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We used a thiol-faradaic electrochemical differential pulse voltammetry and impedance spectroscopy on a gold-modified screen-printed carbon electrode to quantify Chrysophsins antimicrobial peptides in the fish mucus without prior extraction. We have developed a specific anti-Chrysophsins polyclonal antibody and used ferrocene as a transducing system. The platform has a sensitivity of 30.5 nA.mL.ng -1 (11.28 nA.nM -1 ) in a linear range of 0.1-1 µg.mL -1 and a limit of detection of 0.227 ng.mL -1 (84.15 pM). Selectivity, accuracy, repeatability and stability were validated to meet the guidelines of ligand binding assays. Mean Chrysophsins levels in mucus pools from healthy and thermally unstressed Argyrosomus regius, Dicentrarchus labrax and Sparus aurata fish were 8.763 µM (± 0.007), 7.296 µM (± 0.023) and 8.296 (± 0.044) respectively, within the range of mass spectrometry gill values and below the minimum inhibitory concentration (MIC) of Chrysophsins for fish pathogens. The multi-infected D. labrax pool shows a significant decrease in concentration compared to the healthy and thermally stressed pools (p < 0.0262) with 2.8 µM (± 0.024). The thermally stressed A. regius pool is not significantly different from the other pools with 8.763 µM (± 0.007). This electrochemical platform is a flexible tool for real-time targeting of peptide biomarkers in the real matrix and is suitable for mucosal fluids for early fish welfare monitoring., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025 Elsevier B.V. All rights reserved.)

Background: Vaccination is the most effective way to prevent influenza infection and adverse outcomes; despite global recommendations to vaccinate pregnant persons, access to influenza vaccines remains low. We explored knowledge, attitudes, and practices of pregnant persons to inform actions to improve vaccine uptake., Methods: We pooled data from cross-sectional surveys assessing pregnant persons' attitudes toward influenza vaccines in 8 low- and middle-income countries. Countries used standard methods to measure attitudes and intents toward influenza vaccination. We stratified by presence/absence of a national influenza vaccination program, income group, geographic region, and individual-level factors., Results: Our analysis included 8556 pregnant persons from 8 countries. Most pregnant persons (6323, 74%) were willing to receive influenza vaccine if it was offered for free. Willingness differed by presence of an existing influenza vaccination program; acceptance was higher in countries without programs (2383, 89%) than in those with programs (3940, 67%, P < .001)., Conclusions: Most pregnant persons in middle-income countries, regardless of influenza vaccination program status, were willing to be vaccinated against influenza if the vaccine was provided free of charge. National investments in influenza vaccination programs present an opportunity to avert illness both in pregnant persons themselves and in their newborn babies., Competing Interests: Potential conflicts of interest. All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Introduction: Tree bacterial diseases are a threat in forestry due to their increasing incidence and severity. Understanding tree defence mechanisms requires evaluating metabolic changes arising during infection. Metabolite extraction affects the chemical diversity of the samples and, therefore, the biological relevance of the data. Metabolite extraction has been standardized for several biological models. However, little information is available regarding how it influences wood extract's chemical diversity., Objectives: This study aimed to develop a methodological approach to obtain extracts from different tree species with the highest reproducibility and chemical diversity possible, to ensure proper coverage of the trees' metabolome., Methods: A full factorial design was used to evaluate the effect of solvent type, extraction temperature and number of extraction cycles on the metabolic profile, chemical diversity and antibacterial activity of four tree species., Results: Solvent, temperature and their interaction significantly affected the extracts' chemical diversity, while the number of extraction cycles positively correlated with yield and antibacterial activity. Although 60% of the features were recovered in all the tested conditions, differences in the presence and abundance of specific chemical classes per tree were observed, including organooxygen compounds, prenol lipids, carboxylic acids, and flavonoids., Conclusions: Each tree species has a unique metabolic profile, which means that no single protocol is universally effective. Extraction at 50 °C for three cycles using 80% methanol or chloroform/methanol/water showed the best results and is suggested for studying wood metabolome. These observations highlight the need to tailor extraction protocols to each tree species to ensure comprehensive metabolome coverage for metabolic profiling., Competing Interests: Declarations. Ethical approval: This article does not contain any studies with human and/or animal participants. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

This study aimed to investigate the antibacterial activity of essential oils from Cupressus sempervirens (CSEO) and Eucalyptus globulus (EGEO) against clinical isolates of colistin-resistant E. coli and methicillin-resistant M. sciuri recovered from diseased food-producing animals in Tunisia. The chemical compositions of both essential oils were analyzed using Gas chromatography-mass spectrometry (GC-MS). Antibacterial activity was determined through disk diffusion and microdilution assays, while the fractional inhibitory concentration index (FICI) was used to measure the synergistic effect of CSEO and colistin. The major constituents of CSEO were α-pinene (59.03%), δ-3 Carene (21.48%), D-limonene (5.62%) and α-thujene (3.08%), while EGEO contained eucalyptol (52.1%), α-pinene (26.51%), γ-terpinene (4.81%) and D-limonene (3.54%). The inhibition zone diameters (IZDs) against E. coli and M. sciuri ranged from 6 to 29 mm for CSEO and 9.3 to 28.6 mm for EGEO. Minimum inhibitory concentrations (MICs) showed a range of 4 to 512 µg/mL for CSEO against M. sciuri and 2 to 426.66 µg/mL for EGEO. For E. coli, MICs ranged from 170.66 to 512 µg/mL for CSEO and 53.33 to 512 µg/mL for EGEO. The checkerboard test demonstrated a synergistic effect between CSEO and colistin against two colistin-resistant E. coli isolates, with FICI 3- to 32-fold lower than the MICs of the individual compounds. This study highlights the potency of essential oils from CSEO and EGEO against antibiotic-resistant M. sciuri and E. coli strains from animals in Tunisia and provides evidence of the synergistic effect between CSEO and colistin against clinically relevant colistin-resistant E. coli isolates., Competing Interests: Declarations. Conflict of interest: The authors have not disclosed any competing interests. Ethical Approval: This study was subjected to ethical review and given approval by the ethics committee in animal experimentation (CEEA- ENMV) at the National School of Veterinary Medicine of Sidi Thabet, Ariana, Tunisia, ref: 07. 2024/ISBST. Consent to Participate: Not applicable. Consent for Publication: Not applicable., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Integrated pest management based on the use of biopesticides is largely applied. Experimental bioassays are critical to assess biopesticide biosafety at the ecotoxicological level. In this study, we investigated the effects of the new Bacillus thuringiensis subsp. kurstaki ( Btk )-formulated-based biopesticides BLB1 and Lip, efficiently tested in field assays (IPM-4-CITRUS EC project no. 734921) on two aquatic non-target organisms, precisely the water flea Daphnia magna and the bioluminescent bacteria Aliivibrio fischeri . Acute toxicity studies, carried out in a comparative manner with Delfin ® as the reference bioproduct and the lactose-based Blank formulation, show that no significant toxicity was observed up to 1 g/L. Our results indicated that BLB1- and Lip-formulated new bioproducts are far less toxic than the Delfin ® reference bioproduct.

Background: Major histocompatibility complex class-1-related protein (MR1), unlike human leukocyte antigen (HLA) class-1, was until recently considered to be monomorphic. MR1 presents metabolites in the context of host responses to bacterial infection. MR1-restricted TCRs specific to tumor cells have been described, raising interest in their potential therapeutic application for cancer treatment. The diversity of MR1-ligand biology has broadened with the observation that single nucleotide variants (SNVs) exist within MR1 and that allelic variants can impact host immunity., Methods: The TCR from a MR1-restricted T-cell clone, MC.7.G5, with reported cancer specificity and pan-cancer activity, was cloned and expressed in Jurkat E6.1 TCRαβ- β2M- CD8+ NF-κB:CFP NFAT:eGFP AP-1:mCherry cells or in human donor T cells. Functional activity of 7G5.TCR-T was demonstrated using cytotoxicity assays and by measuring cytokine release after co-culture with cancer cell lines with or without loading of previously described MR1 ligands. MR1 allele sequencing was undertaken after the amplification of the MR1 gene region by PCR. In vivo studies were undertaken at Labcorp Drug Development (Ann Arbor, MI, USA) or Epistem Ltd (Manchester, UK)., Results: The TCR cloned from MC.7.G5 retained MR1-restricted functional cytotoxicity as 7G5.TCR-T. However, activity was not pan-cancer, as initially reported with the clone MC.7.G5. Recognition was restricted to cells expressing a SNV of MR1 (MR1*04) and was not cancer-specific. 7G5.TCR-T and 7G5-like TCR-T cells reacted to both cancer and healthy cells endogenously expressing MR1*04 SNVs, which encode R9H and H17R substitutions. This allelic specificity could be overcome by expressing supraphysiological levels of the wild-type MR1 (MR1*01) in cell lines., Conclusions: Healthy individuals harbor T cells reactive to MR1 variants displaying self-ligands expressed in cancer and benign tissues. Described "cancer-specific" MR1-restricted TCRs need further validation, covering conserved allomorphs of MR1. Ligands require identification to ensure targeting MR1 is restricted to those specific to cancer and not normal tissues. For the wider field of immunology and transplant biology, the observation that MR1*04 may behave as an alloantigen warrants further study. ., Competing Interests: Authors TC, NM, SC, EL, TL, RW, AL, KW, ED, GM, PQY, CM, SD, SK, JS, LW, SP, DH, JD were employed by Enara Bio Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. JLN declared that they were an editorial board member of Frontiers at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Cornforth, Moyo, Cole, Lam, Lobry, Wolchinsky, Lloyd, Ward, Denham, Masi, Qing Yun, Moore, Dhaouadi, Besra, Veerapen, Illing, Vivian, Raynes, Le Nours, Purcell, Kundu, Silk, Williams, Papa, Rossjohn, Howie and Dukes.)

Treating plant bacterial diseases is notoriously difficult because of the lack of available antimicrobials. Pseudomonas syringae pathovar syringae (Pss) is a major pathogen of cherry (Prunus avium) causing bacterial canker of the stem, leaf and fruit, impacting productivity and leading to a loss of trees. In an attempt to find a treatment for this disease, naturally occurring bacteriophage (phage) that specifically target Pss is being investigated as a biocontrol strategy. However, before using them as a biocontrol treatment, it is important to both understand their efficacy in reducing the bacterial population and determine if the bacterial pathogens can evolve resistance to evade phage infection. To investigate this, killing curve assays of five MR phages targeting Pss showed that phage resistance rapidly emerges in vitro, even when using a cocktail of the five phages together. To gain insight to the changes occurring, Pss colonies were collected three times during a 66-h killing curve assay and separately, Pss and phage were also coevolved over 10 generations, enabling the measurement of genomic and fitness changes in bacterial populations. Pss evolved resistance to phages through modifications in lipopolysaccharide (LPS) synthesis pathways. Bacterial fitness (growth) and virulence were affected in only a few mutants. Deletion of LPS-associated genes suggested that LPS was the main target receptor for all five MR phages. Later generations of coevolved phages from the coevolution experiment were more potent at reducing the bacterial density and when used with wild-type phages could reduce the emergence of phage-resistant mutants. This study shows that understanding the genetic mechanisms of bacterial pathogen resistance to phages is important for helping to design a more effective approach to kill the bacteria while minimizing the opportunity for phage resistance to manifest., (© 2024 The Author(s). Microbial Biotechnology published by John Wiley & Sons Ltd.)

Tenascin-C (TNC) is a matricellular and multimodular glycoprotein highly expressed under pathological conditions, especially in cancer and chronic inflammatory diseases. Since a long time TNC is considered as a promising target for diagnostic and therapeutic approaches in anti-cancer treatments and was already extensively targeted in clinical trials on cancer patients. This review provides an overview of the current most advanced strategies used for TNC detection and anti-TNC theranostic approaches including some advanced clinical strategies. We also discuss novel treatment protocols, where targeting immune modulating functions of TNC could be center stage., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The injudicious usage of antibiotics during infections caused by Gram-negative bacteria leads to the emergence of β-lactamases. Among them, the NDM-1 enzyme poses a serious threat to human health. Developing new antibiotics or inhibiting β-lactamases might become essential to reduce and prevent bacterial infections. Nanobodies (Nbs), the smallest antigen-binding single-domain fragments derived from Camelidae heavy-chain-only antibodies, targeting enzymes, are innovative alternatives to develop effective inhibitors. The biopanning of an immune VHH library after phage display has helped to retrieve recombinant antibody fragments with high inhibitory activity against recombinant-NDM-1 enzyme. Nb02NDM-1, Nb12NDM-1, and Nb17NDM-1 behaved as uncompetitive inhibitors against NDM-1 with K i values in the nM range. Remarkably, IC 50 values of 25.0 nM and 8.5 nM were noted for Nb02NDM-1 and Nb17NDM-1, respectively. The promising inhibition of NDM-1 by Nbs highlights their potential application in combating particular Gram-negative infections.

Background: In response to the COVID-19 pandemic, multiple vaccines to protect against COVID-19 disease have been developed rapidly. Precise estimates of vaccine effectiveness (VE) vary according to studies design, outcomes measured and circulating variants. The aim of this study was to evaluate the anti-covid-19 vaccine effectiveness in Tunisia., Methods: We conducted a matched case-control study from 2nd to 15th August 2021. Cases and controls were subjects over 60 years of age, selected from the National testing database, regardless vaccine status. A standardized questionnaire was administered for cases and controls to collect information about vaccination status. For cases, vaccination status was defined based on the number of doses received before becoming ill and excludes doses received during the previous two weeks. For matched controls, a reference date based on the case's date of illness onset was defined in order to look at the control's vaccination status before its corresponding case became ill. The odds-ratio was calculated using simple conditional logistic regression. The VE (95 % confidence intervals) was calculated as (1 - odds ratio for vaccination) × 100 %., Results: A sample of 977 matched peers for age and Gender, were included between August 2, and August 15, 2021. The overall vaccine effectiveness (VE) was 70 % [95 % CI 62.8-75.8 %]. Among our sample, 68.1 % of the male population and 56.4 % of the female population were vaccinated with a VE of 73 % [95 % CI 62.9-80.3 %] and 67 % [95 % CI 55.8-75.3 %] respectively, regardless vaccine scheme (complete or incomplete). VE was higher for the age group [60-70 years[ (72.3 % [95 % CI 62.8-79.3 %]). VE was 77.6 % [95 % CI 70.9-82.8 %] to prevent both symptomatic and asymptomatic forms of the disease. Moreover, in prevention from severe forms (treated with oxygen-therapy or admission to an Intensive-care-unit) VE was 86.6 % [95 % CI 75.6-92.7 %] and 98.4 % [95 % CI [79.2-99.8 %] in prevention from COVID-19 deaths with a complete anti-Covid vaccination scheme., Conclusion: The results of our study showed that the anti-Covid-19 vaccines used in Tunisia are efficient to prevent both SARS-COV-2 infections and severe forms related to the disease. This study provided important data on the performance of vaccines in real-world settings that guide decisions about vaccine sustained use., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Due to the emergence of the SARS-CoV-2 B.1.1.7 (Alpha) variant in the UK in 2020 and its risk of increased transmission, the Ministry of Health in Tunisia implemented a sequencing surveillance strategy for SARS-CoV-2. The aim of this study was to analyze SARS-CoV-2 genomic surveillance data in Tunisia (January 2021-February 2022) and to assess the implementation of the sequencing strategy for SARS-CoV-2 in accordance with national recommendations and the guidance for SARS-CoV-2 genomic surveillance for public health goals. A descriptive study of all sequenced RT-PCR samples sequenced (January 2021-February2022). An internal audit was also done to assess the compliance against standards covering national recommendations and the Guidance for SARS-CoV-2 genomic surveillance for public health goals. A total of 12 simple or composite requirements related to the following areas were included in the audit standards: sampling (one requirements); data collection/analysis (six requirements); partnership (one requirement); and ethical considerations (one requirement). A total of 4819 samples were sent to laboratories and 4278 samples were sequenced. A total of 3648 samples were classified. Positive variants of concern (VOC) samples were 80.92%, differentiated as follows: Alpha, 40.24%; Beta, 0.24%; Gamma, 0.03%; Delta, 45.26%; and Omicron, 14.19%. Three principal phases of VOCs per ISO-week were shown: Alpha 3/2021-25/2021; Delta 26/2021-2/2022; and Omicron 3/2022-6/2022. Levels of compliance were identified; from a total of 12 requirements, 7 were considered as "not met", 4 as "partially met", and 1 as "fully met" but including not totally achieved objectives. In conclusion, the internal audit of the national SARS-CoV-2 sequencing strategy revealed an overall "not met" level of compliance. The results offered a trigger to collaborate with all stakeholders to develop a surveillance strategy for early detection and response to outbreaks caused by VOCs.

The extent of the SARS-CoV-2 circulation and the COVID-19 epidemic in Tunisia three months after virus circulation was unknown. The aim of this study was to determine the extent of SARS-CoV-2 infection among household contacts of confirmed COVID-19 cases living in Hot spot areas of Great Tunis, Tunisia by estimating the seroprevalence of antibodies anti SARS-CoV-2 and to identify factors associated to seroprevalence at the first stage of the pandemic in order to guide decision making and to constitute a baseline for further longitudinal analysis of protective immunity to SARS-CoV-2. The National Observatory of New and Emerging Diseases (ONMNE), Ministry of Health Tunisia (MoH), with the support of the Office of the World Health Organization Representative in Tunisia and the WHO Regional Office for the Eastern Mediterranean (EMRO)), conducted a household cross-sectional survey on April 2020 in Great Tunis (Tunis, Ariana, Manouba and Ben Arous). The study was based on the WHO seroepidemiological investigation protocol for SARS-CoV-2 infection. SARS-CoV-2 specific antibodies (IgG and IgM) were qualitatively detected using a lateral immunoassay that detect SARS-CoV-2 nucleocapsid protein and administered by the interviewers. The included subjects were confirmed COVID-19 cases and their households contacts resided in hot spot areas (cumulative incidence rate ≥ 10 cases/100,000 inhabitants) of Great Tunis. Results: In total, 1165 subjects were enrolled: 116 confirmed COVID-19 cases (43 active cases and 73 convalescents cases) and 1049 household contacts resided in 291 households. The median age of participants was 39.0 with 31 years' interquartile range (Min = 8 months; Max = 96 years). The sex ratio (M/F) was 0.98. Twenty-nine per cent of participants resided in Tunis. The global crude seroprevalence among household contacts was 2.5% (26/1049); 95% CI 1.6-3.6%, 4.8%; 95% CI 2.3-8.7% in Ariana governorate and 0.3%; 95% CI 0.01%-1.8% in Manouba governorate. In multivariate analysis, the associated factors independently related to seroprevalence were age ≥25 years (aOR = 5.1; 95% CI 1.2-22.0), history of travel outside Tunisia since January 2020 (aOR = 4.6; 95% CI 1.7-12.9), symptomatic illness in the previous four months (aOR = 3.5; 95% CI 1.4-9.0) and governorate of residence ( p = 0.02). The low seroprevalence estimated among household contacts in Great Tunis reflect the effect of public health measures early taken (national lockdown, borders closed, remote work), the respect of non-pharmaceutical interventions and the efficacy of COVID-19 contact-tracing and case management at the first stage of the pandemic in Tunisia.

Background: Escherichia coli ( E. coli ) is one of the main etiological agents responsible for bovine mastitis (BM), neonatal calf diarrhea (NCD), and avian colibacillosis (AC). This study aimed to assess resistance and virulence genes content, biofilm-forming ability, phylogenetic groups, and genetic relatedness in E. coli isolates recovered from clinical cases of BM, NCD, and AC., Materials/methods: A total of 120 samples including samples of milk ( n = 70) and feces ( n = 50) from cows with BM and calves with NCD, respectively, were collected from different farms in Northern Tunisia. Bacterial isolation and identification were performed. Then, E. coli isolates were examined by disk diffusion and broth microdilution method for their antimicrobial susceptibility and biofilm-forming ability. PCR was used to detect antimicrobial resistance genes (ARGs), virulence genes (VGs), phylogenetic groups, and Enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) for their clonal relationship., Results: Among the 120 samples, 67 E. coli isolates (25 from BM, 22 from AC, and 20 from NCD) were collected. Overall, 83.6% of isolates were multidrug resistant. Thirty-six (53.73%) isolates were phenotypically colistin-resistant (CREC), 28.3% (19/67) were ESBL producers (ESBL-EC), and forty-nine (73.1%) formed biofilm. The bla TEM gene was found in 73.7% (14/19) of isolates from the three diseases, whilst the bla CTXM-g-1 gene was detected in 47.3% (9/19) of isolates, all from AC. The most common VG was the fim A gene (26/36, 72.2%), followed by aer (12/36, 33.3%) , cnf1 (6/36, 16.6%) , pap C (4/36, 11.1%), and stx 1 and stx 2 genes (2/36; 5.5% for each). Phylogenetic analysis showed that isolates belonged to three groups: A (20/36; 55.5%), B2 (7/36; 19.4%), and D (6/36; 16.6%). Molecular typing by ERIC-PCR showed high genetic diversity of CREC and ESBL E. coli isolates from the three animal diseases and gave evidence of their clonal dissemination within farms in Tunisia., Conclusion: The present study sheds new light on the biofilm-forming ability and clonality within CREC and ESBL-EC isolated from three different animal diseases in Tunisian farm animals.

Introduction: the purpose of this study was to describe the clinical and epidemiological features of COVID-19-related deaths in Tunisia notified at the ONMNE (National Observatory of New and emerging Diseases) between 2 nd March 2020 and 28 th February 2021 and to compare COVID-19-related deaths recorded in Tunisia with the international data., Methods: we conducted a national prospective longitudinal descriptive study of data collected from the National Surveillance System of SARS-CoV-2 infection of the ONMNE, Ministry of Health. All COVID-19-related deaths that occurred in Tunisia between March 2020 and February 2021 were included in this study. Data were collected from hospitals, municipalities and regional health departments. Death notifications were collected from multiple data sources (triangulation): The Regional Directorate of Basic Health Care, the ShocRoom (Strategic Health Operations Center), public and private health facilities, the Crisis Unit of the Presidency of the Government, the Directorate for Hygiene and Environmental Protection, the Ministry of Local Affairs and the Environment, as part of the follow-up of confirmed cases by the ONMNE team, positive RT-PCR / TDR post mortem results., Results: during this study, 8051 deaths were recorded, corresponding to a proportional mortality of 10.4%. The median age was 73 years, with an interquartile range of 17 years. Sex-ratio (M/F) was 1.8. The crude death rate was 69.1/100 000 inhabitants and fatality rate was 3.5%. The analysis of the epidemic curve showed 2 peaks of deaths on 29 th October 2020 and 22 nd January 2021, with 70 and 86 deaths notified respectively. The spatial distribution of mortality showed that the southern Tunisian region had the highest mortality rate. Patients aged 65 and over were most affected (73.7% of cases) with a crude mortality rate of 570.9/100,000 inhabitants and a fatality rate of 13.7%., Conclusion: prevention strategy based on public health measures must be reinforced by the rapid deployment of anti-COVID-19 vaccination, especially for people at risk of death., Competing Interests: Les auteurs ne déclarent avoir aucun conflit d´intérêt., (Copyright: Sonia Dhaouadi et al.)

Objectives: In this study, we aimed to assess the extent of dissemination of methicillin-resistant Mammaliicoccus sciuri in animal farms in Tunisia and evaluate the distribution of virulence and methicillin resistance genes in the M. sciuri population., Methods: Staphylococci and mammaliicocci isolated from unhealthy animals and healthy humans from adjacent farms in Tunisia were characterized for antimicrobial susceptibility, biofilm formation, agglutination, and hemolysis abilities. Mammaliicoccus sciuri relatedness and content in antibiotic resistance and virulence genes were analyzed by whole-genome sequencing (WGS)., Results: Mammaliicoccus sciuri was the most prevalent species (46.2%), showing the highest resistance rates to fusidic acid (94.6%), oxacillin (73%), penicillin (40.5%), clindamycin (37%), ciprofloxacin (27%), and cefoxitin (24.3%). Some isolates carried genes encoding resistance to nine different antibiotic classes. mecA was found in 35% of M. sciuri and mecC in 16.2%. All isolates carrying mecC were of S. sciuri subspecies carnaticus and carried the hybrid element SCCmec-mecC. Mammaliicoccus sciuri were able to produce strong biofilm (27%) and have clumping ability (16%). Additionally, they carried genes for capsule production (cap8, 100%), iron-regulated surface determinants (isdE, 24%; isdG, 3%), and virulence regulation (clpC and clpP, 100%). Single nucleotide polymorphisms (SNPs) analysis showed that 17 M. sciuri cross-transmission events probably occurred between different animal species and farms. Moreover, SCCmec was estimated to have been acquired five times by S. sciuri subsp. carnaticus., Conclusion: Multidrug resistant and pathogenic M. sciuri were frequently disseminated between different animal species within the farm environment. mecA and mecC can be disseminated by both frequent acquisition of the SCCmec element and clonal dissemination., Competing Interests: Competing interests None declared, (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Scorpion envenoming is a severe health problem in many regions causing significant clinical toxic effects and fatalities. In the Middle East/North Africa (MENA) region, Buthidae scorpion stings are responsible for devastating toxic outcomes in human. The only available specific immunotherapeutic treatment is based on IgG fragments of animal origin. To overcome the limitations of classical immunotherapy, we have demonstrated the in vivo efficacy of NbF12-10 bispecific nanobody at preclinical level. Nanobodies were developed against BotI analogues belonging to a distinct structural and antigenic group of scorpion toxins, occurring in the MENA region. From Buthus occitanus tunetanus venom, BotI-like toxin was purified. The 41 N-terminal amino acid residues were sequenced, and the LD 50 was estimated at 40 ng/mouse. The BotI-like toxin was used for dromedary immunization. An immune VHH library was constructed, and after screening, two nanobodies were selected with nanomolar and sub-nanomolar affinity and recognizing an overlapping epitope. NbBotI-01 was able to neutralize 50% of the lethal effect of 13 LD 50 BotI-like toxins in mice when injected by i.c.v route, whereas NbBotI-17 neutralized 50% of the lethal effect of 7 LD 50 . Interestingly, NbBotI-01 completely reduced the lethal effect of the 2 LD 50 of BotG50 when injected at 1:4 molar ratio excess. More interestingly, an equimolar mixture of NbBotI-01 with NbF12-10 neutralized completely the lethal effect of 7 and 5 LD 50 of BotG50 or AahG50, at 1:4 and 1:2 molar ratio, respectively. Hence, NbBotI-01 and NbF12-10 display synergic effects, leading to a novel therapeutic candidate for treating Buthus occitanus scorpion stings in the MENA region., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ben Abderrazek, Ksouri, Idoudi, Dhaouadi, Hamdi, Vincke, Farah, Benlasfar, Majdoub, El Ayeb, Muyldermans and Bouhaouala-Zahar.)

The extracellular matrix molecule Tenascin-C (TNC) promotes cancer and chronic inflammation by multiple mechanisms. Recently, TNC was shown to promote an immune suppressive tumor microenvironment (TME) through binding soluble chemoattracting factors, thus retaining leukocytes in the stroma. TNC also binds to fibronectin (FN) and other molecules, raising the question of a potential common TNC binding mechanism. By sequence comparison of two TNC-interacting domains in FN, the fifth (FN5) and thirteenth (FN13) fibronectin type III domains we identified a MAtrix REgulating MOtif "MAREMO" or M-motif that is highly conserved amongst vertebrates. By sequence analysis, structural modeling and functional analysis we found also putative M-motifs in TNC itself. We showed by negative staining electron microscopic imaging that the M-motif in FN mediates interactions with FN as well as with TNC. We generated two M-motif mimetic peptides P5 and P13 resembling the M-motif in FN5 and FN13, respectively. By using structural information we modelled binding of these M-motif mimetics revealing a putative MAREMO binding site MBS in FN5 and TN3, respectively overlapping with the M-motif. We further demonstrated that the M-motif mimetic peptides blocked several functions of TNC, such as binding of TNC to FN, cell rounding on a mixed FN/TNC substratum, FN matrix expression and subsequent assembly, TNC-induced signaling and gene expression, TNC chemokine binding and dendritic cell retention, thus providing novel opportunities to inhibit TNC actions. Our results suggest that targeting the MAREMO/MBS interaction could be exploited for reducing inflammation and matrix functions in cancer and fibrosis., Competing Interests: Declaration of competing interests The discovery of MAREMO has been protected by patent No. WO2021233766A1 on “Compounds Binding for use in the treatment of diseases”., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: The uptake and acceptance of the influenza vaccine (IV) among pregnant women remain unknown in Tunisia despite the increased influenza-related complications and death. The present study aimed to assess the IV uptake and acceptability and to describe related knowledge and attitudes among pregnant women in Tunisia., Methods: A cross-sectional study was conducted in 84 Tunisian healthcare facilities over a period of three months (from March to May 2019). All pregnant women aged ≥18 years who sought antenatal care in related health structures were included in this study based on a multistage self-weighted sampling. We measured knowledge and attitudes towards the IV and assessed factors related to willingness for its uptake., Results: The questionnaire was completed by 1157 pregnant women. More than half of the participants (60.2%; 95% confidence interval [CI] [57.3%-63.0%]) reported awareness about the IV. Among included PW, only 4.6%; 95% CI [3.5%-6.1%] received it during their current pregnancy. However, (36.8%; 95% CI [34.0%-39.6%]) declared their willingness to receive the vaccine in the next pregnancy. Recommendation by healthcare providers, identified to be the most trustful source of information, was the main reason for acceptance. However, the intention to accept the IV by pregnant women was significantly associated with such recommendation and perceived safety and effectiveness of this vaccine., Conclusion: Antenatal care visits are a precious opportunity that should not be missed by health care providers and especially gynecologists to promote the IV uptake by pregnant women in Tunisia., Competing Interests: We have no conflicts of interest to disclose.

Background: The aim of this study was to characterize the transmission chains and clusters of COVID-19 infection in Tunisia., Methods: All cases were confirmed by Reverse Transcriptase Polymerase Chain Reaction of a nasopharyngeal specimen. Contact tracing is undertaken for all confirmed cases in order to identify close contacts that will be systematically screened and quarantined. Transmission chains were identified based on field investigation, contact tracing, results of screening tests and by assessing all probable mode of transmission and interactions., Results: As of May 18, 2020, 656 cases out of a total of 1043 confirmed cases of Coronavirus disease 2019 belong to 127 transmission chains identified during the epidemic (mean age 42.36 years, Standard deviation 19.56 and sex ratio 0.86). The virus transmission is the most concentrated in the governorate of Tunis (31.5%), Ariana (10.2%) and Ben Arous (10.2%). Virus transmission occurred 50 times (9.72% of secondary transmission events) between two different governorates. A maximum of seven generations of secondary infection was identified, whereas 62% of these secondary infections belong the first generation. A total of 11 "super spreader" cases were identified in this investigation. Four large clusters have been identified. The evolution of secondary cases highlighted two peaks: one in 2nd April and a second in 16 th April whereas imported cases caused local transmission of virus during the early phase of the epidemic., Conclusion: Correct contact tracing and early active case finding is useful to identify transmission chains and source of infection in order to contain the widespread transmission in the community.

The extracellular matrix (ECM) molecule Tenascin-C (TNC) is well-known to promote tumor progression by multiple mechanisms. However, reliable TNC detection in tissues of tumor banks remains limited. Therefore, we generated dromedary single-domain nanobodies Nb3 and Nb4 highly specific for human TNC (hTNC) and characterized the interaction with TNC by several approaches including ELISA, western blot, isothermal fluorescence titration and negative electron microscopic imaging. Our results revealed binding of both nanobodies to distinct sequences within fibronectin type III repeats of hTNC. By immunofluroescence and immunohistochemical imaging we observed that both nanobodies detected TNC expression in PFA and paraffin embedded human tissue from ulcerative colitis, solid tumors and liver metastasis. As TNC impairs cell adhesion to fibronectin we determined whether the nanobodies abolished this TNC function. Indeed, Nb3 and Nb4 restored adhesion of tumor and mesangial cells on a fibronectin/TNC substratum. We recently showed that TNC orchestrates the immune-suppressive tumor microenvironment involving chemoretention, causing tethering of CD11c+ myeloid/dendritic cells in the stroma. Here, we document that immobilization of DC2.4 dendritic cells by a CCL21 adsorbed TNC substratum was blocked by both nanobodies. Altogether, our novel TNC specific nanobodies could offer valuable tools for detection of TNC in the clinical practice and may be useful to inhibit the immune-suppressive and other functions of TNC in cancer and other diseases., Competing Interests: MM was employed by Colzyx AB. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Dhaouadi, Ben Abderrazek, Loustau, Abou-Faycal, Ksouri, Erne, Murdamoothoo, Mörgelin, Kungl, Jung, Ledrappier, Benlasfar, Bichet, Chiquet-Ehrismann, Hendaoui, Orend and Bouhaouala-Zahar.)

Background: The outbreak of coronavirus disease (COVID-19) continues to constitute an international public health concern. Few data are available on the duration and prognostic factors of the disease. We aimed to study the recovery time among a Tunisian cohort of COVID-19 confirmed patients and identify the prognostic factors., Methods: A retrospective, nationwide study was conducted from March 2 to May 8, 2020, recruiting all patients who were diagnosed with COVID-19, by RT-PCR methods, in Tunisia. Data were collected via phone call interview. Kaplan-Meir Methods and Cox proportional hazards regression models were, respectively, used to study the recovery time and estimate its prognostic factors., Results: One thousand and thirty patients with COVID-19 (aged 43.2 ± 18.2 years, 526 female (51.1%)) were enrolled. Among them 141 (14.8%) were healthcare professionals. Out of 173 patients (17.8%) admitted to the hospital, 47 were admitted in an intensive care unit. Among 827 patients who didn't require specialized care, 55.5% were self-isolated at home, while the rest were in specialized centers. Six hundred and two patients were symptomatic. A total of 634 (61.6%) patients have recovered and 45 (4.4%) patients died. The median duration of illness was estimated to be 31 days (95% CI: [29-32]). Older age (HR = 0.66, CI:[0.46-0.96], P = 0.031) and symptoms (HR = 0.61, CI:[0.43-0.81], P = 0.021) were independently associated with a delay in recovery time. Being a healthcare professional (HR = 1.52, CI: [1.10-2.08], P = 0.011) and patients in home isolation compared to isolation centers (HR = 2.99, CI: [1.85-4.83], P < 10¯ 3 ) were independently associated with faster recovery time., Conclusion: The duration of illness was estimated to be 1 month. However, this long estimated duration of illness may not equate to infectiousness. A particular attention must to be paid to elderly and symptomatic patients with closer monitoring.

Background: A wide range of neurological manifestations has been described in COVID-19., Methods: In this nationwide retrospective observational study, patients in Tunisia diagnosed with COVID-19 between the 2nd of March and the 16th of May 2020 were contacted by telephone. We collected demographic and clinical data and specified characteristics and evolution of main neurological symptoms., Results: Of 1034 confirmed COVID-19 patients, 646 were included (mean age 42.17 years old) and 466 (72.1%) had neurological symptoms. Neurological symptoms were isolated 22.7% (n = 106). Headache was the most frequent neurological symptom (n = 279, 41.1%): mainly frontotemporal (n = 143, 51.1%) and mild or moderate (n = 165, 59.1%). When associated with fever (n = 143, 51.3%), headache was more likely to be severe and present at onset. Recovery was reported in 83.2%. Smell and taste impairment were found in 37.9% (n = 245) and 36.8% (n = 238) respectively. Among them, 65.3% (156/239) were anosmic and 63.2% (146/231) were ageusic. A complete improvement was found in 72.1% (174/240) of smell impairment and in 76.8% (179/233) of taste impairment. Myalgia (n = 241, 37.3%) and sleep disturbances (n = 241, 37.3%) were also frequent. Imported cases had more neurological symptoms (p = 0.001). In 14.5%, neurological symptoms preceded the respiratory signs (RS). RS were associated with more frequent (p = 0.006) and numerous (p < 0.001) neurological symptoms., Conclusions: Neurological symptoms in COVID-19 are frequent, can be isolated and present at onset. A total recovery is the most recorded outcome. RS are predictive of neurological symptoms. Studies in to virus and host genetics should be considered to understand the different phenotypes.

Introduction: Metabolic syndrome (MetS) is a worldwide major public health problem. In Tunisia, few studies have focused on MetS in general population and in military setting in particular., Aim: To determine the prevalence of the MetS in a military population at Bizerte garrison (Tunisia) and to identify its associated factors., Methods: An analytical cross sectional study was conducted in Bizerte military garrison during the period 2015-2017. The study population was the active military persons who were presents during the time of the survey and assigned at the three main navy, air force and army units of Bizerte garrison. The adopted definition for the diagnosis of MetS was the International Diabetes Federation 2005 (IDF 2005). Multivariate analysis using a binary logistic regression model to identify independent factors to MetS (variable of interest) was performed. Data entry and analysis were performed using SPSS 20.0 Software., Results: During the study period, 2500 active military were enrolled among them 2418 men. The mean age was 36.6 ± 9.1 years [20.0 - 59.0]. The prevalence of MetS was 17.7%. MetS was significantly higher among participants aged 40 and above, (23.8% vs 13.1%), single (21.6% vs 13.7%), diabetic (38.5% vs 15.5%), and assigned in the air force (23.1% vs 14.6%) compared to the rest of the study population. The independent factors identified in multivariate analysis were age, armed forces' affiliation and marital status., Conclusion: The prevalence of MetS in our military population was relatively high despite of the medical criteria imposed before incorporation. Population and targeted approach are needed to prevent this scourge and to protect from the complications.

Aim: To describe the burden of breast and gynecological cancers in Tunisia in 2017, to analyze the trend between 1990 and 2017 and to performit's prediction by 2030., Methods: This was a study using data estimated by the Institute for Health Metrics and Evaluation (IHME) for years of life by breast cancer and gynecological cancers: ovary, cervix and uterus (endometrium and other unspecified parts of the uterus) in Tunisia in 2017.The trend analysis of age standardized DALYrate between 1990 and 2017 was analyzed by using Join Point software.The projection of number of DALY and YLL by 2030 was conducted by SPSS software using Age-Period-Cohort (APC) models., Results: In 2017, the age standardized DALYs rate for breast and gynecological cancers was 538.5/100,000 women. Breast cancer proved observed the highest rate of standardized DALY rate with 378.5/100,000 women followed by ovarian cancer (rate standardized DALY=72.7/100,000 women).The standardized DALY rate increased between 1990 and 2017 except for cervix cancer and uterine cancer which have been on a downward trend. According to the same conditions between 1990 and 2017, the age standardized DALY rate in 2030 will reach 674.6/100,000 women (95% CI=667.2/100,000-682.1/100,000)., Conclusion: Strengthening prevention strategy against cancer in general and women's cancers in particularis strongly recommended to reduce cancer burden and to changeits trend.

Background: Describing transmission dynamics of the outbreak and impact of intervention measures are critical to planning responses to future outbreaks and providing timely information to guide policy makers decision. We estimate serial interval (SI) and temporal reproduction number (R t ) of SARS-CoV-2 in Tunisia., Methods: We collected data of investigations and contact tracing between March 1, 2020 and May 5, 2020 as well as illness onset data during the period February 29-May 5, 2020 from National Observatory of New and Emerging Diseases of Tunisia. Maximum likelihood (ML) approach is used to estimate dynamics of R t ., Results: Four hundred ninety-one of infector-infectee pairs were involved, with 14.46% reported pre-symptomatic transmission. SI follows Gamma distribution with mean 5.30 days [95% Confidence Interval (CI) 4.66-5.95] and standard deviation 0.26 [95% CI 0.23-0.30]. Also, we estimated large changes in R t in response to the combined lockdown interventions. The R t moves from 3.18 [95% Credible Interval (CrI) 2.73-3.69] to 1.77 [95% CrI 1.49-2.08] with curfew prevention measure, and under the epidemic threshold (0.89 [95% CrI 0.84-0.94]) by national lockdown measure., Conclusions: Overall, our findings highlight contribution of interventions to interrupt transmission of SARS-CoV-2 in Tunisia.

The genus Rhodococcus contains several species with agricultural, biotechnological and ecological importance. Within this genus, many phyllosphere, rhizosphere and endosphere strains are plant growth promoting bacteria, whereas strains designated as R. fascians are plant pathogens. In this study, we isolated 47 Rhodococcus strains from a range of herbaceous and woody plant species. Phylogenetic analysis based on 16S rDNA, gyr B and alk B genes was used to compare our strains with type strains of Rhodococcus . For most of our strains, sequence similarity of the 16S rDNA, gyr B and alk B regions to type strains ranged from 98-100 %. Results of the concatenated gene sequence comparisons identified 18 strains of R. fascians and three strains of R. kroppenstedtii . The remaining strains were unclassified, and may represent novel species of Rhodococcus . Phylogenetic analysis based on gyr B sequences provided a more precise classification of our strains to species level than 16S rDNA sequences, whereas analysis of alk B sequences was unable to identify strains with orange-coloured colonies to species level.

New Delhi Metallo-β-lactamase-1 (NDM-1) is the most prevalent type of metallo-β-lactamase, able to hydrolyze almost all antibiotics of the β-lactam group, leading to multidrug-resistant bacteria. To date, there are no clinically relevant inhibitors to fight NDM-1. The use of dromedary polyclonal antibody inhibitors against NDM-1 represents a promising new class of molecules with inhibitory activity. In the current study, immunoreactivities of dromedary Immunoglobulin G (IgG) isotypes containing heavy-chain and conventional antibodies were tested after successful immunization of dromedary using increasing amounts of the recombinant NDM-1 enzyme. Inhibition kinetic assays, performed using a spectrophotometric method with nitrocefin as a reporter substrate, demonstrated that IgG1, IgG2, and IgG3 were able to inhibit not only the hydrolytic activity of NDM-1 but also Verona integron-encoded metallo-β-lactamase (VIM-1) (subclass B1) and L1 metallo-β-lactamase (L1) (subclass B3) with inhibitory concentration (IC 50) values ranging from 100 to 0.04 μM. Investigations on the ability of IgG subclasses to reduce the growth of recombinant Escherichia coli BL21(DE3)/codon plus cells containing the recombinant plasmid expressing NDM-1, L1, or VIM-1 showed that the addition of IgGs (4 and 8 mg/L) to the cell culture was unable to restore the susceptibility of carbapenems. Interestingly, IgGs were able to interact with NDM-1, L1, and VIM-1 when tested on the periplasm extract of each cultured strain. The inhibitory concentration was in the micromolar range for all β-lactams tested. A visualization of the 3D structural basis using the three enzyme Protein Data Bank (PDB) files supports preliminarily the recorded inhibition of the three MBLs.

Tenascin-C (TNC) and tenascin-W (TNW), large hexameric glycoproteins overexpressed in the tumor microenvironment, are useful tumor biomarkers for theranostic applications. For now, polyclonal and monoclonal antibodies, as well as aptamers targeting TNC and TNW have been developed. However, the immunostaining sensitivity of antibodies is very heterogenous. The main aim of this study was to generate antibodies in dromedary that detect TNC and TNW, respectively. We show that immune sera from immunized dromedaries are able to specifically bind native TNC and TNW by ELISA and also to detect TNC and TNW in matrix tracks of mammary tumors by immunostaining. Furthermore, we demonstrate that purified IgG subtypes are able to interact specifically with TNC or TNW by ELISA and immunostaining. These camelid antibodies are a good basis to develop tools for the detection of TNC and TNW in the tumor microenvironment and could potentially have a broader application for early diagnosis of solid cancers., Competing Interests: Declaration of competing interest On behalf of the following co-authors, Balkiss Bouhaouala-Zahar, corresponding author from Laboratory of Venoms and Therapeutic Molecules, Institut Pasteur Tunis, 13 Place Pasteur, BP74, 1002 Tunis, Tunisia, declare No conflict of Interest. Kinetics of anti-mTNC/mTNW (A) and anti-hTNC/hTNW (C) immune responses elicited in D1 and D2 dromedaries, respectively (1/8000) and measurement at the indicated time points upon immunization. (B) The titer of antibodies raised against hTNC (0.5 μg/ml) in the tested serial dilutions (1:2000 to 1:256 000). (D) Cross-antigenic reactivity between S1 and S2 sera in comparison to the serum taken as control collected from non immunized dromedary (i.e. S1, S2 and C, respectively) towards hTNC and mTNC (0. 5 μg/ml). Tissues from mammary gland tumors of NeuNT (A, G), NeuNT-TNCKO (B,H) mice and tissues from NT193WT/shC (C,I) and NT193KO/shTNC tumors (D,J) were stained with MTn12 (red) and dromedary S1 and S2 sera (green). NT193WT/shC (E) and NT193KO/shTNC tumors (F) were stained with MTn12 (red), S1 serum (green) and anti-mTNW (cyan). S1 serum from dromedary (D1) immunized also against mTNW, recognizes murine TNW which is similarly detected by anti-mTNW (E, F). (A, B) S1 dilution: 1:5000, MTn12 dilution: 1:400. (C, D) S1 dilution: 1:2000, MTn12 dilution: 1:400. (E, F) S1 dilution: 1:2000, MTn12 dilution:1:400, anti-mTNW antibody dilution: 1:200. (G, H) S2 dilution: 1:500, MTn12 dilution: 1:400. (I, J) S2 dilution: 1:1000, MTn12 dilution: 1:400. Scale Bar: 7 μm., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Objectives: Colibacillosis caused by avian pathogenic Escherichia coli (APEC) is considered a major hindrance in poultry farming worldwide. This study aimed to characterize the genetic content and the relatedness between multidrug-resistant E. coli isolates from broiler chickens died due to colibacillosis from three farms from Tunisia., Methods: One hundred samples were collected from chickens' fresh carcasses from three poultry farms in Tunisia. E. coli isolation and identification were performed. Then, antimicrobial susceptibility regarding antibiotics, the ability to produce β-lactamases and minimum inhibitory concentration for colistin were determined according to Clinical and Laboratory Standards Institute guidelines. β-Lactam and non-β-lactam antimicrobial resistance genes, integrons, virulence genes, and phylogenetic groups were investigated using polymerase chain reaction. The genetic relatedness of the E. coli isolates was analysed by pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST)., Results: A high infection rate of E. coli (50%) in infected organs of chickens was observed. The majority of E. coli isolates were multidrug resistant (96%); among them, 24% were colistin resistant and 30% were ESBL producing. Seven of 12 colistin-resistant isolates harboured the mcr-1 gene; among them, 10 were ESBL producing and carried bla CTX-M-1 , bla TEM , and bla SHV β-lactamase-encoding genes. E. coli isolates were assigned to different phylogroups but most of them (74%) belonged to the pathogenic phylogroup B2. Molecular typing by PFGE showed that some E. coli isolates harbouring ESBL-mcr-1 genes were clonally related. MLST revealed the presence of four different ST lineages among ESBL- and mcr-1-carrying E. coli: ST4187, ST3882; ST5693, and ST8932 with clonal dissemination of E. coli ST4187 between two of the farms., Conclusion: This is the first report of ESBL-mcr-1-carrying E. coli isolates of a clinically relevant phylogenetic group (B2) from chickens that died due to colibacillosis in Tunisian poultry farms., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)