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Differential elimination of indicator bacteria and pathogenic Vibrio sp. from eastern oysters (Crassostrea virginica Gmelin, 1791) in a commercial controlled purification facility in Maine
... Shellfish harvesting from polluted (category B and C) areas is allowed when shellfish undergo previous treatment, before being commercialized. Bivalve molluscs harvested from growing areas exceeding Category A standards can be placed on the market for human consumption following controlled self-purification in tanks of clean seawater (commercial depuration), prolonged relaying in clean seawater or commercial heat treatment or processing by any other acceptable method (Jones, Howell, & O'Neill, 1991;Lees, 2000;Murchie et al., 2005). Shellfish from category C areas may, if necessary, be depurated before commercialization. ...
... The final product is sealed, labelled for traceability and commercialized giving distributors and consumers the confidence of a safe certified product (Jones et al., 1991;Lees, 2000;Shumway & Rodrick, 2009). ...
... In many countries, these standard guidelines become very important for the regulation of shellfish harvesting and routine monitoring of overlying waters (Jones et al., 1991). However, when authorized shell fishing harvesting areas decrease, non-ethical activities such as illegal harvesting from polluted and restricted areas, wet storage of harvested shellfish in polluted waters, and other violations of legislation become problematic (Jones et al., 1991). ...
Shellfish are a nutritious food source whose consumption and commercial value has risen dramatically worldwide. Although bivalve’s consumption can contribute to a healthy diet, some can cause foodborne illnesses. Microbial contamination is chronic and pervasive in harvesting areas and may be passed on to the consumers. Current food safety programs intend to protect consumers. Nevertheless, bivalve’s microbial contamination is underestimated and undermanaged, which can pose a potential public health risk. We intend to provide an updated overview of the microbial assessment of bivalves and emerging alternatives or complementary perspectives for the elimination of microbial contamination. Further research is needed for the improvement of public health control and the enhancement of shellfish safety.
... Harvesting from polluted (categories B and C) areas is allowed, but shellfish must undergo treatment before being commercialized. In this case, shellfish can be placed on the market for human consumption after controlled self-purification in tanks of clean seawater (commercial depuration), prolonged relaying in a category A area or commercial heat treatment or processing by any other acceptable method (Jones et al., 1991;Lees, 2000;Murchie et al., 2005). Harvesting from category D zones is forbidden. ...
... The depuration process efficiency is directly related to the bivalve's size, siphoning activity and physiological conditions (Jones et al., 1991;Richards, 1988). It is also related to the type and amount of initial contamination, since higher contamination requires longer depuration times and different microorganisms respond differently to the purification process. ...
Infectious human diseases acquired from bivalve shellfish consumption constitute a public health threat. These health threats are largely related to the filter-feeding phenomenon, by which bivalve organisms retain and concentrate pathogenic bacteria from their surrounding waters. Even after depuration, bivalve shellfish are still involved in outbreaks caused by pathogenic bacteria, which increases the demand for new and efficient strategies to control transmission of shellfish infection. Bacteriophage (or phage) therapy represents a promising, tailor-made approach to control human pathogens in bivalves, but its success depends on a deep understanding of several factors that include the bacterial communities present in the harvesting waters, the appropriate selection of phage particles, the multiplicity of infection that produces the best bacterial inactivation, chemical and physical factors, the emergence of phage-resistant bacterial mutants and the life cycle of bivalves. This review discusses the need to advance phage therapy research for bivalve decontamination, highlighting their efficiency as an antimicrobial strategy and identifying critical aspects to successfully apply this therapy to control human pathogens associated with bivalve consumption.
... Harvesting from polluted (categories B and C) areas is allowed, but shellfish must undergo treatment before being commercialized. In this case, shellfish can be placed on the market for human consumption after controlled self-purification in tanks of clean seawater (commercial depuration), prolonged relaying in a category A area or commercial heat treatment or processing by any other acceptable method (Jones et al., 1991;Lees, 2000;Murchie et al., 2005). Harvesting from category D zones is forbidden. ...
... The depuration process efficiency is directly related to the bivalve's size, siphoning activity and physiological conditions (Jones et al., 1991;Richards, 1988). It is also related to the type and amount of initial contamination, since higher contamination requires longer depuration times and different microorganisms respond differently to the purification process. ...
Infectious human diseases acquired from bivalve shellfish consumption constitute a public health threat. These health threats are largely related to the filter-feeding phenomenon, by which bivalve organisms retain and concentrate pathogenic bacteria from their surrounding waters. Even after depuration, bivalve shellfish are still involved in outbreaks caused by pathogenic bacteria, which increases the demand for new and efficient strategies to control transmission of shellfish infection. Bacteriophage (or phage) therapy represents a promising, tailor-made approach to control human pathogens in bivalves, but its success depends on a deep understanding of several factors that include the bacterial communities present in the harvesting waters, the appropriate selection of phage particles, the multiplicity of infection that produces the best bacterial inactivation, chemical and physical factors, the emergence of phage-resistant bacterial mutants and the life cycle of bivalves. This review discusses the need to advance phage therapy research for bivalve decontamination, highlighting their efficiency as an antimicrobial strategy and identifying critical aspects to successfully apply this therapy to control human pathogens associated with bivalve consumption.
... Outbreaks of bacterial and viral diseases in humans associated with bivalve consumption demonstrate that some microorganisms are able to resist degradation and persist in bivalve tissues. Bacterial genera that survive in the tissues of bivalves and resist degradation include Salmonella and Vibrio species (Jones et al. 1991;Wright et al. 1996;Hernroth et al. 2002;Pruzzo et al. 2005). These species are responsible for the most cases of bacterial-caused food poisoning associated with bivalve consumption and are readily isolated from bivalves (Jones et al. 1991;Rippey 1994;Wright et al. 1996;Potasman et al. 2002;Hernroth et al. 2002). ...
... Bacterial genera that survive in the tissues of bivalves and resist degradation include Salmonella and Vibrio species (Jones et al. 1991;Wright et al. 1996;Hernroth et al. 2002;Pruzzo et al. 2005). These species are responsible for the most cases of bacterial-caused food poisoning associated with bivalve consumption and are readily isolated from bivalves (Jones et al. 1991;Rippey 1994;Wright et al. 1996;Potasman et al. 2002;Hernroth et al. 2002). Viruses that survive in the tissues of bivalves and resist degradation are frequently nonenveloped viruses, such as noroviruses (Potasman et al. 2002). ...
Rapid environmental change is linked to increases in aquatic disease heightening the need to develop strategies to manage disease. Filter-feeding species are effective biofilters and can naturally mitigate disease risk to humans and wildlife. We review the role of filter-feeders, with an emphasis on bivalves, in altering disease outcomes via augmentation and reduction. Filtration can reduce transmission by removing pathogens from the water column via degradation and release of pathogens in pseudofeces. In other cases, filtration can increase pathogen transmission and disease risk. The effect of filtration on pathogen transmission depends on the selectivity of the filter-feeder, the degree of infectivity by the pathogen, the mechanism(s) of pathogen transmission and the ability of the pathogen to resist degradation. For example, some bacteria and viruses can resist degradation and accumulate within a filter-feeder leading to disease transmission to humans and other wildlife upon ingestion. Since bivalves can concentrate microorganisms, they are also useful as sentinels for the presence of pathogenic microorganisms. While somewhat less studied, other invertebrates, including ascidians and sponges may also provide ecosystem services by altering pathogen transmission. In all scenarios, climate change may affect the potential for filter-feeders to mitigate disease risk. We conclude that an assessment including empirical data and modeling of system-wide impacts should be conducted before selection of filter-feeders to mitigate disease. Such studies should consider physiology of the host and microbe and risk factors for negative impacts including augmentation of other pathogens.
... Depuration is a process during which shellfish are held in tanks with clean seawater and are allowed to resume their natural filter-feeding activity, purging themselves of contaminants (Blogoslawski & Stewart 1983, Jackson & Ogburn 1999). This process was developed initially in response to a number of outbreaks of shellfish-associated typhoid illness (Richards 1988, Jones et al. 1991). Although depuration is not yet an established practice in India, it is used in many other developed countries such as the United Kingdom, France, Italy, the United States, and others, depending on the microbial quality of shellfishgrowing waters and load of microbes in tissues. ...
... Earlier studies have not examined the depuration time with respect to density of animals in the tank (Chae et al. 2009, Love et al. 2010, Kasai et al. 2011, Phuvasate et al. 2012). The siphoning activity and filtration capacity of oyster is size dependant and this, too, can affect the time taken for depuration (Jones et al. 1991, Oliveira et al. 2011). In the current study, 2-y-old oysters (length, 60–110 mm) were used, and these are the normal sizes at which oysters are harvested. ...
The efficiency of depuration of the Indian backwater oyster Crassostrea madrasensis (Preston, 1916) using the filldraw method (static method) with high-loading density was evaluated in this study. Depuration experiments were conducted with cartridge-filtered and UV-treated seawater at a salinity of 30.3 parts per thousand a pH of 8.3, and a temperature of 29.5 degrees C. The oysters located in trays on the surface and on the bottomwere compared formicrobial loads. Samples were taken at 0 h, 8 h, 16 h, 24 h, 36 h, and 48 h of depuration. The results showed that in winter monsoon-sampled nondepurated oysters, the most probable number of fecal coliforms and Escherichia coli were greater than the limits according to NSSP and European Union regulations. The surface held oysters took 24 h to reduce the coliforms and E. coli levels to below safe limits whereas for bottom held oysters it took 48 h. The species Salmonella was never detected in the oysters sampled, whereas Vibrio spp. were present in the nondepurated oysters and were eliminated completely after 8 h of depuration. Variation in depuration of total coliforms, fecal coliforms, E. coli, total plate count, and fecal streptococci in oysterswere significant (P < 0.05) between surface and bottomoysters. The study results recommend a loading density of 2 oysters/L water stacked in 1 layer as the optimum loading density for commercial depuration completed within 24 h.
... Bacteriile manifestă un grad variabil de sensibilitate faţă de această procedură de epurare (Marino et al, 1999;Olafsen et al, 1993;Perkins et al, 1980;Richards, 1988). De exemplu, unele specii de Vibrio îşi păstrează viabilitatea după ce sunt reţinute de bivalve, fiind capabile să persiste şi să se multiplice în ţesuturile lor (Jones et al, 1991;Murphree & Tamplin, 1992;Shumway, 1992). Lucrarea prezintă date asupra densităţii şi activităţii microbiene în zona de influenţă a unei coloniii de midii din apropierea Staţiunii Zoologice Marine "Prof. ...
... Hemolimfa scoicilor conţine atât hemocite -responsabile de mecanismele celulare de apărare (fagocitoză) -cât şi factori de apărare umorali, cum ar fi lectinele şi enzimele hidrolitice (Anderson, 1980;Fryer & Bayne, 1996;Glinski & Jarosz, 1997;Leclerc, 1996;Prieur et al, 1990;Renwranz, 1990;Tripp, 1992aTripp, , 1992bVasta et al 1999).Capacitatea unor bacterii de a supravieţui la acţiunea microbicidă a hemolimfei depinde de sensibilitatea lor faţă de acţiunea simultană a mai multor factori. Până în prezent, nu se ştie cu claritate de ce unele bacterii sunt mai sensibile decât altele, deşi există o serie de studii ce descriu activitatea bactericidă a hemolimfei bivalvelor marine (Birkbeck & Gallacher, 1993;Jones et al, 1991;Murphree & Tamplin, 1992); doar puţini autori au examinat în detaliu structurile de suprafaţă a celulelor bacteriene, structuri ce influenţeaza interacţiunea cu hemocitele (Genthner et al, 1999;Harris-Young et al, 1995). ...
F. Aonofriesei1 Rodica Stanciu1
1UNIVERSITATEA "OVIDIUS" CONSTANŢA,
FACULTATEA DE ŞTIINTE ALE NATURII
BD. MAMAIA 124, 900 527 CONSTANŢA
Email: aonofrieseif@yahoo.com
Summary
Bivalves are widespread invertebrates in marine environment, having an important role in energy flux in some marine ecosystems. These organisms can retain and use as food a significant proportion of microorganisms, thus influencing their abundance in less or more extended areas. At the same time, the release of organic matter by bivalves stimulates the growth of bacterioplankton. In order to understand the relationship between mussels and bacteria, the paper analyzes the spatial distribution of total count, abundance of proteolytic bacteria, and dehydrogenase activity in the water surrounding the colony. Samples were collected during summer 2003 near the "Prof. I Borcea" Zoological Station Agigea. Water samples were taken by diver from 5-6 m depth at five points; one from the upper layer of waters adjacent to colony, three with variable distance from the mussel colony (1 m, 5 m, 10 m), and the fifth from an uninfluenced region (30 m). The total count of heterotrophic bacteria and abundance of proteolytic bacteria were maximal in the region with mussels, but the number of bacteria decreased with the increase of distance from colony. This spatial pattern suggested the occurrence of a gradient of organic matter generated by the mussels’ colony. Most probably, the amount of organic nutrients lowered as distance increased and the low nutrient supply thus restricted the growth of bacterioplankton. The mussels consume a significant proportion of bacteria, but their abundance remains however high due to nutrient enrichment around the colony. Dehydrogenase activity was low around the mussels, but it increased at distance from colony, being maximal at points with the lowest abundance of culturable bacteria.These data suggested that decrease in organic nutrients at distance from colony was also accompanied by changes of their quality. Thus, it can be assumed that a strong downshift in organic nitrogen might occur at points with low bacterial abundance and this change stimulated the existing bacteria to use other nutrients (carbohydrates) by increasing their dehydrogenase activity.
Analele SNBC, A. Ardelean, C. Craciun (edit), III: 361-365
... Several depuration (controlled purification) studies have shown that oysters do not eliminate all microorganisms equally. Enteric bacteria (e.g., Escherichia coli, fecal coliforms, Salmonella spp.) are eliminated quite effectively (Barrow & Miller 1969, Son & Fleet 1980, Jones et al. 1991, whereas viruses (Metcalf et al. 1979, Richards 1988, Power & Collins 1989) and indigenous estuarine vibrios (Greenberg et al. 1982, Eyles & Davey 1984, Fletcher et al. 1991, Jones et al. 1991, Tamplin & Capers 1992 are less consistently eliminated. In fact, Tamplin & Capers (1992) reported that Vibrio vulnificus in oysters held at 23°C can persist and replicate at a rate that allows the release of 10 5 bacteria oyster -1 h -1 . ...
... Several depuration (controlled purification) studies have shown that oysters do not eliminate all microorganisms equally. Enteric bacteria (e.g., Escherichia coli, fecal coliforms, Salmonella spp.) are eliminated quite effectively (Barrow & Miller 1969, Son & Fleet 1980, Jones et al. 1991, whereas viruses (Metcalf et al. 1979, Richards 1988, Power & Collins 1989) and indigenous estuarine vibrios (Greenberg et al. 1982, Eyles & Davey 1984, Fletcher et al. 1991, Jones et al. 1991, Tamplin & Capers 1992 are less consistently eliminated. In fact, Tamplin & Capers (1992) reported that Vibrio vulnificus in oysters held at 23°C can persist and replicate at a rate that allows the release of 10 5 bacteria oyster -1 h -1 . ...
Ingestion of bacteria by oysters Crassostrea virginica and bactericidal activity of oyster hemocytes were studied using 4 environmental isolates (shellfish) and 3 clinical isolates (fecal) of Vibrio parahaemolyticus, Clinical isolates (2030, 2062, 2107) were obtained from the feces of patients with gastroenteritis who became ill during the 1998 food poisoning outbreak traced to consumption of raw oysters from Galveston Bay, Texas. This outbreak was the first reported occurrence in the United States of the virulent serotype O3:K6, Environmental isolates were from oysters (1094, 1100), crab (1163) and sardines (ATCC 17802). All isolates possessed the thermolabile direct hemolysin (tlh) gene, whereas only the clinical isolates possessed the thermostable direct hemolysin (tdh) gene, a virulence determinant. On average, environmental isolates were more susceptible than clinical isolates to killing by oyster hemocytes, as determined by an in vitro dye reduction assay. Isolate 2062 was the most susceptible of the clinical isolates; it lacked identifiable capsular material present in the other clinical isolates and displayed the most diffuse colony morphology on nutrient agar plates. When oysters were exposed in vivo to mixtures of a clinical (2030) and an environmental (1163) isolate, more clinical than environmental isolates were found in the tissues and hemolymph.
... Several depuration (controlled purification) studies have shown that oysters do not eliminate all microorganisms equally. Enteric bacteria (e.g., Escherichia coli, fecal coliforms, Salmonella spp.) are eliminated quite effectively (Barrow & Miller 1969, Son & Fleet 1980, Jones et al. 1991), whereas viruses (Metcalf et al. 1979, Richards 1988, Power & Collins 1989 ) and indigenous estuarine vibrios (Greenberg et al. 1982, Eyles & Davey 1984, Fletcher et al. 1991, Jones et al. 1991, Tamplin & Capers 1992 ) are less consistently eliminated . In fact, Tamplin & Capers (1992) reported that Vibrio vulnificus in oysters held at 23°C can persist and replicate at a rate that allows the release of 10 5 bacteria oyster –1 h –1 . ...
... Several depuration (controlled purification) studies have shown that oysters do not eliminate all microorganisms equally. Enteric bacteria (e.g., Escherichia coli, fecal coliforms, Salmonella spp.) are eliminated quite effectively (Barrow & Miller 1969, Son & Fleet 1980, Jones et al. 1991), whereas viruses (Metcalf et al. 1979, Richards 1988, Power & Collins 1989 ) and indigenous estuarine vibrios (Greenberg et al. 1982, Eyles & Davey 1984, Fletcher et al. 1991, Jones et al. 1991, Tamplin & Capers 1992 ) are less consistently eliminated . In fact, Tamplin & Capers (1992) reported that Vibrio vulnificus in oysters held at 23°C can persist and replicate at a rate that allows the release of 10 5 bacteria oyster –1 h –1 . ...
... Depuration rates of shellfish impacted by many factors including size, siphoning activity, physiological conditions (Richards, 1988;Jones et al., 1991), type and amount of contamination, water quality parameters such as temperature and salinity (Chalek, 2013), shellfish to water ratio, flow rate, oxygenation, species and duration (jones et al., 1991; Barile et al., 2009;Cozzi et al., 2009;Lee et al., 2010;Anacleto, 2014). ...
The edible clams from Lake Timsah are exposed to different industrial
wastes which may reflect the reason for the high concentration of heavy metals in studied species. The current study aims to evaluate the effect of size classes on the elimination of heavy metals (Cu, Fe, Pb, Co, Ni, and Zn) in some commercial bivalves Ruditapes decussatus, Venerupis pullastra and Paphia undulata. Negative correlations were found between
the sizes of studied species for all heavy metals (except Cu which showed a positive correlation with size in V. pullastra). The concentrations of all heavy metals (Cu, Fe, Pb, Co, and Zn) in the studied species were higher
than those in water and sediment. The highest depuration rate for all studied species was recorded in small clam classes.
... The depurating process represents a metabolic effort/fitness that could compromise bivalve' viability, especially when the suspended food particles are scarce or under stressful conditions (Antunes et al., 2010). Moreover, this ability may explain why artificially contaminated mollusks depurate more rapidly than environmentally contaminated ones (Jones et al., 1991;Schneider et al., 2009). Generally, bacteria are rapidly reduced in shellfish by using seawater only. ...
The effect of superchilled storage at -1°C on the microbial safety of oyster depurated with 0.2, 0.4, and 0.6 mg/L ozone was studied for 14 days. Fecal coliforms (4,100–16,000 MPN/100 g), Escherichia coli (1,500–3,650 MPN/100 g), Vibrio cholerae non-O1/non-O139 (13.0–102.0 MPN/g), and Salmonella spp. (2.270–3.035 × 103 CFU/g) were initially present in raw oysters. After 6 h depuration, fecal coliform counts decreased (P < 0.05) to 300, 20 and 20 MPN/100 g for 0.2, 0.4, and 0.6 mg/L treatments, while a 0.3 log decrease in control oysters was observed. Initial E. coli counts decreased (P < 0.05) in oysters to 50, 20, and 20 MPN/100 g for 0.2, 0.4, and 0.6 mg/L treatments, respectively. A 1 log reduction in V. cholerae non-O1/non-139 levels were observed in 0.4 and 0.6 mg/L-treatments after 2 and 4 h depuration. Salmonella spp. was not detected in oyster samples after 6 h depuration in 0.4 and 0.6 mg/L-ozone treatments. Considering the bacterial loads after depuration, at the end of superchilled storage the 0.4 mg/L-ozonated oysters attained lower (P < 0.05) fecal coliform levels (280 MPN/100 g) and E. coli counts in 0.4 and 0.6 mg/L-ozonated oysters (20 and 95 MPN/100 g, respectively). A 2-log decrease in V. cholerae non-O1/non-O139 levels on day 5 in 0.4 and 0.6 mg/L-ozonated oysters (< 0.3 MPN/g) was attained. V. cholerae non-O1/non-O139 counts in control oysters decreased 1 log on day 9 of superchilled storage. Salmonella spp. was not detected in ozonated and superchilled stored oysters. Levels of fecal coliforms, E. coli, Salmonella spp., and V. cholerae non-O1/non-O139 in non-ozone depurated oyster samples were higher than in control, 0.4 and 0.6 mg/L ozonated oyster samples during superchilled storage. The cumulative mortality rates after 14 days of storage for superchilled oysters (22.2%) was higher (P < 0.05) than 0.6 mg/L O3 (7.2%) and 0.4 mg/L O3 (5.8%) treatments, and control oysters (5.6%). pH values in control oysters decreased significantly (P < 0.05) throughout the storage period but not in oysters of both ozone treatments, indicating no detrimental effects on oyster survival. The results of this study suggest that superchilled storage enables ozonated shellstock oysters (0.4 mg/L-6 h) stored for 9 days to be safe human consumption.
... Studies on the removal of bacteria during "traditional" depuration (without phage addition) using bivalves artificially challenged with bacterial cultures show a greater degree of removal than studies using naturally contaminated shellfish (FAO, 2008). The type and quantity of initial contamination is also highly relevant for depuration efficiency, as more contaminated bivalves require longer depuration times and different microorganisms respond differently to the depuration process (Croci et al., 2002;Jones et al., 1991;Richards, 1988). In this study, when naturally contaminated cockles (using 0.6 L of seawater added with phages, but without water recirculation) were depurated with the single suspensions of the two phages tested and the phage cocktail, the maximum reduction of cultivable bacteria (0.7e0.8 log CFU/g) was less than that observed in artificially contaminated cockles (reductions 1.8e2.0 ...
... Studies on the removal of bacteria during "traditional" depuration (without phage addition) using bivalves artificially challenged with bacterial cultures show a greater degree of removal than studies using naturally contaminated shellfish (FAO, 2008). The type and quantity of initial contamination is also highly relevant for depuration efficiency, as more contaminated bivalves require longer depuration times and different microorganisms respond differently to the depuration process (Croci et al., 2002;Jones et al., 1991;Richards, 1988). In this study, when naturally contaminated cockles (using 0.6 L of seawater added with phages, but without water recirculation) were depurated with the single suspensions of the two phages tested and the phage cocktail, the maximum reduction of cultivable bacteria (0.7e0.8 log CFU/g) was less than that observed in artificially contaminated cockles (reductions 1.8e2.0 ...
The aim of this study was to compare the dynamics of three previously isolated bacteriophages (or phages) individually (phSE-1, phSE-2 and phSE-5) or combined in cocktails of two or three phages (phSE-1/phSE-2, phSE-1/phSE-5, phSE-2/phSE-5 and phSE-1/phSE-2/phSE-5) to control Salmonella enterica serovar Typhimurium (Salmonella Typhimurium) in order to evaluate their potential application during depuration. Phages were assigned to the family Siphoviridae and revealed identical restriction digest profiles, although they showed a different phage adsorption, host range, burst size, explosion time and survival in seawater. The three phages were effective against S. Typhimurium (reduction of∼2.0 log CFU/mL after 4h treatment). The use of cocktails was not significantly more effective than the use of single phages. A big fraction of the remained bacteria are phage-resistant mutants (frequency of phage-resistant mutants 9.19×10(-5) - 5.11×10(-4)) but phage-resistant bacterial mutants was lower for the cocktail phages than for the single phage suspensions and the phage phSE-1 presented the highest rate of resistance and phage phSE-5 the lowest one. The spectral changes of S. Typhimurium resistant and phage-sensitive cells were compared and revealed relevant differences for peaks associated to amide I (1620cm(-1)) and amide II (1515cm(-1)) from proteins and from carbohydrates and phosphates region (1080-1000cm(-1)). Despite the similar efficiency of individual phages, the development of lower resistance indicates that phage cocktails might be the most promising choice to be used during the bivalve depuration to control the transmission of salmonellosis.
... Harvesting from polluted (category B and C) areas is allowed but shellfish must undergo treatment, before being commercialized. In this case, shellfish can be placed on the market for human consumption following controlled self-purification in tanks of clean seawater (commercial depuration), prolonged relaying in clean seawater or commercial heat treatment or processing by any other acceptable method (Jones et al., 1991;Lees, 2000;Murchie et al., 2005). Where E. coli concentrations exceed 46,000 per 100 g of FIL (category D) harvesting is forbidden. ...
... Shellfish depuration in controlled waters is used extensively worldwide to decrease the number of unwanted microorganisms to acceptable levels for human consumption. Regarding this depuration procedure, bacteria behave differently (Marino et al., 1999, Olafsen et al., 1993Perkins et al., 1980;Richards, 1988); for instance, some Vibrio species have been reported to be resistant to the process and are able to persist and multiply within shellfish tissues (Jones et al., 1991;Murphree & Tamplin. 1992;Shumway, 1992). ...
The importance of combating infectious diseases has received international attention, p- viding the opportunity for a multidisciplinary approach that combines medicine with other scienti? candtechnologicalcapabilities,notablyinformationtechnology,nanotechnology,and biotechnology. In fact, it has been predicted that the future will bring a merging of these te- nologies with the cognitive and behavioral sciences-major forces that have the potential to balancetheworld'sinequities.Thescienti?ccommunityandworldleadersmustworktogether to use knowledge and its applications to improve the condition of the planet. The connection between infectious diseases and the oceans provides a paradigm for this perspective. A stark global context indisputably frames all human health issues in the twenty-?rst century: the world wide movement of people and goods. Throughout the past half century, international travel has skyrocketed; there are more than 500 million international arrivals per year. The greatest increase has taken place since the mid-1990s. The world has become integrated and global; consequently, the notion that it is possible to successfully eradicate a disease from the face of the planet has become simplistic. Infectious disease is a moving target and climate shifts will affect any disease that has an environmentally sensitive stage or vector. Recognizingsignalsfromclimatemodelsandincorporatingthemintohealthmeasurescanp videnewopportunitiesforproactive-ratherthanreactive-approachestopublichealth.Thus, careful attention to the role of the oceans in human health can offer new avenues of research that will provide new means of predicting and preventing those diseases that are rooted in the environment. In this volume, pathogens in the sea are reviewed by Colin Munn, who provides a broader perspective for the topic of pathogenic microorganisms associated with the world oceans. © 2005 Springer Science+Business Media, Inc. All rights reserved.
... Elimination of bacteria from oyster tissues (depuration) is at least partly achieved through phagocytic action of amebocytes (Rodrick & Ulrich 1984). The ability of oysters to successfully eliminate specific microorganisms partly depends on the species; Vibrio vulnificus, for example, is much more persistent in oysters than fecal pollutants such as Escherichia coli and Salmo-nella (Perkins et al. 1980, Richards 1988, Jones et al. 1991, Tamplin & Capers 1992. Such persistence may depend on whether the bacteria have light or heavy mucopolysaccharide coats that mask the bacteria from amebocyte recognition (Harris-Young et al. 1993, Volety et al. 2001. ...
The distribution of eastern oysters Crassostrea virginica near terrestrial watersheds has led to a general impression that low or variable salinity is imperative for survival. However, freshwater runoff contains numerous mineral elements from geologic deposits that could play significant roles in oyster physiology. Two metals of terrestrial origin, copper and zinc, are accumulated to extremely high concentrations in eastern oysters, even in the absence of anthropogenic sources. As yet, there has been no defendable demonstration of a physiologic function for such high concentrations. Both copper and zinc, however, are accumulated almost exclusively in the amebocytes and calcareous shell of oysters, a unique distribution that implicates a role in the functions of amebocytes. Amebocytes are migratory, diapedetic cells generally recognized to provide nutriment and defense through phagocytosis, killing, and digestion of invading or ingested microorganisms. There is sufficient evidence in existing literature to suggest that copper and zinc directly contribute to these antimicrobial activities. This review presents historical and recent findings that demonstrate a strong affinity of oyster amebocytes for copper and zinc (even in low ambient concentrations), prolonged retention of the metals despite a potential route of elimination, and strong circumstantial evidence of antimicrobial activity by accumulated copper and zinc. It is proposed that oysters actively concentrate copper and zinc as antimicrobial agents to be used in intracellular and extracellular killing (direct toxicity) as well as extracellular clot formation (precipitation of hemolymph). This potential, combined with evidence of amebocyte involvement in deposition of oyster shell, provides an alternative framework for understanding amebocyte functions, defense activities, and coastal distributions of oyster populations. It also affords some resolution to the apparent contradiction of eastern oysters thriving at seemingly polluted locations.
... Harvesting from polluted (category B and C) areas is allowed but shellfish must undergo treatment, before being commercialized. In this case, shellfish can be placed on the market for human consumption following controlled self-purification in tanks of clean seawater (commercial depuration), prolonged relaying in clean seawater or commercial heat treatment or processing by any other acceptable method (Jones et al., 1991;Lees, 2000;Murchie et al., 2005). Where E. coli concentrations exceed 46,000 per 100 g of FIL (category D) harvesting is forbidden. ...
... The experiments in this study represent a range of conditions for treating shellfish from traditional depuration to natural relay. Previous studies have shown traditional depuration to be ineffective at reducing levels of pathogenic vibrios, including studies at the same study site (Jones et al. 1991, Tamplin and Capers 1992, Croci et al. 2002. There have been only a few studies on relaying to reduce vibrio concentrations in shellfish, yet these have shown promising results (Jones 1994;Jones et al. 1995;Motes and DePaola 1996). ...
... Estuarine fish species may also serve as a reservoir for transport of this bacteria species between oyster beds, or as a source of wound infections [9]. In oysters, V. vulnificus resists depuration and other chemical disinfection methods typically used to eliminate enteric pathogens [19]. Cooking shellfish is currently the only reliable method to completely destroy the bacterium. ...
To investigate the possible link between Vibrio vulnificus population size in seawater and water temperature.
We collected incidence and water temperature data in coastal regions of Korea and constructed a mathematical model that consisted of three classes; susceptible fish, infected fish available to humans, and infected humans.
We developed a mathematical model to connect V. vulnificus incidence with water temperature using estimated bacterial population sizes and actual coastal water temperatures.
Increased V. vulnificus population sizes in marine environments may increase the risk of infection in people who eat at coastal restaurants in Korea. Furthermore, we estimated the near-future number of infected patients using our model, which will help to establish a public-health policy to reduce the disease burden.
... Consequently, the occurrence of this bacterium in aquatic environments is a significant concern of the shellfish industry and public health agencies. Depuration has been examined as a means to eliminate V. vulnificus from oysters but has proven largely unsuccessful, even though such treatment is effective in removing many coliforms (2). As a possible aid in removing this potential pathogen from shellfish stock, we recently examined ten FDA-approved food preservatives that might be used as antimicrobial agents against V. vulnificus. ...
Vibrio vulnificus is a bacterium indigenous to estuarine waters and is known to be a significant human pathogen. Infections are generally associated with the consumption of raw shellfish, especially oysters. We have previously determined that a variety of FDA-approved food preservatives were effective agents in causing lethality of V. vulnificus in vitro. In the present study we tested the effects of these compounds in treating natural oysters and found that diacetyl at concentrations of 0.05% or greater could significantly decrease the number of V. vulnificus naturally present in oysters. Lactic acid and BHA were found not to be effective at concentrations up to 0.05%. We conclude that diacetyl is a possible antimicrobial agent for this organism that might be used in shellfish stock.
... Sanitary regulations for shellfish production rely on the isolation of specified bacterial indicators of sewage contamination, to classify shellfish harvesting waters and estimate the efficacy of purification methods, such as relaying and depuration (Anonymous, 1991(Anonymous, , 2002. However, limitations of bacterial standards have been widely acknowledged, particularly the lack of correlation between the presence of bacterial indicators and viral pathogens in shellfish and their harvesting waters (Lees, 2000;Portnoy et al., 1975), and differential elimination rates from shellfish of indicator bacteria compared to viruses and indigenous marine bacteria (Eyles & Davey, 1984;Jones, Howell, & O'Neill, 1991;Lees, 2000). ...
Four types of shellfish, mussels, prawns, scallops and oysters, were pressure treated at 300, 400, 500 and 600 MPa for 2 min at 20 °C and stored for up to 28 days at 2 °C. The shellfish were sampled before and after pressure treatment and at 7-day intervals for total aerobic counts, psychrotrophic counts, pseudomonads and coliforms. Pressure treatment readily inactivated psychrotrophic bacteria, coliforms and pseudomonads. Randomly selected isolates were identified from the shellfish before and after pressure treatment and after storage at 2 °C. The range of bacteria present in the shellfish decreased after pressure treatment. This was predominantly because of inactivation of Gram-negative bacteria leading to an increase in the proportion of Gram-positive species isolated. The main types of bacteria isolated from pressure-treated shellfish, after storage, were Bacillus, Acinetobacter/Moraxella and lactic acid bacteria. Together these made up 96% of the bacteria isolated from all the pressure-treated shellfish.
... Shellfish depuration in controlled waters is used extensively worldwide to decrease the number of unwanted microorganisms to acceptable levels for human consumption. Regarding this depuration procedure, bacteria behave differently (Marino et al., 1999, Olafsen et al., 1993Perkins et al., 1980;Richards, 1988); for instance, some Vibrio species have been reported to be resistant to the process and are able to persist and multiply within shellfish tissues (Jones et al., 1991;Murphree & Tamplin. 1992;Shumway, 1992). ...
The genus Vibrio includes more than 30 species, at least 12 of which are pathogenic to humans and/or have been associated with foodborne diseases
(Chakraborty et al., 1997). Among these species, Vibrio cholerae, serogroups O1 and O139, are the most important, since they are associated with epidemic and pandemic diarrhea outbreaks
in many parts of the world (Centers for Disease Control and Prevention, 1995; Kaper et al., 1995). However, other species
of vibrios capable of causing diarrheal disease in humans have received greater attention in the last decade. These include
Vibrio parahaemolyticus, a leading cause of foodborne disease outbreaks in Japan and Korea (Lee et al., 2001), Vibrio vulnificus, Vibrio alginolyticus, Vibrio damsela, Vibrio fluvialis, Vibrio furnissii, Vibrio hollisae, Vibrio metschnikovii, and Vibrio mimicus (Altekruse et al., 2000; Høi et al., 1997). In the USA, Vibrio species have been estimated to be the cause of about 8000 illnesses annually (Mead et al., 1999).
... Sanitary regulations for shellfish production rely on the isolation of specified bacterial indicators of sewage contamination, to classify shellfish harvesting waters and estimate the efficacy of purification methods, such as relaying and depuration (Anonymous, 1991(Anonymous, , 2002. However, limitations of bacterial standards have been widely acknowledged, particularly the lack of correlation between the presence of bacterial indicators and viral pathogens in shellfish and their harvesting waters (Lees, 2000;Portnoy et al., 1975), and differential elimination rates from shellfish of indicator bacteria compared to viruses and indigenous marine bacteria (Eyles & Davey, 1984;Jones, Howell, & O'Neill, 1991;Lees, 2000). ...
Many commercially important shellfish are filter feeders and, as a consequence, concentrate microbes from the surrounding waters. Shellfish may be relayed or depurated to reduce the level of microbial contamination, but the efficiency of these purification practices, particularly in relation to viruses and indigenous marine bacteria, is questionable. Therefore additional processing is necessary to ensure the safety of shellfish for human consumption. In recent years high pressure (HP) processing has been investigated as an alternative method for food preservation. HP technology allows inactivation of microorganisms while maintaining sensory and nutritional properties of foods. Currently, HP processing has several commercial food applications, including oysters. As well as enhancing safety and extending shelf-life, HP treatment has the additional advantage of shucking or opening shellfish, making this technology particularly beneficial to the shellfish processing industry and consumers alike.Industrial relevanceHigh pressure (HP) processing is increasingly being used in the commercial processing of oysters, due to its minimal effects on sensory and nutritional quality, the opening or shucking of oysters during treatment, and the reduction of levels of Vibrio vulnificus, a pathogen of concern particularly in the US. However, little is known of the efficacy of HP treatment in reducing other pathogens in shellfish such as human enteric viruses, which are the predominant cause of shellfish-borne disease. This article reviews the inactivation of microorganisms of importance to shellfish, particularly viruses, the commercial HP processing of oysters and the advantages of HP technology as they pertain to the seafood industry.
... As such, Vibrio spp. appear to selectively colonize, persist, and may even multiply in oysters between the time they are harvested and consumed (8,11,14,16,21,39). Presently, the precise mechanism involved in their persistence is not understood. ...
Thesis (Ph. D.)--University of Washington, 2005. Vibrio vulnificus is a Gram negative, halophilic bacterium, which is a natural inhabitant of sub tropical and tropical marine estuarine waters. These bacteria concentrate in filter-feeding mollusks such as oysters and unlike fecal contaminants are not easily eliminated during normal shellfish processing. The specific mechanisms by which these bacteria interact with or colonize oysters are unclear. Food-borne infections, mainly by consumption of raw oysters, caused by this bacterium have a high fatality rate of about 50% in susceptible individuals. This bacterium can also cause severe wound infections from handling fish or shellfish. 20--30% of which are fatal.Bacterial adherence to biotic and abiotic surfaces is mediated by several mechanisms including pill, flagella, surface polysaccharide capsules and non-fimbrial adhesions. This study verifies that one of the pilins of V. vulnificus , PilA, of the type IV class of pili, contributes to virulence in a mouse model, adherence to human epithelial cells, and biofilm formation on abiotic surfaces. Loss of PilA also resulted in a decrease in the ability of this bacterium to persist in oysters. A strain with a mutation in the type IV prepilin peptidase, PilD, which is defective in expression of all surface pili and secretion of exoenzymes that are exported by the type II secretion pathway was also less persistent in oysters as compared to the wild type strain. If these factors prove to be responsible for the bacterium's ability to colonize oyster tissue, they may present a unique and specific target(s) for compounds designed to interfere with this attachment, leading to depuration methods that could potentially reduce or eliminate the bacteria from oysters.Examination of the Y. vulnificus pilA sequences from several clinical and non clinical isolates demonstrates that these pilins cluster into a limited number of groups within which the amino acid sequences are almost identical, although the sequences between groups are considerably variable. Preliminary phylogenetic analysis also suggests that the sequence diversity in pilA is similar to that of drE, an Escherichia coli fimbrial adhesin that is both the structural and adhesin gene, raising the possibility that pilA may be evolving in a similar fashion.
... In oysters, V. vulnificus resists depuration and other chemical disinfection methods typically used to eliminate enteric pathogens [28,29]. Cooking shellfish is currently the only reliable method to completely destroy the bacterium. ...
Vibrio vulnificus is capable of causing severe and often fatal infections in susceptible individuals. It causes two distinct disease syndromes, a primary septicemia and necrotizing wound infections. This review discusses the interaction of environmental conditions, host factors, and bacterial virulence determinants that contribute to the epidemiology and pathogenesis of V. vulnificus.
This chapter describes the role of mussels in transmission of disease to humans under the following headings: bacterial infections, viral infections, parasites, biotoxins and industrial pollutants. Methods of bacterial, viral and toxin detection, decontamination procedures, monitoring and food safety measures are also covered. To protect public health, many countries have established sanitation programmes that supervise the production, harvesting and marketing of bivalve species for human consumption. Hazard Analysis and Critical Control Point (HACCP) is a science‐based system that aims to prevent food safety problems from occurring rather than having to react to noncompliance of finished products.
Previous short-duration depuration studies with the eastern oyster (Crassostrea virginica) demonstrated difficulty in achieving significant naturally incurred Vibrio vulnificus population count reductions. The present study used long-duration depuration (14 days) at controlled temperatures (10 or 22°C) and salinities (12, 16, or 20 mg/g). All depuration temperature– salinity combinations significantly reduced V. vulnificus counts, with greatest reductions seen in 12 mg/g, 10°C seawater (2.7-log CFU/g reduction) and in 20 mg/g, 22°C seawater (2.8-log reduction). Mesophilic vibrios dominated the overall microflora of freshly harvested oysters, whereas refrigerated storage selected for psychrotrophic bacteria (Pseudomonas spp., Aeromonas spp., Shewanella spp., Psychrobacter spp.) as well as did depuration at 10°C (Pseudoalteromonas spp., Shewanella spp., Vibrio spp.). Depuration at 22°C retained dominance of mesophilic vibrios, including pathogenic species, followed by Shewanella spp., Pseudoalteromonas spp., and Photobacterium spp. Although aerobic plate counts were lower in 22°C depurated oysters (5.0 log versus 6.0 log) compared with 10°C, depuration at 10°C offered greater V. vulnificus population reductions than depuration at 22°C. This advantage was only seen at 12 mg/g salinity, with no impact at 16 and 20 mg/g salinities. No depuration treatment reduced V. vulnificus counts to nondetectable levels. Use of prolonged depuration may be a helpful intervention to control V. vulnificus populations in oysters.
Vibrio parahaemolyticus is the leading cause of seafood-borne human infections in the United States, and many of these illnesses are associated with consumption of raw molluscan shellfish. V. parahaemolyticus levels in shellfish vary temporally and spatially with environmental conditions in and around production areas. The objective of this study was to study the potential for reducing levels of V. parahaemolyticus in live oysters by relaying them during higher-risk warm weather to a site with elevated salinity and consistently low V. parahaemolyticus levels. The effectiveness of relaying was assessed by analyzing oyster samples collected on days 0, 2, 7, 10, and 14 for V. parahaemolyticus levels using a three-tube most-probable-number enrichment method in conjunction with genetic marker-based quantitative PCR. The salinity at the relay site was always higher than the salinity at the harvest site, with the difference between the two sites ranging from 3.4 to 19.1 ppt (average, 12 ppt) during 2011 to 2014. Oysters relayed during June, July, and August in 2011 and 2012 showed consistently reduced V. parahaemolyticus levels after 14 days, whereas relaying was less successful and V. parahaemolyticus populations changed to include trh-positive strains during 2013. When effective, relay required at least 10 days to reduce V. parahaemolyticus levels. A sample of oysters collected in August 2012, which was temperature abused to increase initial V. parahaemolyticus levels, showed a 4.5-log decrease in V. parahaemolyticus levels after 14 days of relay. These results suggest that relaying oysters to reduce V. parahaemolyticus levels holds promise, but that both microbial community and environmental conditions at relay sites can affect relay success. Further investigation to discover key factors that affect V. parahaemolyticus levels in relayed oysters may aid in developing a consistent approach for reducing V. parahaemolyticus in oysters to eliminate the risk of illness for oyster consumers.
By virtue of their feeding habit, bivalves concentrate and accumulate material from the environment. Generally speaking, the bivalves are not themselves affected by the microorganisms or toxins, merely serving to concentrate and passively transport the etiological agent. In this chapter on public health the role of bivalves in transmission of disease to humans is dealt with under the following headings: bacterial infections, viral infections, biotoxins and industrial pollutants. Methods of bacterial, viral and toxin detection, decontamination procedures and monitoring measures are also covered. To safeguard public health, many countries have established sanitation programmes that oversee the production, harvesting and marketing of bivalve species for human consumption. The capacity of bivalves to concentrate and accumulate bacteria, viruses and biotoxins and pollutants means that special decontamination procedures are often necessary before bivalves can be harvested and marketed. The chapter also discusses the hazard analysis and critical control point (HACCP) system.
The application of natural zeolite for water and wastewater treatment has been carried out and is still a promising technique in environmental cleaning processes. Natural zeolite can be used to improve the purification process of clams (Ruditapes decussatus). Thus, our study aimed at improving the clam purification system in order to reduce Escherichia coli and eliminate Salmonella in samples artificially contaminated with this bacterium using a natural zeolite to replace the biological filter. The results showed that zeolite used in a depuration system improved the clam purification process. Moreover, natural zeolite exhibited high performance in the adsorption of bacteria and allowed to reduce the Escherichia coli abundance in 24 h, thus ensuring purified clams conformity with the ISO 16649-3 standard. These results indicate the beneficial effects of using zeolite in the adsorption of bacteria and the reduction in the abundance of Escherichia coli and set the Salmonella from marine organisms.
The most recent data show that shellfish farming represents approximately 60% of the total aquaculture production in Europe. Bivalves molluscs are internationally recognized as a potential vehicle for foodborne diseases especially when consumed raw or improperly cooked. Bivalves are filter-feeding animals that may accumulate particles present in the surrounding water, including viruses and pathogenic microorganisms. For these reasons consumers have to be careful during purchasing, handling and cooking of this food. In Europe several regulations have been adopted to protect consumers’ health. However in the development of new food safety strategies it is also crucial to find an effective approach to guide authorities’ decisions on priorities for controlling foodborne risks. In the article a study for the selection and ranking of behavioural information on bivalve risks is reported. The “consensus methods” provide a means of synthesising information and of harnessing the insights of experts to enable decisions shared and validated by the scientific community. The Delphi method and Nominal Group technique (NGT) were applied for their capability to assess the level of agreement and to develop consensus among participants. In the article, the NGT is presented as a way to involve relevant stakeholders in the ranking of risky behaviours from the production to the consumption of bivalve molluscs previously selected by scientific experts through the Delphi method. The inclusion of a full range of experts and stakeholders in a community participatory research project regarding food risk management represents an innovative approach in the Italian public health context, especially in the analysis of bivalve risks.
Many Vibrio species are capable of causing infections in humans. Vibrio vulnificus and Vibrio parahaemolyticus are part of the normal microflora of estuaries and have been implicated in diseases from consumption of raw or undercooked shellfish. In the Great Bay Estuary of Maine and New Hampshire, oysters (Crassostrea virginica) and soft-shell clams (Mya arenaria) are harvested for commercial and recreational purposes. Only one incidence of V. parahaemolyticus infection from shellfish consumption has been documented. Traditional methods and a gene probe assay were used to enumerate V. vulnificus in water from sites along salinity gradients from two tributaries to the main water body (Great Bay) of the estuary. V. parahaemolyticus, Escherichia coli, enterococci, fecal coliforms, nitrate, ammonium, orthophosphate, suspended solids, chlorophyll a, dissolved organic carbon (DOC), temperature, and salinity were also measured. Results showed lower salinity and higher concentrations of dissolved nutrients, suspended solids, fecal indicator bacteria, and chlorophyll a in tributaries compared to Great Bay. Both Vibrio sp. were detected more frequently and at higher concentrations in the tributaries. Multiple regression analysis suggested suspended solids were the most significant variable, accounting for ~27% of the variance in V. vulnificus and V. parahaemolyticus concentrations. However, the gene probe results showed DOC was the most significant variable for explaining (44%) the variance in V. vulnificus concentrations. The results suggest that improved detection methods can enhance the understanding of environmental conditions conducive to both growth and inhibition of these pathogens.
Microbial contamination is a challenging and significant issue for the shellfish industry. It is the main public health concern associated with consuming shellfish, and it often limits shellfish harvesting throughout the world. Enteric viruses, pathogenic Vibrio species, and fecal-borne bacterial pathogens are the main causes of shellfish-borne disease. These microorganisms have widely different properties, sources, virulence factors, and fate in the environment, and the current indicators used to classify harvest waters have significant limitations. A great deal of progress is currently being made in the detection of pathogenic microorganisms and in understanding their fate in the environment. With increasing human development in coastal areas, emerging diseases, habitat destruction, and global climate changes, the challenges associated with managing microbial contamination and shellfish safety continues to evolve.
The efficiency of depuration of the Indian backwater oyster Crassostrea madrasensis (Preston, 1916) using the fill-draw method (static method) with high-loading density was evaluated in this study. Depuration experiments were conducted with cartridge-filtered and UV-treated seawater at a salinity of 30.3‰, a pH of 8.3, and a temperature of 29.5°C. The oysters located in trays on the surface and on the bottom were compared for microbial loads. Samples were taken at 0 h, 8 h, 16 h, 24 h, 36 h, and 48 h of depuration. The results showed that in winter monsoon-sampled nondepurated oysters, the most probable number of fecal coliforms and Escherichia coli were greater than the limits according to NSSP and European Union regulations. The surface held oysters took 24 h to reduce the coliforms and E. coli levels to below safe limits whereas for bottom held oysters it took 48 h. The species Salmonella was never detected in the oysters sampled, whereas Vibrio spp. were present in the nondepurated oysters and were eliminated completely after 8 h of depuration. Variation in depuration of total coliforms, fecal coliforms, E. coli, total plate count, and fecal streptococci in oysters were significant (P < 0.05) between surface and bottom oysters. The study results recommend a loading density of 2 oysters/L water stacked in 1 layer as the optimum loading density for commercial depuration completed within 24 h.
The selective effect that refrigeration exerts on the autochthonous microbiota of the Eastern oyster was investigated using culture-independent methods. Oysters from two different locations along the Gulf Coast of Mexico were analyzed concurrently during two weeks refrigeration. Ribosomal intergenic sequence analysis (RISA) generated complex bacterial community fingerprints that provided a strong association between RISA-defined clusters and sample date. Denaturing Gradient Gel Electrophoresis (DGGE) profiles obtained with universal primers did not reflect any association based on date or geographic origin of the samples. During the storage period, Vibrio parahaemolyticus was replaced, at least in overall abundance, with other non-pathogenic Vibrio species better adapted to cold temperatures. This study shows that refrigeration itself does not limit the growth of the bacterial community nor reduces its richness.
The Great Bay/Piscataqua River Estuary in New Hampshire and Maine has an abundant oyster resource in sewage-contaminated water. The only approved area in the Maine por- tion of the Estuary is Spinney Creek, where fecal indicator bacteria are present at reduced levels and Vibrio vulnificus is absent. Spinney Creek Oyster Company (SCOC) of Eliot, Maine, operates relay lagoons and a depuration facility for oysters harvested commercially from restricted areas of the Salmon Falls River in Maine. Oysters naturally contaminated with vibrios and fecal indicator bacteria were used to evaluate depuration and relaying as strategies for eliminating these bacteria from shellfish. Oysters and water were analysed for the presence of total and fecal coliforms, V. vulnificus, V. parahaemolyticus, and total vibrios. Coliforms were always detected at varying levels in water and oyster samples. V. vulnificus was detected consistently during July-October at the harvest site, but never in Spinney Creek water or anywhere else during November-June. Relaying oysters for 7 days to the SCOC relay lagoons consistently decreased levels of fecal coliforms and V. vulnifi- cus. Depuration of oysters for 48 hours significantly reduced total and fecal coliforms, but did not decrease vibrio levels. These results demonstrate the effectiveness of depuration and relaying in reducing fecal contamination in oysters, and a unique, additional benefit of relaying for eliminating otherwise recalcitrant V. vulnificus.
Vibrio vulnificus is an estuarine bacterium which is known to be a significant human pathogen. It occurs in high numbers in oysters and other molluscan shellfish, where it is part of the animals' normal flora. V. vulnificus occurs in two colony morphotypes ; the opaque, encapsulated variety is virulent, whereas the translucent, non encapsulated variety is avirulent. Studies were carried out to determine if the opaque and translucent morphotypes were taken up by oysters (Crassostrea virginica) at different rates, and whe- ther UV - assisted depuration proceeded at different rates. Possible interconversion of the opaque and translucent morphotypes was also examined, as was the possibility that the virulence of Vibrio vulnificus would be modified following passage through the oyster. All studies were carried out using a strain of V. vulnificus which harbors the transposon, TnphoA, which allowed the added cells of V. vulnificus to be readily differentiated from other bacteria naturally present in the oysters. Results indicated little difference in the rate of uptake or depuration of V. vulnificus by
Different strains of Vibrio parahaemolyticus (Vp) in broth cultures and Vp-inoculated live Pacific oysters (Crassostrea gigas) were subjected to high-pressure processing (HPP) at 241, 276, 310, and 345 MPa. Results showed Vp numbers were reduced by HPP in both pure culture and whole oysters. Vp inactivation was dependent on time and pressure. Optimum conditions for reducing Vp in pure culture and oysters to nondetectable levels were achieved at 345 MPa for 30 and 90 s, respectively. Resistance variations were detected between Vp in pure culture and in oysters. HPP proved to be an efficient means of reducing Vp in oysters.
Bacteria that are members of the Vibrio genus are ubiquitous in the marine environment and are part of the natural estuarine microflora. Several Vibrio species can accumulate in shellfish through filter feeding, and pose a significant threat to human health from important fishery resources. Little is known about the interactions of these microorganisms with shellfish that result in significant accumulation of the bacteria. Vibrio vulnificus can cause fatal septicemic infections in individuals who are immunocompromised or suffer from liver disease through consumption of raw shellfish harvested from warm water regions. In addition, the bacterium can cause severe necrotizing wound infections in otherwise healthy people who handle shellfish. Such infections impact the entire U.S. shellfish industry causing significant economic losses. Another vibrio, Vibrio parahaemolyticus, has a wider geographic distribution and is a significant source of shellfish-transmitted gastrointestinal illness in humans, with increased incidence during summer months. Our research is aimed at the characterization of surface structures called pili or fimbriae to ascertain their function in persistence of vibrios in oysters, as well as in human pathogenesis. Pili are thin (ca. 10 nm diameter) fiber-like structures that extend from bacterial cells and are often involved in cell to cell binding or attachment of a bacterium to a variety of biotic and abiotic surfaces. In initial studies on the role of a specific class of pili in V. vulnificus, designated type IV, we have demonstrated their role in adherence to human epithelial cells, biofilm formation, virulence in a mouse model, and persistence in oysters. We are currently examining the function of homologous pili in V. parahaemolyticus. If these factors prove to be responsible for the bacterium's ability to colonize oyster tissue, they may present a unique and specific target(s) for compounds designed to interfere with this attachment, leading to-
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depuration methods that could potentially reduce or eliminate the organisms from oysters
Vibrio vulnificus is an estuarine bacterium which is known to be a significant human pathogen. It occurs in high numbers in oysters and other molluscan shelfish, where it is part of the animals' normal flora. V. vulnificus occurs in two colony morphotypes; the opaque, encapsulated variety is virulent, whereas the translucent, non encapsulated variety is avirulent. Studies were carried out to determine if the opaque and translucent morphotypes were taken up by oysters (Crassostrea virginica) at different rates, and whether UV - assisted depuration proceeded at different rates. Possible interconversion of the opaque and translucent morphotypes was also examined, as was the possibility that the virulence ofVibrio vulnificus would be modified following passage through the oyster. All studies were carried out using a strain of V. vulnificus which harbors the transposon, TnphoA, which allowed the added cells of V. vulnificus to be readily differentiated from other bacteria naturally present in the oysters. Results indicated little difference in the rate of uptake or depuration of V. vulnificus by oysters (Figure 1). Uptake in either case was rapid (saturation appeared within 30-60 minutes), and depuration by the laboratory-infected oysters appeared complete within 48 hours. Conversions for opaque and translucent morphotypes in oysters were not significantly different from in vitro rates (Table 1). No conversion from the translucent (avirulent) to the opaque (virulent) form was seen. LD50 values in mice after oyster passage were unchanged (Table 2). Our results suggest that the presence of the capsule on V. vulnificus cells does not markedly contribute to its ability to be taken up or depurated by oysters, and that oyster passage neither increases nor decreases the virulence of this human pathogen.
The Great Bay/Piscataqua River Estuary in New Hampshire and Maine has an abundant oyster resource in sewage-contaminated water. The only approved area in the Maine portion of the Estuary is Spinney Creek, where fecal indicator bacteria are present at reduced levels and Vibrio vulnificus is absent. Spinney Creek Oyster Company (SCOC) of Eliot, Maine, operates relay lagoons and a depuration facility for oysters harvested commercially from restricted areas of the Salmon Falls River in Maine. Oysters naturally contaminated with vibrios and fecal indicator bacteria were used to evaluate depuration and relaying as strategies for eliminating these bacteria from shellfish. Oysters and water were analysed for the presence of total and fecal coliforms, V. vulnificus, V. parahaemolyticus, and total vibrios. Coliforms were always detected at varying levels in water and oyster samples. V. vulnificus was detected consistently during July-October at the harvest site, but never in Spinney Creek water or anywhere else during November-June. Relaying oysters for 7 days to the SCOC relay lagoons consistently decreased levels of fecal coliforms and V. vulnificus. Depuration of oysters for 48 hours significantly reduced total and fecal coliforms, but did not decrease vibrio levels. These results demonstrate the effectiveness of depuration and relaying in reducing fecal contamination in oysters, and a unique, additional benefit of relaying for eliminating otherwise recalcitrant V. vulnificus.
Vibrio cholerae 01, the causative agent of cholera, is known to persist in estuarine environments as endogenous microflora. The recent introduction of V. cholerae 01 into estuaries of the North and South American continents has stimulated the need to determine the effect of controlled purification on reducing this pathogen in edible molluscan shellfish. Experiments defined parameters for the uptake and retention of V. cholerae 01 in tissues of Crassostrea virginica, and these parameters were compared with those for Escherichia coli and Salmonella tallahassee, bacteria which are usually eliminated from moderately contaminated shellfish within 48 h. Oysters accumulated greater concentrations of V. cholerae 01 than E. coli and S. tallahassee. When V. cholerae 01 was exposed to controlled purification at 15, 19 and 25 degrees C over 48 h, it persisted in oysters at markedly higher levels than E. coli and S. tallahassee. The concentration of a V. cholerae 01-specific agglutinin did not positively correlate with the uptake or retention of V. cholerae 01. These data show that state and federally approved controlled purification techniques are not effective at reducing V. cholerae 01 in oysters.
Vibrio vulnificus is a potentially lethal human pathogen capable of producing septicemia in susceptible persons. Disease is almost always associated with consumption of seafood, particularly raw oysters, or with exposure of wounds to seawater. An oligonucleotide DNA probe (V. vulnificus alkaline phosphatase-labeled DNA probe [VVAP]), previously shown to be highly specific for V. vulnificus, was used to enumerate this species in environmental samples collected from the Chesapeake Bay between April 1991 and December 1992. Total aerobic, heterotrophic, culturable bacteria were enumerated by plate counts on nonselective medium. The number of V. vulnificus organisms was determined by colony lifts of spread plates for subsequent hybridization with VVAP. V. vulnificus was not detected in any samples collected during February and March (water temperature of < 8 degrees C) but was found in 80% of the water samples collected during May, July, September, and December (water temperature of > 8 degrees C), with concentrations ranging from 3.0 x 10(1) to 2.1 x 10(2)/ml (ca. 8% of the total culturable heterotrophic bacteria). In a multiple regression analysis, increased V. vulnificus concentrations were correlated with lower salinities and with isolation from samples collected closer to the bottom. Isolation from oysters was demonstrable when water temperatures were 7.6 degrees C, with concentrations ranging from 1.0 x 10(3) to 4.7 x 10(4)/g (ca. 12% of total culturable bacteria). In samples collected in May and July, V. vulnificus was identified in seven of seven plankton samples and four of nine sediment samples. Our data demonstrate that V. vulnificus is a widespread and important component of the bacterial population of the Chesapeake Bay, with counts that are comparable to those reported from the Gulf of Mexico.
Oysters naturally contaminated with 10(3) to 10(4) most probable numbers (MPN) of Vibrio vulnificus per g were relayed to offshore waters (salinity, 30 to 34 ppt), where they were suspended in racks at a depth of 7.6 m. V. vulnificus counts in oysters were reduced to < 10 MPN/g within 7 to 17 days in five of the six studies. At the end of the studies (17 to 49 days), V. vulnificus levels were reduced further and ranged from a mean of 0.23 to 2.6 MPN/g. Oyster mortalities during relaying were < 6%. The reduction of V. vulnificus in relayed oysters is associated with exposure to high-salinity environments essentially devoid of V. vulnificus. Offshore suspension relaying may be a method that industry can employ to reduce V. vulnificus levels in raw Gulf Coast oysters.
The role of type 1 fimbriae in the interactions betweenEscherichia coli and Mytilus galloprovincialisLam. hemocytes was evaluated. The association of fimbriated strain MG155 with hemocyte monolayers at 18°C was 1.5- and 3-
to 4-fold greater than the association of unfimbriated mutant AAEC072 in artificial seawater and in hemolymph serum, respectively.
Such differences were apparently due to different adhesive properties since MG155 adhered more efficiently than AAEC072 when
hemocytes were incubated at 4°C to inhibit the internalization process. Hemolymph serum increased both association and adherence
of MG155 two- to threefold but did not affect association and adherence of AAEC072. MG155 was also 1.5- to 1.7-fold more sensitive
to killing by hemocytes than AAEC072, as evaluated by the number of culturable bacteria after 60 and 120 min of incubation.
The role of type 1 fimbriae in MG155 interactions with hemocytes was confirmed by the inhibitory effect ofd-mannose. In in vivo experiments MG155 cells were cleared from circulating hemolymph more rapidly than AAEC072 cells were
cleared. These results confirm that surface properties are crucial in influencing bacterial persistence and survival within
mussel hemolymph.
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