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.

Atypical Hemolytic Listeria innocua Isolates Are Virulent, albeit Less than Listeria monocytogenes.

Listeria innocua is considered a nonpathogenic Listeria species. Natural atypical hemolytic L. innocua isolates have been reported but have not been characterized in detail. Here, we report the genomic and functional characterization of representative isolates from the two known natural hemolytic L. innocua clades. Whole-genome sequencing confirmed the presence of Listeria pathogenicity islands (LIPI) characteristic of Listeria monocytogenes species. Functional assays showed that LIPI-1 and inlA genes are transcribed, and the corresponding gene products are expressed and functional. Using in vitro and in vivo assays, we show that atypical hemolytic L. innocua is virulent, can actively cross the intestinal epithelium, and spreads systemically to the liver and spleen, albeit to a lesser degree than the reference L. monocytogenes EGDe strain. Although human exposure to hemolytic L. innocua is likely rare, these findings are important for food safety and public health. The presence of virulence traits in some L. innocua clades supports the existence of a common virulent ancestor of L. monocytogenes and L. innocua.

Pathogenicity, genotyping and antibacterial susceptibility of the Listeria spp. recovered from stray dogs.

The present study aimed to determine the prevalence of Listeria spp. in stray dogs in the Kayseri province of Turkey. In addition, serotyping, genotyping and virulence gene analysis of the isolated Listeria spp. were performed and their pathogenicity and antibacterial susceptibility were investigated. The study included 80 rectal swaps taken from 80 stray dogs of different ages and gender that were sheltered in the Kayseri Municipal Dog Shelter. Listeria selective broth and Listeria selective agar were used for the isolation of Listeria spp. and the isolates were identified using a Microbact 12L (Oxoid, England) identification test kit. 16S rDNA sequencing and species-specific polymerase chain reaction (PCR) were performed for molecular identification of the isolates, multiplex PCR and a serological test were performed for serotyping, and PCR was used for virulence gene analysis. For determining the pathogenicity of L. monocytogenes and L. innocua isolates, a total of 100 mice (50 pregnant and 50 non-pregnant) were used. The mice were infected intraperitoneally; the inoculation dose was 1 × 109 CFU/mL and 0.2 mL was used for each animal. Tissue samples obtained from infected mice were processed for the re-isolation of the Listeria spp. and then stained with hematoxylin eosin and Brown-Brenn Gram stain. The antibiotic susceptibilities of the isolates were determined by the disk diffusion method. Listeria spp. were isolated from 5 (6.25%) of the 80 fecal samples. While 1 of the isolates was identified as L. monocytogenes, 4 of them were identified as L. innocua. Serotyping by serological and molecular methods revealed the isolate of L. monocytogenes to be serotype 1/2a. According to the phylogenetic trees, L. innocua and L. monocytogenes strains were clustered in different groups. The L. monocytogenes isolate was positive for all virulence genes tested. All L. innocua isolates were positive for the plcB gene. While all L. innocua isolates were negative for the lin1068 gene, 3 L. innocua isolates were found to be positive for the lin0558 gene. In mice infected with L. monocytogenes, pathological findings were observed in the uterus, intestines, pancreas, and heart. In mice infected with L. innocua, pathological findings were observed in the stomach, intestines and spleen. L. monocytogenes- or L. innocua-related infections or other inflammatory reactions were not observed in the brains of infected animals. On histopathological examination with Gram stain, an abundance of Listeria spp. was observed in the lesions of the liver, spleen, uterus, and kidney. Moreover, while abortion was observed in all animals infected with L. monocytogenes, it was not observed in any of the animals infected with L. innocua. Antibiotic susceptibility testing revealed that all 5 isolates were sensitive to ampicillin, amoxicillin/clavulanic acid, erythromycin, gentamicin, penicillin G, and trimethoprim-sulfamethoxazole and were resistant to nalidixic acid, streptomycin, and cefuroxime sodium. Considering also the pathogenicity of the isolated microorganisms, it can be suggested that stray dogs as carriers of Listeria spp. are a significant risk to public health. As L. innocua isolates, which are considered apathogenic, led to the occurrence of lesions similar to those caused by L. monocytogenes, detailed studies on the pathogenesis of L. innocua infections caused by L. innocua isolates recovered from various sources are required.

Antibacterial Activity of Neat Chitosan Powder and Flakes.

This study investigates the antibacterial activity of neat chitosan powder and flakes against three different bacterial species, Escherichia coli, Listeria innocua and Staphylococcus aureus, which are frequent causes of food spoilage. The effect of chitosan concentration and purity, as well as the influence of temperature, ionic strength (salt) and impact of a solid physical support in the medium are examined. Results show that the antibacterial activity of neat chitosan: (i) requires partial solubilisation; (ii) can be promoted by environmental factors such as adequate temperature range, ionic strength and the presence of a solid physical support that may facilitate the attachment of bacteria; (iii) depends on bacterial species, with a sensitivity order of E. coli > L. innocua > S. aureus; and (iv) increases with chitosan concentration, up to a critical point above which this effect decreases. The latter may be due to remaining proteins in chitosan acting as nutrients for bacteria therefore limiting its antibacterial activity. These results on the direct use of chitosan powder and flakes as potential antimicrobial agents for food protection at pH values lower than the chitosan pKa (6.2-6.7) are promising.

Study on synergistic effects of curcumin and bixin against foodborne pathogens.

Various studies have shown that natural colorants, in addition to their coloring attributes, have valuable biological effects such as antioxidant, anti-inflammation, and anticarcinogenic properties. Moreover, their use as a food colorant can restrict the potential disadvantages of synthetic additives and turn foods into functional products. In this study, in vitro antimicrobial activities of two natural colorants of bixin and curcumin against some important foodborne pathogens: Staphylococcus aureus (S. aureus), Listeria innocua (L. innocua), and Escherichia coli (E. coli) were investigated by disk diffusion method. Minimum inhibitory concentration and minimum bactericidal concentration values were determined by agar dilution and broth microdilution methods. The synergistic activity of the colorants against selected microorganisms was assayed by the checkerboard microdilution method. The results showed that the inhibitory effects of bixin against S. aureus were more pronounced than E. coli and L. innocua. The lowest concentration of curcumin (0.6 mg/mL) in the disk diffusion method was not inhibited by any tested bacteria. However, it was effective at the higher concentrations against three microorganisms, but its diameter of inhibition zones was lower than gentamicin in all concentrations. Synergetic effects were observed by curcumin and bixin combination against S. aureus (FICI ≤ 0.5), but they act as an antagonist against E. coli and L. innocua. The results of the synergy test were confirmed by the isobologram curves.

Application of Ultrasound Treatments in the Processing and Production of High-Quality and Safe-to-Drink Kiwi Juice.

This study explores the potential of thermosonication as an alternative to traditional heat treatments, such as pasteurization, in the processing of fruit juices. Conventional methods often lead to undesirable quality changes in fruit juices, whereas thermosonication offers promising results regarding microbial inactivation and quality preservation. This work focused on the inactivation kinetics of Listeria innocua 2030c, a surrogate for pathogenic L. monocytogenes, in kiwifruit juice using thermosonication at 45 °C, 50 °C, and 55 °C. These treatments were compared with equivalent heat treatments. Quality attributes of the juice were also evaluated to assess process efficiency. Survival data of L. innocua were fitted with the Weibull model, estimating first decimal reduction times (δ) and shape parameters (n). The results reveal temperature and process dependencies on δ, while n remains mostly temperature and treatment independent. Thermosonication outperforms heat treatment, achieving higher L. innocua reductions while retaining quality attributes like pH, soluble solid content, and total phenolics and chlorophylls. Thermosonication at 55 °C stands out, providing a 6.2-log-cycle reduction in just 3 min with superior quality retention. These findings highlight the synergistic effect of temperature and ultrasound, making mild heat processes feasible while enhancing product quality. Thermosonication, particularly at 55 °C, emerges as an effective alternative to traditional thermal treatments for fruit juices, offering improved microbial safety without compromising product quality.

Genomic characterization of Listeria monocytogenes and Listeria innocua isolated from milk and dairy samples in Ethiopia.

Listeriosis caused by Listeria monocytogenes often poses a significant threat to vulnerable populations. Dairy products have been implicated in outbreaks of listeriosis worldwide. In Ethiopia, studies have identified Listeria spp. and L. monocytogenes in various dairy products, but the genetic diversity and phylogenetic relationships of these bacteria remain largely unknown in the low- and middle-income countries. Therefore, we conducted whole-genome sequencing on 15 L. monocytogenes and 55 L. innocua isolates obtained from different levels of the dairy supply chains across three regions in Ethiopia. Genomes were assembled and used for MLST genotyping and single nucleotide polymorphism (SNP) analysis to infer phylogenetic relationships. We identified a total of 3 L. monocytogenes (i.e., 2, 145, and 18) and 12 L. innocua (i.e., 1489, 1619, 603, 537, 1010, 3186, 492, 3007, 1087, 474, 1008, and 637) MLST sequence types among the studied isolates. Some of these sequence types showed region-specific occurrence, while others were broadly distributed across regions. Through high-quality SNP analysis, we found that among 13 L. monocytogenes identified as ST 2, 11 of them were highly similar with low genetic variation, differing by only 1 to 10 SNPs, suggesting potential selection in the dairy food supply chain. The L. innocua isolates also exhibited low intra-ST genetic variation with only 0-10 SNP differences, except for the ST 1619, which displayed a greater diversity.

Identification of Listeria species and Multilocus Variable-Number Tandem Repeat Analysis (MLVA) Typing of Listeria innocua and Listeria monocytogenes Isolates from Cattle Farms and Beef and Beef-Based Products from Retail Outlets in Mpumalanga and North West Provinces, South Africa.

In this study, Listeria isolates (214) were characterized as follows: L. innocua (77.10%), L. monocytogenes (11.21%), L. welshimeri (5.61%), L. grayi (1.40%), L. seeligeri (0.93%), and L. species (3.73%) that were not identified at the species level, from beef and beef based products from retail and farms in Mpumalanga and North West provinces of South Africa. MLVA was further used to type Listeria innocua isolates (165) and Listeria monocytogenes isolates (24). The L. monocytogenes isolates were also serogrouped using PCR. The MLVA protocol for L. monocytogenes typing included six tandem repeat primer sets, and the MLVA protocol for L. innocua included the use of three tandem repeats primer sets. The L. monocytogenes serogroups were determined as follows: 4b-4d-4e (IVb) (37.50%), 1/2a-3a (IIa) (29.16%), 1/2b-3b (IIb) (12.50%), 1/2c-3c (IIc) (8.33%), and IVb-1 (4.16%). MLVA could cluster isolates belonging to each specie, L. monocytogenes, and L. innocua isolates, into MLVA-related strains. There were 34 and 10 MLVA types obtained from the MLVA typing of L. innocua and L. monocytogenes, respectively. MLVA clustered the L. monocytogenes isolates irrespective of sample category, serogroups, and geographical origin. Similarly, the L. innocua isolates clustered irrespective of meat category and geographical origin. MLVA was able to cluster isolates based on MLVA relatedness. The clustering of isolates from farms and retailers indicates transmission of Listeria spp. MLVA is an affordable, simple, and discriminatory method that can be used routinely to type L. monocytogenes and L. innocua isolates.

Inactivation of Bacteria Using Bioactive Nanoparticles and Alternating Magnetic Fields.

Foodborne pathogens are frequently associated with risks and outbreaks of many diseases; therefore, food safety and processing remain a priority to control and minimize these risks. In this work, nisin-loaded magnetic nanoparticles were used and activated by alternating 10 and 125 mT (peak to peak) magnetic fields (AMFs) for biocontrol of bacteria Listeria innocua, a suitable model to study the inactivation of common foodborne pathogen L. monocytogenes. It was shown that L. innocua features high resistance to nisin-based bioactive nanoparticles, however, application of AMFs (15 and 30 min exposure) significantly potentiates the treatment resulting in considerable log reduction of viable cells. The morphological changes and the resulting cellular damage, which was induced by the synergistic treatment, was confirmed using scanning electron microscopy. The thermal effects were also estimated in the study. The results are useful for the development of new methods for treatment of the drug-resistant foodborne pathogens to minimize the risks of invasive infections. The proposed methodology is a contactless alternative to the currently established pulsed-electric field-based treatment in food processing.

The effect of ultrasound treatment in combination with nisin on the inactivation of Listeria innocua and Escherichia coli.

Ultrasound, alone or in combination with natural antimicrobials, is a novel food processing technology of interest to replace traditional food decontamination methods, as it is milder than classical sterilisation (heat treatment) and maintains desirable sensory characteristics. However, ultrasound efficacy can be affected by food structure/composition, as well as the order in which combined treatments are applied. More specifically, treatments which target different cell components could result in enhanced inactivation if applied in the appropriate order. The microbial properties i.e. Gram positive/Gram negative can also impact the treatment efficacy. This work presents a systematic study of the combined effect of ultrasound and nisin on the inactivation of the bacteria Listeria innocua (Gram positive) and Escherichia coli (Gram negative), at a range of cavitation conditions (44, 500, 1000 kHz). The order of treatment application was varied, and the impact of system structure was also investigated by varying the concentration of Xanthan gum used to create the food model systems (0 - 0.5% w/v). Microbial inactivation kinetics were monitored, and advanced microscopy and flow cytometry techniques were utilised to quantify the impact of treatment on a cellular level. Ultrasound was shown to be effective against E. coli at 500 kHz only, with L. innocua demonstrating resistance to all frequencies studied. Enhanced inactivation of E. coli was observed for the combination of nisin and ultrasound at 500 kHz, but only when nisin was applied before ultrasound treatment. The system structure negatively impacted the inactivation efficacy. The combined effect of ultrasound and nisin on E. coli was attributed to short-lived destabilisation of the outer membrane as a result of sonication, allowing nisin to penetrate the cytoplasmic membrane and facilitate cell inactivation.

Cold plasma treatment of ready-to-eat ham: Influence of process conditions and storage on inactivation of Listeria innocua.

Ready-to-eat (RTE) deli meat has been linked to several Listeria monocytogenes associated recalls. Recent studies demonstrated the potential antimicrobial effects of atmospheric cold plasma treatment on various food surfaces including RTE meat products. However, the influence of intrinsic and extrinsic factors, determining the efficacy of cold plasma to reduce Listeria has not been reported. This study investigated the influence of rosemary extract, salt (% NaCl), and treatment temperature on the efficacy of plasma to reduce numbers of L. innocua on RTE ham. The effect of post-treatment storage on L. innocua inactivation was also investigated. When the cold plasma treatment temperature was 4 °C, we observed a significant reduction in L. innocua of 1.75 and 1.51 log CFU/cm2 on 1% and 3% NaCl ham surface without rosemary extract respectively, after 180 s treatment. At a treatment temperature of 23 °C, the L. innocua cells were reduced by 1.78 and 1.43 log CFU/cm2, respectively on these surfaces after 180 s. No significant effects of salt concentration and treatment temperature were observed on L. innocua inactivation during cold plasma treatment of ham. The post treatment storage at 4 °C for 6 h after 180 s of plasma treatment enhanced further reduction of L. innocua on 1% NaCl ham without rosemary. We also observed the increased concentration of malondialdehyde (MDA) equivalent lipid oxidation of plasma treated samples and was significantly higher (1.53 MDA mg/ kg ham) compared to untreated samples (0.92 MDA mg/kg ham). However, no significant differences in surface color parameters, L* and b* values were observed after plasma treatment, except a significant increase in a* values. The water content of plasma exposed samples decreased significantly for all treatment conditions whereas the water activity values were not changed significantly. In conclusion, the atmospheric cold plasma could be applied as a means for surface decontamination of RTE ham. However, the drying and oxidation of ham should be controlled in an open atmospheric plasma treatment condition.

Physical and Antimicrobial Properties of Starch-PVA Blend Films as Affected by the Incorporation of Natural Antimicrobial Agents.

In this work, active films based on starch and PVA (S:PVA ratio of 2:1) were developed by incorporating neem (NO) and oregano essential oils (OEO). First, a screening of the antifungal effectiveness of different natural extracts (echinacea, horsetail extract, liquid smoke and neem seed oil) against two fungus (P. expansum and A. niger) was carried out. The effect of NO and OEO incorporation on the films' physical and antimicrobial properties was analyzed. Only composite films containing OEO exhibited antibacterial and antifungal activity. Antibacterial activity occurred at low OEO concentration (6.7%), while antifungal effect required higher doses of OEO in the films. Incorporation of oils did not notably affect the water sorption capacity and water vapor barrier properties of S-PVA films, but reduced their transparency and gloss, especially at the highest concentrations. The mechanical response of the S-PVA films was also negatively affected by oil incorporation but this was only relevant at the highest oil ratio (22%). S-PVA films with 6.7% of OEO exhibited the best physical properties, without significant differences with respect to the S-PVA matrix, while exhibiting antibacterial activity. Thus, the use of OEO as a natural antimicrobial incorporated into starch-PVA films represents a good and novel alternative in food packaging applications.