Occurrence and distribution of Vibrio parahaemolyticus in retail oysters in Sao Paulo State, Brazil

Vibrio parahaemolyticus is a potentially pathogenic bacterium that occurs naturally in estuarine environments worldwide, and is often associated with gastroenteritis in humans following consumption of raw bivalve mollusks, especially raw oysters. The occurrence of total and pathogenic V. parahaemolyticus in 74 samples of raw oysters collected in restaurants, supermarkets, groceries and beach huts in Sao Paulo State, was monitored between February 2006 and January 2007. Enumeration of V. parahaemolyticus was performed according to the most probable number (MPN) procedure. Five to ten typical colonies were selected from thiosulfate-citrate-bile salts-sucrose (TCBS) agar plates for confirmation by the presence of the species-specific gene tlh and the virulence genes tdh and trh by multiplex PCR. V. parahaemolyticus was detected in 100% of samples. The densities of total V. parahaemolyticus varied from 1.78 to 6.04 log₁₀ (MPN/g), with higher densities being detected in fall and summer, and lower densities in winter (P < 0.05). There was no statistical difference among densities of V parahaemolyticus regarding the site of collection. None of the 1943 V. parahaemolyticus isolates contained tdh and/or trh. These data provide information for the assessment of exposure to V. parahaemolyticus in oysters consumed in Sao Paulo, State, Brazil.     

Evaluating the use of fatty acid profiles to identify deep-sea Vibrio isolates

Capillary gas chromatography with flame ionisation detection (GC-FID) was used to determine the cellular fatty acid (CFA) profiles of a number of Vibrio strains obtained from ATCC and grown on various media. This initial determination for optimal growth conditions, including type of medium and incubation temperature, for these various ATCC Vibrio species, was important for use in the subsequent evaluation of deep-sea Vibrio strains. The deep-sea Vibrios were obtained from Harbor Branch Oceanographic Institution (Fort Pierce, FL), and GC-FID analysis was used to determine whole cell fatty acid methyl esters (FAMEs) from the cells. The Vibrio strains were cultured for 24 h on a specific medium which included brain heart infusion (BHI) agar, trypticase soy agar (TSA), trypticase soy broth agar (TSBA), and Luria–Bertani (LB) agar. The temperature of incubation was 28 °C for cells grown on TSA, TSBA, and LB and 35 °C for BHI. A data set for each Vibrio species was prepared, using fatty acid profiles for a specific medium. Major fatty acids of the Vibrio strains evaluated in this study were straight-chain C_12:0, C_14:0, C_16:0, and unsaturated summed C_16:1 ω7c/C_16:1 ω6c, C_18:1 ω7c, and summed C_14:0 3-OH/iso-C_16:1. Most of the deep-sea Vibrio isolates were identified as Vibrio parahaemolyticus and Vibrio harveyi. Analysis of FAMEs from Vibro strains grown on a specific medium by this rapid GC-FID method can provide a sensitive procedure for the identification of these organisms, and the differentiation between Vibrio species.     

Effect of X-ray treatments on inoculated Escherichia coli O157: H7, Salmonella enterica, Shigella flexneri and Vibrio parahaemolyticus in ready-to-eat shrimp

This study was conducted to evaluate the inactivation effect of X-ray treatments on Escherichia coli O157: H7, Salmonella enteric (S. enterica), Shigella flexneri (S. flexneri) and Vibrio parahaemolyticus (V. parahaemolyticus) artificially inoculated in ready-to-eat (RTE) shrimp. A mixed culture of three strains of each tested pathogen was used to inoculate RTE shrimp. The shrimp samples were inoculated individually with selected pathogenic bacteria then aseptically placed in sterile plastic cups and air-dried at 22 °C for 30 min (to allow bacterial attachment) in the biosafety cabinet prior to X-ray treatments. The inoculated shrimp samples were then placed in sterilized bags and treated with 0.1, 0.2, 0.3, 0.5, 0.75, 1.0, 2.0, 3.0 and 4.0 kGy X-ray at ambient temperature (22 °C and 60% relative humidity). Surviving bacterial populations were evaluated using a non-selective medium (TSA) with the appropriate selective medium overlay for each bacterium; CT-SMAC agar for E. coli O157: H7, XLD for S. enterica and S. flexneri and TCBS for V. parahaemolyticus. More than a 6 log CFU reduction of E. coli O157: H7, S. enterica, S. flexneri and V. parahaemolyticus was achieved with 2.0, 4.0, 3.0 and 3.0 kGy X-ray, respectively. Furthermore, treatment with 0.75 kGy X-ray significantly reduced the initial microflora on RTE shrimp samples from 3.8 ± 0.2 log CFU g⁻¹ to less than detectable limit (<1.0 log CFU g⁻¹).     

High Salinity Relaying to Reduce Vibrio parahaemolyticus and Vibrio vulnificus in Chesapeake Bay Oysters (Crassostrea virginica)

Cases of Vibrio infections in the United States have tripled from 1996 to 2009 and these infections are most often associated with the consumption of seafood, particularly oysters (Crassostrea virginica). Information is needed on how to reduce numbers of Vibrio parahaemolyticus and Vibrio vulnificus in bi‐valve molluscan shellfish (for example, oysters). The purpose of this study was to evaluate the effectiveness of high salinity relaying or treatment in recirculating aquaculture systems (RASs) as methods to reduce the abundance of V. parahaemolyticus and V. vulnificus in oysters. For relaying field trials, oysters were collected from approved harvest waters, temperature abused outside under a tarp for 4 h, and then transferred to high (29 to 33 ppt.) and moderate (12 to 19 ppt.) salinities. For RAS treatment trial, oysters were transferred to 32 to 34 ppt. salinity at 15 °C. After 7, 14, 21, and in some instances 28 d, oysters were collected and analyzed for V. parahaemolyticus and V. vulnificus levels using multiplex real‐time PCR. Initial levels of V. parahaemolyticus and V. vulnificus ranged from 3.70 to 5.64 log₁₀ MPN/g, and were reduced by 2 to 5 logs after 21 to 28 d in high salinity water (29 to 34 ppt.). Oyster mortalities averaged 4% or less, and did not exceed 7%. Relaying of oysters to high salinity field sites or transfer to high salinity RAS tanks was more effective in reducing V. vulnificus compared with V. parahaemolyticus. These results suggest that high salinity relaying of oysters is more effective in reducing V. vulnificus than V. parahaemolyticus in the oyster species used in this study.     

Reductions of Vibrio parahaemolyticus in Pacific oysters (Crassostrea gigas) by depuration at various temperatures

Consumption of raw oysters has been linked to several outbreaks of Vibrio parahaemolyticus infection in the United States. This study investigated effects of ice storage and UV-sterilized seawater depuration at various temperatures on reducing V. parahaemolyticus in oysters. Raw Pacific oysters (Crassostrea gigas) were inoculated with a mixed culture of five clinical strains of V. parahaemolyticus (10290, 10292, 10293, BE 98-2029 and 027-1c1) at levels of 10⁴⁻⁶ MPN/g. Inoculated oysters were either stored in ice or depurated in recirculating artificial seawater at 2, 3, 7, 10, 12.5, and 15 °C for 4–6 days. Holding oysters in ice or depuration of oysters in recirculating seawater at 2 or 3 °C for 4 days did not result in significant reductions (P > 0.05) of V. parahaemolyticus in the oysters. However, depuration at temperatures between 7 and 15 °C reduced V. parahaemolyticus populations in oysters by >3.0 log MPN/g after 5 days with no loss of oysters. Depuration at refrigerated temperatures (7–15 °C) can be applied as a post-harvest treatment for reducing V. parahaemolyticus in Pacific oysters.     

Validation of high pressure processing for inactivating Vibrio parahaemolyticus in Pacific oysters (Crassostrea gigas)

This study identified and validated high hydrostatic pressure processing (HPP) for achieving greater than 3.52-log reductions of Vibrio parahaemolyticus in the Pacific oysters (Crassostrea gigas) and determined shelf life of processed oysters stored at 5 °C or in ice. Raw Pacific oysters were inoculated with a clinical strain of V. parahaemolyticus 10293 (O1:K56) to levels of 10^4-5 cells per gram and processed at 293 MPa (43 K PSI) for 90, 120, 150, 180 and 210 s. Populations of V. parahaemolyticus in oysters after processes were analyzed with the 5-tube most probable number (MPN) method. Negative results obtained by the MPN method were confirmed with a multiplex PCR detecting genes encoding thermolabile hemolysin (tl), thermostable direct hemolysin (tdh) and TDH-related hemolysin (trh). A HPP of 293 MPa for 120 s at groundwater temperature (8 ± 1 °C) was identified capable of achieving greater than 3.52-log reductions of V. parahaemolyticus in Pacific oysters. Oysters processed at 293 MPa for 120 s had a shelf life of 6-8 days when stored at 5 °C or 16-18 days when stored in ice. This HPP can be adopted by the shellfish industry as a post harvest process to eliminate V. parahaemolyticus in raw oysters.     

Behavior and incidence of Vibro parahaemolyticus in Sydney rock oysters (Crassostrea commercialis)

Vibrio parahaemolyticus grew poorly or not at all during storage of unopened Sydney rock oysters (Crassostrea commercialis) at 15 and 30°C for 2 and 7 days. Although V. parahaemolyticus counts often increased at 30°C, counts above 10⁴/g were not observed. Escherichia coli counts did not usually change substantially under these conditions. V. parahaemolyticus grew more readily during storage of unopened oysters under more severe conditions, with counts approaching or exceeding 10⁶/g after continuous or intermittent storage at 37°C. Opened oysters provided a much more favourable environment than unopened oysters for growth of V. parahaemolyticus. Growth occurred at 15, 30 and 37°C, with counts > 10⁶/g after overnight storage at 30 or 37°C. A survey of 30 market samples of oysters was conducted. Sixteen samples of unopened oysters were collected at the wholesale level and 14 samples of refrigerated opened oysters were purchased from retailers. V. parahaemolyticus was present in all samples of unopened oysters (range 0.4/g?2.3 × 10⁴/g) and in 13 samples of opened oysters (range 4.3/g? > 1.1 × 10³/g).     

Evaluation of a loop-mediated isothermal amplification assay for rapid and simple detection of Vibrio parahaemolyticus in naturally contaminated seafood samples

We investigated the efficacy of a loop-mediated isothermal amplification (LAMP) assay for rapid screening of seafood samples naturally contaminated with Vibrio parahaemolyticus. A total of 171 seafood samples enriched in alkaline peptone water (APW) were assessed by LAMP assay and conventional culture methods, which consist of a combination of APW enrichment culture and plating onto CHROMagar Vibrio and TCBS agars. Compared with V. parahaemolyticus isolation using the conventional culture test, LAMP results showed 100% (30/30) and 90.8% (128/141) sensitivity and specificity, respectively. The conventional culture test required more than 3 days to isolate and identify V. parahaemolyticus in the APW enrichment culture. In contrast, the LAMP assay was markedly faster, requiring less than 60 min from the beginning of DNA extraction to final detection of V. parahaemolyticus. In total, the LAMP assay required 17-19 h from the beginning of enrichment culture to final determination. This is the first report of the LAMP assay for rapid screening of seafood samples naturally contaminated by V. parahaemolyticus.     

一株溶藻弧菌的分离鉴定及其对九孔鲍苗的致病性

为找到导致2002年以来中国南部沿海鲍鱼养殖大量死亡的原因,利用胰蛋白培养基或硫代硫酸盐-柠檬酸盐-胆盐-蔗糖培养基从2003年汕尾发生病害的鲍鱼中分离得到了一株具有致病性的细菌.经生理生化特性试验及与特征溶藻弧菌ATCC17749比较,此菌株为溶藻弧菌.经感染试验证明其对鲍苗的半致死量为1.58x 104 CFU/m...     

An evaluation of the effect of catalase and 3,3-thiodipropionic acid on the recovery of freeze-injured coliform bacteria

Coliform bacteria isolated from ground beef were frozen in 0.1 M phosphate buffer at ?18°C for 72 h. Before and after freezing, colony counts were made in tryptic soy agar (TSA) (Difco) and in TSA supplemented with 680 units of catalase ml^? and 1% 3,3-thiodipropionic acid (TDPA), respectively. The two H₂O₂ scavengers exerted no enhancing effect on cell recovery in the non-selective medium.Catalase and TDPA were also added to the TSA bottom layer in the selective dual-plating repair detection procedure described by Ray and Speck [(1978) Appl. Environ. Microbiol. 35, 883–885] using violet red bile agar (VRBA) as the selective medium. After freezing, approximately 70% of surviving cells could be recovered using TSA and TSA supplemented with catalase or TDPA (TSAC, TSAT).A total of 19 samples of various frozen food products (ice cream, pastries, ground meat and vegetables) were examined for coliform bacteria by the repair detection procedure with unsupplemented TSA and TSA with catalase or TDPA. Coliforms were detected in 15 samples. The logarithmic mean colony count with the TSA/VRBA procedure was 1350 g^?1. With TSAC/VRBA and TSAT/VRBA the counts were 1590 and 1400 g^?, respectively. These counts did not differ significantly from those obtained with unsupplemented TSA/VRBA (P>0.05).     

Antimicrobial activity of 405 nm light-emitting diode (LED) in the presence of riboflavin against Listeria monocytogenes on the surface of smoked salmon

This study investigated the antimicrobial activity of 405 nm light-emitting diode (LED) with and without riboflavin against Listeria monocytogenes in phosphate buffered saline (PBS) and on smoked salmon at different storage temperatures and evaluated its impact on food quality. The results show that riboflavin-mediated LED illumination in PBS 25 °C significantly inactivated L. monocytogenes cells by 6.2 log CFU/mL at 19.2 J/cm², while illumination alone reduced 1.9 log CFU/mL of L. monocytogenes populations at 57.6 J/cm². L. monocytogenes populations on illuminated smoked salmon decreased by 1.0–2.2 log CFU/cm² at 1.27–2.76 kJ/cm² at 4, 12, and 25 °C, regardless of the presence of riboflavin. Although illumination with and without riboflavin caused the lipid peroxidation and color change in smoked salmon, this study demonstrates the potential of a 405 nm LED to preserve the smoked salmon products, reducing the risk of listeriosis.     

Novel inactivation methods of Doenjang (fermented soybean paste) by high pressure and ohmic heating

The changes of the total cell number in doenjang (Korean traditional fermented soybean paste) by conventional conduction heating, high pressure non-thermal treatment, and ohmic heating were compared. A total of (10¹–10²) CFU/g cells were decreased by heating at (100–105) °C for 10 min. The inactivation rate was improved when heated to a temperature higher than 110 °C, but the taste, color of doenjang were severely changed. Inactivation by high pressure at (200–800) MPa was not achieved, because the total cell did not reach a reduction of 10¹ CFU/g. The total bacterial counts of 10³ CFU/g were decreased during ohmic heating at 15 V and 60 Hz for 10 min, and showed the most effective inactivation. Therefore, application of the ohmic heating in doenjang with high viscosity can kill target microorganisms related to quality deterioration, and rapid and uniform ohmic heating leads to reduction in sensory quality damage.     

Comparison of molecular detection methods for Vibrio parahaemolyticus and Vibrio vulnificus

Pathogenic vibrios are a global concern for seafood safety and many molecular methods have been developed for their detection. This study compares several molecular methods for detection of total and pathogenic Vibrio parahaemolyticus and Vibrio vulnificus, in MPN enrichments from oysters and fish intestine samples. This study employed the DuPont Qualicon BAX^® System Real-Time PCR assay for detection of V. parahaemolyticus and V. vulnificus. Multiplex real-time PCR detection of total (tlh+), tdh+, and trh+ V. parahaemolyticus was conducted on the Cepheid SmartCycler II. Total (rpoD) and tdh+ V. parahaemolyticus were also detected using LAMP. V. vulnificus detection was performed using real-time PCR methods developed for the SmartCycler and the AB 7500 Fast. Recommended template preparations were compared to BAX^® lysis samples for suitability. There was no significant difference in detection of V. parahaemolyticus and V. vulnificus using the BAX^® or SmartCycler assays. The AB assay showed no difference from other methods in detection of V. vulnificus unless boiled templates were utilized. There was a significant difference in detection of tdh+ V. parahaemolyticus between SmartCycler and LAMP assays unless the total (tlh+) V. parahaemolyticus gene target was omitted from the SmartCycler assay; a similar trend was observed for trh+ V. parahaemolyticus.     

Evaluation of a Double Layer Agar Plate For Direct Enumeration of Vibrio parahaemolyticus

A double layer agar plate (DLAP) was developed according to the thin agar layer (TAL) method as a 1‐step procedure for direct enumeration of injured Vibrio parahaemolyticus cells based on the formation of unique purple colonies by V. parahaemolyticus. The DLAP was prepared by overlaying an equal volume (10 mL) of a nonselective medium (tryptic soy agar supplemented with 1.5% NaCl) onto a selective medium (Bio‐Chrome Vibrio medium). The DLAP was capable of detecting V. parahaemolyticus in mixed cultures containing non‐Vibrio bacteria. Production of purple colonies by V. parahaemolyticus on DLAP was not affected by the growth of other bacteria, even when V. parahaemolyticus was only a small fraction (5%) of the entire bacterial population. Direct plating on DLAP was found as effective as the most probable number (MPN) method for recovering heat‐and cold‐injured V. parahaemolyticus cells, which could not be detected by direct plating on Bio‐Chrome Vibrio medium or thiosulfate‐citrate‐bile salts‐sucrose agar. The DLAP offers an alternative to the MPN method for detecting injured V. parahaemolyticus cells and can be used as a simple 1‐step procedure for quick screening of V. parahaemolyticus in foods.     

Recovery of hesperidin and narirutin from waste Citrus unshiu peel using subcritical water extraction aided by pulsed electric field treatment

The objective of this study was to identify whether the efficacy of extracting hesperidin and narirutin from Citrus unshiu peel by-products can be increased by combining pulsed electric field (PEF) and subcritical water extraction (SWE). The samples were treated with a PEF at a strength of 3 kV/cm for 60 and 120 s. Subsequent SWE was conducted at extraction temperatures of 110–190 °C for 3–15 min. The concentration of hesperidin was highest at 46.96 ± 3.37 mg/g peel (dry basis) after PEF treatment at 120 s, combined with SWE at 150 °C for 15 min, while that of narirutin peaked at 8.76 ± 0.83 mg/g after PEF treatment at 120 s, integrated with SWE at 190 °C for 5 min. The concentrations of both hesperidin and narirutin increased with PEF treatment time. The PEF increased the amounts of hesperidin and narirutin extracted by 22.1% and 33.6%, respectively. This study demonstrate the potential of PEF pretreatment for enhancing the SWE of flavonoids from C. unshiu peel.     

Thermal inactivation of Yersinia enterocolitica in pork slaughter plant scald tank water

The objective of this study was to establish the time–temperature combinations required to ensure the thermal inactivation of Yersinia enterocolitica during scalding of pork carcasses. A 2 strain cocktail of Y. enterocolitica (bioserotypes 2/O:5,27 and 1A/O:6,30) was heat treated at 50, 55 and 60 °C in samples of scald tank water obtained from a commercial pork slaughter plant. Samples were removed at regular intervals and surviving cells enumerated using (i) Cefsulodin–Irgasan–Novobiocin Agar (CIN) supplemented with ampicillin and arabinose and (ii) Tryptone Soya Agar (TSA), overlaid with CIN agar with ampicillin and arabinose. The data generated was used to estimate D- and z-values and the formula D_x = log^− 1(log D₆₀ − ((t₂ − t₁)/z)) was applied to calculate thermal death time–temperature combinations from 55 to 65 °C. D₅₀, D₅₅ and D₆₀-values of 45.9, 10.6 and 2.7 min were calculated from the cell counts obtained on CIN agar, respectively. The corresponding D-values calculated from the TSA/CIN counts were 45.1, 11 and 2.5 min, respectively. The z-value was 7.8. It was concluded that a time–temperature combination of 2.7 min at 60 °C is required to achieve a 1 log reduction in Y. enterocolitica in pork scald tank water. The predicted equivalent at 65 °C was 0.6 min. This study provides data and a model to enable pork processors to identify and apply parameters to limit the risk of carcass cross-contamination with Y. enterocolitica in pork carcass scald tanks.     

Pasteurization of grapefruit juice using a centrifugal ultraviolet light irradiator

Studies are lacking on the nonthermal pasteurization of liquid foods using UV irradiators that centrifugally form very thin films to overcome the problem of limited penetration depth of UV. Grapefruit juice inoculated with Escherichia coli or Saccharomyces cerevisiae was processed at the following conditions: UV dose 4.8–24 mJ/cm²; treatment time 3.2 s, cylinder rotational speed 450–750 rpm, cylinder inclination angle 15–45°, outlet temperature 11 °C, and flow rate 300 ml/min, and was stored for 35 days. Appropriate dilutions of the samples were pour plated with TSA and TSA + 3% NaCl for E. coli and Sabouraud dextrose agar (SDA) and SDA + 5% NaCl for S. cerevisiae. Nonthermal UV processing at 19 mJ/cm², 450 rpm and 15° reduced E. coli in grapefruit juice by 5.1 log₁₀. A dose of 14 mJ/cm² reduced S. cerevisiae by 6.0 log₁₀. Inactivation increased linearly with increasing UV dose. The inactivations at 600 and 750 rpm were similar, and were better than at 450 rpm. The results at 30° and 45° were similar, and were better than at 15°. The occurrence of sublethal injury in either microorganism was not detected. Storing UV processed grapefruit juice at 4 and 10 °C reduced the surviving E. coli to below 1 log₁₀ cfu/ml in 14 days. Processing UV juice reduced the population of S. cerevisiae to less than 1 log₁₀ cfu/ml where it remained for 35 days during refrigerated storage. These results suggest that grapefruit juice may be pasteurized using a nonthermal UV irradiator that centrifugally forms a thin film.     

Incidence and Enumeration of Vibrio parahaemolyticus in Shellfish from Two Retail Sources and the Genetic Diversity of Isolates as Determined by RAPD-PCR Analysis

A total of 83 shellfish samples from two local retail sources (A and B) yielded 38 samples positive for the presence of Vibrio parahaemolyticus based on 3 tube MPN enrichments and isolation from thiosulfate-citrate-bile salts-sucrose agar (TCBS) and biochemical tests. The 38 positive samples yielded 133 biochemically presumptive isolates of V. parahaemolyticus. Among these 133 presumptive isolates, 104 were confirmed by the polymerase chain reaction (PCR), which yielded more reliable identification results than the biochemical tests. The 38 biochemically presumptive samples yielded 29 samples that were confirmed by PCR to be positive for the presence of V. parahaemolyticus. RAPD analysis with three random primers was performed to examine the genetic diversity of 64 strains among the PCR confirmed V. parahaemolyticus isolates from both retail sources. 52 of 56 composite RAPD types consisted of single strains, indicating that most of the V. parahaemolyticus isolates were genetically quite heterogeneous. No strains representing the same RAPD type occurred in both retail outlets, implying that contamination of the shellfish by V. parahaemolyticus from the 2 retail sources was from different environmental locals and shellfish harvesting areas. Eight genomic clusters were generated at the 25% similarity level in a dendrogram based on RAPD profiles. With few exception, isolates with close genetic relationships grouping into an individual cluster tended to be derived from the same retail source.     

Detection,Identification, and Prevalence of Pathogenic Vibrio parahaemolyticus in Fish and Coastal Environment in Jordan

Vibrio parahaemolyticus is widely distributed in the marine environments and considered the leading cause of human gastroenteritis in Asian countries. A total of 150 marketed fish and 50 water and sediment samples from the Gulf of Aqaba were examined for the prevalence of pathogenic strains of V. parahaemolyticus. A total of 132 typical isolates obtained from the primary selective medium (thiosulfate‐citrate bile salt sucrose agar) and showed positive biochemical properties were subjected to confirmation by polymerase chain reaction targeting the gyrB and toxR genes. These genes were confirmed at rates of 82% (108 isolates) and 72% (95 isolates), respectively. The toxR positive isolates were tested for the presence of thermolabile hemolysin (tlh), thermostable direct hemolysin (tdh), and tdh‐related hemolysin (trh) virulence genes. Accordingly, the prevalence rates of pathogenic V. parahaemolyticus were 4%, 8%, and 12% in sediment, water, and fish samples, respectively. The 16S rRNA amplification and sequences were conducted for confirmation of the isolates and showing the relatedness among these isolates. The results showed that both 16S rRNA and toxR assays had same sensitivity and tested isolates had high nucleotide similarity irrespective of their sources.     

A predictive model for assessment of decontamination effects of lactic acid and chitosan used in combination on Vibrio parahaemolyticus in shrimps

Vibrio parahaemolyticus is a major causative agent of human gastroenteritis in seafood products including shrimps. Lactic acid and chitosan are natural antimicrobials for food decontamination in the washing process of seafood. In this research, a 4-factor response surface model based on the Box–Behnken experimental design was developed to evaluate the effects of lactic acid (1%, 2%, and 3%, v/v), chitosan (0.4%, 1%, and 1.6%, w/v), rotational rate (90, 110, and 130 rpm) and washing time (10, 20, and 30 min) on reduction of V. parahaemolyticus inoculated in raw shrimps. These treatments achieved 2.2 to 4.3 log₁₀ CFU/g reduction of V. parahaemolyticus in shrimps. Stepwise stratification led to a simplified model that has a satisfactory performance as evidenced by statistical indices (R² = 0.92; p < 0.0001; RMSE = 0.196) and external validation parameters [bias factor (B_f) = 1.01; accuracy factor (A_f) = 1.05]. The model generated an optimum treatment combination (3% lactic acid, 1.6% chitosan, and rotational rate at 110 rpm) that could achieve greatest bacterial reduction of 4.5 log₁₀ CFU/g. Among the four factors, lactic acid and chitosan were the major contributors for bacterial decontamination. Analysis of variances showed a significant interactive inactivation effect (p < 0.05) from combined use of lactic acid and chitosan. The treatments did not have adverse effects on the quality attributes such as color and pH of the shrimps.