Effect of detergents as antibacterial agents on biofilm of antibiotics-resistant Vibrio parahaemolyticus isolates

Vibrio parahaemolyticus (V. parahaemolyticus) is a halophilic, Gram-negative human pathogen known as a leading cause of seafood-derived food poisoning. Due to high contamination rate of seafood in Asian countries, V. parahaemolyticus is considered as a food safety concern. V. parahaemolyticus is able to produce biofilm which is more resistant toward disinfectants and antibodies than its planktonic form. Thirty six V. parahaemolyticus isolates from seafood were tested for their susceptibility using 18 different antibiotics. Two V. parahaemolyticus isolates were resistant to bacitracin, chloramphenicol, rifampin, ampicillin, vancomycin, nalidixic acid, penicillin and spectinomycin. Fourteen V. parahaemolyticus isolates were found to be resistant to bacitracin, tetracycline, rifampin, ampicillin, vancomycin, penicillin and spectinomycin. The remaining two isolates were resistant to more than 2 antibiotics. Majority of the V. parahaemolyticus isolates (97.2%) showed MAR index > 0.2, indicating that these isolates were originated from high risk sources. To investigate effect of three common detergents on antibacterial-resistant V. parahaemolyticus, 16 V. parahaemolyticus isolates resistant to more than 7 antibiotics were selected. V. parahaemolyticus (ATCC 17802) was used as reference strain. Detergents were tested for their minimum inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) and time–kill curves were constructed to assess the concentration between MIC and bactericidal activity of detergents. Detergents D1 (Linear alkyl benzene based) was found to be the most effective with MIC and MBC ranged between 97.656 and 1562.5 μg/ml and 781.25–3125 μg/ml, respectively. The time–kill curves demonstrated that the bactericidal endpoint for resistant V. parahaemolyticus isolates reached after 30 min incubation with D1 at concentration 8 × MIC. The isolate VP003 was killed at 8 × MIC within 0.5 h and the reduction in CFU/ml was 3 log units (99.9%). V. parahaemolyticus biofilms were formed in 96 wells microtiter plates at 37 °C and 24 h-old biofilm were used to test antibacterial activity of detergents. Results showed that biofilm-producing ability of antibacterial-resistant V. parahaemolyticus isolates were inhibited at 1562.5–6250 μg/ml of D1 and eradicated at 3125 – ≥50,000 μg/ml of D1. Detergents showed potential antimicrobial activity against V. parahaemolyticus     

The efficacy of grape seed extract,citric acid and lactic acid on the inactivation of Vibrio parahaemolyticus in shucked oysters

The efficacy of grape seed extract (GE), citric acid (CA) or lactic acid (LA) on the inactivation of Vibrio parahaemolyticus in shucked oysters was studied. The minimum inhibitory concentration (MIC) of GE, CA or LA against V. parahaemolyticus in TSB-1% NaCl was also determined. The shucked oysters were artificially inoculated with V. parahaemolyticus, the inoculated shucked oysters (25 g) were then dipped in solution of GE (0.0, 10.0, 20.0, 50.0, 100, 200, 300 and 500 mg mL⁻¹), CA (0.0, 5.0, 10.0, 15.0, 20.0, 50.0, 100, 200 and 300 mg mL⁻¹) or LA (0.0, 1.0, 5.0, 10.0, 15.0, 20.0, 50.0, 100 and 150 mg mL⁻¹) for 10 min. The population of V. parahaemolyticus in shucked oysters was determined. The MICs of GE, CA or LA against V. parahaemolyticus were 10.0, 5.0 or 1.0 mg mL⁻¹, respectively. A 500, 300 or 150 mg mL⁻¹ GE, CA or LA solutions were needed to reduce the population of V. parahaemolyticus to below the detection level (1.0 log g⁻¹) in shucked oysters.     

Antibacterial activity and mechanism of cinnamon essential oil against Escherichia coli and Staphylococcus aureus

Cinnamon essential oil (EO) exhibited effective antibacterial activity against foodborne spoilage and pathogenic bacteria in model systems using Escherichia coli and Staphylococcus. The minimum inhibition concentration (MIC) of cinnamon EO was similar for both bacteria (1.0 mg/ml) while the minimum bactericide concentration (MBC) were 4.0 mg/ml and 2.0 mg/ml for E. coli and Staphylococcus aureus. GC–MS analysis confirmed that cinnamaldehyde was the major constituent in cinnamon EO (92.40%). Much effort was focused on elucidating the mechanism of antibacterial action of cinnamon EO against E. coli and S. aureus by observing the changes of cell microstructure using scanning electron microscope, determination of cell permeability, membrane integrity and membrane potential. After adding cinnamon EO at MIC level, there were obvious changes in the morphology of bacteria cells indicating cell damage. When cinnamon EO were added at MBC levels, the cells were destroyed. Cinnamon EO led to leakage of small electrolytes, causing rapid increase in the electric conductivity of samples at the first few hours. The values for E. coli and S. aureus reached 60% and 79.4% respectively at 7 h. Moreover, the concentration of proteins and nucleic acids in cell suspension also rose with increased cinnamon EO. Bacterial metabolic activity was decreased 3–5 folds as reflected by the results of membrane potential. Overall, S. aureus was more susceptible to cinnamon EO than E. coli.     

Assessment of antibacterial activity of fractions from burdock leaf against food-related bacteria

The chemical composition and antibacterial activities of burdock leaves fractions against six food-related bacteria (Staphylococcus aureus, Streptococcus pneumoniae, Bacillus subtilis, Escherichia coli, Shigella dysenteriae and Salmonella typhimurium) were first investigated. The burdock leaves extract was fractioned with ethyl acetate, n-butanol and water, named as EF, BF and WF. The data from minimum inhibitory concentration (MIC) values showed that EF and other fractions effectively inhibited the growth of all test bacterial pathogens, the antibacterial activity of EF being much greater than BF and WF. The time–kill study indicated that EF exhibited significant bactericidal activity against all the six pathogens. At 12 h, EF was bactericidal for 4/6, 6/6 and 6/6 strains at 1× MIC, 3× MIC and 5× MIC, respectively. For all the fractions, the antibacterial capacity had a significant correlation with total phenolic content, suggesting that the activities were probably due to the combined action of phenolic compounds. The phenolic compounds of the fractions were then identified for the first time. After activity-guide fractionation and separation by flash chromatogram, two purified active compounds (chlorogenic acid, rutin) were obtained, and identified by UPLC–MS/MS. The ethyl acetate fraction based on its lower MIC values, concentration and time-dependent antibacterial and bactericidal activities could be useful in control of foodborne bacterial pathogens.     

Chemical composition,antibacterial activity of Cyperus rotundus rhizomes essential oil against Staphylococcus aureus via membrane disruption and apoptosis pathway

The chemical composition, antibacterial activity and mechanism of essential oil from Cyperus rotundus rhizomes against Staphylococcus aureus were investigated in this study. Results showed that α-cyperone, cyperene and α-selinene were the major components of the essential oil. The essential oil exhibited strong antibacterial activity against S. aureus with the minimum inhibitory concentration (MIC) and minimum bactericide concentration (MBC) were 10 and 20 mg/mL respectively, and the antibacterial effects increased with increasing essential oil concentrations and treatment time. The electric conductivity, cell membrane integrity, NPN uptake, and membrane potential assays demonstrated that essential oil disrupted the membrane integrity of S. aureus. Electron microscope observations further confirmed that essential oil destroyed cell membrane. Moreover, we found that essential oil could induce cells death of S. aureus through apoptosis pathway based on apoptosis analysis. These findings suggested that essential oil mainly exerted antibacterial activity by damaging cell membrane and membrane-mediated apoptosis pathway.     

Chemical composition,antibacterial activity and mechanism of action of essential oil from seeds of fennel (Foeniculum vulgare Mill.)

Fennel (Foeniculum vulgare Mill.) is widely cultivated and used as a culinary spice. In this work, the chemical composition of the essential oil obtained by hydrodistillation of fennel seeds was analyzed by gas chromatography-mass spectrometry (GC–MS), and 28 components were identified. Trans-anethole (68.53%) and estragole (10.42%) were found to be the major components. The antibacterial activity, minimum inhibitory concentration (MIC), and minimum bactericide concentration (MBC) of essential oil against several food-borne pathogens were evaluated. The results showed that the gram positive and gram negative strains of bacteria had different sensitivities to essential oil of fennel seeds, the essential oil exhibited antibacterial activity against Staphylococcus albus, Bacillus subtilis, Salmonella typhimurium, Shigella dysenteriae and Escherichia coli according to the results of MIC and MBC. Among these bacteria, S. dysenteriae was the most sensitive to essential oil, showing the lowest MIC and MBC values of 0.125 and 0.25 mg/mL respectively. In addition, kill-time assay also showed that the essential oil had a significant effect on the growth rate of surviving S. dysenteriae. We concluded that the mechanism of action of the essential oil against S. dysenteriae might be described as essential oil acting on membrane integrity according to the results of the leakage of electrolytes, the losses of contents (proteins, reducing sugars and 260 nm absorbing materials) assays and electron microscopy observation.     

Antibacterial activity and mechanism of bifidocin A against Listeria monocytogenes

Bifidocin A, produced by Bifidobacterium animalis BB04, is a novel bacteriocin with antimicrobial activity against a wide range of gram-positive and gram-negative foodborne bacteria. The objective of this study was to investigate the antibacterial activity and mechanism of action of bifidocin A against Listeria monocytogenes, one of the most susceptible bacteria to this bacteriocin. The minimum inhibitory concentration (MIC) of bifidocin A for L. monocytogenes 35152 was 0.029 mg/mL. Time-kill assays showed that bifidocin A effectively inhibited the growth of L. monocytogenes in a time-and concentration-dependent manner. The mechanism of action of bifidocin A was studied by analyzing its effects at a MIC on the cell morphology, intracellular organization, membrane permeability, membrane integrity, and membrane proton motive force (PMF) of L. monocytogenes. Scanning and transmission electron microscopy analyses showed that bifidocin A induced alterations in the morphology and intracellular organization of L. monocytogenes cells. Confocal laser scanning microscopy images showed that L. monocytogenes cells treated with bifidocin A took up propidium iodide. Bifidocin A treatment also induced the leakage of K⁺ and inorganic phosphate, the hydrolysis and release of ATP, and a collapse of the transmembrane electrical potential and pH gradient in L. monocytogenes cells. These results suggested that bifidocin A exerted its anti-Listeria monocytogenes effect through the dissipation of the cytoplasmic membrane PMF, increased membrane permeability, cell membrane pore formation, destruction of membrane integrity, and ultimately complete disintegration of the cells.     

Effects of green tea extract on reducing Vibrio parahaemolyticus and increasing shelf life of oyster meats

This study investigated effects of tea extract on growth of pathogenic Vibrio parahaemolyticus and potential utilization in post-harvest treatment to extend shelf life of Pacific oysters (Crassostrea gigas). Longjing Tea, which exhibited strong bactericidal activity against V. parahaemolyticus, was selected to use in this study. Tea extract containing equal or higher than 4.6 g/l total phenolic contents (TPC) as gallic acid equivalents (GAE) determined by Folin-Ciocalteau method could reduce a mixture of five clinical V. parahaemolyticus strains in tryptic soy broth plus 1.5% NaCl from 4.5 log CFU/ml to non-detectable level (<1 log CFU/ml) within 8 h. A treatment of shucked oysters with tea extract containing 9.1 g/l TPC as GAE for 2 h at 23 ± 1 °C with oyster/tea extract ratio of approximate 0.9 g/ml resulted in greater (p < 0.05) V. parahaemolyticus reductions (0.8 log MPN/g) compared to controls (0.2 log MPN/g). The following shelf life study indicated that green tea treatment at oyster/tea extract ratio of approximate 0.7 g/ml could enhance reducing V. parahaemolyticus while retarding the growth of total bacteria in oysters during 5 ± 1 °C storage. Therefore, green tea might be utilized as a natural antimicrobial agent to inactivate V. parahaemolyticus in oysters and extend the shelf life during refrigeration storage.     

Production and characterization of a monoclonal antibody against recombinant thermolabile hemolysin and its application to screen for Vibrio parahaemolyticus contamination in raw seafood

Thermolabile hemolysin gene (tlh) is regarded as a species-specific marker for Vibrio parahaemolyticus. To assess the utility of the tlh gene product, thermolabile hemolysin (TLH), as a marker to screen for V. parahaemolyticus-contamination in raw seafood, we generated a monoclonal antibody (MAb) against recombinant TLH and established with an enzyme-linked immunosorbent assay using the MAb (TLH-ELISA). TLH-ELISA testing of broth cultures for 78 V. parahaemolyticus strains showed positive results all around. In contrast, most broth cultures of 53 non-V. parahaemolyticus species tested yielded negative results.We devised a screening method using TLH-ELISA to check for low-level contamination of V. parahaemolyticus in raw seafood within 24 h and evaluated its ability. In testing of V. parahaemolyticus-spiked raw seafood, results suggested that our screening method can detect 100 most-probable-number (MPN) of V. parahaemolyticus/g. Further, on testing 119 commercial raw seafood samples with our screening method, 117 samples were determined to contain less than 100 MPN of V. parahaemolyticus/g. All of the 117 samples were also estimated by the MPN method to contain less than 100 MPN of V. parahaemolyticus/g. Taken together, these results suggest that our screening method using TLH-ELISA is useful to check for low-level (<100 MPN/g) of V. parahaemolyticus in raw seafood.     

PMA based real-time fluorescent LAMP for detection of Vibrio parahaemolyticus in viable but nonculturable state

Vibrio parahaemolyticus is a crucial foodborne pathogen. The viable but non-culturable (VBNC) state of V. parahaemolyticus cannot be detected by traditional culture methods. The objective of this study was to develop and evaluate a method that combines propidium monoazide (PMA) treatment with real-time fluorescent loop-mediated isothermal amplification (RF-LAMP) to detect and quantify VBNC cells of V. parahaemolyticus. Different states of cells were treated with PMA in dark for 5 min and subsequently exposed to a 650 W halogen lamp for 5 min. The cells were collected and DNA was amplified by RF-LAMP. The primers which targeted six distinct regions in the tlh gene of V. parahaemolyticus were used for the PMA-RF-LAMP assay. The results indicated that the treatment with 4 μg/mL of PMA and a further exposure to light for 5 min was suitable for PMA-RF-LAMP to distinguish viable cells from dead cells of V. parahaemolyticus. The developed assay exhibited remarkably high specificity, sensitivity and rapidity, without any cross reaction with the tested non-V. parahaemolyticus strains. There was good linear correlation between Ct values and log copy/mL in the range of 6.8–6.8 × 10⁷ copy/mL, with the reaction endpoints less than 30 cycles (30 min). It could detect as low as 14 copy/g of V. parahaemolyticus in spiked seafood samples (pomfret, shrimp, scallop, oyster and salted fish) without any interference from food matrices, dead cells and other bacteria.     

Pomegranate peel (Punica granatum L) extract and Chinese gall (Galla chinensis) extract inhibit Vibrio parahaemolyticus and Listeria monocytogenes on cooked shrimp and raw tuna

Vibrio parahaemolyticus and Listeria monocytogenes are bacterial pathogens associated with raw or ready-to-eat seafood products. Many compounds extracted from plant material have shown promise for inhibiting bacterial pathogens when applied to some foods. In this study, aqueous methanol extracts from pomegranate peel (Punica granatum L.) and Chinese gallnut (Galla chinensis) were tested against V. parahaemolyticus and L. monocytogenes on cooked shrimp and raw homogenized tuna. The extracts were applied to the shrimp by soaking for 2 min (5 mg/ml). The extracts (1.7 mg ml) were added to homogenized tuna and stirred. The antimicrobial assay on V. parahaemolyticus was conducted at 12 °C, and the assay on tuna was conducted at both 4° and 12 °C. Both Chinese gall and pomegranate peel extracts significantly inhibited the growth of V. parahaemolyticus in both shrimp and tuna. Only Chinese gall extract significantly inhibited growth of L. monocytogenes. Overall V. parahaemolyticus was more sensitive to both plant extracts compared with L. monocytogenes. Both plant extracts had stronger antimicrobial activity on shrimp compared with the tuna. Neither extract completely inhibited the growth of V. parahaemolyticus or L. monocytogenes.     

Efficacy of the combined application of oregano and rosemary essential oils for the control of Escherichia coli,Listeria monocytogenes and Salmonella Enteritidis in leafy vegetables

This study assessed the effect of the combined application of essential oils (EOs) from Origanum vulgare L. – oregano (OVEO) and Rosmarinus officinalis L. – rosemary (ROEO), alone or in combination at subinhibitory concentrations, against three pathogenic bacteria that are associated with fresh leafy vegetables: Listeria monocytogenes (L. monocytogenes), Escherichia coli (E. coli) and Salmonella enterica Serovar Enteritidis (S. Enteritidis). The inhibitory effects were evaluated by determining the minimum inhibitory concentration (MIC) and the fractional inhibitory concentration index (FICI) and assessing the viable cell counts in vegetable broth and artificially infected vegetables over time. Still, the effects of the EOs on native spoilage native flora were assessed. The MIC of OVEO was 0.6 μL/mL against the test strains either in single and mixed inoculum. The MIC of ROEO was 5 μL/mL against L. monocytogenes and E. coli and 10 μL/mL against S. Enteritidis in single inocula, whereas it was 10 μL/mL against the mixed inoculum. The FICI of the combined EOs was 0.5 against the mixed bacterial inoculum, which suggested a synergic interaction. The incorporation of OVEO and ROEO alone (MIC) or combined at different subinhibitory concentrations in vegetable broth resulted in a decrease in the viable cell counts of all test strains over 24 h. Similarly, the EOs alone or in the tested combinations reduced the viable cell counts of all test strains in experimentally infected fresh vegetables, besides to decrease the counts of spoiling native flora (mesophilic bacteria, enterobacteria and fungi). These findings reinforce the rational for the use of OVEO and ROEO in combination at subinhibitory concentrations to guarantee the safety and extend the shelf life of fresh vegetables.     

Epidemiology of foodborne disease outbreaks caused by Vibrio parahaemolyticus during 2010–2014 in Zhejiang Province,China

Vibrio parahaemolyticus is one of the most commonly reported species causing foodborne diseases in China, to gain understanding of epidemiology characteristics of V. parahaemolyticus outbreaks in Zhejiang Province, China, we summarized data on V. parahaemolyticus outbreaks from 2010 to 2014 in Zhejiang Province reported to China National Foodborne Diseases Surveillance Network. A food item was implicated if V. parahaemolyticus was isolated from food or based on epidemiologic evidence. From 2010 to 2014, 71 outbreaks due to V. parahaemolyticus were reported, resulting in 933 illnesses and 117 hospitalizations without death. The median annual incidence rate of confirmed V. parahaemolyticus outbreaks reported was 2.75 per million population (rang, 1.11–5.60). About 90.1% (64/71) of outbreaks caused by V. parahaemolyticus occurred during May and October. Aquatic products (27 outbreaks, 38.0%), meat and meat products (9 outbreaks, 12.7%), plant-based foods (6 outbreaks, 8.4%), mixed foods (5 outbreaks, 7.0%) were the most commonly implicated foods. Outbreaks most frequently occurred in restaurants (50.7%), private residents (21.1%), and cafeteria (12.7%). Cross contamination (39.4%), improper cooking (21.1%), improper storage (16.9%) were main reasons. To prevent V. parahaemolyticus outbreaks caused by cross contamination, improper cooking and improper storage in high-temperature seasons, regulations for seafood safety from the production stage to the consumption stage should be strengthened.     

Antifungal activity of citral,octanal and α-terpineol against Geotrichum citri-aurantii

This study investigated the antifungal activity and potential antifungal mechanisms of three volatile compounds (i.e., citral, octanal, and α-terpineol) against Geotrichum citri-aurantii, one of the main postharvest pathogens in citrus. Results showed that the volatile compounds exhibited strong antifungal activity against the targeted pathogens, with minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of 0.50 μL/mL and 1.00 μL/mL for citral, 0.50 μL/mL and 2.00 μL/mL for octanal, and 2.00 μL/mL and 4.00 μL/mL for α-terpineol. The volatile compounds alter the morphology of G. citri-aurantii hyphae by causing loss of cytoplasm content and distortion of the mycelia. The membrane permeability of the G. citri-aurantii increased with increasing concentrations of the three volatile compounds, as evidenced by cell constituent release, extracellular conductivity, and pH. Moreover, the volatile compounds induced a decrease in the total lipid content of G. citri-aurantii cells, indicating the destruction of cell membrane structures. These results suggest that the antifungal activity of citral, octanal, and α-terpineol against G. citri-aurantii could be attributed to the disruption of cell membrane integrity and leakage of cell components.     

Selective turn-on fluorescence detection of Vibrio parahaemolyticus in food based on charge-transfer between CdSe/ZnS quantum dots and gold nanoparticles

Vibrio parahaemolyticus (V. parahaemolyticus) is one of the most common food-borne pathogens found along the east coast of China, Japan, and several southeast Asian countries, where marine foods are frequently consumed. Thus it is of great interest to establish a fast, sensitive and robust analytical assay for V. parahaemolyticus detection. Herein, we report a V. parahaemolyticus-specific egg yolk antibody (IgY), extracted from the egg yolks of immunized hens with inactivated V. parahaemolyticus bacteria. By immobilizing IgY onto the surface of gold nanoparticles (AuNPs) through electrostatic self-assembly, a detection system was developed based on charge-transfer quenching fluorescence, which contains core/shell CdSe/ZnS quantum dots (QDs), IgY-AuNPs and free AuNPs. In this strategy, the IgY-AuNPs were induced to aggregate quickly in the presence of target V. parahaemolyticus, resulting in a rapid fluorescence response of the QDs. More importantly, the convenient turn-on fluorescent detection system exhibited excellent selectivity over the other bacteria. Furthermore, this facile one-step method can detect V. parahaemolyticus at low concentrations of 10 cfu/mL without pre-enrichment and can be applicable for the detection of V. parahaemolyticus in real food samples. With the advantage of simplicity, rapidness, sensitivity, and specificity, this proposed approach shows promise as a successful application of V. parahaemolyticus in bioanalysis.     

In vitro control of multiplication of some food-associated bacteria by thyme, rosemary and sage isolates

Antibacterial activity of thyme, rosemary and sage isolates obtained by supercritical fluid extraction and hydrodistillation was investigated on Geobacillus stearotermophillus, Bacillus cereus, Bacillus subtilis var. niger, Enterococcus faecium, Salmonella enteritidis and Escherichia coli strains. Bacillus species were the most susceptible to all tested isolates. The thyme isolates showed the strongest antibacterial activity against tested bacteria with MIC values of 40-640 μg/ml, followed by rosemary (MIC = 320-1280 μg/ml) and sage (MIC = 160-2560 μg/ml) isolates. Therefore, the antibacterial activity of the most abundant components found in the thyme isolates, thymol, p-cymene and their mixture was investigated as well. The thyme isolates, especially supercritical extract, showed stronger antibacterial activity against Bacillus strains compared to the single components and their mixture, which indicated synergetic effect of the other components. Results of this study indicated thyme as a valuable source of natural antibacterial agents and supercritical fluid extraction as an efficient isolation method.     

Prevalence and antimicrobial susceptibility of Vibrio parahaemolyticus isolated from retail shellfish in Shanghai

Vibrio parahaemolyticus is a marine and estuarine bacterium that poses the greatest threat to human health worldwide. It has been the leading bacterial cause of seafood-borne illness. This study investigated the prevalence and drug resistance of V. parahaemolyticus isolated from retail shellfish in Shanghai. A total of 140 shellfish samples were collected from February 2014 to February 2015. The occurrence of V. parahaemolyticus in shellfish was 34.3%, which has increased compared to previous reports. In addition, discernible differences of total presumptive V. parahaemolyticus counts (TPVPC) were also observed in shellfish between market A and B. The results from PCR assays indicated that thermostable direct hemolysin (tdh) gene was positive in two isolates (2.1%), and the thermostable direct hemolysin-related hemolysin (trh) gene was not detected in all isolates. Antibiotic resistance profiles of those isolates were as follows: ampicillin (87.5%), cephazolin (31.3%), cephalothin (6.3%), amoxicillin/clavulanic acid (6.3%), piperacillin (6.3%), and amikacin (3.2%). Thirty-three out of 96 isolates were resistant to two or more antimicrobial agents. It is suggested that V. parahaemolyticus in retail shellfish could be a potential risk to consumers in Shanghai.     

Antibacterial activity of a grape seed extract and its fractions against Campylobacter spp.

In this study, the antibacterial activity of a grape seed extract (GSE) was examined against different Campylobacter strains. Growth inhibition was in the range from 5.08 to 6.97 log CFU/ml, demonstrating the strong capacity of the GSE to inhibit Campylobacter growth. Further dilution of the extract showed a minimal inhibitory concentration (MIC) of 20 mg/l and a minimal bactericidal concentration (MBC) of 60 mg/l against Campylobacter jejuni. GSE was fractioned by RP-HPLC and phenolic composition was determined by HPLC-DAD and HPLC-MS. The phenolic profile of GSE mainly consisted on flavonols, phenolic acids, catechins and proanthocyanidins, and anthocyanins. Among them, catechins and proanthocyanidins were the major compounds, representing 77.6% of total phenolic compounds determined. The analysis of the antibacterial activity against C. jejuni of the collected fractions showed that phenolic acids, catechins and proanthocyanidins were the main responsible of the behavior observed. These results showed that identification and quantification of the individual phenolic compounds of GSE could be feasible to standardize the production process to obtain an enriched extract potentially useful to control Campylobacter in the food chain.     

Application of grape seed extract in depuration for decontaminating Vibrio parahaemolyticus in Pacific oysters (Crassostrea gigas)

This study investigated the potential application of grape seed extract (GSE) in depuration to increase its efficacy in reducing Vibrio parahaemolyticus populations in Pacific oysters (Crassostrea gigas). Pacific oysters were inoculated with five clinical strains of V. parahaemolyticus to 10⁴⁻⁵ MPN/g and depurated in UV-irradiated artificial seawater (ASW) containing GSE at a level of 1.0% (1.8 mg/mL total phenolic contents as gallic acid equivalents) or 1.5% (3.1 mg/mL total phenolic contents as gallic acid equivalents) at 12.5 °C for up to 5 days. Changes of V. parahaemolyticus populations in oysters during depuration were analyzed every 24 h using the three-tube most probable number (MPN) method. The populations of V. parahaemolyticus in inoculated oysters decreased by 3.06 and 3.71 log MPN/g, respectively, after 4 and 5 days of depuration in ASW at 12.5 °C. However, populations of V. parahaemolyticus in oysters were reduced by 3.01 and 4.18 log MPN/g after 2 days of depuration at 12.5 °C in ASW containing 1.0 and 1.5% GSE, respectively. Further studies confirmed that depuration using ASW containing 1.5% GSE with 3.1 mg/mL total phenolic contents as gallic acid equivalents at 12.5 °C for two days was capable of achieving >3.52 log MPN/g reductions of V. parahaemolyticus in Pacific oysters.     

Synergistic inhibition of bacteria associated with minimally processed vegetables in mixed culture by carvacrol and 1,8-cineole

This study assessed the effect of the combined application of carvacrol (CAR) and 1,8-cineole (CIN) against bacteria associated with minimally processed vegetables (MPV), namely Listeria monocytogenes, Aeromonas hydrophila and Pseudomonas fluorescens, in mixed culture. For this, Fractional Inhibitory Concentration (FIC) index, time-kill in vegetable-based broth, vegetable matrices and confocal and scanning electron microscopy analysis were carried out. CAR and CIN displayed Minimum Inhibitory Concentrations (MIC) of 1.25 and 40 μL/mL, respectively. FIC index of the combined compounds was 0.25 against the mixed inoculum, suggesting a synergic interaction. The application of CAR and CIN alone (MIC) or combined (1/8 MIC + 1/8 MIC) in vegetable-based broth caused a decrease (p < 0.05) in viable cell counts over 24 h. CAR and CIN in combination reduced (p < 0.05) the mixed inoculum in vegetable broth and in experimentally inoculated fresh vegetables. The exposure to these compounds changed the membrane permeability and caused ultrastructural changes in surfaces of bacterial cells. The enhancing inhibitory effects observed for subinhibitory concentrations of CAR and CIN in combination against a mixed culture of bacteria associated with MPV suggest that the combination of these compounds is effective for controlling bacterial growth and survival in these foods.