Analysis of Escherichia coli cell damage induced by HPCD using microscopies and fluorescent staining

Cellular damage of Escherichia coli (E. coli) induced by high pressure carbon dioxide (HPCD) at 37-57 °C and 10-30 MPa for 5-75 min was investigated using scanning electronic microscopy (SEM), transmission electronic microscopy (TEM), confocal laser scanning microscopy (CLSM), and fluorospectrophotometer (FSM). HPCD-induced alterations in the morphology and the intracellular organization of E. coli cells was more susceptible to HPCD. A vast majority of HPCD-treated E. coli cells with seemingly intact morphology sustained severe damage in their intracellular organization. CLSM suggested that initial disruption of the outer membrane and later permeabilization of the cytoplasmic membrane of HPCD-treated E. coli cells was a consecutive and progressive process. These results were confirmed by FSM with the probes PI and SYTO 9. The membrane fluidity of HPCD-treated E. coli cells decreased as suggested by increased fluorescence polarization using FSM with the probe 1,6-diphenyl-1,3,5-hexatriene (DPH). The temperatures of 37, 42 and 47 °C alone showed no impact on the outer membrane and membrane fluidity of E. coli cells whereas 57 °C alone had greater impact on them. Combined with HPCD, the temperatures of 37, 42 and 47 °C disrupted the outer membrane of E. coli cells without damage to the cytoplasmic membrane and of 57 °C damaged the cytoplasmic membrane, but all these temperatures decreased the membrane fluidity of E. coli cells. Higher temperature increased HPCD-induced outer membrane disruption and the cytoplasmic membrane damage and decreased the membrane fluidity.     

Hurdle Effect of Antimicrobial Activity Achieved by Time Differential Releasing of Nisin and Chitosan Hydrolysates from Bacterial Cellulose

We investigated the combined antimicrobial effect of nisin and chitosan hydrolysates (CHs) by regulating the antimicrobial reaction order of substances due to differential releasing rate from hydroxypropylmethylcellulose‐modified bacterial cellulose (HBC). The minimum inhibitory concentration of nisin against Staphylococcus aureus and that of CHs against Escherichia coli were 6 IU and 200 μg/mL, respectively. Hurdle and additive effects in antimicrobial tests were observed when nisin was used 6 h before CH treatment against S. aureus; similar effects were observed when CH was used before nisin treatment against E. coli. Simultaneously combined treatment of nisin and CHs exhibited the low antimicrobial effect. HBC was then selected as the carrier for the controlled release of nisin and CHs. A 90% inhibition in the growth of S. aureus and E. coli was achieved when 30 IU‐nisin‐containing HBC and 62.5 μg/mL‐CH‐containing HBC were used simultaneously. The controlled release of nisin and CHs by using HBC minimized the interaction between nisin and CHs as well as increased the number of microbial targets.     

Membrane permeabilization and cellular death of Escherichia coli, Listeria monocytogenes and Saccharomyces cerevisiae as induced by high pressure carbon dioxide treatment

In this study, the relationship between (irreversible) membrane permeabilization and loss of viability in Escherichia coli, Listeria monocytogenes and Saccharomyces cerevisiae cells subjected to high pressure carbon dioxide (HPCD) treatment at different process conditions including temperature (35–45 °C), pressure (10.5–21.0 MPa) and treatment time (0–60 min) was examined. Loss of membrane integrity was measured as increased uptake of the fluorescent dye propidium iodide (PI) with spectrofluorometry, while cell inactivation was determined by viable cell count. Uptake of PI by all three strains indicated that membrane damage is involved in the mechanism of HPCD inactivation of vegetative cells. The extent of membrane permeabilization and cellular death increased with the severity of the HPCD treatment. The resistance of the three tested organisms to HPCD treatment changed as a function of treatment time, leading to significant tailing in the survival curves, and was dependent on pressure and temperature. The results in this study also indicated a HPCD-induced damage on nucleic acids during cell inactivation. Transmission electron microscopy showed that HPCD treatment had a profound effect on the intracellular organization of the micro-organisms and influenced the permeability of the bacterial cells by introducing pores in the cell wall.     

Inactivation of natural microorganisms in litchi juice by high-pressure carbon dioxide combined with mild heat and nisin

The individual and combined effects of high-pressure carbon dioxide (HPCD), mild heat (MH) and nisin (200 ppm) on the inactivation of natural microorganisms, including aerobic bacteria (AB), yeasts and molds (Y&;M), in litchi juice were evaluated. The samples were treated at a pressure of 10 MPa and temperatures of 32, 42 or 52 °C for 5, 10, 15, 20, 25 or 30 min. Temperature played a prominent role in the inactivation of both AB and Y&;M when combined with HPCD, particularly for AB at 52 °C and Y&;M at temperatures ≥42 °C. Nisin increased the susceptibility of AB to the combined treatment of HPCD and MH (HPCD + MH). A reduction of 4.19 log cycles was achieved by HPCD + MH at 52 °C for 15 min, and complete inactivation of AB was obtained by combination of HPCD, MH and nisin (HPCD + MH + nisin). No significant effect of nisin was found on the inactivation of Y&;M.     

Potential use of niosomes for encapsulation of nisin and EDTA and their antibacterial activity enhancement

Different formulations of niosomes were prepared using Span 80 with sodium stearoyl lactate (SSL) and PEG400 as additives. The corresponding niosomes were used for encapsulation of antibacterial agents including nisin and EDTA. Characterization of unloaded and loaded niosomes was carried out by means of size, zeta-potential, and also encapsulation efficiency and antibacterial activity specifically for loaded niosomes. Unloaded niosomes were stable at least 2 months when temperature challenge was applied at 4 °C, RT, and 45 °C. An addition of 0.5 mM SSL, both with and without PEG was found to enhance stability of loaded niosomes throughout 2-months of storage. Effect of niosome formulation to zeta potential and encapsulation efficiency of nisin and EDTA was discussed in this paper. A 96-well plate assay was used to assess the antibacterial activity between free and encapsulated forms of nisin and EDTA against Staphylococcus aureus and Escherichia coli. A higher and longer-lasting inhibition effect of encapsulated nisin and EDTA against S. aureus as compared to their free forms was observed in an encapsulation using niosome formulations S_0.5s and S_1.0s, both with and without PEG. To our knowledge, this work is the first report on applying niosomes for encapsulation of nisin and EDTA. The results in this study offer an alternative approach to encapsulation of nisin as good as or better than liposomes.     

Enhancement Antimicrobial Activity of Hyphothiocyanite Using Carrot Against Staphylococcus Aureus and Escherichia Coli

Hypothiocyanite has been known as antimicrobial agent that was generated from lactoperoxidase system (LPOS) but its antimicrobial activity was low against pathogenic bacteria in milk. This research has been done to enhance the antimicrobial activity of hypothiocyanite against pathogenic bacteria commonly found in milk: Staphylococcus aureus and Escherichia coli by addition of carrot extract. The result showed that carrot extract was able to enhance the antimicrobial activity of hypothiocyanite strongly against E. coli, however less enhancement has been found in the antibacterial activity of hypothiocyanite against S. aureus.     

Inactivation of high pressure resistant Escherichia coli by lysozyme and nisin under high pressure

We studied the inactivation (by high hydrostatic pressure at 20°C) of Escherichia coli MG1655 and the selected pressure-resistant mutants that were derived previously from this strain, and which are the most pressure-resistant vegetative cells described to date. The natural antimicrobial peptides, lysozyme (50 μg/ml) and nisin (100 IU/ml), enhanced considerably the inactivation of the target bacteria under pressure. However, kinetic inactivation experiments in the presence of these compounds revealed pronounced tailing, which limited the level of inactivation that could be achieved under mild conditions of pressure and temperature. Interrupted pressure treatments enhanced the effectiveness of lysozyme and nisin, allowing a reduction by at least 6 logs of all strains at 400 MPa. A hypothetical mechanism of ‘pressure-promoted uptake’ is proposed to explain E. coli outer membrane permeabilization for lipophilic and cationic peptides like lysozyme and nisin under pressure.     

Antimicrobial properties and action of galangal (Alpinia galanga Linn.) on Staphylococcus aureus

The ethanol extracts of the Zingiberaceae family (galangal, ginger, turmeric and krachai) were evaluated for antimicrobial action on Staphylococcus aureus 209P and Escherichia coli NIHJ JC-2 by using an agar disc diffusion assay. The galangal extract had the strongest inhibitory effect against S. aureus. The minimum inhibitory concentration (MIC) of the galangal extract was 0.325 mg/ml and the minimum bactericidal concentration (MBC) at 1.3 mg/ml using the broth dilution method. Transmission electron microscopy clearly demonstrated that the galangal extract caused both outer and inner membrane damage, and cytoplasm coagulation. The disruption of the cytoplasmic membrane properties was determined by the releasing of cell materials including nucleic acid which absorbed UV/VIS spectrophotometer at 260 nm. The major compound of the extract was d,l-1′-acetoxychavicol acetate which was identified by GC-MS and NMR.     

Synergetic activity of nisin with cell-free supernatant of Bacillus licheniformis ZJU12 against food-borne bacteria

Antibacterial activity of nisin and cell-free supernatant of Bacillus licheniformis ZJU12 (CFSb), separately and in combination, against three food-borne bacteria: Micrococcus flavus, Bacillius cereus and Staphylococcus aureus was determined. M. flavus was more sensitive to nisin and CFSb than other two indicator strains in the MIC tests. Synergetic activity of nisin and CFSb was found against all the three indicator strains in the checkerboard microtiter test and the time-kill test. The combination had the most notable antibacterial activity against B. cereus as determined by the fractional inhibitory concentration index (FICI). The antibacterial activity of nisin and CFSb separately began within the first 30 min, while their synergetic activity was observed after 4 h.     

Influence of farming methods on microbiological contamination and prevalence of resistance to antimicrobial drugs in isolates from beef

The presence of Escherichia coli, Staphylococcus aureus, Listeria monocytogenes and Salmonella spp. was determined in 75 samples of conventional beef and in 75 samples of organic beef. All samples came from cattle slaughtered and processed in the same slaughterhouse and quartering room. A total of 180 E. coli, 180 S. aureus and 98 L. monocytogenes strains were analyzed by an agar disk diffusion assay for their resistance to 11 antimicrobials, for the case of E. coli and S. aureus, or 9 antimicrobials, for the case of L. monocytogenes. Salmonella spp. were not isolated from any of the beef samples. No significant differences in prevalence were obtained for any of the bacterial species tested between organic and conventional beef. E. coli isolated from organic beef exhibited significant differences in antimicrobial resistance against 5 of the 11 antimicrobials tested as compared to isolates recovered from conventional beef. In the case of S. aureus, these differences were only found for 3 of the 11 antimicrobials tested and for L. monocytogenes, no differences were obtained between isolates obtained from organic or conventional beef. Although no significant differences were obtained in microbiological contamination, E. coli and S. aureus isolates from organically farmed beef samples showed significantly lower rates of antimicrobial resistance in E. coli and S. aureus isolates.     

Effect of vacuum-thermosonication on the inactivation of Escherichia coli,Staphylococcus aureus,polyphenol oxidase and the quality parameters of soursop puree

The combined effect of vacuum, heat and ultrasound (vacuum-thermosonication, VTS) on soursop puree was investigated with regard to the viability of Escherichia coli and Staphylococcus aureus, inactivation of polyphenol oxidase and sensory quality. The VTS conditions were: vacuum (8.46, 11 and 16.93 kPa), heat (40, 45 and 50 °C), 1–3 intermittent vacuum pulses and ultrasound (24 kHz and 0.34 W/g of acoustic energy density) during 10 min. According to response surface methodology, the best conditions to obtain the highest microbial inactivation of ≥7 log CFU of inoculated E. coli and S. aureus, and reduction in polyphenol oxidase activity (94%) were 16.5 kPa vacuum, 50 °C, and three intermittent vacuum pulses for 10 min with ultrasound. The best VTS conditions did not negatively affect the quality parameters, and there were no significant changes in sensory attributes of the puree (a panel of 50 untrained judges). Therefore, we conclude that VTS appears a viable option in the processing of soursop.     

Prevalence and characterization of foodborne bacteria from meat products in Korea

In this study, the distribution of Salmonella spp., Escherichia coli, Staphylococcus aureus, Campylobacter jejuni, and Vibrio parahaemolyticus in raw meat products in Korea were investigated. A total of 155 meat products consisting of 52 beef, 62 pork, and 41 chicken were purchased randomly from 41 stores located in 5 different Korean provinces. E. coli and S. aureus were detected in 37.4 and 33.5% of the samples. Salmonella spp., C. jejuni, and V. parahaemolyticus were not detected. More than 30% of S. aureus were found to be enterotoxin producers and these organisms primarily possessed type A toxin genes. Conversely, verocytotoxin producing E. coli were not detected. Taken together, these results indicate that consumption of raw meat products may pose a risk of foodborne disease and that good hygienic practices should be required to ensure public health.     

Component release after exposure of Staphylococcus aureus cells to pulsed electric fields

The objective of this work was to get further insights on the mechanism of inactivation of bacterial cells by pulsed electric fields (PEF) through the study of the release of intracellular components after exposing Staphylococcus aureus cells in McIvlaine buffer (pH 7.0, 2 mS/cm) to PEF treatments of different intensity (18 and 25 kV/cm) and treatment times (from 20 to 400 μs). Release of most compounds, except proteins, was almost immediate after the treatment, but the relative amount released depended on the molecule studied. A good correlation between the release of the smallest components studied (particularly ions) and membrane permeabilization (as measured by NaCl sensitization and PI entry) was observed. On the other hand, results obtained suggested that S. aureus inactivation by PEF would be related to the exit of cytoplasmic proteins of a molecular weight higher than 6 kDa. Results obtained in this work indicated that increasing PEF treatment time would reduce the capability of S. aureus cells to repair the electropores formed and suggested that this might be due to the formation of pores of a larger size, which S. aureus cells would be unable to reseal in a situation of homeostasis loss.Industrial relevanceResults reported here can help to design more effective treatments for microbial inactivation using PEF on food, and therefore facilitate its industrial implementation.     

Effects of CO2-assisted high-pressure processing on microbiological and physicochemical properties of Chinese spiced beef

The objective of this study was to evaluate the effect of CO₂-assisted high-pressure processing (CO₂-HPP) on microbiological and physicochemical properties of Chinese spiced beef. The presence of CO₂ induced extra reduction of Staphylococcus aureus, Escherichia coli and Listeria innocua under 150–450 MPa. The maximum extra reduction of S. aureus and E. coli were found at 450 MPa. The Listeria innocua cells were more resistant to CO₂-HPP than the Staphylococcus aureus and Escherichia coli cells. CO₂–450 MPa extended the shelf-life of Chinese spiced beef by one week than 450 MPa. CO₂–450 MPa didn't significantly increased the thiobarbituric acid reactive substances (TBARS) value of Chinese spiced beef compared with 450 MPa. CO₂–450 MPa limited 450 MPa induced color change. During cold storage, CO₂–450 MPa better maintained the firmness of Chinese spiced beef compared with 450 MPa, and the TBARS value of CO₂–450 MPa treated samples was significantly lower than 450 MPa-treated samples.Industrial relevanceHigh-pressure processing (HPP) is the most successful commercialized non-thermal technique in food industry, which could extend the shelf-life of Chinese spiced beef to 3 weeks. Nevertheless, HPP may induce color change, decrease firmness and increase lipid oxidation. Addition of carbon dioxide to HPP was shown to reduce lipid oxidation, limit color and texture changes, and slow down the growth of microorganisms in Chinese spiced beef during refrigerated storage. Our study determined the optimal condition. Furthermore, CO₂ inflation can be achieved with commercially available modified atmosphere packaging machine. When CO₂-HPP is industrialized, in addition to safety and nutrient preservation, the shelf-life of products will also increase.     

Inactivation of Staphylococcus aureus by high pressure processing: An overview

Food safety is a major concern of consumers, food industry and governments, with 25 million foodborne diseases occurring annually worldwide. Staphylococcus aureus, is an extremely versatile opportunistic pathogen being responsible for staphylococcal food poisoning due to enterotoxic strains. With increasing demands for safer food, new food preservation technologies are increasingly gaining interest. In the last two decades, high pressure processing (HPP) appeared as an alternative non-thermal food preservation method promoting inactivation of some spoilage and pathogenic microorganisms, while maintaining food characteristics. Factors that modulate its efficiency will be revised, firstly based on the state-of-the art described for bacteria in general and afterwards, when studies exist, for S. aureus specifically. S. aureus inactivation by HPP, like in other microorganisms, is conditioned by cell structures and biomolecules, matrix, HPP processing conditions, the use of antimicrobials and is also dependent of the strain and growth phase. Cell membrane is the most pressure sensitive structure of S. aureus, being the lipids and proteins the most important target molecules. However, monomeric proteins such as staphylococcal enterotoxins (SE) are not affected by HPP, and strains with SE appear to be more efficiently inactivated than those without. Other phenotypic and genotypic characteristics of S. aureus strains, such as pigmentation and the presence of σ^B factor are extremely important factors determining the efficacy of HPP treatments. Inactivation of S. aureus by HPP to ensure food safety still remains a current challenge regarding the understanding of its particular barotolerance and its inactivation kinetics profile that often deviates from the simpler first order decay. Thus, this review provides state-of-the-art information for researchers interested in studying HPP inactivation of S. aureus.Industrial relevanceThis review gives an insight on the importance of Staphylococcus aureus as a foodborne versatile opportunistic pathogen and its importance from the food safety point, its barotolerance and the main reasons for this resistant behavior to high pressure processing and the mechanisms of S. aureus inactivation by HPP.     

Influence of alanine uptake on Staphylococcus aureus surface charge and its susceptibility to two cationic antibacterial agents,nisin and low molecular weight chitosan

Low molecular weight chitosan (LMWC) and nisin, recognized as cationic antibacterial agents (CAAs), inhibit bacterial growth by interacting with the anionically charged cell wall. In this study, alanine uptake significantly reduced the anionic cell surface charge, as determined by the zeta potential (ZP) measurements, of Staphylococcus aureus, resulting from the incorporation of d-alanine into the cell wall. Minimum inhibitory concentration (MIC) tests and growth inhibition curves revealed that LMWC and nisin possessed inverse antibacterial activity against three strains of S. aureus, depending on the strains’ net charge. A twofold reduction in the MIC value of nisin was obtained against S. aureus, inoculated in a 1.0% d- or l-alanine-augmented trypticase soy broth medium. A flocculation test demonstrated that neutralizing the anionic surface charge using d-alanine reduced the adsorption of S. aureus onto LMWC. Furthermore, the reduced surface net charge could enhance the colonization capacity of S. aureus on glass.     

The influence of temperature on the adhesion of mixed cultures of Staphylococcus aureus and Escherichia coli to polypropylene

The adhesion of Staphylococcus aureus and Escherichia coli, in mixed cultures, to polypropylene surfaces was evaluated at 12°C and 30°C. The micro-organisms were isolated from a chicken carcass and cultured in an aqueous extract, prepared from the same carcass, for the production of biofilms on polypropylene coupons. Adhered cells were counted by epifluorescence microscopy with acridine orange staining. Escherichia coli adhered in greater numbers to the coupons than S. aureus at both temperatures. Staphylococcus aureus adhered better at 12°C than at 30°C, while the reverse was true for E. coli. At 30°C, there was no increase in the number of adherent cells ofS. aureus over 8 h, while E. coli increased from a median of 5·0–19·0 per microscope field. At 12°C, the major increase in adherent cell numbers for both species occurred between 2 and 4 h, so that leaving cleaning until 8 h, as is common, would not result in greatly increased biofilms, 2-hourly cleansing is clearly unrealistic. However, total adherent cell numbers were the same at 12° and 30°C between 4 and 6 h incubation. Hence it seems that reduced temperature has little to offer for restricting biofilm formation on polypropylene work surfaces in a well-run food processing plant.     

Effect of PEF on microbial inactivation and physical-chemical properties of green tea extracts

The effects of pulsed electric fields (PEF) on (1) the inactivation of Escherichia coli and Staphylococus aureus in green tea beverage, and (2) the color, green tea polyphenols (GTP) content, and total free amino acids in green tea extracts were investigated. Green tea extract samples inoculated with E. coli and S. aureus were treated using a bench-scale PEF system at electric field strengths of 18.1, 27.4, and 38.4 kV/cm and total treatment times of 40, 80, 120, 160 and 200 μs. The inactivation of E. coli and S. aureus by PEF treatment at 38.4 kV/cm for 160 and 200 μs reached 5.6 and 4.9 log reductions, respectively. PEF processing caused no considerable changes in color, GTP and total free amino acids. The storage tests at 4 °C showed that synergistic effect of low temperature storage and the antimicrobial functionality of GTP resulted in a considerable reduction in the microoganisms of the PEF-treated tea beverage, extending its shelf-life to over 6 months at 4 °C.     

Antimicrobial effect and mode of action of chlorogenic acid on Staphylococcus aureus

Natural products have been developed as an alternative source of novel antibacterials in combating food-borne pathogens such as Staphylococcus aureus (S. aureus), Salmonella, Listeria monocytogenes. The aims of this study were to investigate the antimicrobial effect of chlorogenic acid (CA) on S. aureus and to elucidate its mechanism of action. Minimum inhibitory concentrations of CA were determined using the agar dilution method. Intracellular pH (pH_in), membrane potential, intracellular and extracellular ATP concentrations, and the release of cell constituents were measured to elucidate antibacterial mechanism. Morphological changes induced by CA were examined by electron microscopy. CA induced a decrease in the intracellular ATP concentrations, but no proportional increase in the extracellular ATP. And the release of cell constituents increased significantly when treated with CA. A reduction in pH_in and cell membrane hyperpolarization were observed in S. aureus after CA treatment. Electron microscopic observations showed that the cell membrane of S. aureus was damaged by CA. It is concluded that CA inhibited the proliferation of S. aureus and destroyed the permeability of the cell membrane. The findings indicated that CA has the potential to be developed as an alternative to control S. aureus and the diseases associated with this microorganism.     

Antimicrobial Activity of Thyme (Tymus vulgaris) and Oregano (Origanum vulgare) Essential Oils against Some Food-borne Microorganisms

The aim of this study was to investigate antibacterial effects of oregano and thyme essential oils (EOs) on some food-borne bacteria. GC-MS analysis of EOs was performed in order to determine their composition and phenols were predominant constituents. The investigation of the antibacterial effects of EOs was performed on Salmonella Enteritidis, Salmonella Thyphimurium, Staphylococcus aureus, methicillin resistant Staphylococcus aureus, Escherichia coli and Bacillus cereus, and MICs were determined by broth microdilution method. EOs exhibited antibacterial activity against all tested microorganisms.