Vibrio Parahaemolyticus In Oysters Research Articles

Serodiversity, antibiotic resistance, and virulence genes of Vibrio parahaemolyticus in oysters collected in coastal areas of northwestern Mexico between 2012 and 2020

This study aimed to determine the prevalence of V. parahaemolyticus in oysters from the northwestern coast of Mexico and to identify the serotypes, virulence factors, and antibiotic resistance of the strains. Oyster samples were collected from 2012 to 2020 from the northwest coast of Mexico; biochemical and molecular methods were used to identify V. parahaemolyticus from oysters; antiserum reaction to determine V. parahaemolyticus serotypes, and PCR assays were performed to identify pathogenic (tdh and/or trh) or pandemic (toxRS/new, and/or orf8) strains and antibiotic resistance testing. A total of 441 oyster samples were collected and tested for V. parahaemolyticus. Forty-seven percent of oyster samples were positive for V. parahaemolyticus. Ten different O serogroups and 72 serovars were identified, predominantly serotype O1:KUT with 22.2% and OUT:KUT with 17.3%. Twenty new serotypes that had not been previously reported in our region were identified. We detected 4.3% of pathogenic clones but no pandemic strains. About 73.5% of strains were resistant to at least one antibiotic, mainly ampicillin and ciprofloxacin; 25% were multi-drug resistant. In conclusion, the pathogenic strains in oysters and antibiotic resistance are of public health concern, as the potential for outbreaks throughout northwestern Mexico is well established.

Modeling the risk of Vibrio parahaemolyticus in oysters in Taiwan by considering seasonal variations, time periods, climate change scenarios, and post-harvest interventions

Vibrio parahaemolyticus is a halophilic gram-negative bacterium commonly found in marine environments, particularly in warm coastal waters. This pathogen has been reported as a common cause of foodborne illness associated with the consumption of raw or undercooked seafood. The presence and density of this bacterium in seafood are often associated with the climatological conditions of the marine environment. Herein, we developed the quantitative risk assessment model for Vibrio parahaemolyticus in oysters in Taiwan by considering seasonal variations, time periods, climate change scenarios, and post-harvest interventions. This study showed that season, time period, shared socioeconomic pathway (SSP), and post-harvest intervention significantly influenced the risk level of becoming ill from consuming oysters. The mean estimates of risk in winter, spring, summer, and fall were estimated to be 9.1 × 10−5, 2.0 × 10−3, 2.0 × 10−2, 6.9 × 10−3 per serving, respectively. Our models predict that, if global temperatures continue to increase in the coming decades due to climate change, the risk per serving of oysters is likely to increase by 18-145% by 2041-2060 and by 18-718% by 2081-2100, depending on the season and SSP. The application of thermal processing or high hydrostatic pressure processing was found to be the most effective approach in reducing risk, even under the threat of increasing global temperatures.

Predicting the Growth of Vibrio parahaemolyticus in Oysters under Varying Ambient Temperature

Temperature is a critical factor that influences the proliferation of pathogens in hosts. One example of this is the human pathogen Vibrio parahaemolyticus (V. parahaemolyticus) in oysters. Here, a continuous time model was developed for predicting the growth of Vibrio parahaemolyticus in oysters under varying ambient temperature. The model was fit and evaluated against data from previous experiments. Once evaluated, the V. parahaemolyticus dynamics in oysters were estimated at different post-harvest varying temperature scenarios affected by water and air temperature and different ice treatment timing. The model performed adequately under varying temperature, reflecting that (i) increasing temperature, particularly in hot summers, favors a rapid V. parahaemolyticus growth in oysters, resulting in a very high risk of gastroenteritis in humans after consumption of a serving of raw oysters, (ii) pathogen inactivation due to day/night oscillations and, more evidently, due to ice treatments, and (iii) ice treatment is much more effective, limiting the risk of illness when applied immediately onboard compared to dockside. The model resulted in being a promising tool for improving the understanding of the V. parahaemolyticus–oyster system and supporting studies on the public health impact of pathogenic V. parahaemolyticus associated with raw oyster consumption. Although robust validation of the model predictions is needed, the initial results and evaluation showed the potential of the model to be easily modified to match similar systems where the temperature is a critical factor shaping the proliferation of pathogens in hosts.

Inactivation of Vibrio parahaemolyticus and retardation of quality loss in oyster (Crassostrea gigas) by ultrasound processing during storage

The health problems caused by foodborne pathogens of raw oysters have been widely concerned. Traditional heating methods tend to lead the loss of the original nutrients and flavors, in this study, the nonthermal ultrasound technology was applied to inactivate Vibrio parahaemolyticus on raw oysters, and the retardation effects on microbial growth and quality loss of oysters stored at 4 ℃ after ultrasonic treatment were also investigated. After treated by 7.5 W/mL ultrasound for 12.5 min, the Vibrio parahaemolyticus in oysters was reduced by 3.13 log CFU/g. By measuring total aerobic bacteria and total volatile base nitrogen, the growth trend after ultrasonic treatment was delayed compared with heat treatment, and the shelf life of oysters was prolonged. At the same time, ultrasonic treatment delayed the changes of color difference and lipid oxidation of oysters during cold storage. Texture analysis showed that ultrasonic treatment helped maintain the good textural structure of oysters. Histological section analysis also demonstrated that muscle fibers were still tightly packed after ultrasonic treatment. Low-field nuclear magnetic resonance (LF-NMR) illustrated that the water in the oysters was well maintained after ultrasonic treatment. In addition, gas chromatograph – ion mobility spectrometer (GC-IMS) showed that ultrasound treatment could better preserve the flavor of oysters during cold storage. Therefore, it is believed that ultrasound can inactivate foodborne pathogens of raw oysters and keep its freshness and original taste better during storage.

Vibrio vulnificus and Vibrio parahaemolyticus in Oysters under Low Tidal Range Conditions: Is Seawater Analysis Useful for Risk Assessment?

Human-pathogenic Vibrio bacteria are acquired by oysters through filtering seawater, however, the relationships between levels of these bacteria in measured in oysters and overlying waters are inconsistent across regions. The reasons for these discrepancies are unclear hindering our ability to assess if -or when- seawater samples can be used as a proxy for oysters to assess risk. We investigated whether concentrations of total and human pathogenic Vibrio vulnificus (vvhA and pilF genes) and Vibrio parahaemolyticus (tlh, tdh and trh genes) measured in seawater reflect concentrations of these bacteria in oysters (Crassostrea virginica) cultured within the US lower Chesapeake Bay region. We measured Vibrio spp. concentrations using an MPN-qPCR approach and analyzed the data using structural equation modeling (SEM). We found seawater concentrations of these bacteria to predictably respond to temperature and salinity over chlorophyll a, pheophytin or turbidity. We also inferred from the SEM results that Vibrio concentrations in seawater strongly predict their respective concentrations in oysters. We hypothesize that such seawater-oyster coupling can be observed in regions of low tidal range. Due to the ease of sampling and processing of seawater samples compared to oyster samples, we suggest that under low tidal range conditions, seawater samples can foster increased spatial and temporal coverage and complement data associated with oyster samples.

A climate-driven model for predicting the level of Vibrio parahaemolyticus in oysters harvested from Taiwanese farms using elastic net regularized regression

This study aimed at, and developed, a climate-driven model for predicting the abundance of V. parahaemolyticus in oysters based on the local climatological and environmental conditions in Taiwan. The predictive model was constructed using the elastic net machine learning method, and the most influential predictors were evaluated using a permutation-based approach. The abundance of V. parahaemolyticus in oysters in different seasons, time horizons, and representative concentration pathways (RCPs) were predicted using the Elastic-net machine learning model. The results showed: (1) the variation in wind speed or gust wind speed, sea surface temperature, precipitation, and pH influenced the prediction of V. parahaemolyticus concentration in oysters, and (2) the level of V. parahaemolyticus in oysters in Taiwan was projected to be increased by 40–67% in the near future (2046–2065) and by 39–86% by the end of twentieth-century (2081–2100) if the global temperature continues to increase due to climate change. The findings in this study may be used as inputs for quantifying the V. parahaemolyticus infection risk from eating this seafood in Taiwan.

Combination of Direct Viable Count and Fluorescent In Situ Hybridization (DVC-FISH) as a Potential Method for Identifying Viable Vibrioparahaemolyticus in Oysters and Mussels.

Vibrio parahaemolyticus is a human food-borne pathogen with the ability to enter the food chain. It is able to acquire a viable, non-cultivable state (VBNC), which is not detected by traditional methods. The combination of the direct viable count method and a fluorescent in situ hybridization technique (DVC-FISH) makes it possible to detect microorganisms that can present VBNC forms in complex samples The optimization of the in vitro DVC-FISH technique for V. parahaemolyticus was carried out. The selected antibiotic was ciprofloxacin at a concentration of 0.75 μg/mL with an incubation time in DVC broth of 5 h. The DVC-FISH technique and the traditional plate culture were applied to detect and quantify the viable cells of the affected pathogen in artificially contaminated food matrices at different temperatures. The results obtained showed that low temperatures produced an important logarithmic decrease of V. parahaemolyticus, while at 22 °C, it proliferated rapidly. The DVC-FISH technique proved to be a useful tool for the detection and quantification of V. parahaemolyticus in the two seafood matrices of oysters and mussels. This is the first study in which this technique has been developed to detect viable cells for this microorganism.

Development of enhanced selective media for detection of Vibrio parahaemolyticus in oysters.

The online version contains supplementary material available at (10.1007/s10068-021-00877-0).

Establishment and Validation of RNA-Based Predictive Models for Understanding Survival of Vibrio parahaemolyticus in Oysters Stored at Low Temperatures.

This study developed RNA-based predictive models describing the survival of Vibrio parahaemolyticus in Eastern oysters (Crassostrea virginica) during storage at 0, 4, and 10°C. Postharvested oysters were inoculated with a cocktail of five V. parahaemolyticus strains and were then stored at 0, 4, and 10°C for 21 or 11 days. A real-time reverse transcription-PCR (RT-PCR) assay targeting expression of the tlh gene was used to evaluate the number of surviving V. parahaemolyticus cells, which was then used to establish primary molecular models (MMs). Before construction of the MMs, consistent expression levels of the tlh gene at 0, 4, and 10°C were confirmed, and this gene was used to monitor the survival of the total V. parahaemolyticus cells. In addition, the tdh and trh genes were used for monitoring the survival of virulent V. parahaemolyticus Traditional models (TMs) were built based on data collected using a plate counting method. From the MMs, V. parahaemolyticus populations had decreased 0.493, 0.362, and 0.238 log10 CFU/g by the end of storage at 0, 4, and 10°C, respectively. Rates of reduction of V. parahaemolyticus shown in the TMs were 2.109, 1.579, and 0.894 log10 CFU/g for storage at 0, 4, and 10°C, respectively. Bacterial inactivation rates (IRs) estimated with the TMs (-0.245, -0.152, and -0.121 log10 CFU/day, respectively) were higher than those estimated with the MMs (-0.134, -0.0887, and -0.0732 log10 CFU/day, respectively) for storage at 0, 4, and 10°C. Higher viable V. parahaemolyticus numbers were predicted using the MMs than using the TMs. On the basis of this study, RNA-based predictive MMs are the more accurate and reliable models and can prevent false-negative results compared to TMs.IMPORTANCE One important method for validating postharvest techniques and for monitoring the behavior of V. parahaemolyticus is to establish predictive models. Unfortunately, previous predictive models established based on plate counting methods or on DNA-based PCR can underestimate or overestimate the number of surviving cells. This study developed and validated RNA-based molecular predictive models to describe the survival of V. parahaemolyticus in oysters during low-temperature storage (0, 4, and 10°C). The RNA-based predictive models show the advantage of being able to count all of the culturable, nonculturable, and stressed cells. By using primers targeting the tlh gene and pathogenesis-associated genes (tdh and trh), real-time RT-PCR can evaluate the total surviving V. parahaemolyticus population as well as differentiate the pathogenic ones from the total population. Reliable and accurate predictive models are very important for conducting risk assessment and management of pathogens in food.

Eating oysters without risk of vibriosis: Application of a bacteriophage against Vibrio parahaemolyticus in oysters

Vibrio parahaemolyticus is a major cause of foodborne illness and related with the consumption of raw contaminated seafood, especially oysters. To evaluate the effectiveness of various applications of a bacteriophage (phage), pVp-1, against a multiple-antibiotic-resistant V. parahaemolyticus pandemic strain (CRS 09-17), we designed artificial contamination models that are most likely to be encountered during oyster processing. When live oysters were treated with bath immersion with pVp-1 after CRS 09-17 challenge, the growth of bacterial strain was significantly reduced. After 72h of phage application with bath immersion, bacterial growth reduction was observed to be 8.9×106CFU/ml (control group) to 1.4×10CFU/ml (treatment group). When pVp-1 was surface-applied on the flesh of oysters after CRS 09-17 inoculation, bacterial growth was properly inhibited. After 12h of phage application on the surface of oysters, bacterial growth inhibition was revealed to be 1.44×106CFU/ml (control group) to 1.94CFU/ml (treatment group). This is the first report, to the best of our knowledge, of oyster surface-application of a phage against a multiple-antibiotic-resistant V. parahaemolyticus pandemic strain, and our successful phage application to various situations emphasizes the potential use of the phage to avoid V. parahaemolyticus infection from aquaculture to consumption.

Impacts of Lactobacillus plantarum in Depuration for Reducing Vibrio parahaemolyticus in Pacific Oysters (Crassostrea gigas)

This study investigated potential application of lactic acid bacteria (LAB) in depuration for reducing Vibrio parahaemolyticus in oysters. Lactobacillus plantarum ATCC 8014, which exhibited strong bactericidal effects against V. parahaemolyticus in vitro, was added to artificial seawater for depuration of Pacific oysters (Crassostrea gigas) inoculated with V. parahaemolyticus BE 98-2029 (O3:K6) to levels of about 104 MPN/g at 15 ± 1 and 10 ± 1°C. Application of L. plantarum ATCC 8014 treatment (107 CFU/mL) in oyster depuration did not enhance reductions of V. parahaemolyticus in oysters depurated at 15 ± 1°C but significantly decreased (p < 0.05) levels of V. parahaemolyticus in oysters depurated at 10 ± 1°C after 5 days (3.40 log reductions) when compared with controls (2.75 log reductions). It is not clear if a competitive exclusion by LABs to compete with V. parahaemolyticus binding sites in oyster tissues plays a role in the reduction of V. parahaemolyticus in the oysters. Further studies utilizing different types of LABs in oyster depuration might provide additional knowledge for application of LAB in depuration for decontaminating V. parahaemolyticus in oysters.

Reductions of Vibrio parahaemolyticus in oysters after bacteriophage application during depuration

Vibrio parahaemolyticus is closely associated with oysters in China, which are normally consumed raw or lightly cooked. Bacteriophages are a safe bio-controlling agent, and have been recognized in aquaculture for their pathogen reduction properties. This study investigated the potential application of the bacteriophage VPp1 during depuration to reduce V. parahaemolyticus in oysters at different multiplicity of infection (MOI) and temperature levels. Oysters were infected with 105, 106, and 107 colony-forming units (CFU)/mL of V. parahaemolyticus in the seawater and each infected group was treated with three different MOI values: 10, 1, and 0.1. Infected oysters were depurated in non-recirculating seawater at 22°C, 20°C, 16°C, and 12°C for 36h. Results revealed that temperatures <20°C were safe for oyster rearing. Depuration at 16°C with 0.1 MOI was the best condition for reducing V. parahaemolyticus in oysters, which decreased by 2.35–2.76logCFU/g within 36h. This study provides the basis for the use of bacteriophages in depuration techniques to eliminate V. parahaemolyticus in oysters.

The relationship of Vibrio parahaemolyticus densities and water quality factor during oyster culture

Vibrio parahaemolyticus(VP)is a pathogen of bacterial food poisoning associated with shellfish.VP,which is detected both in human and fish,has even higher concentrations in oysters and other filter-feeding shellfish.In order to study the relationship of VP densities versus water quality factor including water salinity,temperature,pH and dissolved oxygen in the course of oyster culture,we detected total VP densities in the oyster and water quality factor at the oyster farm in Yangxi,Guangdong from March to November in 2009.The results showed that the detection ratio of total VP in oysters was 94.38%.The water temperature was correlated positively with total VP in oysters,but the salinity was correlated negatively with total VP in oysters.The dissolved oxygen and pH didn't show correlation with total VP in oysters.Based on the regression analysis,a quadratic relationship for VP densities versus water temperature and salinity was found.The regression equation of VP densities versus water temperature was:LgVP=0.093T-0.685(R2=0.336);The regression equation of VP densities versus salinity was:LgVP=1.199+0.116S-0.004S2(R2=0.217);The best fit regression equation of VP densities versus water temperature and salinity was LgVP=-1.941+0.116T+0.058S-0.001S2(R2=0.414).The results of this paper may provide some reference data for the risk analysis of VP during oyster culture.

Influence of growth conditions on pressure resistance of Vibrio parahaemolyticus in oysters and the optimization of postpressure treatment recovery conditions.

Vibrio parahaemolyticus ATCC 43996 was grown at 15°C for 53 h, 20°C for 24 h, 25°C for 12 h, 30°C for 9 h, 35°C for 9 h, or 40°C for 6 h to early stationary phase. Oyster meats were blended, autoclaved at 121°C for 15 min, inoculated with V. parahaemolyticus, and pressure treated at 250 MPa for 2 and 3 min and at 300 MPa for 1 and 2 min at 21°C. Overall, growth temperatures of 20 and 40°C yielded the greatest pressure resistance in V. parahaemolyticus. The effects of salt concentration and H(2)O(2)-degrading compounds on the recovery of V. parahaemolyticus also were investigated. Sterile oyster meats were inoculated with V. parahaemolyticus and treated at 250 MPa for 1, 2, or 3 min at 21°C. These meats were then blended with 0.1% peptone water supplemented with 0.5 to 1.5% NaCl and plated on tryptic soy agar (TSA) supplemented with 0 to 3.5% NaCl. For recovery of pressure-injured cells, peptone water with 1% NaCl and TSA with 0.5% NaCl were the best diluent and plating medium, respectively. Addition of sodium pyruvate (0.05 to 0.2%) or catalase (8 to 32 U/ml) did not increase the recovery of V. parahaemolyticus after pressure treatment. The effect of incubation temperature and gas atmosphere on the recovery of V. parahaemolyticus after pressure treatment also was determined. Aerobic incubation at 30°C resulted in the highest recovery of V. parahaemolyticus in sterile oyster meats. The 30°C incubation temperature was also the optimum temperature for recovery of V. parahaemolyticus in pressure-treated live oysters. The results of this study indicate that the growth conditions for V. parahaemolyticus before and after high hydrostatic pressure treatment should be taken into consideration when assessing the efficacy of pressure inactivation.

Correlation between environmental factors and prevalence of Vibrio parahaemolyticus in oysters harvested in the southern coastal area of Sao Paulo State, Brazil.

The presence of Vibrio parahaemolyticus in 123 oyster samples collected from an estuary on the southern coast of Sao Paulo state, Brazil, was investigated. Of the 123 samples, 99.2% were positive with densities ranging from < 3 to 10(5) most probable number (MPN)/g. Densities correlated significantly with water temperature (r = 0.48; P < 0.001) but not with salinity (r = -0.09; P = 0.34). The effect of harvest site on counts was not significant (P > 0.05). These data provide information for the assessment of exposure of V. parahaemolyticus in oysters at harvest.

Occurrence of Vibrio parahaemolyticus in oysters from Korean retail outlets

The occurrence of Vibrio parahaemolyticus in 72 samples of raw Korean oysters obtained monthly from April to December from retail outlets was monitored, and the virulence of the strains isolated from 48 oyster samples investigated. V. parahaemolyticus was isolated in oysters from May to November with the highest level in August and September. Multiplex PCR showed that none of the V. parahaemolyticus strains isolated contained the gene for thermostable direct haemolysin ( tdh), although one strain was positive for TDH-related haemolysin ( trh). These data can be utilized for a risk-management plan to establish Food Safety Objectives (FSO) for V. parahaemolyticus in oysters at retail outlets in Korea.

Bactericidal Effects of Wine on Vibrio parahaemolyticus in Oysters

The bactericidal effects of wines on Vibrio parahaemolyticus in oysters were studied to evaluate potential inactivation of V. parahaemolyticus in contaminated oysters by wine consumption. Shucked whole oyster and oyster meat homogenate were inoculated with V. parahaemolyticus and mixed with red or white wine. Survivals of V. parahaemolyticus in inoculated oysters were determined at 7 and 25°C. Populations of V. parahaemolyticus in inoculated whole oysters (5.52 log most probable number [MPN] per g) decreased slightly to 4.90 log MPN/g (a 0.62-log reduction) after 24 h at 7°C but increased to 7.37 log MPN/g over the same period at 25°C. However, the populations in wine-treated whole oysters decreased by >1.7 and >1.9 log MPN/g after 24 h at 7 and 25°C, respectively. Both red and white wines were more effective in inactivating V. parahaemolyticus in oyster meat homogenate than in whole oyster. Populations of V. parahaemolyticus in oyster meat homogenate (7.8 × 103 MPN/g) decreased rapidly to nondetectable levels (<3 MPN/g) after 30 min of mixing with wine at 25°C (a 3.89-log MPN/g reduction). These results suggest that chewing oysters before swallowing when eating raw oysters may result in greater inactivation of V. parahaemolyticus if wine is consumed. More studies are needed to determine the bactericidal effects of wine on V. parahaemolyticus in the complicated stomach environment.

Seasonal variation in abundance of total and pathogenic Vibrio parahaemolyticus bacteria in oysters along the southwest coast of India.

The seasonal abundance of Vibrio parahaemolyticus in oysters from two estuaries along the southwest coast of India was studied by colony hybridization using nonradioactive labeled oligonucleotide probes. The density of total V. parahaemolyticus bacteria was determined using a probe binding to the tlh (thermolabile hemolysin) gene, and the density of pathogenic V. parahaemolyticus bacteria was determined by using a probe binding to the tdh (thermostable direct hemolysin) gene. Furthermore, the prevalence of V. parahaemolyticus was studied by PCR amplification of the toxR, tdh, and trh genes. PCR was performed directly with oyster homogenates and also following enrichment in alkaline peptone water for 6 and 18 h. V. parahaemolyticus was detected in 93.87% of the samples, and the densities ranged from <10 to 10(4) organisms per g. Pathogenic V. parahaemolyticus could be detected in 5 of 49 samples (10.2%) by colony hybridization using the tdh probe and in 3 of 49 samples (6.1%) by PCR. Isolates from one of the samples belonged to the pandemic serotype O3:K6. Twenty-nine of the 49 samples analyzed (59.3%) were positive as determined by PCR for the presence of the trh gene in the enrichment broth media. trh-positive V. parahaemolyticus was frequently found in oysters from India.

Detection of Vibrio parahaemolyticus and Vibrio cholerae in oyster, Crassostrea rhizophorae, collected from a natural nursery in the Cocó river estuary, Fortaleza, Ceará, Brazil.

Oysters are edible organisms that are often ingested partially cooked or even raw, presenting therefore a very high risk to the consumers' health, especially in tropical regions. The presence of Vibrio cholerae and Vibrio parahaemolyticus in oysters sampled at an estuary in the Brazilian northeastern region was studied, with 300 oysters tested through an 8-months period. The salinity of the water at the sampling point varied between 3% and 27. V. cholerae was the most frequently detected species (33.3% of the samples), and of the 22 V. cholerae isolates, 20 were identified as non-O1/non-O139, with two of the colonies presenting a rough surface and most of remaining ones belonging to the Heiberg II fermentation group. V. parahaemolyticus was isolated from just one of the samples. Other bacteria such as Providencia spp., Klebsiella spp. and Morganella morganii were also isolated.

Rapid detection of Vibrio parahaemolyticus in oysters by immunofluorescence microscopy

A method of indirect immunofluorescence microscopy was developed for the rapid detection of Vibrio parahaemolyticus in oysters. Affinity-purified antibodies against two outer membrane proteins (molecular mass 36 and 34 kDa, respectively) of V. parahaemolyticus were used as the primary antibodies to label the vibrio, and fluorescein isothiocyanate-conjugated goat anti-rabbit immunoglobulin G was used to reveal the antigen-antibody reaction. Of 85 strains of V. parahaemolyticus tested, all produced strong fluorescence under the fluorescence microscope. However, for 63 strains (including 27 vibrios) of other bacteria tested, 6 produced weak to moderate fluorescence. The sensitivity and specificity of the immunostaining technique were 100 and 90.5%, respectively. In the inoculation experiments, as low as 1.7 CFU g of V. parahaemolyticus spiked in oysters could be detected by the immunostaining method after 18-h enrichment in alkaline peptone water containing 3% NaCl. The immunofluorescence microscopy is recommended for rapid screening of V. parahaemolyticus in oysters, a presumptive positive result could be obtained within 24 h.