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Antimicrobial-resistant Escherichia coli isolates have emerged in various ecologic compartments and evolved to spread globally. We sought to (1.) investigate the occurrence of ESBL-producing E. coli (ESBL-Ec) in feces from free-range chickens in a rural region and (2.) characterize the genetic background of antimicrobial resistance and the genetic relatedness of collected isolates. Ninety-five feces swabs from free-range chickens associated with two households (House 1/House 2) in a rural region in northern Tunisia were collected. Samples were screened to recover ESBL-Ec, and collected isolates were characterized for phenotype/genotype of antimicrobial resistance, integrons, and molecular typing (pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST)). Overall, 47 ESBL-Ec were identified, with the following genes detected: 35 blaCTX-M-1, 5 blaCTX-M-55, 5 blaCTX-M-15, 1 blaSHV-2, and 1 blaSHV-12. Resistance to fluoroquinolones, tetracycline, sulfonamides, and colistin was encoded by aac(6′)-Ib-cr (n = 21), qnrB (n = 1), and qnrS (n = 2); tetA (n = 17)/tetB (n = 26); sul1 (n = 29)/sul2 (n = 18); and mcr-2 (n = 2) genes, respectively. PFGE and MLST identified genetic homogeneity of isolates in House 1; however, isolates from House 2 were heterogeneous. Notably, among nine identified sequence types, ST58, ST69, ST224, and ST410 belong to pandemic high-risk clonal lineages associated with extrapathogenic E. coli. Minor clones belonging to ST410 and ST471 were shared by chickens from both households. The virulence genes fyuA, fimH, papGIII, and iutA were detected in 35, 47, 17, and 23 isolates, respectively. Findings indicate a high occurrence of ESBL-Ec in free-range chickens and highlight the occurrence of pandemic zoonotic clones.

Graphical abstract Abstract 7-Ketocholesterol (or 7-oxocholesterol) is an oxysterol essentially formed by cholesterol autoxidation. It is often found at enhanced levels in the body fluids and/or target tissues of patients with age-related diseases (cardiovascular, neuronal, and ocular diseases) as well as in subjects concerned with civilization diseases (type 2 diabetes, bowel diseases, and metabolic syndrome). The involvement of increased 7-ketocholesterol levels in the pathophysiology of these diseases is widely suspected. Indeed, 7-ketocholesterol at elevated concentrations is a powerful inducer of oxidative stress, inflammation, and cellular degeneration which are common features of all these diseases. It is important to better know the origin of 7-ketocholesterol (diet, incidence of environmental factors, and endogenous formation (autoxidation and enzymatic synthesis)) and its inactivation mechanisms which include esterification, sulfation, oxidation, and reduction. This knowledge will make it possible to act at different levels to regulate 7-ketocholesterol level and counteract its toxicity in order to limit the incidence of diseases associated with this oxysterol. These different points as well as food and biomedical applications are addressed in this review.

Peroxisomes play an important role in regulating cell metabolism and RedOx homeostasis. Peroxisomal dysfunctions favor oxidative stress and cell death. The ability of 7β-hydroxycholesterol (7β-OHC; 50 μM, 24 h), known to be increased in patients with age-related diseases such as sarcopenia, to trigger oxidative stress, mitochondrial and peroxisomal dysfunction was studied in murine C2C12 myoblasts. The capacity of milk thistle seed oil (MTSO, 100 μg/mL) as well as α-tocopherol (400 µM; reference cytoprotective agent) to counteract the toxic effects of 7β-OHC, mainly at the peroxisomal level were evaluated. The impacts of 7β-OHC, in the presence or absence of MTSO or α-tocopherol, were studied with complementary methods: measurement of cell density and viability, quantification of reactive oxygen species (ROS) production and transmembrane mitochondrial potential (ΔΨm), evaluation of peroxisomal mass as well as topographic, morphologic and functional peroxisomal changes. Our results indicate that 7β-OHC induces a loss of cell viability and a decrease of cell adhesion associated with ROS overproduction, alterations of mitochondrial ultrastructure, a drop of ΔΨm, and several peroxisomal modifications. In the presence of 7β-OHC, comparatively to untreated cells, important quantitative and qualitative peroxisomal modifications were also identified: a) a reduced number of peroxisomes with abnormal sizes and shapes, mainly localized in cytoplasmic vacuoles, were observed; b) the peroxisomal mass was decreased as indicated by lower protein and mRNA levels of the peroxisomal ABCD3 transporter; c) lower mRNA level of Pex5 involved in peroxisomal biogenesis as well as higher mRNA levels of Pex13 and Pex14, involved in peroxisomal biogenesis and/or pexophagy, was found; d) lower levels of ACOX1 and MFP2 enzymes, implicated in peroxisomal β-oxidation, were detected; e) higher levels of very-long-chain fatty acids, which are substrates of peroxisomal β-oxidation, were found. These different cytotoxic effects were strongly attenuated by MTSO, in the same range of order as with α-tocopherol. These findings underline the interest of MTSO and α-tocopherol in the prevention of peroxisomal damages (pexotherapy).

Prenatal ethanol exposure provokes teratogenic effects, due to oxidative stress and massive neuronal apoptosis in the developing brain that result in lifelong behavioral abnormalities. PACAP exerts anti-oxidative and neuroprotective activities on neuronal cells, and prevents ethanol neurotoxicity. The present study focused on the ability of PACAP to protect the brain of 30-day-old mice (P30) from prenatal alcohol exposure induced oxidative damage and toxicity. Pregnant mice were divided randomly into 4 groups, i.e. control group, ethanol group (1.5 g/kg ip daily injection), PACAP group (5 μg intrauterine daily injection) and an ethanol plus PACAP group. Offspring prenatally exposed to ethanol had decreased body weight and reduced cell survival. Moreover, production of ROS was sharply enhanced in the brain of prenatal ethanol-exposed animals, associated with an elevation in the activity of the antioxidant enzymes, and an increase of oxidative damages as shown by the accumulation of the lipid oxidation marker malondialdehyde and of protein carbonyl compounds. Intrauterine administration of PACAP during the gestational period restored the endogenous antioxidant system, prevented ROS overproduction and promoted the survival of dissociated cells from animals prenatally exposed to ethanol. Behavioral tests revealed that P30 animals exposed to ethanol during the prenatal period exhibited reduced motor activity, altered exploratory interest and increased anxiety. However, PACAP treatment significantly attenuated these behavioral impairments. This study demonstrates that PACAP exerts a potent neuroprotective effect against alcohol toxicity during brain development, and indicates that PACAP and/or PACAP analogs might be a useful tool for treatment of alcohol intoxication during pregnancy.

This study deals with the genetic characterisation of two enterocin-producing Enterococcus lactis strains named Q1 and 4CP3 using Randomly Amplified Polymorphic DNA (RAPD)ePCR and the effect of their separate additions in the control of Listeria monocytogenes in refrigerated chicken breast meat. These strains demonstrated interesting in vitro antimicrobial activity towards L. monocytogenes EGDe 107776 by agar assay. To evaluate the in situ effect of E. lactis strains against L. monocytogenes in white meat model, chicken breast were artificially contaminated with 105 CFU/g of L. monocytogenes and inoculated with E. lactis Q1 and 4CP3 of each at 107 CFU/ g. The pathogen counts were monitored during cold storage for 28 days. The anti-listerial effect of the enterococcal cultures was examined using linear (ANOVA) and general (ANCOVA) models and it has been shown that both of them controlled L. monocytogenes by decreasing (P < 0.05) and suppressing the pathogen growth in refrigerated chicken meats during 28 days of storage. The present study shows that our E. lactis strains have great potential as protective cultures and might be used for preventing the growth of pathogenic bacteria such as L. monocytogenes in meat products during their refrigerated storage

Little is known about the role of forestland and non-fertilized agriculture soils as reservoirs of extended-spectrum beta-lactamase (ESBL) and plasmid-borne AmpC (pAmpC)-producing Escherichia coli isolates. Thus, in the present study, 210 soil samples from various origins (forest of Oued Zen (Ain Drahem), non-agriculture soils from different park gardens in Tunis City, cereal culture soils and home gardens) were investigated to characterize cefotaxime-resistant E. coli isolates. A total of 22 ESBL/pAmpC-producing E. coli were collected, and all harbored variants of the blaCTX-M gene (15 blaCTX-M-1, 5 blaCTX-M-55 and 2 blaCTX-M-15). A total of seven and two isolates harbored also blaEBC and blaDHA-like genes, respectively. Resistances to tetracycline, sulfonamides and fluoroquinolones were encoded by tetA (n = 4)/tetB (n = 12), sul1 (n = 17)/sul2 (n = 19) and aac(6’)-Ib-cr (n = 2)/qnrA (n = 1)/qnrS (n = 1) genes, respectively. A total of seven isolates were able to transfer by conjugation cefotaxime-resistance in association or not with other resistance markers. PFGE showed that ten and two isolates were clonally related (pulsotypes P1 and P2). The 10 P1 isolates had been collected from forestland, cereal culture soils and an urban park garden in Tunis City, arguing for a large spread of clonal strains. Our findings highlight the occurrence of ESBL/pAmpC-E. coli isolates in soils under limited anthropogenic activities and the predominance of CTX-M enzymes that are largely disseminated in E. coli from humans and animals in Tunisia.

Eight lactic acid bacterial (LAB) strains were newly isolated from fresh shrimps (Penaeus vannamei and Palaemon serratus) and characterised by phenotypic and molecular tests. These isolates, identified as Enterococcus lactis producing the enterocins A, B and/or P, displayed interesting antagonistic activities against pathogenic bacteria and fungi. Regarding biotechnological properties, E. lactis strains had generally great redox potentials, good proteolytic activities (P < 0.05) and medium acidification rates. Evaluated by the API ZYM system, two strains named Q1 and 4CP3 exhibited good enzymatic activities: leucine, valine and cystine arymalidases, Naphthol-ASBI- phosphohydrolase and ?-galactosidase. For probiotic assessment, the examined strains were found to resist low pH and bile salts with presence of gastric and digestive enzymes. Likewise, they indicated potentialities of adhesion and colonisation in the intestines. All strains were sensitive to clinically important antibiotics and no PCR signals were detected for any of the common antibiotic resistance genes such as vanA and vanB for vancomycin. Furthermore, virulence traits including the presence of virulence factors, haemolysin and gelatinase, were not detected. This work suggests that our E. lactis strains can be considered as promising candidates for future applications as a starter, adjunct or protective cultures or could constitute new potential probiotics

Graphical abstract, 7-Ketocholesterol, which is one of the earliest cholesterol oxidization products identified, is essentially formed by the auto-oxidation of cholesterol. In the body, 7-ketocholesterol is both provided by food and produced endogenously. This pro-oxidant and pro-inflammatory molecule, which can activate apoptosis and autophagy at high concentrations, is an abundant component of oxidized Low Density Lipoproteins. 7-Ketocholesterol appears to significantly contribute to the development of age-related diseases (cardiovascular diseases, age-related macular degeneration, and Alzheimer's disease), chronic inflammatory bowel diseases and to certain cancers. Recent studies have also shown that 7-ketocholesterol has anti-viral activities, including on SARS-CoV-2, which are, however, lower than those of oxysterols resulting from the oxidation of cholesterol on the side chain. Furthermore, 7-ketocholesterol is increased in the serum of moderately and severely affected COVID-19 patients. In the case of COVID-19, it can be assumed that the antiviral activity of 7-ketocholesterol could be counterbalanced by its toxic effects, including pro-oxidant, pro-inflammatory and pro-coagulant activities that might promote the induction of cell death in alveolar cells. It is therefore suggested that this oxysterol might be involved in the pathophysiology of COVID-19 by contributing to the acute respiratory distress syndrome and promoting a deleterious, even fatal outcome. Thus, 7-ketocholesterol could possibly constitute a lipid biomarker of COVID-19 outcome and counteracting its toxic effects with adjuvant therapies might have beneficial effects in COVID-19 patients.

International audience; Screening for lactic acid bacteria (LAB) from fresh shrimp samples (Penaeus vannamei) collected from retail seafood markets in the Tunisian's coast, resulted in the isolation of an Enterococcus strain termed Q1. This strain was selected for its antagonistic activity against pathogenic bacteria such as Listeria monocytogenes, Pseudomonas aeruginosa, Lactococcus garvieae and against fungi (Aspergillus niger and Fusarium equiseti). The Q1 strain was characterised using standard morphological and biochemical tests, growth assays at different temperatures, pH and salinity. 16S rRNA, rpoA and pheS gene sequencing, as well as the 16S-23S rRNA intergenic spacer analyses, were combined to identify strain Q1 as a strain of Enterococcus lactis. The bacteriocin produced by E. lactis Q1 is thermostable, active in the pH range from 4.0 to 9.0 and has a bactericidal mode of action. The enterocin P structural gene was detected by specific PCR in strain E. lactis Q1, which is in good agreement with SDS-PAGE data of the purified bacteriocin. A lack of significant antibiotic resistance genes and virulence determinants was confirmed by specific PCRs. This work provides the first description of an enterocin P producer E. lactis strain isolated from a fresh shrimp. Based on its safety properties (absence of haemolytic activity, virulence factors and antibiotic resistance genes), this strain has the potential to be used as a natural additive or adjunct protective culture in food biopreservation and/or probiotic culture.

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Lactic acid bacteria produce various antibacterial peptides such as bacteriocins that are active against pathogenic and spoilage microorganisms. Very little attention has been paid to the production of lysozyme as an antimicrobial enzyme. The present work represents one of the few studies reporting lysozyme production by enterococci. Indeed, this study was first conducted to evaluate the antimicrobial activity of Enterococcus lactis Q1, an enterocin P-producing strain previously isolated from fresh shrimp (Penaeus vannamei), against multidrug-resistant clinical isolates. Results showed significant inhibitory activity (P

In this manuscript, a multiple enterocin-producing Enterococcus lactis strain named 4CP3 was used to control the proliferation of Listeria monocytogenes in refrigerated raw beef meat model. Also, the intraspecific genetic differentiation of 4CP3 strain was assessed by Random Amplified Polymorphic DNA Polymerase Chain Reaction (RAPD-PCR) analysis. E. lactis 4CP3 strain was found to produce the enterocins A, B, and P. It displayed activity against L. monocytogenes EGDe 107776 by agar-well diffusion method. The application of E. lactis 4CP3 culture at 107 CFU/g in raw beef meat was evaluated using both ANOVA and ANCOVA linear models in order to examine its effect on the growth of the pathogen L. monocytogenes during refrigerated storage. Hence, a very interesting result in decreasing (P L. monocytogenes in refrigerated raw beef meat was shown during 28 days of storage. In conclusion, E. lactis 4CP3 strain might be useful for prevention of the proliferation and survival of L. monocytogenes in raw meat during refrigerated storage.

This study aims to characterise a potential bacteriocinogenic lactic acid bacterial strain isolated from a raw pink shrimp (Palaemon serratus) and evaluate its safety aspect. The strain designated as 4CP3 was noted to display antibacterial activities (P < 0.05) against Gram-positive and Gram-negative foodborne pathogens (Listeria monocytogenes and Pseudomonas aeruginosa) and some filamentous fungi (e.g. Aspergillus niger A79). Phenotypic and molecular techniques as well as phylogenetic analysis identified the isolate 4CP3 as Enterococcus lactis. Its produced antimicrobial substance was determined as a bacteriocin that was stable over a wide range of pH (2-10) and after heating at 100 °C for 15 min. The maximum bacteriocin production was 1400 AU/ml recorded after 12 h of incubation in de Man, Rogosa and Sharpe (MRS) broth medium at 30 °C. The mode of action of the bacteriocin produced by 4CP3 strain was identified as bactericidal against L. monocytogenes EGDe 107776 and P. aeruginosa ATCC 27853. By specific PCR amplifications, E. lactis 4CP3 was shown to produce the enterocins A, B and P. To our knowledge, this feature is newly described for E. lactis strain isolated from raw shrimps. Regarding safety aspect of E. lactis 4CP3, it has been demonstrated that this strain was not haemolytic, gelatinase negative, sensitive to vancomycin, and free of common antibiotic resistance genes and virulence factors. Therefore, it may be useful as a safe natural agent in preservation of foods or as a new probiotic strain in food and feed.

The aims of this study are to isolate new bacteriocinogenic lactic acid bacterial strains from white (Penaeus vannamei) and pink (Palaemon serratus) raw shrimps and evaluate their technological and probiotic potentialities. Seven strains were selected, among fifty active isolates, as producing interesting antimicrobial activity. Identified as Enterococcus lactis, these isolates were able to produce enterocins A, B and/or P. The safety aspect, assessed by microbiological and molecular tests, demonstrated that the strains were susceptible to relevant antibiotics such as vancomycin, negative for haemolysin and gelatinase activities, and did not harbour virulence and antibiotic resistance genes. The assessment of potential probiotic and technological properties showed a low or no lipolytic activity, moderate milk-acidifying ability, high reducing power, proteolytic activity and tolerance to bile (P < 0.05) and good autoaggregation and coaggregation capacities. Two strains designated as CQ and C43 exhibiting high enzymatic activities and bile salt hydrolase activity were found to display high survival under simulated in vitro oral cavity and gastrointestinal tract conditions caused by presence of lysozyme, pepsin, pancreatin, bile salts and acidic pH. This study highlights safe Enterococcus lactis strains with great technological and probiotic potentials for future application as new starter, adjunct, protective or probiotic cultures in food industry.

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Aims: To assess the antimicrobial properties of lactic acid bacteria from Tunisian raw bovine milk. Methods and Results: A bacteriocin-producing Enterococcus faecium strain was isolated from raw cow milk with activity against Gram-positive and Gramnegative bacteria. Antimicrobial substances produced by this strain were sensitive to proteolytic enzymes and were thermostable and resistant to a broad range of pH (2–10). Mode of action of antimicrobial substances was determined as bactericidal. Maximum activity was reached at the end of the exponential growth phase when checked against Listeria ivanovii BUG 496 (2366� 62 AU ml � 1 ). However, maximum antimicrobial activity against Pseudomonas aeruginosa 28753 was recorded at the beginning of the exponential growth phase. Enterococcus faecium GGN7 was characterized as free from virulence factors and was susceptible to tested antibiotics. PCR analysis of the micro-organism’s genome revealed the presence of genes coding for enterocins A and B. Mass spectrometry analysis of RP-HPLC active fractions showed molecular masses corresponding to enterocins A (4835� 77 Da) and B (5471� 56 Da), and a peptide with a molecular mass of 3215� 5 Da active only against Gram-negative indicator strains. The latter was unique in the databases. Conclusions: Enterococcus faecium GGN7 produces three bacteriocins with different inhibitory spectra. Based on its antimicrobial properties and safety, Ent. faecium GGN7 is potentially useful for food biopreservation. Significance and Impact of the Study: The results suggest the bacteriocins from GGN7 strain could be useful for food biopreservation.

Use of lactic acid bacteria (LAB) as probiotics may provide an alternative to the use of antibiotics in aquaculture. Lstrains isolated from wild fish viscera and skin were evaluated for bacteriocin production and safety aspects (lack of antibiotic resistance, production of virulence factors). 16S rRNA gene sequences revealed the presence of Enterococcus faecium (13 isolates) and Lactococcus lactis (3 isolates) from fish samples. Pulsed-field gel electrophoresis analyses of the 13 enterococci isolates showed that they were all clustered, with greater than 95% similarity. However, RAPD analysis revealed significant molecular diversity between enterococci strains. Six enterococci strains were chosen and evaluated for their antibacterial activities. These strains produced a bacteriocin-like substance and exhibited a broad spectrum of inhibition against pathogenic bacteria isolated from diseased fish, including Streptococcus parauberis, Vagococcus spp., and Carnobacterium maltaromaticum, and in particular against the Gram-negative bacteria Flavobacterium frigidarium, Vibrio pectenicida, V. penaeicida, and Photobacterium damselae. The inhibition activity towards bacterial indicator strains was at a maximum when bacteria were grown at 37 degrees C. However, bacteriocin production was observed at 15 degrees C after 12 h of incubation. Only structural genes of enterocins A and B were detected by PCR in the 6 enterococci strains, suggesting the production of these enterocins. In addition, these strains did not harbor any virulence factors or any significant antibiotic resistance, and they tolerated bile. Our results suggest that enterococci are an important part of the bacterial flora of fish and that some strains have the potential to be used as probiotics.