Gram-negative Foodborne Pathogens Research Articles

Thymoquinone as a potent antimicrobial agent against Yersinia enterocolitica: Mechanisms of action and potential food safety applications.

Yersinia enterocolitica (Y. enterocolitica) is a Gram-negative foodborne pathogen associated with potentially fatal diseases. Herein, the antibacterial activity and possible mechanism of thymoquinone (TQ) against Y. enterocolitica were explored. The minimum inhibitory concentration and the minimum bactericidal concentration of TQ against Y. enterocolitica were determined to be 0.10 and 0.20mg/mL, respectively. Treatment with TQ increased the lag phase period and reduced the growth rate of Y. enterocolitica. TQ was also effective in preventing Y. enterocolitica contamination of pork. Treatment of Y. enterocolitica with TQ caused a decrease in intracellular ATP, membrane depolarization and an increase in the level of intracellular reactive oxygen species. In addition, TQ caused damage to DNA, a reduction in protein content, loss of membrane integrity and abnormal cell morphology. These findings suggest that TQ exhibits antimicrobial activity against Y. enterocolitica and may be a suitable compound to reduce Y. enterocolitica growth in food.

Development of Alginate Hydrogels Incorporating Essential Oils Loaded in Chitosan Nanoparticles for Biomedical Applications.

A hybrid alginate hydrogel-chitosan nanoparticle system suitable for biomedical applications was prepared. Chitosan (CS) was used as a matrix for the encapsulation of lavender (Lavandula angustifolia) essential oil (LEO) and Mentha (Mentha arvensis) essential oil (MEO). An aqueous solution of an acidic Natural Deep Eutectic Solvent (NADES), namely choline chloride/ascorbic acid in a 2:1 molar ratio, was used to achieve the acidic environment for the dissolution of chitosan and also played the role of the ionic gelator for the preparation of the chitosan nanoparticles (CS-NPs). The hydrodynamic diameter of the CS-MEO NPs was 130.7 nm, and the size of the CS-LEO NPs was 143.4 nm (as determined using Nanoparticle Tracking Analysis). The CS-NPs were incorporated into alginate hydrogels crosslinked with CaCl2. The hydrogels showed significant water retention capacity (>80%) even after the swollen sample was kept in the aqueous HCl solution (pH 1.2) for 4 h, indicating a good stability of the network. The hydrogels were tested (a) for their ability to absorb dietary lipids and (b) for their antimicrobial activity against Gram-positive and Gram-negative foodborne pathogens. The antimicrobial activity of the hybrid hydrogels was comparable to that of the widely used food preservative sodium benzoate 5% w/v.

Development of closed dumbbell mediated isothermal amplification assay for rapid and on-site detection of Vibrio parahaemolyticus

Vibrio parahaemolyticus (V. parahaemolyticus), a gram-negative food-borne pathogen, exists widely in the global marine environment and poses a grave threat to human. Thus, simple, accurate and rapid detection of V. parahaemolyticus is vital for food safety and effective treatment. However, existing methods are difficult to meet the requirements of detection limit, sensitivity and specificity for rapid V. parahaemolyticus diagnosis. The aim of this study was to develop a nucleic acid-based assay by the newly-established closed dumbbell mediated isothermal amplification (CDA) to detect V. parahaemolyticus with high sensitivity and specificity. The suitable inner primers of CDA were designed and screened to specially amplify the conserved target gene of V. parahaemolyticus. Besides, outer primers were designed to accelerate and optimize the CDA reaction process of V. parahaemolyticus gene amplification. And the detection limit of the developed CDA assay for V. parahaemolyticus was 1 copy/μL, which was 10-fold lower than real-time quantitative PCR. Moreover, the CDA assay coupled with Hydroxy naphthol blue (HNB) indicator realized endpoint judgement by naked eye, which would be helpful for onsite diagnosis. In conclusion, the developed real-time fluorescence and visual V. parahaemolyticus CDA assays were suitable for point of care test, holding great potential in food security, environment monitoring and clinical testing.

Potential Use and Chemical Analysis of Some Natural Plant Extracts for Controlling Listeria spp. Growth In Vitro and in Food.

Listeria are Gram-negative intracellular foodborne pathogens that can cause invasive infections with high mortality rates. In this work, the antibacterial activity of ten essential oils, infusion extracts, and decoction extracts of some medicinal plants was tested against Listeria monocytogenes and listeria ivanovii strains. The effects of different physical conditions including temperature, pH, sodium chloride, and some organic acids were studied. The results showed that the water extracts gave the maximum bacterial inhibition, while ethanolic extract was inactive against the tested Listeria spp. The antibiotic sensitivity of L. monocytogenes LMG10470 and L. ivanovii LMZ11352 was tested against five antibiotics including imipenem, levofloxacin, amikacin, ampicillin, and amoxicillin. Imipenem was the most effective antibiotic, resulting in inhibition zones of 40 mm and 31 mm for L. monocytogenes and L. ivanovii, respectively. When imipenem mixed with Syzygium aromaticum oil, Salvia officinalis oil, Pimpinella anisum infusion, and Mentha piperita infusion each, the water extract of Moringa oleifera leaves and seeds against LMG10470 and LMZ11352 resulted in broader antibacterial activity. The antimicrobial activity of both Pimpinella anisum and Mentha piperita plant extracts is related to a variety of bioactive compounds indicated by gas chromatography-mass spectrometry analysis of these two plant extracts. These two plant extracts seemed to contain many chemical compounds elucidated by gas chromatography-mass spectrometry (GC-MS) and infrared radiation spectra. These compounds could be classified into different chemical groups such as ethers, heterocyclic compounds, aromatic aldehydes, condensed heterocyclic compounds, ketones, alicyclic compounds, aromatics, esters, herbicides, saturated fatty acids, and unsaturated fatty acids. The use of these natural compounds seems to be a useful technological adjuvant for the control of Listeria spp. in foods.

Enhancing food preservation and safety: Synergistic effects of Allium-derived organosulfur compounds and outer membrane permeabilization peptide L-11

Ready-to-eat (RTE) foods, characterized by the lack of a terminal heating step prior to consumption, offer enhanced organoleptic qualities compared to their cooked counterparts, as they retain more of their natural flavors, textures, and nutritional content. However, these untreated products pose a risk for foodborne illnesses if not promptly consumed and/or adequately stored under refrigeration or in a frozen state. This investigation delves into the synergistic antimicrobial effects of a novel human-mimetic peptide, L-11, in conjunction with organosulfur compounds derived from Allium species—namely allicin, diallyl sulfide (DAS), dipropyl sulfide (DPS), propyl propane thiosulfinate (PTS), and propyl propane thiosulfonate (PTSO)—on the inhibition and control of Gram-negative foodborne pathogens across various food matrices. Notably, despite the minimal individual antimicrobial activity of peptide L-11 and the evaluated organosulfur compounds against target bacteria (Escherichia coli, Salmonella enterica, Yersinia enterocolitica, and Shigella sonnei), their combinations exhibited remarkable synergistic effects in vitro with until 32-fold reduction for some combinations. Specifically, combinations including PTS and PTSO demonstrated up to a 99% reduction in bacterial proliferation within RTE food models such as salmorejo, aioli, pumpkin cream, and spinach cream. These findings highlight the potential applications of these synergistic combinations in extending the shelf-life and enhancing the safety of RTE foods. To the best of our knowledge, this represents the inaugural report of such synergistic interactions between organosulfur compounds and peptides in extending the shelf-life of RTE foods.

Development and characterisation of agar/silver nanoparticles-based film from Gracilaria canaliculata for food packaging

Gracilaria canaliculata was used as a novel source to extract agar, and for the green synthesis of silver nanoparticles. Silver nanoparticles (0.1 - 0.3%) were incorporated into the agar matrix to develop an environment-friendly food packaging material using the solution casting method. The concentration of nanoparticles affected the physical, thermal, and mechanical properties of the films. Films containing higher concentrations of silver nanoparticles exhibited promising properties, and demonstrated strong antimicrobial activity against both Gram-negative and Gram-positive foodborne pathogens. The film formulation exhibited no cytotoxic effect on HepG2 cells, with cell viability exceeding 94%. These findings suggested that the film possessed favourable characteristics for use as a food packaging material, presenting a potential alternative to plastics.

Antioxidant and antimicrobial activity of extracts from selected Mediterranean agro-food by-products, their mutual interaction and interaction with phenolic compounds

The by-products of Mediterranean agriculture that remain after the production of fruit wines, juices and extracts represent a large source of raw materials rich in phenolic compounds. As a contribution to the current state of knowledge, this study investigated the biological activity and phenolic composition of extracts obtained from blackberry, chokeberry and juniper by-products, as well as the interactions of the extracts in mixtures. The total phenolic content of the samples ranged from 44.42 to 250.28 mg gallic acid equivalents (GAE)/g dry extract, with the extracts from blackberry leaves and juniper needles being the highest. The highest concentration of phenolics detected by high performance liquid chromatography (HPLC) was also found in blackberry leaves (41.30 mg/g dry extract), with rutin dominating. The antioxidant activity of the samples, as measured by ferric reducing antioxidant power (FRAP), oxygen radical absorbance capacity (ORAC) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity, ranged from 3.4 to 26.8 mM Trolox equivalents (TE), 15.8–58.4 mM TE and 80.1%–91.8%, respectively. Among the extract mixtures, the highest synergistic interaction was observed between blackberry leaves and juniper by-products extracts at 1:2 and 2:1 ratios. The antimicrobial activity of the extracts was tested against Gram-positive and Gram-negative foodborne pathogens and spoilage bacteria, with the lowest minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) values against Staphylococcus aureus, Listeria innocua, Bacillus cereus, Campylobacter jejuni, Pseudomonas spp. and Shewanella spp. On the other hand, the antimicrobial activity of blackberry leaf extract in combination with selected phenolic compounds (vanillic acid, catechin, rutin and apigenin) showed no synergy against the tested microorganisms. Overall, the results suggest that the tested by-products extracts could be utilized for the production of natural antioxidants and antimicrobials for further use in the food industry.

Comparative analysis of the application efficiency of gold nanoparticles-based SERS in four foodborne pathogenic microbes

It has been widely reported that SERS can detect foodborne pathogenic bacteria, but SERS has only been limited to the detection of purified single bacteria. However, there’s few single bacterial infections in the natural state were found at the most time, and rare comparative investigations of different Gram-negative and Gram-positive bacteria by SERS exist. At the present study, we are aiming at the comparative analysis of the detection efficiency toward different foodborne pathogenic microbes by a conventional citrate-reduced gold colloid, including Escherichia coli, Salmonella enteritidis, Staphylococcus aureus and Listeria monocytogenes. The SERS spectra of samples were collected with sterile 0.5% NaCl solution as aggregating compound. First, the stability of each bacterial were detected with gold nanoparticles. Afterward, SERS signals of the four bacteria were compared at different concentrations, and it was found that Gram-positive and Gram-negative bacteria have different minimum sensitivity and detection stability. Next, the mixed bacterial SERS detection was applied to classify and identify the similar Raman spectra. Spectral data were analyzed using multivariate data analysis methods, including principal component analysis (PCA) and hierarchical cluster analysis (HCA). The SERS procedure we proposed, which utilized gold nanoparticles adsorbing on bacteria and multivariate analysis, allowed differentiation of species. The results of this pilot study demonstrate the potential of SERS to rapidly identify Gram-negative and Gram-positive foodborne pathogens.

D-tryptophan, an eco-friendly natural, safe, and healthy compound with antimicrobial activity against food-borne pathogens: A systematic review.

Recently, the use of D-amino acids as food preservatives has attracted considerable attention because these natural compounds do not have adverse effects on human health. In addition, D-amino acids such as D-tryptophan can reduce the harmful effects of other treatments. For instance, the use of D-tryptophan in food reduces the requirement for high temperatures and their damaging effects on nutrients such as proteins and vitamins. The purpose of this systematic review was to investigate the antimicrobial effect of D-tryptophan on food-borne pathogens invitro and in food models. To identify related studies, scientific digital databases such as PubMed, Science Direct, and Google Scholar were searched from January 2000 to February 2023. The results of the studies showed that when D-tryptophan was used with other stresses such as using different salt concentrations, refrigeration, or high temperatures, it showed significant antimicrobial effects on Gram-positive and Gram-negative food-borne pathogens, and antibiofilm impacts were also observed with D-tryptophan. Since studies have shown that the antimicrobial activity of D-tryptophan depends on several factors, including the pathogen strain, the type of stress, and the concentration of D-tryptophan, and every article has focused on one of these factors, there is a need for a systematic review that summarizes and concludes the effect of all these factors on the antimicrobial activity of D-tryptophan against food-borne pathogens.

Inhibition of Food-Borne Pathogen Growth and Biogenic Amine Synthesis by Spice Extracts

Food-borne pathogens and their toxins cause significant health problems in humans. Formation of biogenic amines (BAs) produced by microbial decarboxylation of amino acids in food is undesirable because it can induce toxic effects in consumers. Therefore, it is crucial to investigate the effects of natural additives with high bioactivity like spice extracts to inhibit the growth of these bacteria and the formation of BAs in food. In the present study, the antibacterial effects of diethyl ether spice (sumac, cumin, black pepper, and red pepper) extracts at doses of 1% (w/v) on Gram-positive (Staphylococcus aureus and Enterococcus faecalis) and Gram-negative (Klebsiella pneumoniae, Pseudomonas aeruginosa, Campylobacter jejuni, Aeromonas hydrophila, Salmonella Paratyphi A, and Yersinia enterocolitica) food-borne pathogen bacterial strains (FBP) were established. In addition, the accumulation of ammonia (AMN), trimethylamine (TMA), and biogenic amines (BAs) in tyrosine decarboxylase broth (TDB) was investigated by using high performance liquid chromatography (HPLC). Sumac extract exhibited the highest antibacterial potential against all FBPs, followed by cumin and peppers. AMN (570.71 mg/L) and TMA (53.66 mg/L) production were strongly inhibited by sumac extract in the levels of 55.10 mg/L for Y. enterocolitica and 2.76 mg/L for A. hydrophila, respectively. With the exception of S. aureus, black pepper dramatically reduced the synthesis of putrescine, serotonin, dopamine, and agmatine by FBP especially for Gram-negative ones. Furthermore, sumac extracts inhibited histamine and tyramine production by the majority of FBP. This research suggests the application of sumac extracts as natural preservatives for inhibiting the growth of FBPs and limiting the production of AMN, TMA, and BAs.

Effects and molecular mechanism of sugar transporter ESA_RS15745 on desiccation resistance, motility, and biofilm formation of Cronobacter sakazakii.

Cronobacter sakazakii, an important Gram-negative foodborne pathogen, can cause neonatal meningitis and sepsis with high rates of infection and death. Gene ESA_RS15745 encodes a sugar transporter protein, which is not only essential for osmotic pressure maintenance during bacterial growth and reproduction but also associated with their desiccation tolerance, motility, and biofilm formation. Here, a mutant strain of ESA_RS15745 (ΔESA_RS15745) and the complementation strain (cpESA_RS15745) were constructed using a suicide vector knockout and gene complementation. ΔESA_RS15745 was found to have a decrease in its ability to transport maltose and trehalose and resist desiccation, whereas an increase in the ability of motility and biofilm formation, implying that ESA_RS15745 may positively regulate sugar transport and desiccation tolerance and negatively regulate motility and biofilm formation. To further investigate the molecular mechanisms underlying the function of related genes, RNA-seq was performed to explore the differentially expressed genes in the mutants. RNA-seq results showed the upregulation of 114 genes (mainly including those regulating chemotaxis and flagellar motility) and the downregulation of 22 genes (mainly including those regulating sugar transport). qRT-PCR analysis supported the RNA-seq results and showed that ESA_RS15745 may influence the dehydration tolerance though decreasing the intracellular trehalose content and negatively regulate the motility though the chemotactic signaling pathway. In addition, the biofilm formation of C. sakazakii should also be speculated to negatively regulate by ESA_RS15745 by consuming the extracellular carbohydrates concentration and then downregulating the intracellular cyclic diguanosine monophosphate. This study offers a reference for comprehending the molecular mechanism of gene ESA_RS15745 in C. sakazakii.

Antibacterial potential of silver nanoparticles (SP-AgNPs) synthesized from Syzygium polyanthum (Wight) Walp. against selected foodborne pathogens

Foodborne diseases continue to pose a significant global health concern, causing a considerable number of deaths worldwide. In response to concerns surrounding bacterial resistance and the limitations of traditional antibiotics, there is a growing interest in exploring natural antibacterial agents as potential alternatives for addressing foodborne pathogens. Nowadays efforts are being made on exploring the potential of natural antibacterial agents against foodborne pathogens. In this study, the antibacterial efficacy of silver nanoparticles synthesized using S. polyanthum leaves extract (SP-AgNPs) against selected Gram-negative and Gram-positive foodborne pathogens was investigated by using multiple assays, including the well diffusion assay (WDA), minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and time-kill assay. The well diffusion assay demonstrated the inhibitory potential of SP-AgNPs against all tested foodborne pathogens, with inhibition zones ranging from 10.16 + 1.25 to 13.16 + 1.52 mm. Furthermore, the MIC values ranged from 0.008 to 0.125 mg/mL, indicating the potent antibacterial activity of SP-AgNPs across a broad spectrum of foodborne pathogens. The MBC values, ranging from 0.008 to 0.250 mg/mL, further confirming the bactericidal ability of SP-AgNPs against these pathogens. In the time-kill experiment, most foodborne pathogens were entirely killed after 4 h of incubation at 4 × MIC concentration. However, only E. coli, K. pneumoniae, and S. Typhimurium showed a reduction in population to 3 Log10 CFU/mL, indicating a strong bactericidal effect of SP-AgNPs on most tested pathogens. In conclusion, this study provides compelling evidence that SP-AgNPs exhibit potent antibacterial activity against selected foodborne pathogens. The findings suggest that SP-AgNPs synthesized using S. polyanthum leaves extract hold great promise as a novel antibacterial agent for effectively controlling foodborne pathogens. These findings contribute to continuing efforts in developing alternative strategies to prevent foodborne diseases and enhance food safety.

Identification of VP0143 gene which modulates biofilm formation of Vibrio parahaemolyticus

Vibrio parahaemolyticus is a Gram-negative halophilic foodborne pathogen associated with seafood consumption and can cause acute inflammatory gastroenteritis. The biofilm formed by V. parahaemolyticus facilitates its colonization and persistence in the food production chain, posing a continuous threat to food safety. In this study, through transposon mutants screening, we identified a novel gene, VP0143, which positively regulated biofilm formation of V. parahaemolyticus. The VP0143 deletion mutant (ΔVP0143) exhibited less biofilm formation as determined by crystal violet staining assay and showed decreased viability of biofilm cells by XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) assay compared to the wild type strain. The ΔVP0143 strain also showed weakened flagella-mediated motility and surface hydrophobicity. Moreover, VP0143 deletion significantly affected the production of autoinducer-2 and exopolysaccharides in biofilm, and reduced Raman peak intensity of biofilm components. Microscopic analysis confirmed that biofilm formed by ΔVP0143 strain showed loosened structure. In addition, ΔVP0143 strain exhibited compromised ability to form biofilm on silicon wafers, glass and stainless-steel plates, as well as on shrimp and crab surfaces. All these defects were reversed in the complementation strain ΔVP0143C. RT-qPCR further indicated that expression of genes associated with biofilm formation was decreased after VP0143 deletion. Therefore, our data suggest that VP0143 is involved in regulating biofilm formation of V. parahaemolyticus and extend our understanding of genetic determinants of V. parahaemolyticus biofilm formation.

Isolation, antibacterial characterization, and alternating tangential flow–based preparation of viable cells of Lacticaseibacillus paracasei XLK 401: Potential application in milk preservation

It is desirable to obtain high levels of viable Lacticaseibacillus paracasei, a widely used food probiotic whose antibacterial activity and potential application in milk remain largely uninvestigated. Here, we isolated and purified the L. paracasei strain XLK 401 from food-grade blueberry ferments and found that it exhibited strong antibacterial activity against both gram-positive and gram-negative foodborne pathogens, including Staphylococcus aureus, Salmonella paratyphi B, Escherichia coli O157, and Shigella flexneri. Then, we applied alternating tangential flow (ATF) technology to produce viable L. paracasei XLK 401 cells and its cell-free supernatant (CFS). Compared with the conventional fed-batch method, 22 h of ATF-based processing markedly increased the number of viable cells of L. paracasei XLK 401 to 12.14 ± 0.13 log cfu/mL. Additionally, the CFS exhibited good thermal stability and pH tolerance, inhibiting biofilm formation in the abovementioned foodborne pathogens. According to liquid chromatography-mass spectrometry analysis, organic acids were the main antibacterial components of XLK 401 CFS, accounting for its inhibition activity. Moreover, the CFS of L. paracasei XLK 401 effectively inhibited the growth of multidrug-resistant gram-positive Staph. aureus and gram-negative E. coli O157 pathogens in milk, and caused a reduction in the pathogenic cell counts by 6 to 7 log cfu/mL compared with untreated control, thus considerably maintaining the safety of milk samples. For the first time to our knowledge, ATF-based technology was employed to obtain viable L. paracasei on a large scale, and its CFS could serve as a broad-spectrum biopreservative for potential application against foodborne pathogens in milk products.

Grafting bacterial cellulose nanowhiskers into whey protein/essential oil film composites: Effect on structure, essential oil release and antibacterial properties of films

The development of bio-based primary packaging is imperative to improve food safety and quality, along with mitigating the environmental pollution resulting from the increasing production of packaging wastes. Whey protein concentrate (WPC) has received significant attention as an alternative raw material for edible films production, whereas an area of many ongoing researches has been the inclusion of bio-based compounds to formulate functional edible films. This study elaborated the development of WPC edible films fortified with different essential oils (EO) to assess antimicrobial activity against gram-positive (Staphylococcus aureus and Listeria monocytogenes) and gram-negative (Salmonella enteritidis and Pseudomonas aeruginosa) food-borne pathogens. Subsequently, the effect of grafting bacterial cellulose nanowhiskers (BCNW) in film composites was evaluated for WPC/oregano EO (WPC/OEO) films, which showed the highest antibacterial activity. Notably, increasing the concentration of grafted BCNW entailed a proportional increase on the inhibitory activity. In specific, antibacterial activity increased up to 80% when 10% BCNW was grafted in WPC/OEO films. Inclusion of BCNW and homogenous distribution of OEO into the film blend was pointed out through Raman analysis. In addition, AFM and SEM analysis revealed that inclusion of BCNW and OEO into the film matrix led to the formation of rough films with pores. This structural change enhanced the release capacity of OEO, that was further confirmed through a release kinetic experiment. This study indicated that BCNW addition positively affected whey protein films properties, thereby envisaging the potential development of sustainable food packaging materials with tailor-made properties.

Inhibitory effect of resveratrol on swimming motility and adhesion ability against Salmonella enterica serovar Typhimurium infection

Salmonella enterica serovar Typhimurium (S. typhimurium) is a common Gram-negative foodborne pathogen that threatens public health and hinders the development of livestock industry. Resveratrol, an important component in grape fruits and seeds, has been shown to possess multiple biological activities, but its potential effects on S. typhimurium-mediated virulence have been rarely reported. In this study, we investigated the effect of resveratrol on S. typhimurium flagella -mediated virulence. The results showed that resveratrol significantly reduced the transcription of flagella genes and swimming motility of S. typhimurium, and also inhibited the transcription of T3SS-related virulence genes with varying degrees inhibiting bacterial growth. Simultaneously, resveratrol significantly reduced the adhesion of S. typhimurium to HeLa cells. Unfortunately, resveratrol does not improve the survival rate of S. typhimurium-infected mice, but it reduces the bacterial load in the liver and spleen of infected mice, and it also has a certain degree of anti-inflammatory activity. In summary, these results indicated that resveratrol has the potential to be developed as an alternative drug or antibacterial agent to prevent Salmonella infection.

Inactivation of Salmonella enteritidis on the surface of eggs by air activated with gliding arc discharge plasma

Salmonella enteritidis, a Gram-negative foodborne pathogen, causes inflammation of the stomach and intestines. Poultry and poultry products are considered to be the main hosts of S. enteritidis, especially the most common edible eggs, and increase the risk of Salmonella infection. Therefore, developing the inactivation strategy of S. enteritidis on the surface of eggs is the key step during the egg production process. Gas plasma could effectively inactivate bacteria and fungi and is developed as a potential non-thermal sterilization method in the food industry. In this study, the plasma-activated air (PAA) prepared by the air activated with gliding arc discharge contained a variety of reactive species. When the PAA activated at the power of 50 W and 100 W was inducted into the incubation chamber, the numbers of S. enteritidis on egg fragments or the whole eggs were reduced below the detection limit. After PAA treatment, the eggshells exhibited no change in structure demonstrated by scanning electron microscope, the color of eggshells became a little decrease in red, the internal quality of both the raw egg and the cooked egg also exhibited no change, and trace reactive species remained in the albumen. Further, the PAA was also feasible for the batch treatment of stacked eggs. Therefore, this study supplied an effective and safe strategy for aseptic processing in the poultry industry.

Antimicrobial Activities and Biopreservation Potential of Lactic Acid Bacteria (LAB) from Raw Buffalo (Bubalus bubalis) Milk.

The aim of this study was to investigate the antimicrobial and biopreservation potential of lactic acid bacteria. The potential probiotic culture inhibited the growth of gram-positive and gram-negative foodborne pathogens in agar spot assay with inhibition zones ranging from 10 to 21 mm in diameter. The strains showed coaggregation capabilities ranging from 7 to 71% with tested food pathogens including Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, and Salmonella enterica subsp. enterica serovar Typhimurium. The effect of cell-free supernatants on the release of 260 nm absorbing material, especially nucleic acids, was evaluated and indicated the antagonistic activity on foodborne pathogens, the highest being Lactobacillus paraplantarum against E. coli (3.77) and S. aureus (3.86) after 60 min. The effect of cell-free supernatant (CFS) on the growth of pathogens showed that Lactobacillus paraplantarum 11 and L. pentosus 93 had the highest inhibitory activity against tested strains. The biopreservation assay indicated that the potential probiotic strains Lactobacillus paraplantarum 11 (BT), Lactiplantibacillus plantarum 19, Lactobacillus pentosus 42, Limosilactobacillus fermentum 60, Lactobacillus pentosus 93, and Limosilactobacillus reuteri 112 were effective in reducing the Listeria monocytogenes population in raw buffalo milk. Complete Listeria monocytogenes inhibition was observed after 6-8 days. This study showed that probiotic LAB from buffalo milk have antimicrobial and biopreservation potential; these strains have the potential to be utilized as biopreservative agents in food products.

Diarrheagenic Escherichia coli and Their Antibiotic Resistance Patterns in Dairy Farms and Their Microbial Ecosystems

Ruminants are the largest reservoir for all types of Escherichia coli, including the pathogenic ones, which can potentially be transmitted to humans via the food chain and environment. A longitudinal study was performed to estimate the prevalence and antibiotic-resistant pattern of pathogenic E. coli (pE.coli) strains in dairy farm environments. A total of 846 environmental samples (water, lagoon slurry, bedding, feed, feces, soil, and compost) were collected in summer over two years from five dairy farms in Maryland, USA. An additional 40 soil samples were collected in winter and summer seasons for evaluating microbiome composition. Collected environmental samples were screened for the presence of pE.coli, which was isolated using a selective culture medium, for later confirmation and virotyping using PCR with specific primers. The overall prevalence of pE.coli in dairy farms was 8.93% (71/846), with the most common virotype identified in isolates being ETEC, followed by STEC. The highest pE.coli prevalence were recorded in lagoon slurry (21.57%) while the lowest was in compost heap (2.99%). Among isolates, 95.87% of the virotypes were resistant to 9 classes of antibiotics whereas only 4.12% were sensitive. The highest proportion (68.04%) of resistance was found for quinolones (e.g., ciprofloxacin). The resulting metagenomic analysis at the phylum and genus levels of the grazing land soil suggests that climatic conditions actively influence the abundance of bacteria. Proteobacteria, which contains many Gram-negative foodborne pathogens (including pE.coli), was the most predominant phylum, accounting for 26.70% and 24.93% of soil bacteria in summer and winter, respectively. In addition to relative abundance, there was no significant difference in species diversity between seasons when calculated via Simpson (D) and Shannon (H) index. This study suggests that antibiotic-resistant E. coli virotypes are present in the dairy farm environment, and proper steps are warranted to control its transmission irrespective of seasonality.

A novel bacteriocin against multiple foodborne pathogens from Lacticaseibacillus rhamnosus isolated from juice ferments: ATF perfusion-based preparation of viable cells, characterization, antibacterial and antibiofilm activity

Foodborne pathogens and their biofilms pose a risk to human health through food chain. However, the bacteriocin resources combating this threat are still limited. Here, Lacticaseibacillus rhamnosus, one of the most used probiotics in food industry, was prepared on a large scale using alternating tangential flow (ATF) perfusion-based technology. Compared to the conventional fed-batch approach, ATF perfusion remarkably increased the viable cells of L. rhamnosus CLK 101 to 11.93 ± 0.14 log CFU/mL. Based on obtained viable cells, we purified and characterized a novel bacteriocin CLK_01 with a broad spectrum of activity against both Gram-positive and Gram-negative foodborne pathogens. LC-MS/MS analysis revealed that CLK_01 has a molecular mass of 701.49 Da and a hydrophobic amino acid composition of I–K–K–V-T-I. As a novel bacteriocin, CLK_01 showed high thermal stability and acid-base tolerance over 25–121 °C and pH 2–10. It significantly reduced cell viability of bacterial pathogens (p < 0.001), and strongly inhibited their biofilm formation. Scanning electron microscopy demonstrated deformation of pathogenic cells caused by CLK_01, leading to cytoplasmic content leakage and bacterial death. Summarily, we employed ATF perfusion to obtain viable L. rhamnosus, and presented that bacteriocin CLK_01 could serve as a promising biopreservative for controlling foodborne pathogenic bacteria and their biofilms.