International Journal of Food Microbiology 163 (2013) 146–152

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International Journal of Food Microbiology

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Effects of pre- or post-processing storage conditions on high-hydrostaticpressure inactivation of Vibrio parahaemolyticus and V. vulnificus in oysters

Mu Ye a, Yaoxin Huang a, Joshua B. Gurtler b, Brendan A. Niemira b, Joseph E. Sites b, Haiqiang Chen a,⁎a Department of Animal and Food Sciences, University of Delaware, Newark, DE 19716, USAb USDA-ARS, Eastern Regional Research Center, Wyndmoor, PA 19038, USA

⁎ Corresponding author. Tel.: +1 302 831 1045; fax:E-mail address: haiqiang@udel.edu (H. Chen).

0168-1605/$ – see front matter © 2013 Elsevier B.V. Alhttp://dx.doi.org/10.1016/j.ijfoodmicro.2013.02.019

a b s t r a c t

a r t i c l e i n f o

Article history:Received 16 November 2012Received in revised form 17 February 2013Accepted 18 February 2013Available online 5 March 2013

Keywords:High-hydrostatic pressureVibrio parahaemolyticusVibrio vulnificusCold storageOyster

The effects of storage conditions on subsequent high-hydrostatic pressure (HHP) inactivation of Vibrioparahaemolyticus and Vibrio vulnificus in oysters were investigated. Live oysters were inoculated withV. parahaemolyticus or V. vulnificus to ca. 7–8 log MPN/g by feeding and stored at varying conditions (i.e.,21 or 35 °C for 5 h, 4 or 10 °C for 1 and 2 days and −18 °C for 2 weeks). Oyster meats were then treatedat 225–300 MPa for 2 min at 4, 21 or 35 °C. HHP at 300 MPa for 2 min achieved a >5-log MPN/g reductionof V. parahaemolyticus, completely inactivating V. vulnificus (negative by enrichment) in oysters. Treatmenttemperatures of 4, 21 and 35 °C did not significantly affect pressure inactivation of V. parahaemolyticus orV. vulnificus (P > 0.05). Cold storage at −18, 4 and 10 °C, prior to HHP, decreased V. parahaemolyticus orV. vulnificus populations by 1.5–3.0 log MPN/g, but did not increase their sensitivity to subsequent HHP treat-ments. The effects of cold storage after HHP on inactivation of V. parahaemolyticus in oysters were also deter-mined. Oysters were inoculated with V. parahaemolyticus and stored at 21 °C for 5 h or 4 °C for 1 day. Oystermeats were then treated at 250–300 MPa for 2 min at 21 or 35 °C and stored for 15 days in ice or in a freezer.V. parahaemolyticus populations in HHP-treated oysters gradually decreased during post-HHP ice or frozenstorage. A validation study using whole-shell oysters was conducted to determine whether the presence ofoyster shells influenced HHP inactivation of V. parahaemolyticus. No appreciable differences in inactivationbetween shucked oyster meat and whole-shell oysters were observed. HPP at 300 MPa for 2 min at 21 °C,followed by 5-day ice storage or 7-day frozen storage, and HPP at 250 MPa for 2 min at 21 °C, followedby 10-day ice or 7-day frozen storage, completely inactivated V. parahaemolyticus in whole-shell oysters(>7 log reductions). The combination of HHP at a relatively low pressure (e.g., 250 MPa) followed byshort-term frozen storage (7 days) could potentially be applied by the shellfish industry as a post-harvestprocess to eliminate V. parahaemolyticus in oysters.

© 2013 Elsevier B.V. All rights reserved.

1. Introduction

Vibrio parahaemolyticus and Vibrio vulnificus are important foodbornepathogens of public health concern. Outbreaks with these pathogenshave mainly been associated with consumption of raw or undercookedoysters (CDC, 1998, 1999, 2006). V. vulnificus can induce primary septi-cemia with mortality rates up to 50% (Linkous and Oliver, 1999) and isresponsible for 95% of all seafood-related deaths in the United States(Oliver and Kaper, 2001). V. parahaemolyticus is a leading cause ofacute human gastroenteritis from the consumption of contaminatedseafood, with an estimated 35,000 annual cases in the United States(Scallan et al., 2011). The consumption of shellfish, including oysters,led to a 2006 outbreak of gastrointestinal illnesses in which 177 cases(72 culture-confirmed) were reported in three states (CDC, 2006).

To mitigate foodborne illness risks associated with Vibrio spp.,harvesting and post-harvest handling of oysters should meet specified

+1 302 831 2822.

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criteria to prevent multiplication of Vibrio to potentially hazardouslevels. In addition, the use of a post-harvest process (PHP) to reduceVibrio bacteria is also recommended. The National Shellfish SanitationProgram (NSSP) established a “Guide for Control ofMolluscan Shellfish”which requires a PHP to reduce Vibrio by >3.52 log to non-detectablelevels (b30 MPN/g) (FDA, 2009). Examples of acceptable PHPs includedepuration (Kelly and Dinuzzo, 1985), cold treatments (Cook andRuple, 1992; Melody et al., 2008), mild-heat treatment (Andrews etal., 2000; Cook and Ruple, 1992), irradiation (Jakabi et al., 2003;Mahmoud, 2009) and high-hydrostatic pressure (HHP) (Calik et al.,2002; Cook, 2003; Kural and Chen, 2008). HHP can inactivate microor-ganisms and sometimes enzymes with only minor deleterious changesto flavor, color, and nutritional quality (San Martin et al., 2002). HHP isbecoming increasingly acknowledged as the processing method ofchoice for oysters by virtue of its overriding safety, quality and econom-ic benefits (He et al., 2002; Martin and Hall, 2006). With HHP, a satis-factory kill can be achieved due to the relatively high sensitivity ofV. parahaemolyticus and V. vulnificus to pressure (Ye et al., 2012). Coldstorage is commonly used to limit the growth of Vibrio spp. in shellfish

147M. Ye et al. / International Journal of Food Microbiology 163 (2013) 146–152

following harvest. Numerous studies have reported that refrigerationor frozen storage is capable of achieving certain reductions ofV. parahaemolyticus and V. vulnificus in seafood (Liu et al., 2009;Oliver, 1981; Parker et al., 1994; Thompson et al., 1976). The NSSP hasestablished time–temperature matrices, requiring strict temperaturecontrol of oysters during transportation, processing and storage. In theV. parahaemolyticus control plan, the time oysters spend betweenharvest and refrigeration temperatures is not to exceed 5 h (FDA,2009). The Interstate Shellfish Sanitation Conference (ISSC) hasadopted freezing combined with frozen storage as an acceptablepostharvest treatment to control V. vulnificus and V. parahaemolyticusin oysters. However, it is generally agreed that the inactivation effectsof cold storage are limited, and complete elimination of both pathogensin oysters during cold storage is unlikely (Cook and Ruple, 1992;Johnson et al., 1973; Liu et al., 2009; Parker et al., 1994).

Depending on the harvest season and methods of transportationand storage, oysters can be exposed to a variety of temperaturesprior to pressure treatment (Cook et al., 2002). The harvest watertemperature can vary from b0 °C to >30 °C, depending on the timeand location of harvest. Oysters may be left on harvest vessels ex-posed to the sun without cooling for several hours or until the vesseldocks. Moreover, oysters may also be held in cold seawater for up to1 day in processing plants prior to processing. However, there is noinformation available regarding whether varying storage conditionsbefore HHP could affect the subsequent pressure inactivation ofVibrio. Furthermore, to our knowledge, the fate of Vibrio survival inoysters during post-HHP cold storage has not been addressed.

The objectives of this studywere to 1) determine the effect of storageconditions on the subsequent pressure inactivation ofV. parahaemolyticusand V. vulnificus in oysters, 2) determine the fate of Vibrio survivors dur-ing post-HHP cold storage aswell as 3) evaluatewhether frozen storagebefore and after HHP treatment could be used as an additional hurdle toenhance HHP inactivation of Vibrio.

2. Materials and methods

2.1. Cultures

Two pressure-resistant Vibrio strains (V. parahaemolyticus ATCC43996 and V. vulnificus MLT 403) were used in this study (Kural andChen, 2008; Kural et al., 2008). Stock cultures of V. parahaemolyticusand V. vulnificuswere maintained on tryptic soy agar (Difco Laborato-ries, Sparks, MD) (TSA) plus 0.5% NaCl (Fisher Scientific, Fair Lawn,NJ) (TSAS0.5%) at room temperature (ca. 21 °C). To prepare workingcultures, a loopful of V. parahaemolyticus or V. vulnificus was trans-ferred from a TSAS0.5% plate to a tube of 10 ml tryptic soy broth(Difco Laboratories) plus 0.5% NaCl (TSBS0.5%) and incubated at35 °C for 9 h, allowing cells to reach stationary phase, as previouslyvalidated in Ye et al. (2011). A loopful of culture was then transferredto 100 ml of fresh TSBS0.5% and incubated at 35 °C for 9 h, to producea cell density of ca. 108–109 CFU/g.

2.2. Oysters

Live oysters (Crassostrea virginica) were obtained from the Collegeof Earth, Ocean, and Environment at the University of Delaware.Oysters were cleaned, placed and maintained in an aerated, circulat-ing seawater tank at room temperature and fed with algae (ReedMariculture Inc., San Jose, CA). The salinity of the seawater wasmaintained at an optimal level of 1.5–2%.

2.3. Inoculation of oysters with V. parahaemolyticus or V. vulnificus

Live oysterswere inoculatedwithV. parahaemolyticus or V. vulnificusas described by Kural and Chen (2008). Briefly, ca. 40 live oysters wereremoved from the seawater tank, placed into an autoclavable plastic

tray filled with 4 l of fresh seawater. To inoculate, 80 ml of the cultureof V. parahaemolyticus or V. vulnificus was poured into the tray andmixed well. The tray was covered with a piece of aluminum foil andkept at room temperature for 24 h.

2.4. Effect of storage conditions before HHP on inactivation ofV. parahaemolyticus or V. vulnificus in oysters

Inoculated oysters were removed from seawater and subjected todifferent storage conditions: 1) 21 or 35 °C for 5 h in air, 2) 4 °C for 1and 2 days or 10 °C for 1 day in seawater, or 3) −18 °C in a freezerfor 2 weeks. Oysters were then shucked. For frozen oysters thatwere used, they were shucked and thawed. The contents (meat andjuice) were placed into sterile plastic pouches (polyethylene; FisherScientific, Pittsburg, PA). Pouches were double-sealed and double-bagged. Samples were pressure-treated at 225, 250, 275 or 300 MPafor 2 min using a high-pressure unit with water as the hydrostaticmedium (model Avure PT-1; Avure Technologies, Kent, WA). Experi-ments were conducted at initial sample temperatures of 4, 21 or35 °C. The pressure come-up rate was ca. 22 MPa/s and the pressurerelease time was b4 s. The compression heating factors were 1.9,2.2 and 2.9 °C/100 MPa with initial sample temperatures of 4, 21and 35 °C, respectively. Pressurization time reported in this studydid not include the pressure come-up or release times. Populationsof V. parahaemolyticus and V. vulnificus in oysters before storage,after storage, and after pressure treatment were determined.

2.5. Effects of cold storage after HHP on inactivation ofV. parahaemolyticus in oysters

Oysters were inoculated with V. parahaemolyticus as describedabove. Inoculated samples were stored at 21 °C for 5 h in air or at4 °C for 1 day in seawater. Oysters were then shucked and the con-tents pressurized with 250 MPa at 21 and 35 °C, or with 300 MPa at21 °C for 2 min. Inoculated oysters with or without HHP treatmentwere stored either in a cooler covered with ice or in a freezer at−18 °C for up to 15 days. V. parahaemolyticus populations insamples were determined 1) following inoculation, 2) just prior toHHP, 3) immediately after HHP and 4) at selected time intervals duringcold storage.

2.6. Validation of HHP on whole-shell live oysters

2.6.1. Effects of HHP on inactivation of V. parahaemolyticus inwhole-shell oysters

Whole-shell oysters were inoculated with V. parahaemolyticus asdescribed above. Oysters were then banded, packaged, and treatedwith 250 or 300 MPa at room temperature (22–24 °C) for 2 minusing a 2 l HHP unit (Avure Technologies, Kent, WA) with water asa hydrostatic medium at the Eastern Regional Research Center ofthe U.S. Department of Agriculture. The 2 l HHP unit used for theseexperiments is larger than the PT-1 HHP unit used for the experi-ments described above. The increased capacity allowed treatment ofintact, whole-shell oysters, rather than oyster meat only. The pressurecome-up rate for the 2-l HHP unit was ca. 20 MPa/s and the pressurerelease time was b4 s. Inoculated oysters with or without HHP treat-ment were stored either in a cooler covered with ice or in a freezer at−18 °C for up to 15 days. Counts of V. parahaemolyticus in the sam-ples were determined after inoculation, just prior to HHP, followingHHP, and at selected time intervals during cold storage. Each sampleconsisted of two whole-shell oysters.

2.6.2. Microbial shelf life of HHP-treated whole-shell oystersUn-inoculated live whole-shell oysters were packaged and treated

at 250 or 300 MPa at room temperature (22–24 °C) for 2 min usingthe 2-l HHP unit. Oysters with or without HHP treatment were either

Table 1Effect of storage conditions on subsequent HHP inactivation of V. parahaemolyticus in oys-ter meat. Oysters were inoculated with V. parahaemolyticus to 7.8 ± 0.3 log MPN/g byfeeding and stored at different conditions before being shucked. Oyster meats were thentreated at 225–300 MPa for 2 min at initial sample temperatures of 4, 21 and 35 °C.Data represent mean log survivors (MPN/g) ± standard deviation. For each of six storageconditions, data in the same columnhaving the same lower case letter are not significantlydifferent (P > 0.05). The detection limit by plating was 3 MPN/g.

Pre-HHP storage HHP @ 225 MPa 250 MPa 275 MPa 300 MPa

Stored at 21 °C for5 h (7.3 ± 0.3)⁎

4 °C 5.2 ± 0.2a 4.4 ± 0.2a 3.0 ± 0.2a 1.3 ± 0.3ab

21 °C 5.7 ± 0.3a 4.4 ± 0.4a 3.4 ± 0.2a 1.6 ± 0.2a

35 °C 5.2 ± 0.2a 4.3 ± 0.4a 2.9 ± 0.5a 0.8 ± 0.2b

Stored at 35 °C for5 h (7.7 ± 0.3)⁎

4 °C 5.9 ± 0.2a 4.1 ± 0.9a 2.1 ± 0.2a 1.1 ± 0.5a

21 °C 6.0 ± 0.4a 4.6 ± 0.5a 3.4 ± 0.5b 2.0 ± 0.4b

35 °C 5.1 ± 0.4b 3.6 ± 0.9a 2.8 ± 0.7ab 1.3 ± 0.7ab

Stored at 4 °C for1 day (6.3 ± 0.2)⁎

4 °C 3.7 ± 0.3a 2.8 ± 0.8a 2.3 ± 0.3a 1.0 ± 0.4a

21 °C 5.2 ± 0.2b 3.7 ± 0.3a 2.5 ± 0.5a 1.2 ± 0.6a

35 °C 4.0 ± 0.4a 3.0 ± 0.3a 2.2 ± 0.4a 1.0 ± 0.4a

Stored at 4 °C for2 days (6.1 ± 0.7)⁎

4 °C 4.0 ± 0.6ab 2.7 ± 0.2a 1.9 ± 0.5a 1.1 ± 0.4a

21 °C 5.0 ± 0.7a 3.2 ± 0.1b 2.4 ± 0.5a 1.2 ± 0.1a

35 °C 3.7 ± 0.5b 2.9 ± 0.4ab 1.4 ± 0.7a 0.9 ± 0.6a

Stored at 10 °C for1 day (6.2 ± 0.5)⁎

4 °C 4.9 ± 0.3a 2.7 ± 0.5a 2.0 ± 0.4a 1.1 ± 0.2a

21 °C 5.3 ± 0.7a 3.3 ± 0.8a 2.0 ± 0.5a 1.1 ± 0.5a

35 °C 3.7 ± 0.3b 2.8 ± 0.5a 1.6 ± 0.6a 0.8 ± 0.5a

Stored at −18 °C for2 weeks (5.6 ± 0.4)⁎

4 °C 3.5 ± 1.1ab 2.8 ± 0.4a 1.7 ± 0.3a 0.6 ± 0.0a

21 °C 4.4 ± 0.4a 2.3 ± 1.0ab 2.1 ± 0.9a 0.7 ± 0.3a

35 °C 3.2 ± 0.7b 1.8 ± 0.5b 1.0 ± 0.1b 0.6 ± 0.0a

⁎ The numbers included in the “pre-HPP storage” column are MPN counts afterstorage and prior to HPP.

Table 2Effect of storage conditions on subsequent HHP inactivation of V. vulnificus in oystermeat. Oysters were inoculated with V. vulnificus to a level of 8.4 ± 0.6 log MPN/g byfeeding and stored at different conditions before being shucked. Oyster meats werethen treated at 225–300 MPa for 2 min at initial sample temperatures of 4, 21 and35 °C. Data represent mean log survivors (MPN/g) ± standard deviation. Numbers infractions represent the number of samples testing positive after enrichment out of atotal of 3 trials. For each of six storage conditions, data in the same column havingthe same lower case letter are not significantly different (P > 0.05). The detectionlimit by plating was 3 MPN/g. The detection limit by enrichment was 0.1 MPN/g.

Pre-HHP storage HHP @ 225 MPa 250 MPa 275 MPa

Stored at 21 °C for5 h (8.2 ± 0.7)⁎

4 °C 3.2 ± 0.7a 2.0 ± 0.4a 0.7 ± 0.3a

21 °C 3.9 ± 0.2a 2.7 ± 0.3a 0.9 ± 0.5a

35 °C 3.0 ± 0.7a 1.1 ± 0.9a b0.5 (2/3)Stored at 35 °C for5 h (8.1 ± 0.2)⁎

4 °C 3.7 ± 0.4a 1.6 ± 0.6ab 0.9 ± 0.5a

21 °C 3.2 ± 0.4a 2.3 ± 0.4a 1.0 ± 0.4a

35 °C 3.0 ± 0.8a 1.1 ± 0.4b 0.5 ± 0.5a

Stored at 4 °C for1 day (5.4 ± 0.2)⁎

4 °C 1.7 ± 0.2a 1.1 ± 0.2a b0.5 (1/3)21 °C 2.7 ± 0.4b 1.1 ± 0.2a b0.5 (2/3)35 °C 2.4 ± 0.7b 1.0 ± 0.0a b0.5 (0/3)

Stored at 4 °C for2 days (6.0 ± 0.5)⁎

4 °C 2.2 ± 0.5a 0.7 ± 0.2a b0.5 (2/3)21 °C 1.8 ± 0.8a 1.1 ± 0.4a b0.5 (2/3)35 °C 1.7 ± 0.7a 0.7 ± 0.2a b0.5 (0/3)

Stored at 10 °C for1 day (6.5 ± 0.4)⁎

4 °C 3.3 ± 0.4a 1.7 ± 0.3a b0.5 (1/3)21 °C 3.7 ± 0b 2.1 ± 0.5a b0.5 (3/3)35 °C 1.8 ± 0.3c 0.8 ± 0.5b b0.5 (0/3)

Stored at −18 °C for2 weeks (5.5 ± 0.5)⁎

4 °C 0.7 ± 0.2a 0.7 ± 0.3a b0.5 (1/3)21 °C 1.4 ± 0.2b 0.9 ± 0.2a b0.5 (2/3)35 °C 0.7 ± 0.2a b0.5 (2/3) b0.5 (0/3)

⁎ The numbers included in the “pre-HPP storage” column are MPN counts afterstorage and prior to HPP.

148 M. Ye et al. / International Journal of Food Microbiology 163 (2013) 146–152

stored in a cooler covered with ice or in a freezer at −18 °C for up to15 days. Total aerobic plate counts (APC) and psychrotrophic platecounts (PPC) in the samples were determined before HHP, afterHHP, and at selected time intervals during cold storage. Each sampleconsisted of three whole-shell oysters.

2.7. Microbiological analysis

2.7.1. Enumeration of V. parahaemolyticus and V. vulnificusCounts of V. parahaemolyticus and V. vulnificus in un-treated and

treated samples were determined using the most probable number(MPN) method as described in the FDA Bacteriological AnalyticalManual (FDA, 2004) with slight modifications. Briefly, samples wereserially diluted with phosphate buffered saline (PBS, pH = 7.4) inten-fold dilutions. Three × 1 ml portions of each dilution were inocu-lated into 3 tubes containing 10-ml alkaline peptone water (APW,pH = 8.5). APW tubes for V. parahaemolyticus-inoculated sampleswere incubated at 30 °C overnight, which was determined to be theoptimal recovery temperature for V. parahaemolyticus following pres-surization (Ye et al., 2011). A loop of enriched APW from the top1.0 cm of a turbid tube was streaked onto thiosulfate citrate bilesalts sucrose (TCBS) agar and incubated at 35 °C overnight. APWtubes for V. vulnificus samples were incubated at 35 °C overnightand a loop of the APW enrichment was streaked onto cellobiose colis-tin (CC) agar and incubated at 40 °C overnight to inhibit the growthof many other marine bacteria. After incubation, plates were evaluat-ed for typical colonies to confirm the presence of V. parahaemolyticusor V. vulnificus in MPN tubes. Positive tubes were counted and the3-tube-MPN tables were employed for final enumeration of the path-ogens. Enrichment of undiluted samples was conducted for treat-ments where low counts were expected. For enrichments, 100 ml ofAPW was added into the remaining meats and incubated overnightat 35 °C along with the tubes. Enrichments were streaked onto TCBSor CC agar plates and the plates were evaluated for typical coloniesto confirm the presence of V. parahaemolyticus or V. vulnificus after in-cubation, as described above.

2.7.2. APC and PPCAt each sampling point, 3 oysters were shucked and homogenized,

representing one sample. Samples were serially diluted with PBS inten-fold dilutions. Bacterial counts in oyster samples were deter-mined by spread-plating onto TSA. The APC and PPC were determinedby incubating TSA plates at 35 °C for 48 h and 7 °C for 10 days, re-spectively (Cousin et al., 2001).

2.8. Statistical analysis

Three independent trials were conducted for each treatment.Colony counts were converted to log MPN/g and means and standarddeviations were calculated. Statistical analyses were conducted usingJMP (SAS Institute, Cary, NC). Tukey's one-way multiple comparisonswere used to determine significant differences among treatments(P b 0.05).

3. Results

3.1. Effects of storage conditions before HHP on inactivation ofV. parahaemolyticus and V. vulnificus in oysters

Effects of storage conditions before HHP on inactivation ofV. parahaemolyticus and V. vulnificus in oysters are shown in Tables 1and 2, respectively. Initial counts of V. parahaemolyticus and V. vulnificusin the untreated oysters were 7.8 and 8.4 log MPN/g, respectively. Afterstorage at 21 or 35 °C for 5 h, populations of V. parahaemolyticus andV. vulnificus remained practically unchanged. V. parahaemolyticusproved to be cold-sensitive, as refrigerated storage at 4 or 10 °C for 1

or 2 days decreased the populations of V. parahaemolyticus by 1.5–1.7 log MPN/g. The strain of V. vulnificus was even more sensitiveto cold storage, as one-day storage at 4 °C or 10 °C reduced its pop-ulation by 3.0 and 1.9 log, respectively. Frozen storage for 2 weeks re-duced V. parahaemolyticus and V. vulnificus by 2.2 and 2.9 log MPN/g,respectively.

The V. vulnificus strain was more sensitive to HHP than theV. parahaemolyticus strain used in the present study. For example,after oysters were stored at 21 °C for 5 h, pressure treatmentsof 225, 250 and 275 MPa for 2 min at 21 °C reduced V. vulnificusby 4.3, 5.5 and 7.3 log MPN/g, respectively; whereas the same

149M. Ye et al. / International Journal of Food Microbiology 163 (2013) 146–152

treatments only resulted in 1.6, 2.9 and 3.9 log MPN/g reductions ofV. parahaemolyticus, respectively. The combinations of prior cold stor-age at 4, 10 or −18 °C and HHP treatment of 275 MPa for 2 min at35 °C completely eliminated V. vulnificus (negative enrichment re-sults). However, none of the 300 MPa treatments inactivated allV. parahaemolyticus cells in oysters, even following prior cold storage.Under the same storage conditions, HHP conducted at 35 and 4 °Cachieved higher reductions of both V. parahaemolyticus and V. vulnificusthan at 21 °C for the majority of HHP treatments. Nevertheless, in mostcases, the differences in log reduction among the three HHP treatmenttemperatures (viz., 4, 21 and 35 °C) were not statistically significant(P > 0.05).

Although both V. parahaemolyticus and V. vulnificus strains werecold sensitive and cold storage reduced their counts (1.5–2.2 log forV. parahaemolyticus and 1.9–3.0 log for V. vulnificus), cold storagedid not increase their sensitivity to subsequent HHP treatments. Forexample, reductions achieved by HHP alone (= counts after stor-age − counts after HHP) at 250 MPa and 21 °C were 2.9, 3.1, 2.6,2.9 and 3.3 log when V. parahaemolyticus was stored at 21, 35, 4, 10and−18 °C, respectively, while therewas no statistical difference in in-activation results among these storage conditions. For V. vulnificus, itseems that prior refrigerated storage at 4 and 10 °C increased its resis-tance to subsequent HHP treatments. Inactivation of V. vulnificus byHHP alone at 225–275 MPa was lower for oysters stored at 4 and10 °C than for oysters stored at 21 and 35 °C. For example, reductionsachieved by HHP alone at 225 MPa and 21 °C were 4.3, 4.9, 2.7, 4.2and 2.8 log when V. vulnificus was stored at 21 °C, 35 °C, 4 °C for1 day, 4 °C for 2 days, and 10 °C, respectively.

3.2. Effect of cold storage after HHP on inactivation ofV. parahaemolyticus in oysters

Since V. parahaemolyticuswas more resistant to both cold tempera-ture and HHP than V. vulnificus, only V. parahaemolyticuswas examinedfor response to post-processing as well as in the whole-shell oysterstudy. Populations of V. parahaemolyticus in oysters stored at room tem-perature for 5 h followed byHHP at 250 MPa or 300 MPawere reducedto ca. 3 log and 0.7 MPN/g, respectively (Table 3). During subsequent iceand frozen storages, populations of V. parahaemolyticus in HHP-treatedand un-treated oysters were further reduced. V. parahaemolyticuscounts in unpressurized samples decreased to 3.7 and 3.5 log MPN/gafter 15 days ice and frozen storages, respectively. Ice and frozen stor-ages of the HHP-treated samples resulted in up to 2.7 log greater reduc-tionswhen comparedwith inactivation assessed immediately followingHHP treatments. During the ice and frozen storages following HHPtreatment, similar results were obtained at each sampling time pointfor the pressure treatment of 250 MPa conducted at two initial sampletemperatures of 21 and 35 °C. The two 250 MPa treatments were

Table 3Effect of cold storage after HHP on inactivation of V. parahaemolyticus in oyster meat. Oystersfor 1 day before being shucked. Oyster meats were then treated at 250–300 MPa for 2 minsurvivors (MPN/g) ± standard deviation. Numbers in fractions represent the number of sV. parahaemolyticus in oysters after inoculation was 7.2 ± 0.2 log MPN/g. The detection lim

Day 0 Ice

Day 1 Day 2 Day

Stored at 21 °C for 5 h prior to HHPNo HHP 6.8 ± 0.2 5.3 ± 0.6 4.7 ± 0.4 4.7250 MPa at 21 °C 3.0 ± 0.2 1.8 ± 0.3 1.5 ± 0.3 0.8250 MPa at 35 °C 3.2 ± 0.1 1.7 ± 0.3 0.9 ± 0.4 0.7300 MPa at 21 °C 0.7 ± 0.2 0.3 ± 0.6 b0.5 (1/3) b0.5

Stored at 4 °C for 1 day prior to HHPNo HHP 5.6 ± 0.4 5.3 ± 0.1 4.5 ± 0.6 5.0250 MPa at 21 °C 2.2 ± 0.2 1.6 ± 0.2 1.0 ± 0.4 0.7250 MPa at 35 °C 2.1 ± 0.4 1.7 ± 0.3 1.0 ± 0.4 0.5300 MPa at 21 °C b0.5 (1/3) b0.5 (1/3) b0.5 (1/3) b0.5

capable of completely eliminating V. parahaemolyticus after 15 days ofice or frozen storage. The 300 MPa treatment was more effective thanthe two 250 MPa treatments, eliminating V. parahaemolyticus after5 days of ice storage or after 7 days of frozen storage.

Although HHP treatments with prior cold storage at 4 °C for 1 dayresulted in lower counts of V. parahaemolyticus immediately follow-ing pressure treatments (data for day 0) than the correspondingHHP treatments with 5 h of prior storage at 21 °C, the benefits of priorcold storage to enhance V. parahaemolyticus inactivation disappearedduring subsequent ice and frozen storages. That is, at each samplingtime point during subsequent cold storage, there were no significant dif-ferences in populations of V. parahaemolyticus in oysters subjected to thesame pressure treatment when comparing oysters exposed to priorwarm storage at 21 °C and prior cold storage at 4 °C (P > 0.05). There-fore, in the following validation study, oysters were only exposed towarm storage at 21 °C prior to HHP.

3.3. Validation of HHP with whole-shell oysters

3.3.1. Effect of HHP on inactivation of V. parahaemolyticus in whole-shelloysters

A validation study using whole-shell oysters was conductedto determine whether HHP had the same inactivation effect ofV. parahaemolyticus in oyster meat as in whole-shell oysters. Resultsfor whole-shell oysters (Table 4) were very comparable to that of oys-ter meat (Table 3). V. parahaemolyticus populations were slightlylower in whole-shell oysters than in oyster meat, probably due tothe longer pressure come-up time of the 2-l unit; however, differ-ences were not statistically significant for any sampling point(P > 0.05). HHP treatments at 1) 250 MPa followed by 10-day icestorage, 2) at 300 MPa followed by 5-day ice storage, and 3) at bothpressure levels followed by 7-day frozen storage completely eliminat-ed V. parahaemolyticus in whole-shell oysters.

3.3.2. Fluctuations in bacterial populations during storageThe total aerobic mesophilic and psychrotrophic bacterial counts

in pressure-treated and un-treated whole-shell oysters during stor-age in ice are shown in Fig. 1. Live oysters had initial APC counts of4.6 log CFU/g and slightly higher PPC levels at 5.0 log CFU/g, whichwere indicative of the high quality of oysters, as fresh bivalve mol-luscs are generally considered of good quality with APC counts ofb5 × 105 (i.e., 5.7 log) CFU/g (ICMSF, 1980). APC counts in oysterswere significantly reduced by ≥2 log CFU/g following pressure treat-ments (P b 0.05). Treatments at 250 and 300 MPa for 2 min reducedthe PPC to 3.3 and 1.5 log CFU/g, respectively. Both APC and PPC grad-ually increased during ice storage. At the end of 15-days of storage,APC and PPC counts in HHP-treated and un-treated oysters wereb7 log CFU/g. Since oysters are generally considered spoiled when

were inoculated with V. parahaemolyticus by feeding and stored at 21 °C for 5 h or 4 °Cat 21 or 35 °C and stored for 15 days in ice or in a freezer. Data represent mean log

amples testing positive after enrichment out of a total of 3 trials. The initial count ofit by plating was 3 MPN/g. The detection limit by enrichment was 0.1 MPN/g.

Frozen

5 Day 10 Day 15 Day 7 Day 15

± 0.1 3.3 ± 0.3 3.7 ± 0.6 4.0 ± 0.4 3.5 ± 0.1± 0.4 b0.5 (0/3) b0.5 (0/3) b0.5 (1/3) b0.5 (0/3)± 0.2 b0.5 (1/3) b0.5 (0/3) b0.5 (1/3) b0.5 (0/3)(0/3) b0.5 (0/3) b0.5 (0/3) b0.5 (0/3) b0.5 (0/3)

± 0.4 4.2 ± 0.8 3.4 ± 0.9 3.4 ± 0.2 3.6 ± 0.2± 0.2 b0.5 (0/3) b0.5 (0/3) b0.5 (1/3) b0.5 (0/3)± 0.1 b0.5 (0/3) b0.5 (0/3) b0.5 (1/3) b0.5 (0/3)(0/3) b0.5 (0/3) b0.5 (0/3) b0.5 (0/3) b0.5 (0/3)

Table 4Effect of cold storage after HHP on inactivation of V. parahaemolyticus in whole-shell oysters. Oysters were inoculated with V. parahaemolyticus by feeding and stored at 21 °C for5 h. They were then treated at 250–300 MPa for 2 min at 22–24 °C and stored for 15 days in ice or in a freezer. Data represent mean log survivors (MPN/g) ± standard deviation.Numbers in fractions represent the number of samples testing positive after enrichment out of 3 trials. The detection limit by plating was 3 MPN/g. The detection limit by enrich-ment was 0.1 MPN/g.

Day 0 Ice Frozen

Day 1 Day 5 Day 10 Day 15 Day 7 Day 15

No HHP 7.0 ± 0.4 5.5 ± 0.2 5.0 ± 0 4.4 ± 0.2 4.4 ± 0.1 5.1 ± 0.2 3.6 ± 0.3250 MPa 2.8 ± 0.4 2.0 ± 0.5 0.5 ± 0.5 b0.5 (0/3) b0.5 (0/3) b0.5 (0/3) b0.5 (0/3)300 MPa 0.6 ± 0.1 b0.5 (2/3) b0.5 (0/3) b0.5 (0/3) b0.5 (0/3) b0.5 (0/3) b0.5 (0/3)

150 M. Ye et al. / International Journal of Food Microbiology 163 (2013) 146–152

APC increases to >107 CFU/g (Kim et al., 2002), the HHP-treated andun-treated oysters had a shelf life of at least 15 days when stored inice. When HHP-treated and un-treated oysters were kept frozen dur-ing storage, APC and PPC counts were significantly lower than thosein oysters stored in ice after 15 days (P b 0.05) (Table 5).

4. Discussion

The effectiveness of HHP on inactivating Vibrio depends on pro-cessing parameters such as pressure level, holding time, temperatureand the physiological state of the bacteria (Berlin et al., 1999; Calik etal., 2002; Cook, 2003; Koo et al., 2006; Kural and Chen, 2008; Kural etal., 2008; Ma and Su, 2011; Ye et al., 2012). Generally speaking, Vibriospp. are sensitive to HHP and can be inactivated by pressures rangingfrom 200 to 350 MPa. Cook (2003) found that a pressure treatment of250 MPa for 2 min at 27–29 °C reduced V. vulnificus in oysters by ca. 6log. However, V. parahaemolyticus serotype O3:K6 in oysters requireda pressure of 300 MPa for 3 min at 24–25 °C for a 5-log reduction.Kural and Chen (2008) reported that pressure treatment needs to

Fig. 1. Effect of HHP and ice storage on total aerobic plate counts (APC) (a) andpsychrotrophic plate counts (PPC) (b) in whole-shell oysters. Un-inoculated whole-shell oysters were treated at 250–300 MPa for 2 min at 22–24 °C and stored for15 days in ice. Data represent mean log survivors (CFU/g). Error bars represent 1 stan-dard deviation.

be conducted at levels of ≥250 MPa to achieve a 5-log reduction ofV. vulnificus in live oysters. They later reported that achieving a5-log reduction of V. parahaemolyticus in live oysters requires≥350 MPa for 2 min at temperatures between 1 and 35 °C (Kural etal., 2008). Our results agree well with those of previous studies.

Treatment temperature is known to influence the effectiveness ofHHP onmicrobial inactivation. It has been demonstrated that slightly el-evated temperatures enhance pressure inactivation of Escherichia coliO157:H7 (Neetoo and Chen, 2010), Salmonella (Neetoo and Chen,2010), Listeria monocytogenes (Chen, 2007), and V. parahaemolyticus(Kural et al., 2008). Kural and Chen (2008) reported that pressure inacti-vation of V. vulnificuswas enhanced as the temperature decreased below20 °C and increased above 30 °C. In the present study, between the treat-ment temperatures of 4, 21, and 35 °C, there were no substantial differ-ences in HHP inactivation of V. parahaemolyticus and V. vulnificus.

In previous studies reported in the literature, the effects of storageconditions prior to HHP treatment on the pressure inactivation ofVibrio was not taken into consideration. In studies conducted byCalik et al. (2002), Cook (2003), Hu et al. (2005), Koo et al. (2006),Kural et al. (2008), and Kural and Chen (2008), oysters were inoculat-ed with V. parahaemolyticus or V. vulnificus through feeding; however,different storage conditions for inoculated oysters prior to HHP wereused for each study. Prior to HHP treatment, inoculated oysters werestored at 10 °C (length of time unreported) by Calik et al. (2002), atroom temperature for 24 h by Cook (2003), at refrigerated tempera-ture (length of time unreported) by Hu et al. (2005), and at refriger-ated temperature for b24 h by Koo et al. (2006). Kural et al. (2008)and Kural and Chen (2008) treated oysters by HHP immediatelyafter inoculation. In reality, oysters could be exposed to various stor-age temperatures after contamination and prior to HHP treatment.Oyster harvest water temperatures can vary from b0 °C to >30 °C,depending on the time and location of harvest. Oysters are knownto be left on decks of harvest vessels in the sun without cooling forseveral hours or held in cold seawater for up to 1 day in processingplants before HHP. In the present study, different storage conditionswere applied to oysters before HHP to simulate oysters being left ondecks of harvest vessels without cooling for several hours, or held incold seawater. Although cold storage at 4, and 10 °C resulted inlower Vibrio counts in oysters than warm storage at 21 and 35 °C, itdid not affect the sensitivity of V. parahaemolyticus to subsequentHHP treatments and even increased the pressure resistance of

Table 5Effect of HHP and frozen storage on APC and PPC in whole-shell oysters. Un-inoculatedoysters were treated at 250–300 MPa for 2 min at 22–24 °C and stored for 15 days in afreezer. Data represent mean log survivors (CFU/g) ± standard deviation. Data in thesame row having the same upper case letter are not significantly different(P > 0.05). Data in the same column having the same lower case letter are not signif-icantly different (P > 0.05).

APC PPC

Initial Frozen Initial Frozen

No HHP 4.6 ± 0.4Aa 3.8 ± 0.1Ba 5.0 ± 0.1Aa 4.3 ± 0.1Ba

250 MPa 2.6 ± 0.5Ab 3.3 ± 0.2Bb 3.3 ± 0.3Ab 4.1 ± 0.4Bb

300 MPa 2.1 ± 0.6Ab 2.6 ± 0.0Bc 1.5 ± 0.2Ac 3.6 ± 0.1Bc

151M. Ye et al. / International Journal of Food Microbiology 163 (2013) 146–152

V. vulnificus. The reason for this increased pressure resistance ofV. vulnificus with prior cold storage is unknown. It is possible that ex-posure to cold temperatures before processing increases the percent-age of polyunsaturated fatty acid in cell membranes, and thereforethe resistance to HHP processing.

Oysters could potentially be contaminated with high populations ofnaturally occurring V. parahaemolyticus (> 6 log MPN/g) upon expo-sure to elevated temperatures, producing potentially greater healthhazards (Gooch et al., 2001; Lorca et al., 2001). It has been demonstrat-ed that the current industry practice of HHP (≤300 MPa) is not capableof completely inactivating V. parahaemolyticus in oysters whenthe contamination level is high (Ye et al., 2012). Therefore, it is impor-tant to know the fate of V. parahaemolyticus survivors after HHP tounderstand the risks associated with excessive pathogen levels in oys-ters. In the present study, icing was chosen as the storage condition fol-lowing HHP treatment to model a common retail market practice foroysters after harvest or processing. We found that following HHP,V. parahaemolyticus populations gradually decreased during ice storage.HHP at 250 MPa, followed by 10-day ice storage, or 300 MPa treatmentfollowed by 5-day ice storage were sufficient to completely eliminateV. parahaemolyticus in whole-shell oysters (>7 log reduction). It ispossible that cold storage after HHP might capitalize on cell damageinduced by pressure treatment or could inhibit the recovery of thesub-lethally injured cells, leading to subsequent death. Although thecombination of prior cold storage and HHP resulted in greater inactiva-tion of V. parahaemolyticus than the combination of prior warm storageand HHP, there was no statistically significant difference in populationsof V. parahaemolyticus between those two treatments during post-HHPcold storage. Therefore, storage conditions prior to HHP might not be aconcern when designing HHP processing parameters, which wouldallow significant flexibility for oyster harvest, processing and storage.

A number of oyster producers use frozen storage to provide highquality frozen oysters to consumers for raw consumption. The com-mon practice is to hold oysters at −21 ± 2 °C for 3 to 6 months be-fore the oysters are distributed to consumers (Drake et al., 2007; Liuet al., 2009). In the present study, frozen storage before or afterHHP was used as a means to help achieve complete inactivation ofV. parahaemolyticus in oysters. While using higher pressures mayguarantee safer products, it is not always desirable since it would in-crease processing costs and might adversely affect the sensory qualityof oysters. Although frozen storage at −18 °C for 2 weeks reducedV. parahaemolyticus populations by 2.2 log, the combination of frozenstorage followed by 300 MPa did not completely inactivate it. Incontrast, frozen storage after HHP enhanced the inactivation ofV. parahaemolyticus in oysters. HHP at 250 or 300 MPa for 2 min at22–24 °C, followed by 7-day frozen storage, were able to completelyeliminate V. parahaemolyticus in whole-shell oysters (Table 4). Simi-larly, Black et al. (2010) investigated the fate of E. coli O157:H7 inground beef during post-HHP frozen storage. HHP at 400 MPa for10 min at 20 °C reduced E. coli O157:H7 in ground beef by 3 logCFU/g, while an extra 2–3 log reduction was observed during the sub-sequent 30-day frozen storage. A recent study in our laboratory alsodemonstrated that post-HHP frozen storage substantially enhancedthe inactivation of E. coli O157:H7 and Salmonella spp. in strawberrypuree (Huang et al., 2013).

The inactivation of V. parahaemolyticus in oysters by HHP wasstudied with shucked oyster meat and validated with whole-shelloysters. Since pressure is transmitted instantaneously and uniformlythroughout the pressure chamber, the process is independent offood shape, size or geometry (Farr, 1990; Knorr, 1999). Thus, no ap-preciable differences of inactivation between shucked oyster meatand whole-shell oysters were expected. This was confirmed by ourresults, which showed that the presence of shells did not influenceHHP inactivation of V. parahaemolyticus within oysters. It is advanta-geous to use shucked oyster meat to run HHP experiments for inocu-lation studies since shucked meat is easier to handle and package. In

addition, using shucked meat would minimize the possibility of equip-ment contamination by pathogens as a result of sharp oyster shellspuncturing packages leading to leakage of contaminated contents.

The shelf life of oysters stored at refrigeration temperatures variesand is dependent on the initial bacterial load. A decrease (2–3 logCFU/g) in total microflora after HHP treatment of oysters in therange of 200–600 MPa has previously been reported, and HHPappeared to delay the onset of bacterial growth during subsequent re-frigerated storage (He et al., 2002; Linton et al., 2003; Lopez-Caballeroet al., 2000). It has been reported that HHP at 207–310 MPa for1–2 min could extend the shelf life of oysters stored on ice from 9to 20 days (He et al., 2002), while oysters processed at 400 MPafor 5 min at 20 °C and stored on ice had a shelf life of 21 days(Cruz-Romero et al., 2008). Our results agree with previous studies,which indicated that HHP significantly reduced background microor-ganisms and that the microbial shelf life of HHP oysters wasmaintained for at least 15 days when stored in ice.

In summary, the present study showed that HHP was an effectiveoyster PHP for the control of V. parahaemolyticus and V. vulnificus andprior cold storage did not increase the sensitivity of V. parahaemolyticusor V. vulnificus to subsequent HHP treatments. HHP at 300 MPa for2 min at 21 °C, followed by 5-day ice storage or 7-day frozen storage,completely eliminated V. parahaemolyticus in whole-shell oysters.HHP-treated oysters maintained a microbial shelf life of at least15 days when stored in ice. HHP followed by ice storage could be ap-plied by the seafood industry as a PHP to inactivate V. parahaemolyticusand V. vulnificus in raw oysters and HHP combined with frozen storagecould improve the microbial safety of frozen oysters.

Acknowledgment

This project was supported by the Agriculture and Food ResearchInitiative Competitive Grants Program of the USDA National Instituteof Food and Agriculture, NIFA award no: 2011-68003-30005. The au-thors are grateful to Dr. PeggyM. Tomasula for the use of the HHP unitat the USDA-ERRC.

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