Antimicrobial resistance and prevalence of Listeria species from raw milk and dairy products in Burdur, Turkey.

OBJECTIVES: Worldwide, but especially in emerging nations, concerns about food safety pose a serious obstacle to societal and economic progress. This research aimed to examine the prevalence of Listeria spp. in raw milk and dairy products in Burdur, as well as the presence of genes associated with biofilm formation and antibiotic resistance in the isolates. METHODS: A total of 185 samples, including raw milk, curd, cream, butter, yogurt and cheese, were randomly collected in Burdur. The enrichment and isolation methods specified by the United States Department of Agriculture was used to identify Listeria species in milk and dairy product samples. Culture-positive strains were identified as Listeria genus and as species by PCR. Antibiotic susceptibility of the isolates was evaluated against 14 antibiotics using the disc diffusion technique (EUCAST). RESULTS: Of them, 2.2% (4/185) were positive for Listeria spp. Listeria species were isolated from cheese and yogurt samples. Two of them were Listeria innocua 1.1% (2/185), one was Listeria ivanovii 0.5% (1/185) and the other was Listeria welshimeri 0.5% (1/185). As a result of multiplex PCR of the biofilm genotypic marker luxS and flaA genes, the flaA gene was detected in three of four isolates, the luxS gene was detected in one isolate, and these two genes were not found in one isolate. Although all isolates were resistant to gentamicin and rifampicin, they also showed multidrug resistance. CONCLUSION: This study revealed that the diversity of prevalence of Listeria spp. in Burdur requires microbial risk assessment in the milk and dairy products value chain and the need to focus on the problem of multiple antibiotic resistance.

Antimicrobial Resistance Profiles of Listeria Species Recovered from Retail Outlets in Gauteng Province, South Africa.

The study determined the antimicrobial resistance (AMR) profiles of Listeria spp. (L. monocytogenes, L. innocua, and L. welshimeri) recovered from beef and beef products sold at retail outlets in Gauteng Province, South Africa. A total of 112 isolates of Listeria spp., including L. monocytogenes (37), L. innocua (65), and L. welshimeri (10), were recovered from beef and beef products collected from 48 retail outlets. Listeria spp. was recovered by direct selective plating following selective enrichment, and PCR was used to confirm and characterize recovered isolates. The disc diffusion method determined the resistance to 16 antimicrobial agents. All 112 isolates of Listeria spp. exhibited resistance to one or more antibiotics (P < 0.05). The prevalence of AMR in Listeria isolates was high for nalidixic acid (99.1%) and cefotaxime (80.4%) but low for gentamycin (2.7%), sulfamethoxazole-trimethoprim (3.6%), azithromycin (5.4%), and doxycycline (6.3%). Overall, for the three species of Listeria, the prevalence of resistance varied significantly only for streptomycin (P = 0.016) and tetracycline (P = 0.034). Multidrug-resistant isolates were detected in 75.7% (28/37), 61.5% (40/65), and 80% (8/10) isolates of L. monocytogenes, L. innocua, and L. welshimeri, respectively. The prevalence of AMR was significantly affected by the location and size of retail outlets, type of beef and beef products, and serogroups of L. monocytogenes. The high prevalence of AMR, particularly among the L. monocytogenes isolates, poses potential therapeutic implications for human consumers of contaminated beef products. There is, therefore, a need to regulate and enforce the use of antimicrobial agents in humans and animals in South Africa.

Antioxidant and antimicrobial extracts from grape stalks obtained with subcritical water. Potential use in active food packaging development.

In order to valorise winemaking grape stalks, subcritical water extraction at 160 and 180 °C has been carried out to obtain phenolic-rich extracts useful for developing active food packaging materials. Red (R) and white (W) varieties (from Requena, Spain) were used, and thus, four kinds of extracts were obtained. These were characterised as to their composition, thermal stability and antioxidant and antibacterial activity. The extracts were incorporated at 6 wt% into polylactic acid (PLA) films and their effect on the optical and barrier properties of the films and their protective effect against sunflower oil oxidation was analysed. Carbohydrates were the major compounds (25-38%) in the extracts that contained 3.5-6.6% of phenolic compounds, the R extracts being the richest, with higher radical scavenging capacity. Every extract exhibited antibacterial effect against Escherichia coli and Listeria innocua, while PLA films with extracts preserved sunflower oil against oxidation.

Coating with phenolic branched-chain fatty acid reduces Listeria innocua populations on apple fruit.

An antimicrobial coating was produced by mixing phenolic branched-chain fatty acid (PBC-FA) with glycerol and a carboxymethyl cellulose solution (CMC) at pH 7. The resulting PBC-FA-CMC solution formed an emulsion with an average droplet size of 77 nm. The emulsion in the coating solution was stable for at least 30 days at 20 °C. The in vitro antimicrobial activity of the film formed from the PBC-FA emulsion was tested against a mixture of 3 strains of Listeria innocua (7 log CFU/mL). Film with a concentration of 1000 µg/mL of PBC-FA effectively reduced the population of L. innocua below the limit of detection (<1.48 log CFU/mL) in vitro. The effect of the 1000 µg/mL PBC-FA-CMC coating formulation was then evaluated against L. innocua inoculated on "Gala" apples. Results showed that compared with the non-coated control, the coating reduced L. innocua populations by ~2 log CFU/fruit and ~6 log CFU/fruit on the apple when enumerated on tryptic soy agar and selective media (PALCAM), respectively, indicating that PBC-FA applied as a coating on apples resulted in the sub-lethal injury of bacterial cells. When L. innocua was inoculated onto PBC-FA-coated apples, the L. innocua population decreased by ~4 log CFU/fruit during 14 days of shelf-life at 20 °C. The PBC-FA coating lowered the moisture loss but did not affect the color, firmness, or soluble solids content of apples during the 14-day at 20 °C. Overall, this study revealed that there is a potential that PBC-FA can be used as an antimicrobial coating to inactivate Listeria and preserve the quality of apples.

Phenotypic and genotypic antimicrobial resistance of Listeria spp. in Spain.

Listeriosis is a zoonotic disease caused by Listeria monocytogenes and Listeria ivanovii. The genus Listeria currently includes 27 recognized species and is found throughout the environment. The number of systematic studies on antimicrobial resistance in L. monocytogenes isolates from domestic farms using antimicrobial substances is limited. Importantly, dairy ruminant farms are reservoir of hypervirulent lineage I L. monocytogenes isolates, previously associated with human clinical cases. Considering that the classes of antibiotics used in food-producing domestic animals are frequently the same or closely related to those used in human medicine, studies about the impact of antibiotic use on the acquisition of antibiotic resistance in Listeria spp. in domestic animal farms are, therefore, of high importance. Here, susceptibility to 25 antibiotics was determined. Eighty-one animal-related, 35 food and 21 human pathogenic Listeria spp. isolates and 114 animal-related non-pathogenic Listeria spp. isolates were tested. Whole genome sequencing data was used for molecular characterization. Regarding L. monocytogenes, 2 strains from the clinical-associated linage I showed resistance to erythromycin, both related to dairy ruminants. Acquired resistance to one antibiotic was exhibited in 1.5% of L. monocytogenes isolates compared with 14% of non-pathogenic Listeria spp. isolates. Resistance to tetracycline (7.9%), doxycycline (7.9%), penicillin (4.4%), and ampicillin (4.4%) were the most frequently observed in non-pathogenic Listeria spp. While resistance to two or more antibiotics (5.6%) was most common in Listeria spp., isolates, resistance to one antibiotic was also observed (1.6%). The present results show that non-pathogenic Listeria spp. harbour antimicrobial resistance genes.

Angicin, a novel bacteriocin of Streptococcus anginosus.

As a conserved defense mechanism, many bacteria produce antimicrobial peptides, called bacteriocins, which provide a colonization advantage in a multispecies environment. Here the first bacteriocin of Streptococcus anginosus, designated Angicin, is described. S. anginosus is commonly described as a commensal, however it also possesses a high pathogenic potential. Therefore, understanding factors contributing to its host colonization and persistence are important. A radial diffusion assay was used to identify S. anginosus BSU 1211 as a potent bacteriocin producer. By genetic mutagenesis the background of bacteriocin production and the bacteriocin gene itself were identified. Synthetic Angicin shows high activity against closely related streptococci, listeria and vancomycin resistant enterococci. It has a fast mechanism of action and causes a membrane disruption in target cells. Angicin, present in cell free supernatant, is insensitive to changes in temperature from - 70 to 90 °C and pH values from 2 to 10, suggesting that it represents an interesting compound for potential applications in food preservation or clinical settings.

Antimicrobial susceptibility, multilocus sequence typing, and virulence of listeria isolated from a slaughterhouse in Jiangsu, China.

BACKGROUND: Listeria monocytogenes is one of the deadliest foodborne pathogens. The bacterium can tolerate severe environments through biofilm formation and antimicrobial resistance. This study aimed to investigate the antimicrobial susceptibility, resistance genes, virulence, and molecular epidemiology about Listeria from meat processing environments. METHODS: This study evaluated the antibiotic resistance and virulence of Listeria isolates from slaughtering and processing plants. All isolates were subjected to antimicrobial susceptibility testing using a standard microbroth dilution method. The harboring of resistant genes was identified by polymerase chain reaction. The multilocus sequence typing was used to determine the subtyping of the isolates and characterize possible routes of contamination from meat processing environments. The virulence of different STs of L. monocytogenes isolates was evaluated using a Caco-2 cell invasion assay. RESULTS: A total of 59 Listeria isolates were identified from 320 samples, including 37 L. monocytogenes isolates (62.71%). This study evaluated the virulence of L. monocytogenes and the antibiotic resistance of Listeria isolates from slaughtering and processing plants. The susceptibility of these 59 isolates against 8 antibiotics was analyzed, and the resistance levels to ceftazidime, ciprofloxacin, and lincomycin were as high as 98.31% (L. m 37; L. innocua 7; L. welshimeri 14), 96.61% (L. m 36; L. innocua 7; L. welshimeri 14), and 93.22% (L. m 35; L. innocua 7; L. welshimeri 13), respectively. More than 90% of the isolates were resistant to three to six antibiotics, indicating that Listeria isolated from meat processing environments had high antimicrobial resistance. Up to 60% of the isolates harbored the tetracycline-resistance genes tetA and tetM. The frequency of ermA, ermB, ermC, and aac(6')-Ib was 16.95, 13.56, 15.25, and 6.78%, respectively. Notably, the resistant phenotype and genotype did not match exactly, suggesting that the mechanisms of antibiotic resistance of these isolates were likely related to the processing environment. Multilocus sequence typing (MLST) revealed that 59 Listeria isolates were grouped into 10 sequence types (STs). The dominant L. monocytogenes STs were ST5, ST9, and ST121 in the slaughtering and processing plant of Jiangsu province. Moreover, ST5 subtypes exhibited high invasion in Caco-2 cells compared with ST9 and ST121 cells. CONCLUSION: The dominant L. monocytogenes ST5 persisted in the slaughtering and processing plant and had high antimicrobial resistance and invasion characteristics, illustrating a potential risk in food safety and human health.

Enzyme- and Relative Humidity-Responsive Antimicrobial Fibers for Active Food Packaging.

Active food packaging materials that are sustainable, biodegradable, and capable of precise delivery of antimicrobial active ingredients (AIs) are in high demand. Here, we report the development of novel enzyme- and relative humidity (RH)-responsive antimicrobial fibers with an average diameter of 225 ± 50 nm, which can be deposited as a functional layer for packaging materials. Cellulose nanocrystals (CNCs), zein (protein), and starch were electrospun to form multistimuli-responsive fibers that incorporated a cocktail of both free nature-derived antimicrobials such as thyme oil, citric acid, and nisin and cyclodextrin-inclusion complexes (CD-ICs) of thyme oil, sorbic acid, and nisin. The multistimuli-responsive fibers were designed to release the free AIs and CD-ICs of AIs in response to enzyme and RH triggers, respectively. Enzyme-responsive release of free AIs is achieved due to the degradation of selected polymers, forming the backbone of the fibers. For instance, protease enzyme can degrade zein polymer, further accelerating the release of AIs from the fibers. Similarly, RH-responsive release is obtained due to the unique chemical nature of CD-ICs, enabling the release of AIs from the cavity at high RH. The successful synthesis of CD-ICs of AIs and incorporation of antimicrobials in the structure of the multistimuli-responsive fibers were confirmed by X-ray diffraction and Fourier transform infrared spectrometry. Fibers were capable of releasing free AIs when triggered by microorganism-exudated enzymes in a dose-dependent manner and releasing CD-IC form of AIs in response to high relative humidity (95% RH). With 24 h of exposure, stimuli-responsive fibers significantly reduced the populations of foodborne pathogenic bacterial surrogates Escherichia coli (by ∼5 log unit) and Listeria innocua (by ∼5 log unit), as well as fungi Aspergillus fumigatus (by >1 log unit). More importantly, the fibers released more AIs at 95% RH than at 50% RH, which resulted in a higher population reduction of E. coli at 95% RH. Such biodegradable, nontoxic, and multistimuli-responsive antimicrobial fibers have great potential for broad applications as active and smart packaging systems.

Ubericin K, a New Pore-Forming Bacteriocin Targeting mannose-PTS.

Bovine mastitis infection in dairy cattle is a significant economic burden for the dairy industry globally. To reduce the use of antibiotics in treatment of clinical mastitis, new alternative treatment options are needed. Antimicrobial peptides from bacteria, also known as bacteriocins, are potential alternatives for combating mastitis pathogens. In search of novel bacteriocins against mastitis pathogens, we screened samples of Norwegian bovine raw milk and found a Streptococcus uberis strain with potent antimicrobial activity toward Enterococcus, Streptococcus, Listeria, and Lactococcus. Whole-genome sequencing of the strain revealed a multibacteriocin gene cluster encoding one class IIb bacteriocin, two class IId bacteriocins, in addition to a three-component regulatory system and a dedicated ABC transporter. Isolation and purification of the antimicrobial activity from culture supernatants resulted in the detection of a 6.3-kDa mass peak by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry, a mass corresponding to the predicted size of one of the class IId bacteriocins. The identification of this bacteriocin, called ubericin K, was further confirmed by in vitro protein synthesis, which showed the same inhibitory spectrum as the purified antimicrobial compound. Ubericin K shows highest sequence similarity to the class IId bacteriocins bovicin 255, lactococcin A, and garvieacin Q. We found that ubericin K uses the sugar transporter mannose phosphotransferase (PTS) as a target receptor. Further, by using the pHlourin sensor system to detect intracellular pH changes due to leakage across the membrane, ubericin K was shown to be a pore former, killing target cells by membrane disruption. IMPORTANCE Bacterial infections in dairy cows are a major burden to farmers worldwide because infected cows require expensive treatments and produce less milk. Today, infected cows are treated with antibiotics, a practice that is becoming less effective due to antibiotic resistance. Compounds other than antibiotics also exist that kill bacteria causing infections in cows; these compounds, known as bacteriocins, are natural products produced by other bacteria in the environment. In this work, we discover a new bacteriocin that we call ubericin K, which kills several species of bacteria known to cause infections in dairy cows. We also use in vitro synthesis as a novel method for rapidly characterizing bacteriocins directly from genomic data, which could be useful for other researchers. We believe that ubericin K and the methods described in this work will aid in the transition away from antibiotics in the dairy industry.

Photoactivated Carbon Dots for Inactivation of Foodborne Pathogens Listeria and Salmonella.

Foodborne pathogens have long been recognized as major challenges for the food industry and repeatedly implicated in food product recalls and outbreaks of foodborne diseases. This study demonstrated the application of a recently discovered class of visible-light-activated carbon-based nanoparticles, namely, carbon dots (CDots), for photodynamic inactivation of foodborne pathogens. The results demonstrated that CDots were highly effective in the photoinactivation of Listeria monocytogenes in suspensions and on stainless steel surfaces. However, it was much less effective for Salmonella cells, but treatments with higher CDot concentrations and longer times were still able to inactivate Salmonella cells. The mechanistic implications of the observed different antibacterial effects on the two types of cells were assessed, and the associated generation of intracellular reactive oxygen species (ROS), the resulting lipid peroxidation, and the leakage of nucleic acid and proteins from the treated cells were analyzed, with the results collectively suggesting CDots as a class of promising photodynamic inactivation agents for foodborne pathogens. IMPORTANCE Foodborne infectious diseases have long been recognized as major challenges in public health. Contaminations of food processing facilities and equipment with foodborne pathogens occur often. There is a critical need for new tools/approaches to control the pathogens and prevent such contaminations in food processing facilities and other settings. This study reports a newly established antimicrobial nanomaterials platform, CDots coupled with visible/natural light, for effective and efficient inactivation of representative foodborne bacterial pathogens. The study will contribute to promoting the practical application of CDots as a new class of promising nanomaterial-based photodynamic inactivation agents for foodborne pathogens.

Proteomic Characterization of Antibiotic Resistance in Listeria and Production of Antimicrobial and Virulence Factors.

Some Listeria species are important human and animal pathogens that can be found in contaminated food and produce a variety of virulence factors involved in their pathogenicity. Listeria strains exhibiting multidrug resistance are known to be progressively increasing and that is why continuous monitoring is needed. Effective therapy against pathogenic Listeria requires identification of the bacterial strain involved, as well as determining its virulence factors, such as antibiotic resistance and sensitivity. The present study describes the use of liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) to do a global shotgun proteomics characterization for pathogenic Listeria species. This method allowed the identification of a total of 2990 non-redundant peptides, representing 2727 proteins. Furthermore, 395 of the peptides correspond to proteins that play a direct role in Listeria pathogenicity; they were identified as virulence factors, toxins and anti-toxins, or associated with either antibiotics (involved in antibiotic-related compounds production or resistance) or resistance to toxic substances. The proteomic repository obtained here can be the base for further research into pathogenic Listeria species and facilitate the development of novel therapeutics for these pathogens.

Listeria exploits IFITM3 to suppress antibacterial activity in phagocytes.

The type I interferon (IFN) signaling pathway has important functions in resistance to viral infection, with the downstream induction of interferon stimulated genes (ISG) protecting the host from virus entry, replication and spread. Listeria monocytogenes (Lm), a facultative intracellular foodborne pathogen, can exploit the type I IFN response as part of their pathogenic strategy, but the molecular mechanisms involved remain unclear. Here we show that type I IFN suppresses the antibacterial activity of phagocytes to promote systemic Lm infection. Mechanistically, type I IFN suppresses phagosome maturation and proteolysis of Lm virulence factors ActA and LLO, thereby promoting phagosome escape and cell-to-cell spread; the antiviral protein, IFN-induced transmembrane protein 3 (IFITM3), is required for this type I IFN-mediated alteration. Ifitm3-/- mice are resistant to systemic infection by Lm, displaying decreased bacterial spread in tissues, and increased immune cell recruitment and pro-inflammatory cytokine signaling. Together, our findings show how an antiviral mechanism in phagocytes can be exploited by bacterial pathogens, and implicate IFITM3 as a potential antimicrobial therapeutic target.

A Novel N-Halamine Biocidal Nanofibrous Membrane for Chlorine Rechargeable Rapid Water Disinfection Applications.

Disinfecting pathogenic contaminated water rapidly and effectively on sites is one of the critical challenges at point-of-use (POU) situations. Currently available technologies are still suffering from irreversible depletion of disinfectants, generation of toxic by-products, and potential biofouling problems. Herein, we developed a chlorine rechargeable biocidal nanofibrous membrane, poly(acrylonitrile-co-5-methyl-5-(4'-vinylphenyl)imidazolidine-2,4-dione) (P(AN-VAPH)), via a combination of a free radical copolymerization reaction and electrospun technology. The copolymer exhibits good electrospinnability and desirable mechanical properties. Also, the 5-methyl-5-(4'-vinylphenyl)imidazolidine-2,4-dione (VAPH) moieties containing unique hydantoin structures are able to be chlorinated and converted to halamine structures, enabling the P(AN-VAPH) nanofibrous membrane with rapid and durable biocidal activity. The chlorinated P(AN-VAPH) nanofibrous membranes showed intriguing features of unique 3D morphological structures with large specific surface area, good mechanical performance, rechargeable chlorination capacity (>5000 ppm), long-term durability, and desirable biocidal activity against both bacteria and viruses (>99.9999% within 2 min of contact). With these attributes, the chlorinated P(AN-VAPH) membranes demonstrated promising disinfecting efficiency against concentrated bacteria-contaminated water during direct filtration applications with superior killing capacity and high flowing flux (5000 L m-2 h-1).

Incorporation of Antimicrobial Bio-Based Carriers onto Poly(vinyl alcohol-co-ethylene) Surface for Enhanced Antimicrobial Activity.

A biobased rechargeable antimicrobial modification approach was developed using a covalent immobilization of food grade yeast cell wall particles on a model plastic film. We demonstrate the applications of this modification approach on poly(vinyl alcohol-co-ethylene) surface to inactivate inoculated bacteria with or without the presence of organic content, reducing the cross-contamination between food contact surface and model fresh produce, and inhibiting the growth of biofilms on the film surface. These biobased cell wall particle modified plastic films can enhance the binding of chlorine to the plastic surface in the form of N-halamine, extend the stability of chlorine against high organic content and ambient storage, and improve the rechargeability of the plastic films. Upon charging with chlorine, these modified plastic films inactivated 5 log of model Gram-negative bacteria (Escherichia coli O157:H7) and Gram-positive bacteria (Listeria innocua used as a surrogate of pathogenic Listeria monocytogenes) within 2 min of surface inoculation in water and within 20 min in an organic-rich aqueous environment. The modified plastic films prevented the transfer of bacteria and eliminated cross-contamination from the contaminated films to a spinach leaf surface, while 3 log CFU/leaf of bacteria were transferred from a contaminated native film to a noninoculated spinach surface. In addition, these modified plastic films reduced the adhesion of L. innocua cells by 2.7-3.6 log CFU/cm2 compared with control films during extended incubation for biofilm formation. Overall, this study demonstrates the feasibility of this biobased food grade modification approach to reduce microbial contamination and improve produce safety in the food processing industry.

Influence of molecular weight fractionation on the antimicrobial and anticancer properties of a fucoidan rich-extract from the macroalgae Fucus vesiculosus.

The objective of this study was to investigate the antimicrobial and anticancer properties of a fucoidan extract and subsequent fractions isolated from the macroalgae Fucus vesiculosus. The fractions obtained (>300 kDa, <300 kDa, <100 kDa, <50 kDa and <10 kDa) could inhibit the growth of B. subtilis, E. coli, L. innocua and P. fluorescens when assayed at concentrations between 12,500 and 25,000 ppm. The bacterial growth was monitored by optical density (OD) measurements (600 nm, 24 h) at 30 °C or 37 °C, depending upon on the strain used. The extracted fractions were also tested for cytotoxicity against brain glioblastoma cancer cells using the Alamar Blue assay for 24 h, 48 h and 6 days. The >300 kDa fraction presented the lowest IC50 values (0.052% - 24 h; 0.032% - 6 days). The potential bioactivity of fucoidan as an antimicrobial and anticancer agent was demonstrated in this study. Hence, the related mechanisms of action should be explored in a near future.

Investigation of combinations of rationally selected bioengineered nisin derivatives for their ability to inhibit Listeria in broth and model food systems.

In this study, we examined the ability of nisin A and a rationally assembled bank of 36 nisin derivative producing Lactococcus lactis strains to inhibit Listeria. A broth-based bioluminescence assay for screening single and combinations of bioengineered nisin derivatives using cell-free supernatants (CFS) from nisin derivative producing strains was developed. In this way, we screened 630 combinations of nisin derivative producing strains, identifying two (CFS from M17Q + N20P and M17Q + S29E) which exhibited enhanced anti-listerial activity when used together compared to when used alone, or to the nisin A producing strain. Minimal inhibitory concentration assays performed with purified peptides revealed than when used singly, the specific activities of M17Q, N20P and S29E (3.75-7.5 µM) against L. innocua were equal to, or less than that of nisin A (MIC of 3.75 µM). Broth-based growth curve assays using purified peptides demonstrated that use of the double peptide combinations and a triple peptide combination (M17Q + N20P + S29E) resulted in an extended lag phase of L. innocua, while kill curve assays confirmed the enhanced bactericidal activity of the combinations in comparison to the single derivative peptides or nisin A. Furthermore, the enhanced activity of the M17Q + N20P combination was maintained in a model food system (frankfurter homogenate) at both chill (4 °C) and abusive (20 °C) temperature conditions, with final cell numbers significantly less (1-2 log10 CFU/ml) than those observed with the derivative peptides alone, or nisin A. To our knowledge, this study is the first investigation that combines bioengineered bacteriocins with the aim of discovering a combination with enhanced antimicrobial activity.

Antimicrobial activity of various essential oils and their application in active packaging of frozen vegetable products.

Essential oils (EOs) have potential utility as clean-label food preservatives due to their antimicrobial and antioxidant properties. In this study, various EOs were screened for their antimicrobial activities against Listeria grayi in vitro. The susceptibility of L. monocytogenes to select EOs was compared with that of L. grayi. The effectiveness of the selected EOs in inhibiting the growth of L. grayi on vegetable products was also investigated. The results showed that cinnamon and oregano EOs and carvacrol were effective in the vapor phase in inhibiting the growth of L. grayi as well as L. monocytogenes, with the susceptibility of L. monocytogenes to cinnamon EO being slightly higher than that of L. grayi. The packaging of green peppers with cellulose stickers impregnated with cinnamon EO at 556 µL/Lheadspace reduced the Listeria count to 1 log CFU/g after 2 days of storage as compared to 7.5 log CFU/g for controls.

Synergistic inactivation of bacteria based on a combination of low frequency, low-intensity ultrasound and a food grade antioxidant.

This study evaluated a synergistic antimicrobial treatment using a combination of low frequency and a low-intensity ultrasound (LFU) and a food-grade antioxidant, propyl gallate (PG), against a model gram-positive (Listeria innocua) and the gram-negative bacteria (Escherichia coli O157:H7). Bacterial inactivation kinetic measurements were complemented by characterization of biophysical changes in liposomes, changes in bacterial membrane permeability, morphological changes in bacterial cells, and intracellular oxidative stress upon treatment with PG, LFU, and a combination of PG + LFU. Combination of PG + LFU significantly (>4 log CFU/mL, P < 0.05) enhanced the inactivation of both L. innocua and E. coli O157:H7 compared to PG or LFU treatment. As expected, L. innocua had a significantly higher resistance to inactivation compared to E. coli using a combination of PG + LFU. Biophysical measurements in liposomes, bacterial permeability measurements, and scanning electron microscope (SEM)-based morphological measurements show rapid interactions of PG with membranes. Upon extended treatment of cells with PG + LFU, a significant increase in membrane damage was observed compared to PG or LFU alone. A lack of change in the intracellular thiol content following the combined treatment and limited effectiveness of exogenously added antioxidants in attenuating the synergistic antimicrobial action demonstrated that oxidative stress was not a leading mechanism responsible for the synergistic inactivation by PG + LFU. Overall, the study illustrates synergistic inactivation of bacteria using a combination of PG + LFU based on enhanced membrane damage and its potential for applications in the food and environmental systems.

Identification and Characterization of a Novel Genomic Island Harboring Cadmium and Arsenic Resistance Genes in Listeria welshimeri.

Listeria monocytogenes, the bacterial foodborne pathogen responsible for the severe disease listeriosis, frequently exhibits heavy metal resistance. Concurrent resistance to cadmium and arsenic in L. monocytogenes is strongly associated with the 35-kb chromosomal island LGI2. LGI2 has been encountered repeatedly among L. monocytogenes serotype 4b hypervirulent clones but, surprisingly, not among non-pathogenic Listeria spp. Here we describe a novel LGI2 variant, LGI2-3, in two L. welshimeri strains from an urban aquatic environment. Whole genome sequence analysis revealed that the genomes were closely related except for one prophage region and confirmed a chromosomally integrated LGI2-3. It harbored a cystathionine beta-lyase gene previously only encountered in LGI2-1 of L. monocytogenes clonal complex 1 but was otherwise most closely related to LGI2. LGI2-3 harbored a novel cadAC cassette (cadA7C7) that, like LGI2's cadA4C4, was associated with lower-level tolerance to cadmium (MIC 50 µg/mL) than other cadAC cassettes (MIC ≥ 140 µg/mL). CadA sequence analysis identified two amino acids that may be important for mediating different levels of cadmium tolerance. Our findings clearly demonstrated the potential for LGI2-like islands to be harbored by non-pathogenic Listeria spp. and generate intriguing hypotheses on the genetic diversity mediated by this island and its transfer among Listeria spp.

Combined Antimicrobial Effect of Bio-Waste Olive Leaf Extract and Remote Cold Atmospheric Plasma Effluent.

A novel strategy involving Olive Leaf Extract (OLE) and Cold Atmospheric Plasma (CAP) was developed as a green antimicrobial treatment. Specifically, we reported a preliminary investigation on the combined use of OLE + CAP against three pathogens, chosen to represent medical and food industries (i.e., E. coli, S. aureus and L. innocua). The results indicated that a concentration of 100 mg/mL (total polyphenols) in OLE can exert an antimicrobial activity, but still insufficient for a total bacterial inactivation. By using plain OLE, we significantly reduced the growth of Gram positive S. aureus and L. innocua, but not Gram-negative E. coli. Instead, we demonstrated a remarkable decontamination effect of OLE + CAP in E. coli, S. aureus and L. innocua samples after 6 h. This effect was optimally maintained up to 24 h in S. aureus strain. E. coli and L. innocua grew again in 24 h. In the latter strain, OLE alone was most effective to significantly reduce bacterial growth. By further adjusting the parameters of OLE + CAP technology, e.g., OLE amount and CAP exposure, it could be possible to prolong the initial powerful decontamination over a longer time. Since OLE derives from a bio-waste and CAP is a non-thermal technology based on ionized air, we propose OLE + CAP as a potential green platform for bacterial decontamination. As a combination, OLE and CAP can lead to better antimicrobial activity than individually and may replace or complement conventional thermal procedures in food and biomedical industries.