Rumi and Pasteurized Kareish Cheeses Are a Source of ß-Lactam-Resistant Salmonella in the Nile Delta Region of Egypt: Insights into Their Incidence, AMR Pattern, Genotypic Determinants of Virulence and ß-Lactam Resistance.

The spread of superbugs in dairy products can jeopardize global public health. To date, information on the incidence rates of virulent and ß-lactams-resistant (BLR) Salmonella in cheeses from rural areas of Egypt has been lacking. Biochemical, serological, antibiotic susceptibility, and multiplex PCR (M-PCR) tests were performed to identify and characterize Salmonella isolates. In this study, 44 (15.71%) Salmonella isolates of eight different serotypes were recovered from 280 samples of Rumi and pasteurized Kariesh cheeses across the Nile Delta region of Egypt. The most predominant serotypes were S. Typhimurium, S. Enteritidis, and S. Infantis. The virulence genes (invA, stn, and hilA) were identified in all isolates. However, spvC was only detected in S. Typhimurium. The highest resistance was developed against Erythromycin and Clindamycin (90.91%), followed by Ceftazidime and Cephalothin (84.09%). Meropenem and colistin were the most effective antibiotics. A high proportion (79.55%) of multi-drug resistance (MDR) isolates carried narrow spectrum (NS), extended-spectrum (ES), and AmpC-BLR genes. The blaOXA-1, blaOXA-2, blaTEM-1, blaCTX-M, blaCMY-1, and blaCMY-2 BLR genes were positive in 37.04%, 29.63%, 25.93%, 14.81%, 37.04%, and 3.70% of isolates, respectively. In conclusion, a high prevalence of virulence and BLR genes harboring Salmonella strains in Egyptian cheeses is considered a great threat to public health.

Effect of Two-Step Sous Vide Cooking and Storage on Microbiological and Oxidative Stability of Chicken Breast.

A two-step sous vide method, which included a low temperature initial stage, was shown to improve texture parameters, increase the solubility of proteins, and decrease the cook loss in chicken breasts. The current work was designed to determine the effect of two-step sous vide and subsequent storage on the microbiological and oxidative stability of chicken breasts. Inoculated chicken breasts were vacuum packaged and cooked at two temperatures, 50 °C and 60 °C, combined in different ratios of the same total cooking time (120 min), and then stored for 21 days at 4 °C, 10 °C, and -20 °C, and compared with the one-step temperature treatment (60 °C for 120 min). One-step sous vide treatment resulted in the total inactivation of Enterococcus faecalis NCAIM B. 01312. Meanwhile, the two-step sous vide treatments resulted in a higher than 3 log reduction in Enterococcus faecalis NCAIM B. 01312, reaching the target pasteurization performance criterion of sous vide for poultry meat. Lipid oxidation and the odor of all chicken breasts remained acceptable for 21 days of storage at 4 °C and -20 °C. Conversely, all chicken breasts had higher lipid oxidation rates and odor after 21 days of storage at 10 °C. Two-step-sous-vide-treated chicken breasts were found to be microbiologically stable regarding Enterococcus faecalis NCAIM B. 01312 and total mesophilic aerobic counts during 21 days of storage at 4 °C and -20 °C, in contrast with those stored at 10 °C. It can be concluded that two-step-sous-vide-cooked chicken breasts had acceptable oxidative and microbiological stability during chilled and frozen storage, similar to one-step sous vide ones. These outcomes highlight that two-step heat treatment can be used as an alternative cooking method to improve the quality properties without compromising the storage life of chicken breasts.

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.

Distribution of bacterial single cell parameters and their estimation from turbidity detection times.

The stochastic growth of homogeneous bacterial populations in the wells of a microtiter plate was studied as a function of the random initial cell number and their random individual lag times. These significantly affected the population growth in the well, while the maximum specific growth rate of the population was constant (or its variance was negligible) for each well. We showed the advantages of the mathematical assumption that a transformation of the single cell lag time, called the single cell physiological state (or, more accurately, that of the sub-population generated by the single cell) follow the Beta distribution. Simulations demonstrated what patterns would such assumption generate for the distribution of the detection times observed in the wells. An estimation procedure was developed, based on the beta-assumption, that resulted in an explicit expression for the expected value of the single cell physiological state as a function of measured "time to detection" values using turbidity experiments. The method was illustrated using laboratory data with Escherichia coli, Salmonella enterica subsp. enterica strains. The results gave a basis to quantify the difference between the studied organisms in terms of their single-cell kinetics.

Effect of α-Terpineol on Chicken Meat Quality during Refrigerated Conditions.

The present study was designed to evaluate the in vitro antimicrobial properties of nine bioactive compounds (BACs). Applying the disc paper and minimum inhibitory concentration (MIC) assays, we found that the BACs with the widest spectrum of in vitro antibacterial activity against the studied bacteria were carvacrol and α-terpineol (αTPN). Subsequently, αTPN was selected and applied at different concentrations into the fresh minced chicken meat. The meat was then vacuum packaged and stored for 14 days at 4 °C. Physicochemical properties, lipid oxidation (thiobarbituric acid reactive substances, TBARS), electronic-nose-based smell detection, and microbiological characteristics were monitored. At day 14, meat treated with higher concentrations of αTPN (MIC-2 and MIC-4) exhibited a significantly increased pH and lightness (L*), increased yellowness (b*), decreased redness (a*), caused a significant decrease in water holding capacity (WHC), and decreased lipid oxidation by keeping TBARS scores lower than the control. Although αTPN showed perceptibly of overlapped aroma profiles, the E-nose was able to distinguish the odor accumulation of αTPN between the different meat groups. During the 2-week storage period, αTPN, particularly MIC-4, showed 5.3 log CFU/g reduction in aerobic mesophilic counts, causing total inhibition to the Pseudomonas lundessis, Listeria monocytogenes, and Salmonella Typhimurium. These promising results highlight that αTPN is exploitable to improve the shelf life and enhance the safety of meat and meat products.

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.

Antibiotics in a wastewater matrix at environmentally relevant concentrations affect coexisting resistant/sensitive bacterial cultures with profound impact on advanced oxidation treatment.

Antibiotic resistance containment strategies at wastewater treatment plants need to be supported by a firm knowledge on the behavior of resistant bacteria within a diverse microbial population in the presence of trace amount of antibiotics. In this study via investigating the population dynamics of resistant/sensitive Staphylococcus aureus co-cultures in several model wastewater matrix systems, valuable insights were obtained into the effect of trace amount of antibiotics (piperacillin and erythromycin) on bacteria, and into the suitability of advanced oxidation treatment (electron beam irradiation) as a remediation measure. It appears that environmentally relevant concentration levels of the antibiotic present in a wastewater matrix leads to a shift in the population in favor of the sensitive subtype, presumably on account of triggering protective biochemical processes in the resistant mutant, which confer no selective advantage since the sensitive strain remains unaffected in this concentration range. The impact of these conditions on the population dynamics can be diminished by using advanced oxidation treatment, considering that degradation products from the wastewater matrix constituents (such as humic acid) might also have an effect. Furthermore, it became also apparent that the presence of trace amount of antibiotics while triggers biological processes in the resistant subtype, concomitantly makes the bacteria more sensitive towards the attack of free radicals during advanced oxidation treatment. The behavior of resistant bacteria under environmental conditions at the cellular and population level clearly merits more attention.

Bioactive Compounds of Nigella Sativa Essential Oil as Antibacterial Agents against Chlamydia Trachomatis D.

Urogenital tract infection caused by obligate intracellular bacterium Chlamydia trachomatis D (CtrD) is a leading cause of sexually transmitted diseases. Essential oil (EO) of Nigella sativa has a broad antimicrobial spectrum. The aim of this study was to evaluate the antimicrobial activity of the bioactive compounds (p-cymene, thymoquinone, carvacrol, and thymol) of N. sativa EO against CtrD. The cytotoxic effects of the compounds were determined by MTT assay. In order to quantify the anti-chlamydial activity of the compounds, HeLa cells were infected with CtrD or CtrD treated previously with the compounds. The titer of the infectious CtrD was determined by indirect immunofluorescence assay. The minimum inhibitory concentrations of the compounds were evaluated by direct quantitative PCR. None of the compounds showed a cytotoxic effect on HeLa cells in the concentrations tested. According to the immunofluorescence assay, all of the compounds significantly inhibited the growth of CtrD. The quantitative PCR revealed that the minimum concentration that exerted anti-chlamydial activity was 3.12 µM in the case of thymoquinone and p-cymene, while that of carvacrol and thymol was 6.25 µM. Therefore, it can be concluded that bioactive compounds of N. sativa EO could be used as effective antimicrobial agents against CtrD.

Nigella sativa essential oil and its bioactive compounds as resistance modifiers against Staphylococcus aureus.

Nigella sativa essential oil (EO) and its compounds (thymoquinone, carvacrol, and p-cymene) have a broad antimicrobial spectrum. The aim of this study was to investigate the antimicrobial and resistance modifying activity of N. sativa EO, thymoquinone, carvacrol, and p-cymene against one methicillin susceptible and one methicillin resistant Staphylococcus aureus strain. N. sativa EO, thymoquinone, carvacrol, and p-cymene were assessed for antimicrobial activity and modulation of antimicrobial resistance (by broth microdilution), inhibition of antimicrobial efflux (by ethidium bromide [EtBr] accumulation assay), relative expression of mepA gene (by real-time reverse transcriptase quantitative polymerase chain reaction), membrane disrupting effect (by LIVE/DEAD BacLight™ Kit), and finally antibiofilm activity (by the crystal violet assay). Both strains of S. aureus were susceptible to N. sativa EO, thymoquinone, and carvacrol. N. sativa EO and carvacrol induced the increase of EtBr accumulated by both S. aureus strains. Membrane integrity of ATCC strain was disrupted by carvacrol and p-cymene, whereas for the methicillin resistant S. aureus (MRSA) strain the membrane integrity was disrupted by each compound. N. sativa EO and its bioactive compounds such as carvacrol and p-cymene could be applied as resistance modifiers in MRSA strains.

Antioxidant and Antibacterial Peptides from Soybean Milk through Enzymatic- and Membrane-Based Technologies.

Enzymatic hydrolysis of soybean milk proteins with cysteine protease papain was performed in an advanced bioreactor, operated with batch mode. In soybean milk protein hydrolysis reaction, enzyme and substrate ratio and reaction temperature were varied, ranging from 0.029:100-0.457:100 and 30-60 °C, respectively. The degree of hydrolysis of soybean milk proteins was increased with increase of enzyme and substrate (soybean milk protein) ratio. However, the degree of hydrolysis was increased due to change of reaction temperature from 30 °C to 60 °C with enzyme and substrate ratio 0.229:100 and was reduced when hydrolysis reaction was performed with enzyme and substrate ratio 0.11:100 at hydrolysis temperature 60 °C. Antioxidant capacity of enzyme-treated milk had a similar trend with degree of hydrolysis. In a later exercise, a membrane bioreactor was adopted for continuous production of antioxidant and antibacterial peptides from soybean milk. The membrane bioreactor was operated for 12 h with constant feeding. Ceramic-made tubular membrane with a pore size 20 nm was used. Application of static turbulence promoter in a membrane separation process was investigated and its positive effects, with respect to higher permeate flux and lower energy consumption in filtration process, were proven. Antioxidant capacity and antibacterial activity against Bacillus cereus of enzyme-hydrolyzed milk and permeate from membrane were confirmed.

Antibacterial and Resistance Modifying Activities of Nigella sativa Essential Oil and its Active Compounds Against Listeria monocytogenes.

BACKGROUND/AIM: N. sativa essential oil (EO) and its compounds (thymoquinone, carvacrol and p-cymene) have a broad antimicrobial spectrum. The aim of this study was to investigate the antimicrobial and resistance modifying activity of N. sativa EO, thymoquinone, carvacrol and p-cymene against Listeria monocytogenes. MATERIALS AND METHODS: N. sativa EO, thymoquinone, carvacrol and p-cymene was assessed for its antimicrobial activity, modulation of antimicrobial resistance, inhibition of antimicrobial efflux and membrane integrity by broth microdilution, ethidium bromide accumulation and LIVE/DEAD BacLight™ assays. RESULTS: L. monocytogenes showed substantial susceptibility toward N. sativa EO, thymoquinone, and carvacrol. A significant reduction in MIC's of EtBr and ciprofloxacin was noticed when tested in combination with N. sativa EO, thymoquinone, carvacrol and reserpine. In the presence of each compound the membrane integrity was disintegrated, and the EtBr accumulation increased which was comparable to positive control reserpine. CONCLUSION: N. sativa EO might have a potential for controlling the antibiotic resistance in Listeria.

Deoxynivalenol in wheat, maize, wheat flour and pasta: surveys in Hungary in 2008-2015.

Among Fusarium mycotoxins, deoxynivalenol (DON) is the most common contaminant in case of cereals and cereal-based foods in Hungary. In this study, Hungarian wheat (n = 305), maize (n = 108), wheat flour (n = 179) and pasta (n = 226) samples were analysed (N = 818). The samples were collected during 2008-2015 in Hungary. Applied methods of analysis were enzyme-linked immunosorbent assay and liquid-chromatography coupled with a mass spectrometer. Results were compared and evaluated with Hungarian weather data. Among cereal samples, in 2011, wheat was contaminated with DON (overall average ± standard deviation was 2159 ± 2818 µg kg-1), which was above the maximum limit (ML). In case of wheat flour and pasta, no average values above ML were found during 2008-2015, but higher DON contamination could be observed in 2011 as well (wheat flour: 537 ± 573 µg kg-1; pasta: 511 ± 175 µg kg-1).

Production of Hypoallergenic Antibacterial Peptides from Defatted Soybean Meal in Membrane Bioreactor: A Bioprocess Engineering Study with Comprehensive Product Characterization.

Hypoallergenic antibacterial low-molecular-mass peptides were produced from defatted soybean meal in a membrane bioreactor. In the first step, soybean meal proteins were digested with trypsin in the bioreactor, operated in batch mode. For the tryptic digestion of soybean meal protein, optimum initial soybean meal concentration of 75 g/L, temperature of 40 °C and pH=9.0 were determined. After enzymatic digestion, low-molecular-mass peptides were purified with cross-flow flat sheet membrane (pore size 100 µm) and then with tubular ceramic ultrafiltration membrane (molecular mass cut-off 5 kDa). Effects of transmembrane pressure and the use of a static turbulence promoter to reduce the concentration polarization near the ultrafiltration membrane surface were examined and their positive effects were proven. For the filtration with ultrafiltration membrane, transmembrane pressure of 3·105 Pa with 3-stage discontinuous diafiltration was found optimal. The molecular mass distribution of purified peptides using ultrafiltration membrane was determined by a liquid chromatography-electrospray ionization quadrupole time-of-flight mass spectrometry setup. More than 96% of the peptides (calculated as relative frequency) from the ultrafiltration membrane permeate had the molecular mass M≤1.7 kDa and the highest molecular mass was found to be 3.1 kDa. The decrease of allergenic property due to the tryptic digestion and membrane filtration was determined by an enzyme-linked immunosorbent assay and it was found to exceed 99.9%. It was also found that the peptides purified in the ultrafiltration membrane promoted the growth of Pediococcus acidilactici HA6111-2 and they possessed antibacterial activity against Bacillus cereus.

Deoxynivalenol, zearalenone and T-2 in grain based swine feed in Hungary.

Fusarium genera can produce trichothecenes like deoxynivalenol (DON), zearalenone (ZEN) and T-2 toxin, which can occur in feed cereal grains. Enzyme-linked immunosorbent assays (ELISA) tests of different Hungarian swine feedstuff proved that these mycotoxins were present. In this survey, 45 feed samples from 3 significant Hungarian swine feedstuff manufacturers were tested. ELISA methodology validation showed mean recovery rates in ranges from 85.3% to 98.1%, with intermediate precision of 86.9-96.9% and variation coefficients of 3.4-5.7% and 5.9-7.1%, respectively. The results showed that among Fusarium toxins, generally DON was present in the highest concentration, followed by T-2 and finally ZEN in all tested swine feeds. Each of the mycotoxins was found above the limit of detection in all swine feedstuffs. Boars feed's DON (average ± standard deviation was 872 ± 139 µg kg-1) and ZEN (172 ± 18 µg kg-1) results of one of the manufacturers were above the guidance values. It indicates the necessity for efficient monitoring of DON, ZEN and T-2 mycotoxins in swine feeds.

Fusarium mycotoxins in cereals harvested from Hungarian fields.

The Fusarium mycotoxins deoxynivalenol (DON), zearalenone (ZEN) and T-2 frequently contaminate grain crops in Middle and Eastern Europe. In this survey, 116 cereal samples (maize, wheat, barley and oat) were examined for DON, ZEN and T-2 mycotoxins. Samples were collected from different areas in two Hungarian regions (North and South Transdanubia). The method of analysis was indirect competitive ELISA. Maize was the most contaminated grain regarding DON (86%), ZEN (41%) and T-2 (55%) toxins. The average results of the deoxynivalenol and zearalenone tests of maize proved to be significantly higher than those of barley or oat. DON was the most represented Fusarium mycotoxin followed by T-2 and ZEN. The examination of these mycotoxins would be necessary at a larger scale as to re-evaluate permissible levels, so increase of the monitoring programme would be advisable for the future.

Antilisterial activity of bacteriocinogenic Pediococcus acidilactici HA6111-2 and Lactobacillus plantarum ESB 202 grown under pH and osmotic stress conditions.

Bacteriocin producing lactic acid bacteria (LAB) cultures can be used as biopreservatives in fermented food products; thus the food industry is interested in stable cultures that produce bacteriocins consistently. Inhibtion of Listeria spp. by bacteriocinogenic Pediococcus acidilactici and Lactobacillus plantarum (both isolated from fermented meats) was investigated under conditions of stress induced by low pH and high salt concentrations. Listeria monocytogenes serogroup IIb (from cheese), L. monocytogenes serogroup IVb (from cheese), L. monocytogenes serogroup IIb (from ground beef) and Listeria innocua NCTC 11288 were used as target strains. P. acidilactici and Lb. plantarum demonstrated antilisterial activity under the stress conditions investigated (pH 3.5; pH 8.5; 7.5% NaCl). However, activity was dependent on the stress conditions applied and on the target organism. L. monocytogenes serogroup IIb (from ground beef) and L. innocua C 11288 were, respectively the most sensitive and the most resistant to the cell-free supernatants produced by the LAB investigated.

Carvacrol and p-cymene inactivate Escherichia coli O157:H7 in apple juice.

BACKGROUND: Outbreaks of food poisoning associated with drinking un-pasteurised apple juice contaminated with enterohaemorrhagic Escherichia coli O157:H7 are a cause of serious illness and occasionally death. Whilst a well-established heat process (pasteurisation) will readily eliminate the pathogen, some consumers are demanding more fresh-like foods that have not been subjected to processing methods that are perceived as severe and may lead to loss of flavour and vitamins. Therefore, alternative methods are being investigated to replace pasteurisation and improve the safety of minimally-processed juices. The addition of natural antimicrobial substances such as the phenolic substances carvacrol and p-cymene (derived from the essential oils of herbs and spices) provides a potential new route to assure safety and extend the shelf-life of raw fruit juices. The aim of this study was to evaluate the addition of very low concentrations (0.25-1.25 mM) of carvacrol and p-cymene both individually and in combination as a novel means of controlling Escherichia coli O157:H7 in un-pasteurised apple juice. RESULTS: When inoculated at a level of 4 log CFU/ml into un-pasteurised apple juice (pH 3.20 +/- 0.06), Escherichia coli O157:H7 survived for up to 3 and 19 days at 25 degrees and 4 degrees C, respectively. Treatment of the juice with 1.25 mM carvacrol or p-cymene reduced the numbers of E. coli O157:H7 to undetectable levels within 1-2 days at both storage temperatures. The effective concentrations of carvacrol could be reduced even further by combining it at 0.5 mM with cymene at 0.25 mM. The phenolic compounds were biocidal against both spoilage yeasts and E. coli O157:H7 thereby increasing the shelf-life and improving the safety of un-pasteurised apple juice, particularly when stored at chill temperatures. CONCLUSION: The results showed that the natural antimicrobial compounds carvacrol and p-cymene could potentially be used to extend the shelf life and improve the safety margins in un-pasteurised chilled fruit juices.