Estimating the optimal efflux inhibitor concentration of carvacrol as a function of the bacterial physiological state.

Our aim was to find the optimal efflux inhibitor concentration of a natural component, carvacrol, as a function of the physiological state of Escherichia coli. Using fluorescence-based measurements with two strains of E. coli, the effect of carvacrol was assessed at 17 sub-inhibitory concentrations, at which the bacterial efflux mechanism was compromised. The efficacy of carvacrol, as an efflux inhibitor, was compared to synthetic inhibitors and we found carvacrol the most efficient one. We considered the accumulation of Ethidium Bromide (EtBr) as a proxy for drugs spreading in the cell, thus measuring the efflux activity indirectly. The change in membrane integrity caused by the exposure to carvacrol was monitored using the LIVE/DEAD BacLight Bacterial Viability kit. To find the optimal inhibitory concentration of carvacrol, we used predictive microbiology methods. This optimum varied with the bacterial physiological state, as non-growing cultures were less susceptible to the effect of carvacrol than growing cultures were. Moreover, we point out, for the first time, that the efflux-mediated resistance of untreated cultures was also stronger in the non-growing than in the growing phase at population level.

Biomass-Derived Plant Extracts in Macromolecular Chitosan Matrices as a Green Coating for PLA Films.

Due to the growing problem of food and packaging waste, environmental awareness, and customer requirements for food safety, there is a great need for the development of innovative and functional packaging. Among these developments, the concept of active packaging is at the forefront. The shortcoming in this area is that there is still a lack of multifunctional concepts, as well as green approaches. Therefore, this work focuses on the development of active chemical substances of natural origin applied as a coating on polylactic acid (PLA) films. Biopolymer chitosan and plant extracts rich in phenolic compounds (blackberry leaves-Rubus fruticosus, needles of prickly juniper-Juniperus oxycedrus) obtained from plant biomass from Southeastern Europe were selected in this work. In order to increase the effectiveness of individual substances and to introduce multifunctionality, they were combined in the form of different colloidal structural formulations. The plant extracts were embedded in chitosan biopolymer particles and dispersed in a macromolecular chitosan solution. In addition, a two-layer coating, the first of a macromolecular chitosan solution, and the second of a dispersion of the embedded extracts in chitosan particles, was applied to the PLA films as a novel approach. The success of the coatings was monitored by X-ray photoelectron spectroscopy (XPS) and attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR), and the wettability was evaluated by contact angle measurements. Scanning electron microscopy SEM tracked the morphology and homogeneity of the coating. Antioxidation was studied by DPPH and ABTS spectrophotometric tests, and microbiological analysis of the films was performed according to the ISO 22196 Standard. Desorption of the coating from the PLA was monitored by reducing the elemental composition of the films themselves. The successful functionalization of PLA was demonstrated, while the XPS and ATR-FTIR analyses clearly showed the peaks of elemental composition of the extracts and chitosan on the PLA surface. Moreover, in all cases, the contact angle of the bilayer coatings decreased by more than 35-60% and contributed to the anti-fogging properties. The desorption experiments, due to decrease in the concentration of the specific typical element (nitrogen), indicated some migration of substances from the PLA's surface. The newly developed films also exhibited antioxidant properties, with antioxidant ABTS efficiencies ranging from 83.5 to 100% and a quite high inhibition of Gram-positive Staphylococcus aureus bacteria, averaging over 95%. The current functionalization of PLA simultaneously confers antifogging, antioxidant, and antimicrobial properties and drives the development of a biodegradable and environmentally friendly composite material using green chemistry principles.

Campylobacter jejuni Biofilm Control with Lavandin Essential Oils and By-Products.

The food industry is constantly struggling with one of the most prevalent biofilm-forming and food-borne pathogenic bacteria, Campylobacter jejuni. Different approaches are used to control biofilms in the food production chain, but none is fully effective. In this study, we aim to produce and determine the chemical profile of essential oils (EOs), ethanolic extracts of flowers prior to distillation (EFs), and ethanolic extracts of post-distillation waste material (EWMs) from Lavandula × intermedia 'Bila', 'Budrovka' St Nicholas and 'Budrovka', which were further used to reduce C. jejuni intercellular signaling, adhesion, and biofilm formation, as well as to test their antioxidant activity. Glycosides of hydroxycinnamic acids were the major constituents of both types of lavandin ethanolic extract, while linalool, linalyl acetate, 1,8-cineol, and camphor were the major compounds found in lavandin EOs. Tested EOs showed the best antibacterial activity with a minimal inhibitory concentration of 0.25 mg/mL. Lavandin EFs proved more effective in reducing C. jejuni intercellular signaling and adhesion compared to lavandin EOs and EWMs, while lavandin EOs showed a slightly better effect against biofilm formation. Interestingly, the best antioxidant activity was determined for lavandin EWMs. A positive and moderate correlation was found between the reduction of C. jejuni intercellular signaling and adhesion, as well as between adhesion and biofilm formation. These findings mean novel bacterial targets are of interest for biofilm control with alternative natural agents throughout the whole food production chain.

Elucidation of the AI-2 communication system in the food-borne pathogen Campylobacter jejuni by whole-cell-based biosensor quantification.

The food-borne pathogen Campylobacter jejuni produces autoinducer-2 (AI-2) as an interspecies signalling molecule. AI-2 can trigger enhanced colonisation and biofilm formation, and this poses a serious risk to public health. To date, this communication system of C. jejuni is only partially understood, as detection and quantification of such autoinducer signalling molecules in complex media is hard to achieve. We have developed a whole-cell Vibrioharveyi-based biosensor assay to accurately quantify and follow production of AI-2 by C. jejuni 81-176 in a defined growth medium and in a model food system. Several V. harveyi strains were tested, but the most sensitive bioluminescent response to C. jejuni AI-2 was achieved with V. harveyi MM30, likely due to its ability to self-amplify the response to AI-2. The AI-2 concentrations measured by this biosensor were confirmed using an independent analytical method, HPLC-FLD, which we introduced for Campylobacter analytics for the first time. The AI-2 concentration produced by C. jejuni 81-176 in the model food system was ∼5-fold that in the defined growth medium, at the same cell density. Together with the linear increments in AI-2 concentrations with cell density, this suggests that in C. jejuni, AI-2 represents a metabolic by-product rather than a true quorum-sensing molecule. This biosensor method is highly sensitive, as shown by the reduction in the limit of detection (by a factor of 100) compared to HPLC-FLD, and it enables quantification of AI-2 in complex matrices, such as food, which will help to improve the quality and safety of food production.

Determining optimum carvacrol treatment as a cardinal value of a secondary model.

Predictive microbiology methods were used to study the effect of carvacrol on the bacterial resistance to antimicrobials. Our objective was to estimate the optimum dose of carvacrol at concentrations below its MIC value (Minimum Inhibitory Concentration). As a fluorescent marker, ethidium bromide (EtBr) was applied to Escherichia coli to acquire raw data. The accumulation of EtBr was measured by its fluorescence signal (Fs), in the unit of RFU (Relative Fluorescence Unit). The temporal change of the fluorescence values, at a constant concentration of carvacrol, was described by a saturation curve (primary model). The difference, within the observation interval, between the fitted initial and maximum fluorescent values was chosen as the primary parameter to be fitted in the secondary model: a convex, asymmetric, bi-linear function of the carvacrol concentration changing between 0 and 0.5 MIC. Its breakpoint is the optimum value of the carvacrol, a cardinal parameter of the secondary model, where the chosen primary parameter assumes its highest value. This optimum was estimated with high uncertainty for individual experiments, but F-test showed that, with appropriate experimental and numerical procedure, its existence and value can be claimed with confidence. Our results demonstrate that the estimation of the optimum of the secondary model can be robust even if the full secondary model is uncertain.

Adhesion of Campylobacter jejuni Is Increased in Association with Foodborne Bacteria.

The aim of this study was to evaluate Campylobacter jejuni NTCT 11168 adhesion to abiotic and biotic surfaces when grown in co-culture with Escherichia coli ATCC 11229 and/or Listeria monocytogenes 4b. Adhesion of C. jejuni to polystyrene and to Caco-2 cells and Acanthamoeba castellanii was lower for at least 3 log CFU/mL compared to E. coli and L. monocytogenes. Electron micrographs of ultrathin sections revealed interactions of C. jejuni with host cells. In co-culture with E. coli and L. monocytogenes, adhesion of C. jejuni to all tested surfaces was significantly increased for more than 1 log CFU/mL. There was 10% higher aggregation for C. jejuni than for other pathogens, and high co-aggregation of co-cultures of C. jejuni with E. coli and L. monocytogenes. These data show that C. jejuni in co-cultures with E. coli and L. monocytogenes present significantly higher risk than C. jejuni as mono-cultures, which need to be taken into account in risk evaluation. C. jejuni adhesion is a prerequisite for their colonization, biofilm formation, and further contamination of the environment. C. jejuni survival under adverse conditions as a factor in their pathogenicity and depends on their adhesion to different surfaces, not only as individual strains, but also in co-cultures with other bacteria like E. coli and L. monocytogenes.

Effect of Lactobacillus spp. on adhesion, invasion, and translocation of Campylobacter jejuni in chicken and pig small-intestinal epithelial cell lines.

BACKGROUND: Campylobacter spp. are a major cause of bacterial food-borne diarrhoeal disease. This mainly arises through contamination of meat products during processing. For infection, Campylobacter spp. must adhere to epithelial cells of the mucus layer, survive conditions of the gastrointestinal tract, and colonise the intestine of the host. Addition of probiotic bacteria might promote competitive adhesion to epithelial cells, consequently reducing Campylobacter jejuni colonisation. Effect of Lactobacillus spp. (PCS20, PCS22, PCS25, LGG, PCK9) on C. jejuni adhesion, invasion and translocation in pig (PSI cl.1) and chicken (B1OXI) small-intestine cell lines, as well as pig enterocytes (CLAB) was investigated. RESULTS: Overall, in competitive adhesion assays with PSI cl.1 and CLAB cell monolayers, the addition of Lactobacillus spp. reduced C. jejuni adherence to the cell surface, and negatively affected the C. jejuni invasion. Interestingly, Lactobacillus spp. significantly impaired C. jejuni adhesion in three-dimensional functional PSI cl.1 and B1OXI cell models. Also, C. jejuni did not translocate across PSI cl.1 and B1OXI cell monolayers when co-incubated with probiotics. Among selected probiotics, Lactobacillus rhamnosus LGG was the strain that reduced adhesion efficacy of C. jejuni most significantly under co-culture conditions. CONCLUSION: The addition of Lactobacillus spp. to feed additives in livestock nutrition might be an effective novel strategy that targets Campylobacter adhesion to epithelial cells, and thus prevents colonisation, reduces the transmission, and finally lowers the incidence of human campylobacteriosis.

The Anti-Campylobacter Activity and Mechanisms of Pinocembrin Action.

We investigated the anti-Campylobacter activity of pinocembrin and its mechanism of action, as well as Campylobacter responses to pinocembrin treatment at the genetic and phenotypic levels, using C. jejuni NCTC 11168 and a multidrug efflux system repressor mutant (11168ΔcmeR). At its minimal inhibitory concentration, pinocembrin significantly increased cell membrane permeability of Campylobacter. Interestingly, at sub-inhibitory concentrations, pinocembrin did not significantly alter membrane functionality and it increased bacterial fitness. Treatment with pinocembrin evoked decreased expression of ribosomal proteins and down-regulation of several NADH dehydrogenase I chain subunits and proteins involved in iron uptake. This suggests altered protein production and redox cycle and iron metabolism. Interestingly, the chelation of Fe ions during the treatment with pinocembrin increased C. jejuni survival, although there was no increase in the formation of reactive oxygen species. Pre-treatment of C. jejuni with sub-inhibitory concentrations of pinocembrin for 2 h resulted in a 1 log decrease in C. jejuni colony forming units in mice liver at 8 days post-infection, compared to untreated C. jejuni. These findings suggest that pinocembrin modulates the metabolic activity of C. jejuni and that pre-treatment of C. jejuni with pinocembrin influences its virulence potential in mice. This anti-Campylobacter potential of pinocembrin warrants further investigation.

Proteotyping as alternate typing method to differentiate Campylobacter coli clades.

Besides Campylobacter jejuni, Campylobacter coli is the most common bacterial cause of gastroenteritis worldwide. C. coli is subdivided into three clades, which are associated with sample source. Clade 1 isolates are associated with acute diarrhea in humans whereas clade 2 and 3 isolates are more commonly obtained from environmental waters. The phylogenetic classification of an isolate is commonly done using laborious multilocus sequence typing (MLST). The aim of this study was to establish a proteotyping scheme using MALDI-TOF MS to offer an alternative to sequence-based methods. A total of 97 clade-representative C. coli isolates were analyzed by MALDI-TOF-based intact cell mass spectrometry (ICMS) and evaluated to establish a C. coli proteotyping scheme. MLST was used as reference method. Different isoforms of the detectable biomarkers, resulting in biomarker mass shifts, were associated with their amino acid sequences and included into the C. coli proteotyping scheme. In total, we identified 16 biomarkers to differentiate C. coli into the three clades and three additional sub-clades of clade 1. In this study, proteotyping has been successfully adapted to C. coli. The established C. coli clades and sub-clades can be discriminated using this method. Especially the clinically relevant clade 1 isolates can be differentiated clearly.

Quality characteristics of wheat flour dough and bread containing grape pomace flour.

Wheat bread was enriched with 6%, 10% and 15% dried and milled grape pomace flour from two grape cultivars: 'Merlot' and 'Zelen'. Rheological, textural, sensory and antioxidant properties of the enriched dough and bread were evaluated, and compared to control samples. Grape cultivar had significant impact on the rheological characteristics of the dough, and on the sensory and antioxidant properties of the final bread. Development time and dough stability were longer when 'Merlot' grape pomace flour was added compared to 'Zelen' grape pomace flour and the control. Grape pomace flour addition affected bread volume, firmness, crumb and crust colour, and odour and taste intensity. Moreover, grape pomace flour addition resulted in a stickier and less springy crumb texture, and some negative sensorial properties, such as increased intensity of aftertaste and sand feeling in the mouth. The phenolic content and antioxidant activity of bread were positively correlated with grape pomace flour addition ( r = 0.987, p = 0.01 and r = 0.941, p = 0.01 between phenolic content and ferric reducing antioxidant power and phenolic content and 2,2-diphenyl-1-picrylhydrazyl, respectively). The highest total phenolic contents were 5.92 mg gallic acid equivalents (GAE)/g dw for 'Merlot' and 3.65 mg gallic acid equivalents /g dw for 'Zelen', which were seen for the bread prepared with the highest grape pomace flour addition (15%). The highest antioxidant activity determined by the 2,2-diphenyl-1-picrylhydrazyl and ferric reducing antioxidant power assays were seen for the bread prepared with the highest 'Merlot' grape pomace flour addition (15%). Dough characteristic and sensory profile are strongly influenced by cultivar of grape pomace flour. Based on results of sensory profiling, the variety 'Zelen' is suggested for use.

Genetic, Biochemical, Nutritional and Antimicrobial Characteristics of Pomegranate (Punica granatum L.)Grown in Istria.

This study characterises the genetic variability of local pomegranate (Punica granatum L.) germplasm from the Slovenian and Croatian areas of Istria. The bioactive components and antioxidant and antimicrobial properties of ethanol and water extracts of different parts of pomegranate fruit were also determined, along with their preliminary nutritional characterisation. Twenty-six different genotypes identified with microsatellite analysis indicate the great diversity of pomegranate in Istria. The pomegranate fruit ethanol extracts represent rich sources of phenolic compounds (mean value of the mass fraction in exocarp and mesocarp expressed as gallic acid is 23 and 16 mg/g, respectively). The ethanol extracts of pomegranate exocarp and mesocarp showed the greatest antimicrobial activity against Candida albicans, Candida parapsilosis, Rhodotorula mucilaginosa, Exophiala dermatitidis and Staphylococcus aureus, and the same water extracts against S. aureus and Escherichia coli. To the best of our knowledge, this study represents the first report of the characterisation of pomegranate genetic resources from Istria at different levels, including the molecular, chemical, antimicrobial and nutritional properties.

Antibiotic resistance, virulence factors and biofilm formation ability in Escherichia coli strains isolated from chicken meat and wildlife in the Czech Republic.

Attachment of pathogenic bacteria to food contact surfaces and the subsequent biofilm formation represent a serious threat for the food industry, since these bacteria are more resistant to antimicrobials or possess more virulence factors. The main aim of this study was to investigate the correlation between antibiotic resistance against 13 antibiotics, distribution of 10 virulence factors and biofilm formation in 105 Escherichia coli strains according to their origin. The high prevalence of antibiotic resistance that we have found in wildlife isolates could be acquired by horizontal transfer of resistance genes from human or domestic or farm animals. Consequently, these commensal bacteria might serve as indicator of antimicrobial usage for human and veterinary purposes in the Czech Republic. Further, 46 out of 66 resistant isolates (70%) were able to form biofilm and we found out statistically significant correlation between prevalence of antibiotic resistance and biofilm formation ability. The highest prevalence of antibiotic resistance was observed in weak biofilm producers. Biofilm formation was not statistically associated with any virulence determinant. However, we confirmed the correlation between prevalence of virulence factors and host origin. Chicken isolates possessed more virulence factors (66%), than isolates from wildlife (37%). We can conclude that the potential spread of antibiotic resistance pattern via the food chain is of high concern for public health. Even more, alarming is that E. coli isolates remain pathogenic potential with ability to form biofilm and these bacteria may persist during food processing and consequently lead to greater risks of food contamination.

Nutritional, antioxidative, and antimicrobial analysis of the Mediterranean hackberry (Celtis australis L.).

Celtis australis is a deciduous tree commonly known as Mediterranean hackberry or the European nettle tree. The fruit of hackberry are seldom used for nutritional purposes. The nutritional and physicochemical properties of ripe hackberry fruit from Istria (Marasi village near Vrsar, Croatia) were determined, including water, total fiber, protein, vitamin, mineral, and phenolic contents. This analysis demonstrates that the hackberry fruit is a valuable source of dietary fiber, protein, and vitamins, and of pigments such as lutein, ß-carotene, zeaxanthin, and tocopherols. The seasonal differences associated with the different growth stages for the element composition, total phenolic content, and phenolic profile were also determined for hackberry mesocarp and leaves. Water and ethanol extracts were prepared from mesocarp and leaves harvested at different growth stages and their phenolic profiles and antioxidant and antimicrobial activities were investigated. This study demonstrates that water and ethanol extracts of hackberry fruit and leaves collected at different growth stages contain epicatechin, gallic acid, vanillic acid, 3,4-dihydroxybenzaldehyde, delphinidin-3,5-di-O-glucoside, cyanidin-3,5-di-O-glucoside, and pelargonidin-3,5-di-O-glucoside. They also show some antimicrobial and antifungal activities. Further studies are needed to identify and define the active ingredients of these hackberry leaf ethanol extracts.

Effects of natural antimicrobials on bacterial cell hydrophobicity, adhesion, and zeta potential.

Interactions between bacterial cells and contact materials play an important role in food safety and technology. As bacterial strains become ever more resistant to antibiotics, the aim of this study was to analyse adhesion of selected foodborne bacterial strains on polystyrene surface and to evaluate the effects of natural antimicrobials on bacterial cell hydrophobicity, adhesion, and zeta potential as strategies of adhesion prevention. The results showed strain-specific adhesion rate on polystyrene. The lowest and the highest adhesion were found for two B. cereus lines. Natural antimicrobials ferulic and rosmarinic acid substantially decreased adhesion, whereas the effect of epigallocatechin gallate was neglectful. Similar results were found for the zeta potential, indicating that natural antimicrobials reduce bacterial adhesion. Targeting bacterial adhesion using natural extracts we can eliminate potential infection at an early stage. Future experimental studies should focus on situations that are as close to industrial conditions as possible.

Virulence genes and cytokine profile in systemic murine Campylobacter coli infection.

Campylobacter coli are one of the most common bacteria in bacterial gastroenteritis and acute enterocolitis in humans. However, relatively little is known regarding the mechanisms of pathogenesis and host response to C. coli infections. To investigate the influence of genetic changes, we first used PCR to demonstrate the presence of the known virulence genes cadF, virB11, cdtB, cdtC and ceuE in the clinical isolate C. coli 26536, which was isolated from the liver of infected BALB/c mice. Sequence analyses of the cadF, virB11, cdtB and ceuE genes in C. coli 26536 confirmed the stability in these virulence genes during their transmission through the host. We further investigated C. coli infection for the bacterial clearance from the liver and spleen of infected mice, and for their immune response. C. coli persisted well in both organs, with better survival in the liver. We also determined the levels of several pro-inflammatory cytokines (i.e., interleukin [IL]-6, IL-12, interferon-γ, tumor necrosis factor-α) and the anti-inflammatory cytokine IL-10 in plasma and in liver homogenates from the infected mice, using enzyme-linked immunosorbent assays. The lowest levels among these cytokines were for tumor necrosis factor-α in the plasma and IL-6 in the liver on days 1, 3 and 8 post-infection. The most pronounced production was for IL-10, in both plasma (days 1 and 8 post-infection) and liver (day 8 post-infection), which suggests that it has a role in healing of the organ inflammation. Our findings showed dynamic relationships between pro- and anti-inflammatory cytokines and thus contribute toward clarification of the healing processes involved in the resolution of C. coli infections.

Alpinia katsumadai Extracts Inhibit Adhesion and Invasion of Campylobacter jejuni in Animal and Human Foetal Small Intestine Cell Lines.

Alpinia katsumadai is used in traditional Chinese medicine for abdominal distention, pain, and diarrhoea. Campylobacter jejuni is the most common cause of bacterial food-borne diarrhoeal illnesses worldwide. Adhesion to gut epithelium is a prerequisite in its pathogenesis. The antimicrobial, cytotoxic, and anti-adhesive activities of a chemically characterised extract (SEE) and its residual material of hydrodistillation (hdSEE-R) from A. katsumadai seeds were evaluated against C. jejuni. Minimal inhibitory concentrations for SEE and hdSEE-R were 0.5 mg/mL and 0.25 mg/mL, respectively, and there was no cytotoxic influence in the anti-adhesion tests, as these were performed at much lower concentrations of these tested plant extracts. Adhesion of C. jejuni to pig (PSI) and human foetal (H4) small-intestine cell lines was significantly decreased at lower concentrations (0.2 to 50 µg/mL). In the same concentration range, the invasiveness of C. jejuni in PSI cells was reduced by 45% to 65% when they were treated with SEE or hdSEE-R. The hdSEE-R represents a bioactive waste with a high phenolic content and an anti-adhesive activity against C. jejuni and thus has the potential for use in pharmaceutical and food products.

Antibiotic resistance modulation and modes of action of (-)-α-pinene in Campylobacter jejuni.

The aim of the study was to investigate the mode of action of (-)-α-pinene in terms of its modulation of antibiotic resistance in Campylobacter jejuni. Broth microdilution and ethidium bromide accumulation assays were used to evaluate the (-)-α-pinene antimicrobial activity, modulation of antimicrobial resistance, and inhibition of antimicrobial efflux. The target antimicrobial efflux systems were identified using an insertion mutagenesis approach, and C. jejuni adaptation to (-)-α-pinene was evaluated using DNA microarrays. Knock-out mutants of the key up-regulated transcriptional regulators hspR and hrcA were constructed to investigate their roles in C. jejuni adaptation to several stress factors, including osmolytes, and pH, using Biolog phenotypical microarrays. Our data demonstrate that (-)-α-pinene efficiently modulates antibiotic resistance in C. jejuni by decreasing the minimum inhibitory concentrations of ciprofloxacin, erythromycin and triclosan by up to 512-fold. Furthermore, (-)-α-pinene promotes increased expression of cmeABC and another putative antimicrobial efflux gene, Cj1687. The ethidium bromide accumulation was greater in the wild-type strain than in the antimicrobial efflux mutant strains, which indicates that these antimicrobial efflux systems are a target of action of (-)-α-pinene. Additionally, (-)-α-pinene decreases membrane integrity, which suggests that enhanced microbial influx is a secondary mode of action of (-)-α-pinene. Transcriptomic analysis indicated that (-)-α-pinene disrupts multiple metabolic pathways, and particularly those involved in heat-shock responses. Thus, (-)-α-pinene has significant activity in the modulation of antibiotic resistance in C. jejuni, which appears to be mediated by multiple mechanisms that include inhibition of microbial efflux, decreased membrane integrity, and metabolic disruption. These data warrant further studies on (-)-α-pinene to develop its use in the control of antibiotic resistance in Campylobacter.

Stress response and virulence of heat-stressed Campylobacter jejuni.

Thermotolerant Campylobacter spp. frequently cause bacterial gastroenteritis in humans commonly infected through the consumption of undercooked poultry meat. We examined Campylobacter jejuni heat-stress responses in vitro after exposure to 48°C and 55°C. The in vivo modulation of its pathogenicity was also investigated using BALB/c mice intravenously infected with stressed C. jejuni. Regardless of the bacterial growth phase, the culturability and viability of C. jejuni in vitro was reduced after exposure to 55°C. This correlated with the altered protein profile and decreased virulence properties observed in vivo. Heat stress at 48°C elicited the transition to more resistant bacterial forms, independent of morphological changes or the appearance of shorter spiral and coccoid cells. This treatment did not cause marked changes in bacterial virulence properties in vivo. These results indicated that the characteristics and pathogenicity of C. jejuni in response to heat stress are temperature dependent. Further studies on the responses of C. jejuni to stresses used during food processing, as well as the modulation of its virulence, are important for a better understanding of its contamination and infective cycle, and will, thus, contribute to improved safety in the food production chain.

Reduction of microbiological risk in minced meat by a combination of natural antimicrobials.

BACKGROUND: Responsibility for food safety must be taken through the entire food-production chain, to avoid consumer cross-contamination. The antimicrobial activities of an Alpinia katsumadai seed extract and epigallocatechin gallate (EGCG), and their combination, were evaluated against individual food-borne pathogenic strains of Listeria monocytogenes, Escherichia coli and Campylobacter jejuni, individually and as a cocktail, in chicken-meat juice and sterile minced meat as food models, and in minced meat with the naturally present microflora, as an actual food sample. RESULTS: The antimicrobial combination of the A. katsumadai extract and EGCG was the most efficient for C. jejuni growth inhibition, followed by inhibition of L. monocytogenes, which was reduced more efficiently in the bacterial cocktail than as an individual strain. The antimicrobial combination added to minced meat at refrigeration temperatures used in the food chain (8 °C) revealed inhibition of these pathogens and inhibition of the naturally present bacteria after 5 days. CONCLUSIONS: The antibacterial efficiencies of the tested combinations are influenced by storage temperature. Food safety can be improved by using the appropriate combination of natural antimicrobials to reduce the microbiological risk of minced meat.

Phenolic acids profile, antioxidant and antibacterial activity of chamomile, common yarrow and immortelle (Asteraceae).

Chamomile, common yarrow and immortelle ethanolic extracts were chemically analysed with respect to phenolics. Twelve phenolic acids were separated and identified by HPLC-DAD and the presence of rosmarinic acid was additionally confirmed by LC-MS. Five methods were applied for the evaluation of extracts' antioxidant properties (FRAP, DPPH, ABTS, chelating activity, Briggs-Rauscher reaction), while the antibacterial activity was tested against some of the major food-borne pathogens (Campylobacter coli, Escherichia coli, Salmonella Infantis, Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus). Immortelle extract was the richest in phenolics with a dominant flavonoid fraction, while the other two extracts contained higher amount of non-flavonoids. The phenolic acid profile also varied; high concentration of rosmarinic acid was found in chamomile and common yarrow, while caffeic acid was dominant in immortelle. The best antioxidant properties were obtained for chamomile extract, while good antimicrobial activity, especially against Gram-positive bacterial species, was detected for immortelle. The obtained results could be used as a tool for chemotaxonomic classification of the investigated plants or for their potential application as natural antioxidants/antimicrobials.