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Англомовна презентація результатів досліджень для навчання учнів презентувати наукові дослідження англійською мовою. Англійська мова для біологів є важливим складовим компонентом навчання в постійнозмінючому науковому просторі. Набуття вмінь та навичок презентувати результатів своїх досліджень англійською мовою є необхідною для тих, хто братиме участь у міжнародних конференціях або продовжуватиме навчання закордоном.

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Опис презентації окремими слайдами:
Depuration as a method to reduce Vibrio vulnificus populations in live Crassostrea virginica oysters Oleksandr Tokarskyy
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Depuration as a method to reduce Vibrio vulnificus populations in live Crassostrea virginica oysters Oleksandr Tokarskyy

Outline - Vibrio vulnificus in raw oysters and foodborne disease - Interventions against V. vulnificus in raw oysters Objectives of the study Mater...
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Outline - Vibrio vulnificus in raw oysters and foodborne disease - Interventions against V. vulnificus in raw oysters Objectives of the study Materials and methods Results and discussions Conclusions

Eastern oysters – Crassostrea virginica Important commercial species of bivalve mollusks Eastern oysters – the dominant species; 77.8% of all oyste...
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Eastern oysters – Crassostrea virginica Important commercial species of bivalve mollusks Eastern oysters – the dominant species; 77.8% of all oysters landings in 2002 (NMFS, 2004) Louisiana, Texas, Mississippi, and Florida accounted for 88% of Eastern oysters harvest -Filter feeders: accumulate toxins and bacteria from seawater

Vibrio vulnificus in raw oysters -Gulf Coast oysters: high incidence Summer (August, >104 MPN/g) vs winter (February-March, <3 MPN/g) (Birkenhauer ...
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Vibrio vulnificus in raw oysters -Gulf Coast oysters: high incidence Summer (August, >104 MPN/g) vs winter (February-March, <3 MPN/g) (Birkenhauer and Oliver, 2003) - Can enter Viable-But-Non-Culturable state, possibly due to hydrogen peroxide sensitivity (Bogosian et al., 1998) Route of pathogen introduction: cutaneous or foodborne illness

Vibrio vulnificus in raw oysters Foodborne illness: raw oysters consumption - No gastrointestinal disorders - Primary septicemia → death in suscept...
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Vibrio vulnificus in raw oysters Foodborne illness: raw oysters consumption - No gastrointestinal disorders - Primary septicemia → death in susceptible individuals, 60% mortality rate (Rosche et al., 2006)

Vibrio vulnificus pathogenicity, virulence, and pathogenesis Capsule and endotoxin are primary virulence factors; opaque phenotypes are virulent (Y...
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Vibrio vulnificus pathogenicity, virulence, and pathogenesis Capsule and endotoxin are primary virulence factors; opaque phenotypes are virulent (Yoshida et al., 1985) Must survive acidity of stomach to cause illness D-value= 3-4 minutes at pH 4.0 (Koo and Marshall, 2000) Cold-stressed V. vulnificus in VBNC state remains infectious by IV injection (Oliver and Bockian, 1995) 89% of all infections tracked to seawater temperature above 22oC (Shapiro et al., 1998) Only certain strains might cause illness (Jackson et al., 1997)

Factors influencing Vibrio vulnificus VBNC state entry Temperature <10oC (Oliver et al., 1991; Nilsson et al., 1991) Stationary culture vs. exponen...
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Factors influencing Vibrio vulnificus VBNC state entry Temperature <10oC (Oliver et al., 1991; Nilsson et al., 1991) Stationary culture vs. exponentially growing culture (Biosca et al., 1996) Pre-starvation before refrigeration and availability of nutrients (Oliver, 1991) Temperature decrease rate

Vibrio vulnificus identification and physiological assessment Analysis of cellular fatty acid profile – AOAC GC-FAME method to confirm Vibrio vulni...
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Vibrio vulnificus identification and physiological assessment Analysis of cellular fatty acid profile – AOAC GC-FAME method to confirm Vibrio vulnificus isolates (DePaola and Kaysner, 2004) - Bacterial fatty acid profile depends on environmental conditions: membrane fluidity adjustment (Yuk and Marshall, 2005; Linder and Oliver, 1989)

Methods to reduce Vibrio vulnificus numbers in raw oysters -Antimicrobials (electrolyzed water, chitosan, bacteriophages, diacetyl, lactic acid): u...
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Methods to reduce Vibrio vulnificus numbers in raw oysters -Antimicrobials (electrolyzed water, chitosan, bacteriophages, diacetyl, lactic acid): unsuccessful or adverse effects (Ren and Su, 2006; Chhabra et al., 2006; Pelon et al., 2005; Sun and Oliver, 1994) -Refrigeration: generally unsuccessful (Motes et al., 1998; Quevedo et al., 2005 , Melody et al. (2008) Freezing: successful, but changes in texture (Parker et al., 1994, Cook and Ruple,1992)

Methods to reduce Vibrio vulnificus numbers in raw oysters Irradiation, gamma-rays: successful, but customer unacceptability (Andrews et al., 2003)...
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Methods to reduce Vibrio vulnificus numbers in raw oysters Irradiation, gamma-rays: successful, but customer unacceptability (Andrews et al., 2003) High Pressure Processing (HPP): successful, but changes in texture (Kural and Chen, 2008; Cruz-Romero et al., 2008) 48 hours depuration: unsuccessful due to the short duration (Perkins et al., 1980; Jones et al., 1991; Eyles and Davis, 1984)

Objective #1 Influence of prolonged depuration (14 days) in ambient and cold seawater on Vibrio vulnificus counts, Aerobic Plate Count, and microbi...
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Objective #1 Influence of prolonged depuration (14 days) in ambient and cold seawater on Vibrio vulnificus counts, Aerobic Plate Count, and microbiological profile of Gulf Coast oysters Hypothesis: prolonged depuration in cold seawater might reduce V. vulnificus numbers, change microflora to select for psychrotrophic bacteria, and decrease aerobic plate count

1.2 x 2.4 meters 1,514 liters seawater volume UV light recirculation water disinfection Filtration: rock and sand gravity filters + charcoal filter...
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1.2 x 2.4 meters 1,514 liters seawater volume UV light recirculation water disinfection Filtration: rock and sand gravity filters + charcoal filter The maximum depth of shellfish- 7.6 cm 6400 liters of process water per cubic meter of shellfish Depuration Tank Design

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Materials and Methods Depuration studies, Pascagoula, MS Three replications with each salinity (12, 20, 35 ppt) at two temperatures (10 and 22oC) V...
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Materials and Methods Depuration studies, Pascagoula, MS Three replications with each salinity (12, 20, 35 ppt) at two temperatures (10 and 22oC) Vibrio vulnificus counts: FDA BAM method (Kaysner and DePaola, 2004) 3-tube MPN Technique, APW mCPC streak plate to confirm positive APW tubes Confirmation of isolates by API20E bioMerieux strips

Materials and Methods Aerobic plate count: Serial dilutions in PBS; spread plating on TSA; 48 hours at 35oC incubation Oyster microflora identifica...
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Materials and Methods Aerobic plate count: Serial dilutions in PBS; spread plating on TSA; 48 hours at 35oC incubation Oyster microflora identification: Countable TSA plates 18 bacterial isolates from fresh oysters 8 bacterial isolates from other treatments Identification using Fatty Acid profile analysis, GC-FAME

Aerobic Plate Count of oysters
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Aerobic Plate Count of oysters

Vibrio vulnificus count of oysters
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Vibrio vulnificus count of oysters

Isolated bacterial genera from freshly harvested oysters
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Isolated bacterial genera from freshly harvested oysters

Percentage of pathogenic Vibrio species out of total vibrios isolated from fresh oysters
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Percentage of pathogenic Vibrio species out of total vibrios isolated from fresh oysters

Isolated bacterial genera from freshly harvested oysters stored for 2 weeks at 4oC
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Isolated bacterial genera from freshly harvested oysters stored for 2 weeks at 4oC

Depuration: Cold vs. Ambient 10oC 22oC
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Depuration: Cold vs. Ambient 10oC 22oC

Incidence of pathogenic vibrios in oysters depurated for 2 weeks at 10 and 22oC
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Incidence of pathogenic vibrios in oysters depurated for 2 weeks at 10 and 22oC

Objective #2 Influence of day (0, 7, and 14), temperature (4, 10, 25oC) and salinity (12, 20, and 35 ppt) on Vibrio vulnificus survival under starv...
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Objective #2 Influence of day (0, 7, and 14), temperature (4, 10, 25oC) and salinity (12, 20, and 35 ppt) on Vibrio vulnificus survival under starvation conditions in artificial seawater in vitro

Materials and Methods Vibrio vulnificus ATCC 25762 strain Three replications with three salinity levels (12, 20, 35 ppt seawater) at three temperat...
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Materials and Methods Vibrio vulnificus ATCC 25762 strain Three replications with three salinity levels (12, 20, 35 ppt seawater) at three temperatures (4, 10 and 25oC) ~8 logs inoculation level of double-washed stationary grown cells into artificial seawater Following the 2-hour seawater adaptation, tubes were incubated at 4, 10, and 25oC for 14 days Vibrio vulnificus counts: APC: Dilutions in PBS as needed; TSA-2 spread plates Turbidity measurements Fluorescent viability counts

APC of starved Vibrio vulnificus
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APC of starved Vibrio vulnificus

Comparison of APC, OD600, FV for 4oC starved Vibrio vulnificus
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Comparison of APC, OD600, FV for 4oC starved Vibrio vulnificus

Objective #3 Influence of salinity (12, 20, and 35ppt), temperature (4, 10, and 25oC) and storage day (0, 7, and 14) on fatty acid profile changes ...
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Objective #3 Influence of salinity (12, 20, and 35ppt), temperature (4, 10, and 25oC) and storage day (0, 7, and 14) on fatty acid profile changes in starved Vibrio vulnificus in artificial seawater Hypothesis: Increase in short-chained and unsaturated fatty acids in Vibrio vulnificus stored at refrigerated temperatures to adjust membrane fluidity.

Materials and Methods Vibrio vulnificus ATCC 25762 strain Three replications with three salinity levels (12, 20, 35 ppt seawater) at three temperat...
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Materials and Methods Vibrio vulnificus ATCC 25762 strain Three replications with three salinity levels (12, 20, 35 ppt seawater) at three temperatures (4, 10 and 25oC) ~9 logs inoculation level of double-washed stationary grown cells into artificial seawater Following the 2-hour seawater adaptation, tubes were incubated at 4, 10, and 25oC for 14 days Sample centrifugation (7 minutes @ 2,700xg) to obtain cell pellet. Cell lysis, fatty acid saponification, methylation, extraction, alkali wash, and analysis as described previously (Yuk and Marshall, 2005)

Results A noticeable significant change (p<0.05) in 18:1 ω7c fatty acid and 18:1 ω6c fatty acid Possible switch from 18:1 ω7c to 18:1 ω6c through a...
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Results A noticeable significant change (p<0.05) in 18:1 ω7c fatty acid and 18:1 ω6c fatty acid Possible switch from 18:1 ω7c to 18:1 ω6c through a double bond relocation at 25oC Treatment Significance of starvation day/salinity influence on V. vulnificus fatty acid abundance. Bacterial culture Storage temperature 12:0 3OH 14:0 16:1 ω9c 16:0 18:1 ω7c 18:1 ω6c 18:0 sum feature 2 sum feature 3 ATCC 27562 4oC +/- +/- -/- -/- +/- +/+ +/- +/- +/- ATCC 27562 10oC +/- +/- -/- -/- +/+ +/- +/- +/- +/- ATCC 27562 25oC +/+ +/- +/- +/- +/- +/- +/- -/- +/-

4oC 10oC 25oC All seawater salinities combined
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4oC 10oC 25oC All seawater salinities combined

Fatty acid change in Vibrio vulnificus
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Fatty acid change in Vibrio vulnificus

Objective #4 Influence of 7 day Vibrio vulnificus starvation in artificial seawater (in vitro) on its ability to survive in simulated gastrointesti...
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Objective #4 Influence of 7 day Vibrio vulnificus starvation in artificial seawater (in vitro) on its ability to survive in simulated gastrointestinal fluid (SGF) Hypothesis: several studies indicated possibility of certain stress adaptation to induce cross-adaptation to another stress. Low incidence of vulnificus-associated septicemia during winter – either due to low numbers, or due to the higher susceptibility to the gastric acid

Materials and Methods Vibrio vulnificus ATCC 25762 strain Five replications (20 ppt seawater) at three temperatures (4, 10 and 25oC) ~8 logs inocul...
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Materials and Methods Vibrio vulnificus ATCC 25762 strain Five replications (20 ppt seawater) at three temperatures (4, 10 and 25oC) ~8 logs inoculation level of double-washed stationary grown cells into 20 ppt seawater Following the 2-hour seawater adaptation, tubes were incubated at 4, 10, and 25oC for 7 days Samples transferred to SGF and aliquots removed every 3 min for bacterial counts Vibrio vulnificus counts: Dilutions in PBS as needed TSA-2%salt spread plates, 37oC, 48 hours

Survival of V. vulnificus in SGF
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Survival of V. vulnificus in SGF

D-values for V. vulnificus populations
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D-values for V. vulnificus populations

Conclusions -Prolonged depuration in either ambient (22oC) or cold (10oC) seawater significantly (p<0.05) reduced V. vulnificus populations, but no...
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Conclusions -Prolonged depuration in either ambient (22oC) or cold (10oC) seawater significantly (p<0.05) reduced V. vulnificus populations, but not to non-detectable level Prolonged cold-water depuration did not offer significant advantage; APC counts were higher Refrigerated depuration, as well as storage, shifts overall microflora, selecting psychrotrophic microorganisms

Conclusions In vitro studies suggested that temperature (4oC) and salinity (35 ppt) might be the significant factors influencing eventual decrease ...
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Conclusions In vitro studies suggested that temperature (4oC) and salinity (35 ppt) might be the significant factors influencing eventual decrease in pathogen numbers Pathogen numbers decrease in oysters during prolonged depuration might be attributed to UV light treatment and dilution Under starvation V. vulnificus cells do not adjust their fatty acid profile noticeably within investigated time frame with an exception of C18:1 double bond relocation at higher temperatures Cold stressed V. vulnificus has significantly lower resistance (p<0.05) to gastric fluid compared to control

Acknowledgements: Dr. Linda Andrews and Mr. Jeff Echols QUESTIONS?
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Acknowledgements: Dr. Linda Andrews and Mr. Jeff Echols QUESTIONS?

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