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.

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.

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.

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.

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.

Seasonal variations of phenolic compounds and biological properties in sage (Salvia officinalis L.).

The aim was to investigate the phenolic content, antioxidant capacity, and antibacterial activity of Dalmatian sage (Salvia officinalis L.) leaves collected during different vegetation periods. Separation and quantification of the individual phenols were performed by reversed-phase (RP)-HPLC coupled with a PDA (photodiode array) detector and using an internal standard, while the contents of total phenols, flavonoids, flavones, and flavonols were determined spectrophotometrically. The antioxidant properties of the sage leaf extracts were evaluated using five different antioxidant assays (FRAP, DPPH, ABTS, Briggs-Rauscher reaction, and ß-carotene bleaching). The antimicrobial activity of the extracts was tested against two Gram-positive (Bacillus cereus and Staphylococcus aureus) and two Gram-negative (Salmonella Infantis and Escherichia coli) bacterial reference strains. All extracts were extremely rich in phenolic compounds, and provided good antioxidant and antibacterial properties, but the phenophase in which the leaves were collected affected the phenolic composition of the sage extracts and consequently their biological activity. The May Extract, the richest in total flavonoids, showed the best antioxidant properties and the highest antimicrobial activity. Thus, collection of the plants during May seems the best choice for further use of them in the pharmaceutical and food industry.

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.

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.

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.

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.

Attachment, invasion, and translocation of Campylobacter jejuni in pig small-intestinal epithelial cells.

Campylobacters are susceptible to environmental conditions such as starvation, temperature, and oxidative stress. Species such as Campylobacter jejuni have developed a number of mechanisms for responding to these conditions. We conducted a study to investigate whether survival of C. jejuni and pathogen-host cell interactions such as adherence, invasiveness, and intraepithelial survival in pig small-intestinal (PSI) epithelial cells are altered in response to starvation, changes in temperature, and atmospheric oxygen concentration. We assessed the ability of C. jejuni to translocate across polarized intestinal epithelial cell monolayers by measuring transepithelial electrical resistance (TER). Following heat stress, we observed loss of C. jejuni culturability but not viability. Heat-stressed C. jejuni adhered efficiently to pig intestinal epithelial cells, but their invasiveness was significantly impaired when compared with unstressed C. jejuni. Prolonged exposure to atmospheric oxygen reduced the ability of C. jejuni to adhere to intestinal epithelial cells, whereas brief exposure increased invasiveness and intraepithelial survival. By comparison, nutrient limitation reduced adherence, invasiveness, and intracellular survival of C. jejuni. Adherence of C. jejuni strongly affected the pig intestinal epithelium, as reflected by a significant decrease in TER of polarized intestinal epithelial cells. No correlation between TER and the translocation capacity of C. jejuni was observed. Additionally, campylobacters were detected in the basal chamber of a functional small-intestinal epithelial cell model at 3 hours post infection, without a significant reduction in the TER value, suggesting transcellular transport of C. jejuni into the body.

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.

In vitro antimicrobial and antioxidant activity of commercial rosemary extract formulations.

Phenolic plant extracts are sources of natural bioactive compounds, which can inhibit the rate of food spoilage. MIC and MBC concentrations of four oil- or water-soluble rosemary (Rosmarinus officinalis) extracts against gram-positive (Bacillus and Staphylococcus) and gram-negative (Campylobacter and Salmonella) bacteria were determined by using disk diffusion, agar dilution, and broth microdilution methods, as well as bacterial survival kinetics in a macrodilution test. To describe the antioxidant properties of the extracts, the reducing power, free radical scavenging effectiveness, and beta-carotene bleaching test were used. The antimicrobial and antioxidant activity depended on the concentration and chemical nature of the phenolic compounds in the extracts. Gram-positive bacteria were more sensitive than were gram-negative bacteria, especially for oil-soluble extracts with carnosic acid as the major phenolic compound. A microdilution method based on ATP measurement was found to be a useful, rapid technique for determining antibacterial efficiency, and its results correlated well with MICs from survival curve measurement. Reducing power and free radical scavenging effectiveness was higher in water-soluble formulations, according to their higher total phenolic content, but in an aqueous emulsion system of linoleic acid, they exhibited lower antioxidant activity. This correlated well with the higher efficiency of antimicrobial activity of oil-soluble formulations, despite the lower total phenolic content of these extracts.

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.

Insufficient differentiation of live and dead Campylobacter jejuni and Listeria monocytogenes cells by ethidium monoazide (EMA) compromises EMA/real-time PCR.

Recently, ethidium monoazide (EMA) has been proposed as a means of reducing the real-time PCR signal originating from free DNA and dead bacterial cells by selectively entering damaged cells and blocking the DNA for PCR amplification via photoactivation. The present study investigated the effect of EMA on viable and dead bacterial cells using real-time PCR, plate count method and microscopy. The foodborne pathogens Campylobacter jejuni and Listeria monocytogenes were used as a Gram-negative and a Gram-positive model organism, respectively. EMA/real-time PCR analysis of heat-treated cultures of C. jejuni and L. monocytogenes containing 2.6x10(5) and 4x10(5) viable and 3x10(6) and 2x10(6) dead cells/ml, respectively, yielded 2x10(3) and 5.2x10(4) bacterial cell equivalents/ml after EMA treatment, thus underestimating the viable cell count in the samples. Similar results were obtained when analyzing late exponential phase cultures of C. jejuni and L. monocytogenes. Inhibition of growth by EMA was observed. It depended on the concentration of the bacterial cells present in the sample and the EMA concentration used (100-1 microg/ml). An EMA concentration at which dead cells would stain brightly and viable cells would not stain at all or would be very pale was not identified, as revealed by comparison with the results of a commercial live/dead stain. The results suggest that EMA influences not only dead but also viable cells of C. jejuni and L. monocytogenes. Thus EMA/real-time PCR is a poor indicator of cell viability.

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.

Survival and stress induced expression of groEL and rpoD of Campylobacter jejuni from different growth phases.

Although Campylobacter jejuni is the leading cause of bacterial diarrhoeal disease in humans worldwide, its potential to adapt to the stressful conditions and survive in extra-intestinal environment is still poorly understood. We tested the effect of heat shock (55 degrees C, 3 min) and oxidative stress (3 mM H2O2 for 10 min or prolonged incubation at atmosphere oxygen concentration) on non-starved and starved cells of Campylobacter jejuni from different growth phases. Viability as assessed with the Bacterial Viability Kit LIVE/DEAD BacLighttrade mark dying before fluorescent microscopy and culturability of the cells (CFU ml(-1)) from both growth phases showed that starvation increased heat but not oxidative resistance. High temperature and oxidative stress invoked quick transformation from culturable spiral shaped to nonculturable spiral and coccoid cells. Despite physiological changes of the cells we were not able to document clear differences in the expression of heat shock and starvation genes (dnaK, htpG, groEL), oxidative (ahpC, sodB), virulence (flaA) and housekeeping genes (16S rRNA, rpoD) after heat treatment (55 degrees C, 3 min) or oxidative stresses applied. When starving, no induction of expression of any of these genes was noticed, chloramphenicol had no influence on their gene expression. Quantitative real-time PCR analyses showed that at least 10-20 min of heat shock was necessary to evidently increase the amount of groEL and rpoD transcripts.