrations. The modified Han-Levenspiel model reveals that salt inhibition in hydrogen production using V. tritonius starts precisely at the point where 10.2 g/L of NaCl is added, and is critically inhibited at 46 g/L. NaCl concentration greatly affects the substrate consumption which in turn affects both growth and hydrogen production. The NaCl-dependent behavior of fermentative hydrogen production of V. tritonius compared to that of Escherichia coli JCM 1649 reveals the marine-adapted fermentative hydrogen production system in V. tritonius. V. tritonius AM2 is capable of producing hydrogen from seaweed carbohydrate under a wide range of NaCl concentrations (5 to 46 g/L). The optimal salt concentration producing the highest levels of hydrogen, optimal substrate consumption and highest molar hydrogen yield is at 10 g/L NaCl (1.0% (w/v)).
Vibrio cholerae still represents a significant threat to human health worldwide despite the advances in hygiene, consumer knowledge, food treatment and food processing. In Malaysia, statistics in year 2009 have shown that among the food and water borne diseases, food poisoning has the highest incidence rate of 36.17 per 100,000 populations and with a mortality rate of 0.01 per 100,000 populations. In this study, 22 seafood samples comprising of fish, squid, crustacean and mollusks purchased from wet market and supermarket were analyzed. The Most Probable Number (MPN) and real time PCR was used to enumerate the Vibrio cholerae in seafood sample. The results showed that MPN-real time PCR of the samples from wet market had a maximum of >1100 MPN/g compare to 93 MPN/g enumerated from the MPN plate. The MPN-real time PCR in the samples from supermarket indicated 290 MPN/g as compared to 240 MPN/g enumerated from the MPN plate. The standard curves showed that there was a good linear correlation between the Ct values. The minimum level of detection of Vibrio cholerae standard DNA at targeted gene was 3 x 10-5 ng/μl.
Molecular typing methods have been widely applied for many purposes. In this study, such methods were adopted as DNA fingerprinting tools to determine the origin and divergence of virulent Vibrio parahaemolyticus strains found in local seafood. Although not all strain carry virulent tdh and trh gene, increasing prevalence demands an effective fingerprinting scheme which can constantly monitor and trace the sources of such emerging food pathogens. By using ERIC-, RAPD-, and BOX-PCR methods, 33 Vibrio parahaemolyticus isolates from local Malaysia bloody clam (Anadara granosa) and Lala (Orbicularia orbiculata) with confirmed presence of tdh and trh gene were characterised, followed by determination of clonal relatedness among virulent strains using cluster analysis and discriminatory index. This study also involved application of Immunomagnetic Separation (IMS) Method which significantly improved the specificity of strain isolation. Cluster analysis using Unweighted Pair Group Mathematical Averaging (UPGMA) and Dice Coefficient shown clustering according to isolation food source, IMS level and haemolysin gene possessed. Nevertheless, different DNA fingerprinting methods generated different clustering at different similarity cut-off percentage, regardless as individual or as composite dendrograms. ERIC- and RAPD-PCR composite fingerprinting relatively shown the highest discriminatory index at following similarity cutoff percentage: 0.68 at 50%; 0.83 at 65%; and 0.93 at 75%. Discriminatory power increased with similarity cut-off percentage. However, result also suggested that BOX-PCR might be an effective fingerprinting tool, as it generated three clusters with no single-colony isolate at 70% similarity cut-off. This study not only achieved its objective to determine clonal relatedness among virulent strains from local seafood via characterisation, but also speculated the best possible combination of molecular typing methods to effectively do so.
Vibrio parahaemolyticus is a foodborne pathogen and their human infection is regularly associated with the consumption of raw or undercooked seafood and contaminated water supplies. Many conventional biochemical identification and confirmation procedures are performed to detect the presence of this pathogen, both from seafood or environmental samples. However, these procedures not only require two or more days to complete, they do not have the capabilities to determine the number of V. parahaemolyticus cells in any given samples. Thus, in this study we describe the development of a rapid SYBR green based real-time PCR assay, targeting the thermo labile (tl) gene of V. parahaemolyticus for the detection and enumeration of this bacterium from seafood and environmental samples. We report that the real-time PCR assay and the primers designed are highly specific, and only generated the desired amplicons with V. parahaemolyticus DNA samples against other bacteria and fungi species. Our assay is also highly sensitive, and, is able to detect V. parahaemolyticus with high coefficient values in concentrations as low as 1.0 pg/μl DNA for pure genomic DNA solutions and 10 cells/ml in serially diluted cell suspension and spiked samples. This assay can be completed in less than 3 hours and may be used as a tool for rapid determination of V. parahaemolyticus densities in the food industries, environmental risk assessment and for clinical diagnostics purposes.
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.
This study was undertaken to characterize the antibiotic resistance and randomly amplified polymorphic DNA (RAPD) profiles of Vibrio parahaemolyticus isolates from raw vegetable samples. A total of 46 isolates of V. parahaemolyticus recovered from raw vegetables samples and were confirmed by PCR were analyzed in this study. Most of the isolates were resistant to nalidixic acid (93.48%) and were the least resistant towards imipinem (4.35%). The MAR index results also demonstrated high individual and multiple resistances to antibiotics among the isolates. From the RAPD analysis, the size for RAPD fragments generated ranged from 250 bp to 1,500 bp, with most of the strains contained three major gene fragments of 350, 1,000 and 1,350 bp. The RAPD profiles revealed a high level of DNA sequence diversity within the isolates. Antibiotic resistance and RAPD proved to be effective tools in characterizing and differentiating the V. parahaemolyticus strains.
A total of 32 clinical strains of Vibrio cholerae, including members of the 01 and 0139 serogroup
were collected from Klang, Selangor; Penang Island; Samarahan, Sarawak and Miri, Sarawak in Malaysia. In general, all the isolates except the 0139 serotype expressed low resistance to all the antibiotics tested with their Multiple Antibiotic Resistance (MAR) indices ranged from 0.10 to 0.48. The presence of ctx gene that encoded the cholera toxin was confirmed in all these clinical isolates by polymerase chain reaction. The results from the RAPD-PCR were analyzed using the RAPDistance software (Version 1.04). From the dendrogram generated, two main groups were observed which were subdivided into two clusters each. The Selangor’s isolates and the 0139 Penang’s isolates formed one group whereas the Samarahan, Sarawak isolates and the Miri, Sarawak isolates made up the other group, thus delineating their different sources of origin based on their geographical location.
Numerous prevalence studies and outbreaks of Vibrio parahaemolyticus infection have been extensively reported in shellfish and crustaceans. Information on the quantitative detection of V. parahaemolyticus in finfish species is limited. In this study, short mackerels (Rastrelliger brachysoma) obtained from different retail marketplaces were monitored with the presence of total and pathogenic strains of V. parahaemolyticus. Out of 130 short mackerel samples, 116 (89.2%) were detected with the presence of total V. parahaemolyticus and microbial loads of total V. parahaemolyticus ranging from <3 to >10(5) MPN/g. Prevalence of total V. parahaemolyticus was found highest in wet markets (95.2%) followed by minimarkets (89.1%) and hypermarkets (83.3%). Pathogenic V. parahaemolyticus strains (tdh+ and/or trh+) were detected in 16.2% (21 of 130) of short mackerel samples. The density of tdh+ V. parahaemolyticus strains were examined ranging from 3.6 to >10(5) MPN/g and microbial loads of V. parahaemolyticus strains positive for both tdh and trh were found ranging from 300 to 740 MPN/g. On the other hand, antibiotic susceptibility profiles of V. parahaemolyticus strains isolated from short mackerels were determined through disc diffusion method in this study. Assessment of antimicrobial susceptibility profile of V. parahaemolyticus revealed majority of the isolates were highly susceptible to ampicillin sulbactam, meropenem, ceftazidime, and imipenem, but resistant to penicillin G and ampicillin. Two isolates (2.99%) exhibited the highest multiple antibiotic resistance (MAR) index value of 0.41 which shown resistance to 7 antibiotics. Results of the present study demonstrated that the occurrence of pathogenic V. parahaemolyticus strains in short mackerels and multidrug resistance of V. parahaemolyticus isolates could be a potential public health concerns to the consumer. Furthermore, prevalence data attained from the current study can be further used to develop a microbial risk assessment model to estimate health risks associated with the consumption of short mackerels contaminated with pathogenic V. parahaemolyticus.
Serving raw oysters with lemon juice is a delicacy in many restaurants in
Malaysia. Oysters (Crassostrea virginica) live in the seacoast and they share the same
environment as Vibrio parahaemolyticus. Consumption of raw oysters contaminated with V.
parahaemolyticus can lead to severe gastroenteritis. A study was performed to determine
whether lemon (Citrus limon) juice is able to inhibit the growth of V. parahaemolyticus after
being inoculated in raw oysters. Methods: Frozen oysters bought from a local supplier
weighing 6 g each were minced and placed in two bottles using sterile technique.
Approximately 1 ml of 107 CFU of V. parahaemolyticus (ATCC strain 17802) was added and
mixed in both bottles. The mixture was treated with 1 ml of lemon juice in only one of the
bottles and the other bottle served as a control. At every 30 s intervals for 2 min, 1 g of the
sample was taken for enumeration of viable cells onto thiosulphate citrate bile salt sucrose
(TCBS). Results: After 30 s of treatment with the lemon juice, it was observed that the
number of colonies in the treated samples reduced from 7 Log to 3 Log. Subsequently, no
viable V. parahaemolyticus was seen. It was also observed that there were 3 Log reductions
of V. parahaemolyticus after 30 s in untreated samples, however the number of colonies
remained stable until the end of the experiment. Conclusion: This study therefore shows
that lemon juice has some antimicrobial effect on V. parahaemolyticus in raw oysters.
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.
Information on the abiotic stress tolerance and ice-ice disease resistance properties of tissue-cultured Kappaphycus alvarezii is scarce and can pose a big hurdle to a wider use of tissue-cultured seaweed in the industry. Here, we reported on a study of seaweed-associated bacteria diversity in farmed and tissue-cultured K. alvarezii, and ice-ice disease resistance and elevated growth temperature tolerance of tissue-cultured K. alvarezii in laboratory conditions. A total of 40 endophytic seaweed-associated bacteria strains were isolated from 4 types of K. alvarezii samples based on their colony morphologies, Gram staining properties and 16S rRNA gene sequences. Bacteria strains isolated were found to belong to Alteromonas sp., Aestuariibacter sp., Idiomarina sp., Jejuia sp., Halomonas sp., Primorskyibacter sp., Pseudoalteromonas sp., Ruegeria sp., Terasakiella sp., Thalassospira sp. and Vibrio sp. Vibrio alginolyticus strain ABI-TU15 isolated in this study showed agar-degrading property when analyzed using agar depression assay. Disease resistance assay was performed by infecting healthy K. alvarezii with 105 cells/mL Vibrio sp. ABI-TU15. Severe ice-ice disease symptoms were detected in farmed seaweeds compared to the tissue-cultured K. alvarezii. Besides disease resistance, tissue-cultured K. alvarezii showed better tolerance to the elevated growth temperatures of 30 and 35 °C. In conclusion, our overall data suggests that tissue-cultured K. alvarezii exhibited better growth performance than farmed seaweeds when exposed to elevated growth temperature and ice-ice disease-causing agent.
This goal of this study was to investigate the presence of Vibrio cholerae in street food,
namely satar and otak-otak, using Loop-Mediated Isothermal Amplification (LAMP),
multiplex Polymerase Chain Reaction (mPCR) and conventional plating on Thiosulphate
Citrate Bile-Salt Sucrose (TCBS) agar methods. A total of 78 satar and 35 otak-otak were
purchased from different districts of Terengganu (Besut, Setiu, Kuala Terengganu and
Kemaman). V. cholerae was found in satar with LAMP (10.3%), mPCR (10.3%) and
plating (0%). No V. cholerae was found in otak-otak using the three methods. This might
be due to V. cholerae able to survive in satar after grilling due to its thickness which may
contribute to undercooking. This study concluded that low presence of V. cholerae in satar
and otak-otak can be detected by molecular methods but not the conventional plating
method. LAMP assay is a useful tool for rapid detection of pathogens in food due to its
simplicity, highly sensitive and visual interpretation capability. Though the prevalence of
V. cholerae was low in the samples, proper handling of this food will help in reducing the
risk of acquiring infection from V. cholerae in contaminated samples.
Aquaculture production of the Pacific white shrimp is the largest in the world for crustacean species. Crucial to the sustainable global production of this important seafood species is a fundamental understanding of the shrimp gut microbiota and its relationship to the microbial ecology of shrimp pond. This is especially true, given the recently recognized role of beneficial microbes in promoting shrimp nutrient intake and in conferring resistance against pathogens. Unfortunately, aquaculture-related microbiome studies are scarce in Southeast Asia countries despite the severe impact of early mortality syndrome outbreaks on shrimp production in the region. In this study, we employed the 16S rRNA amplicon (V3-V4 region) sequencing and amplicon sequence variants (ASV) method to investigate the microbial diversity of shrimp guts and pond water samples collected from aquaculture farms located in Malaysia and Vietnam. Substantial differences in the pond microbiota were observed between countries with the presence and absence of several taxa extending to the family level. Microbial diversity of the shrimp gut was found to be generally lower than that of the pond environments with a few ubiquitous genera representing a majority of the shrimp gut microbial diversity such as Vibrio and Photobacterium, indicating host-specific selection of microbial species. Given the high sequence conservation of the 16S rRNA gene, we assessed its veracity at distinguishing Vibrio species based on nucleotide alignment against type strain reference sequences and demonstrated the utility of ASV approach in uncovering a wider diversity of Vibrio species compared to the conventional OTU clustering approach.
This study was carried out to know the bacteria population density in the blood cockle (Anadara granosa) and green lipped mussel (Perna viridis), to analyse the bacteria resistance towards antibiotics and antimicrobial activity of isolates against selected pathogen. Samples of blood cockle and green lipped mussel were obtained from five areas in Kedah and Negeri Sembilan. Bacterial population densities in mussels and cockles were 3 x 102 - 8 x 108 cFulmL and 5 x 102 - 5 x 108 cFulmL, respectively. A total of 162 isolates were obtained, of which 131 isolates were from mussels and 31 isolates were from cockles. Vibrio sp. was the most dominant genus in both types of samples. Antibiotic testing of all isolates showed most were resistant to Penicillin (10 U) and most were sensitive to Ciprofloxacin (5 Jig). Most isolates (160/162) showed resistance to at least two antibiotics and 10 isolates were resistant to more than five antibiotics. Multiple antibiotic resistance indices (MAR) were calculated based on the antibiotic resistance results. Most isolates had a MAR index value of 02 which indicated the isolates were not contaminated with antibiotic residues. The highest index value was 0 .7 . Fifteen out of 39 isolates which produced beta-lactamase enzyme were tested for antimicrobial activity against selected pathogen. Results indicated that antimicrobial activity were varies among the isolates. Isolate smii-Ip produced antimicrobial activity against six out of the nine tested pathogen and none of the isolates active against Pseudomonas mirabilis.
Vibrio parahaemolyticus is a foodborne bacterial pathogen that may cause gastroenteritis in humans through the consumption of seafood contaminated with this microorganism. The emergence of antimicrobial and multidrug-resistant bacteria is another serious public health threat worldwide. In this study, the prevalence and antibiotic susceptibility test of V. parahaemolyticus in blood clams, shrimps, surf clams, and squids were determined. The overall prevalence of V. parahaemolyticus in seafood was 85.71% (120/140), consisting of 91.43% (32/35) in blood clam, 88.57% (31/35) in shrimps, 82.86% (29/35) in surf clams, and 80% (28/35) in squids. The majority of V. parahaemolyticus isolates from the seafood samples were found to be susceptible to most antibiotics except ampicillin, cefazolin, and penicillin. The MAR indices of V. parahaemolyticus isolates ranged from 0.04 to 0.71 and about 90.83% of isolates were found resistant to more than one antibiotic. The high prevalence of V. parahaemolyticus in seafood and multidrug-resistant isolates detected in this study could pose a potential risk to human health and hence appropriate control methods should be in place to minimize the potential contamination and prevent the emergence of antibiotic resistance.
This study focuses in investigating the fatty acid contents of surviving infected hybrid grouper fed with oleic acid immunostimulant. After a 6-week feeding trial, Epinephelus fuscoguttatus × Epinephelus lanceolatus fingerlings were infected with Vibrio vulnificus. One week after bacterial challenge, fish oil was extracted from body tissue of surviving infected fingerlings using the Soxhlet extraction method. The extracted samples were then sent for GC-MS analysis. The raw GC-MS data were analyzed using software programs and databases (i.e., MetaboAnalyst, SIMCA-P, NIST Library, and KEGG). A total of 39 metabolites were putatively identified, with 18 metabolites derived from the fatty acid group. Our further analysis revealed that most metabolites were highly abundant in the oleic acid dietary samples, including oleic acid (4.56%), 5,8,11-eicosatrienoic acid (3.45%), n-hexadecenoic acid (3.34%), cis-erucic acid (2.76%), and 9-octadecenoic acid (2.5%). Worthy of note, we observed a greater abundance of α-linoleic acid (15.57%) in the control diet samples than in the oleic acid diet samples (14.59%) with no significant difference in their results. The results obtained from this study revealed that surviving infected hybrid grouper expressed more immune-related fatty acids due to the effect of oleic acid immunostimulant. Therefore, in this study, we propose oleic acid as a potential immunostimulant in enhancing fish immunity in aquaculture industry.
Fresh raw shrimps were dipped for 10, 20, and 30 min at room temperature (25°C ± 1°C) in lactic acid (LA; 1.5%, 3.0%, v/v) to evaluate their antipathogenic effects against Vibrio cholerae, Vibrio parahaemolyticus, Salmonella entreitidis, and Escherichia coli O157:H7 inoculated at a level of 10(5) CFU/g. Significant reductions in the population of all these pathogenic bacteria were recorded after dipping treatments, which were correlated to the corresponding LA concentrations and treatment time. With respect to the microbial quality, 3.0% LA treatment for 10 min was acceptable in reducing the pathogenic bacteria. Additionally, sensory evaluation results revealed a 10-min dip in 3.0% LA to be more acceptable organoleptically compared with 20 and 30 min of treatments. Results of the present study are envisaged to be useful for commercial applications for effective decontamination of shrimp.