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Campylobacter species, especially C. coli and C. jejuni, have been associated with a range of human gastrointestinal diseases. During the last two decades, due to the irrational use of antibiotics in poultry farms, high rates of antimicrobial resistance have been globally reported in C. coli and C. jejuni isolates. Recently, acquired linezolid-resistance mechanisms have been reported in Campylobacter spp. isolates, which is a cause of concern to human health. In this study, we performed a retrospective analysis of 139 C. coli isolates previously collected from broilers (n = 41), laying hens (n = 53), eggs (n = 4), and environment (n = 41) to detect acquired genes implicated in linezolid resistance. Isolates were tested for their susceptibility to antimicrobial agents using the Kirby-Bauer disk diffusion assay. Chloramphenicol- and linezolid-resistant isolates were subjected to PCR screening for the following genes: fexA, fexB, floR, RE-cmeABC, cfrA, and optrA. The genetic relatedness of eight multidrug-resistant isolates was determined by multilocus sequence typing (MLST). Among the 139 C. coli isolates, high rates of resistance (57.55%-100%) were detected toward nalidixic acid, ciprofloxacin, erythromycin, azithromycin, ampicillin, chloramphenicol, linezolid, and kanamycin. Among 135 chloramphenicol-resistant isolates, the optrA, cfr, fexA floR, RE-cmeABC, and fexB genes were detected in 124 (124/135, 91.85%), 108 (80%), 105 (77.7%), 64 (47.4%), 56 (41, 48%), and 27 (20%) isolates, respectively. In addition, the majority of isolates harbored more than one of these genes. The selected eight isolates belonged to the same sequence type ST13450, which is a new sequence type (ST), not belonging to ST828 and ST1150 complexes. In conclusion, the emergence of optrA gene in Campylobacter spp. isolates makes this genus an optrA reservoir and vector to other pathogens such as Staphylococcus aureus and Enterococcus spp., which is a cause of concern for human and animal health.
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The aim of this study is to investigate the occurrence of plasmid mediated quinolone resistance (PMQR) determinants in Campylobacter coli isolates collected from broilers, laying hens and poultry farm environments. One hundred and thirty-nine C. coli isolates were isolated from broilers (n = 41), laying hens (n = 53), eggs (n = 4) and the environment (n = 41) of 23 poultry farms located in northeastern of Tunisia. Antimicrobial susceptibility testing was performed on all isolates according to the recommendation of the European Committee on Antimicrobial Susceptibility Testing guidelines. The detection of PMQR genes: qnrA, qnrB, qnrC, qnrD, qnrS, qepA, and aac(6)-Ib gene was performed using polymerase chain reaction (PCR) and specific primers. aac(6')-Ib amplicons were further analyzed by digestion with BtsCI to identify the aac(6')-Ib-cr variant. Mutations in GyrA and the occurrence of RE-CmeABC efflux pump were determined by mismatch amplification mutation assay (MAMA) PCR and PCR, respectively. In addition, eleven isolates were selected to determine their clonal lineage by MLST. The 139 C. coli isolates were resistant to ciprofloxacin, and 86 (61.8%) were resistant to nalidixic acid. High rates of resistance were also observed toward erythromycin (100%), azithromycin (96.4%), tetracycline (100%), chloramphenicol (98.56%), ampicillin (66.1%), amoxicillin-clavulanic acid (55.39%), and kanamycin (57.55%). However, moderate resistance rates were observed for gentamicin (9.35%) and streptomycin (22.3%). All quinolone-resistant isolates harbored the Thr-86-Ile amino acid substitution in GyrA, and the RE-CmeABC efflux pump was detected in 40.28% of isolates. Interestingly, the qnrB, qnrS, qepA, and aac(6')-Ib-cr were detected in 57.7%, 61.15%, 21.58%, and 10% of isolates, respectively. The eleven isolates studied by MLST belonged to a new sequence type ST13450. This study described for the first time the occurrence of PMQR genes in C. coli isolates in Tunisia and globally.
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BACKGROUND: Allogeneic hematopoietic stem cell transplantation (ASCT) induces acquired immunodeficiency, potentially altering vaccine response. Herein, we aimed to explore the clinical tolerance and the humoral and cellular immune responses following anti-SARS-CoV-2 vaccination in ASCT recipients. METHODS: A prospective, non-randomized, controlled study that involved 43 ASCT subjects and 31 healthy controls. Humoral response was investigated using the Elecsys® test anti-SARS-CoV-2. Cellular response was assessed using the QFN® SARS-CoV-2 test. The lymphocyte cytokine profile was tested using the LEGENDplex™ HU Th Cytokine Panel Kit (12-plex). RESULTS: Adverse effects (AE) were observed in 69% of patients, encompassing pain at the injection site, fever, asthenia, or headaches. Controls presented more side effects like pain in the injection site and asthenia with no difference in the overall AE frequency. Both groups exhibited robust humoral and cellular responses. Only the vaccine transplant delay impacted the humoral response alongside a previous SARS-CoV-2 infection. Noteworthily, controls displayed a Th1 cytokine profile, while patients showed a mixed Th1/Th2 profile. CONCLUSIONS: Pfizer-BioNTech® anti-SARS-CoV-2 vaccination is well tolerated in ASCT patients, inducing robust humoral and cellular responses. Further exploration is warranted to understand the impact of a mixed cytokine profile in ASCT patients.
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Temporomandibular joint ankylosis is an important entity that dentists and maxillofacial surgeons should know about. It clinically manifests through a permanent limitation of mandibular movements coupled with mouth opening inferior to 3 cm. This serious pathology can have serious functional repercussions, such as mastication problems, speech troubles, eating disorders, and jaw growth hindrance, in addition to the psychological difficulties in coping with such a condition in daily life. Herein, we present a radiological and chronological illustration of the evolution of temporomandibular joint ankylosis following an overlooked traumatic fracture of the mandibular condyle. The present case report involves an 8-year-old patient referred for a gradually evolving mouth opening limitation following a car accident. Tomodensitometry was helpful as it revealed an osseous block between the left temporomandibular joint surfaces, showing an ankylosis. Posttraumatic cerebral computed tomography scan was performed. It revealed an undetected fracture of the left condyle. The aim of this paper was to show how a traumatic ankylosis could have been avoided if enough attention was paid to the interpretation of immediate posttraumatic computed tomography scans. A thorough dental examination must be carried out once vital emergency is over. Early diagnosis of temporomandibular joint trauma is a crucial factor in preventing complications, such as ankylosis and its consequent oral dysfunctions. The dentist must automatically suspect condylar fracture when a child presents a history of head trauma, especially a mandibular trauma. This case should be a reminder that although temporomandibular joints are very often left out in patients' vital emergency first examination, temporomandibular joints/they are still a highly important structure which omission, and thus, dysfunction, if lesions are present, can lead to nonnegligible medico-legal consequences/that temporomandibular joints should be taken into account during patients' vital emergency first examination because if they are neglected, in the presence of lesions, they cause dysfunction, thus leading to nonnegligible medico-legal consequences.
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The aac(6')-Ib gene is the most widespread gene encoding aminoglycoside-modifying enzyme and conferring resistance to tobramycin, streptomycin and kanamycin. The variant aac(6')-Ib-cr gene confers resistance to both aminoglycosides and fluoroquinolones (FQ). A total of 132 Campylobacter isolates, including 91 C. jejuni and 41 C. coli, were selected from broiler hens isolates. The aac(6')-Ib gene was amplified using PCR and was subsequently digested with the BtsCI restriction enzyme to identify aac(6')-Ib-cr. Among these isolates, 31 out of 41 C. coli (75.6%) and 1 (0.98%) C. jejuni were positive for the aac(6')-Ib gene, which was identified as the aac(6')-Ib-cr variant in 10 (32.25%) C. coli isolates. This variant was correlated with mutations in gyrA (Thr-86-Ile), as well as resistance to FQs. This study is the first report in Tunisia on Campylobacter coli strains harboring both the aac(6')-Ib and aac(6')-Ib-cr variants. These genes were present in Campylobacter isolates exhibiting resistance to multiple antibiotics, which restricts the range of available treatments.
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Campylobacter coli , Fluoroquinolonas , Animales , Femenino , Fluoroquinolonas/farmacología , Escherichia coli/genética , Campylobacter coli/genética , Pollos , Túnez , Antibacterianos/farmacología , Mutación , Aminoglicósidos/farmacología , Pruebas de Sensibilidad Microbiana , Farmacorresistencia Bacteriana/genéticaRESUMEN
Hackathons are collaborative events that bring together diverse groups to solve predefined challenges. The COVID-19 pandemic caused by SARS-CoV-2 has emphasized the need for portable and reproducible genomics analysis pipelines to study the genetic susceptibility of the human host and investigate human-SARS-CoV-2 protein interactions. To build and strengthen institutional capacities in OMICS data analysis applied to host-pathogen interaction (HPI), the PHINDaccess project organized two hackathons in 2020 and 2021. These hackathons are aimed at developing bioinformatics pipelines related to the SARS-CoV-2 viral genome, its phylodynamic transmission, and the identification of human genome host variants, with a focus on addressing global health challenges, particularly in low- and middle-income countries (LMIC). This paper outlines the preparation, proceedings, and lessons learned from these hackathons, including the challenges faced by participants and our recommendations based on our experience for organizing hackathons in LMIC and beyond.
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COVID-19 , Humanos , COVID-19/epidemiología , SARS-CoV-2/genética , Países en Desarrollo , Pandemias , Interacciones Huésped-Patógeno/genéticaRESUMEN
(1) Background: This study aimed to compare the immunogenicity of the mix-and-match CoronaVac/BNT162b2 vaccination to the homologous CoronaVac/CoronaVac regimen. (2) Methods: We conducted a simple-blinded randomized superiority trial to measure SARS-CoV-2 neutralization antibodies and anti-spike receptor binding domain (RBD) IgG concentrations in blood samples of participants who had received the first dose of CoronaVac vaccine followed by a dose of BNT162b2 or CoronaVac vaccine. The primary endpoint for immunogenicity was the serum-neutralizing antibody level with a percentage of inhibition at 90% at 21-35 days after the boost. A difference of 25% between groups was considered clinically relevant. (3) Results: Among the 240 eligible participants, the primary endpoint data were available for 100 participants randomly allocated to the mix-and-match group versus 99 participants randomly allocated to the homologous dose group. The mix-and-match regimen elicited significantly higher levels of neutralizing antibodies (median level of 96%, interquartile range (IQR) (95-97) versus median level of 94%, IQR (81-96) and anti-spike IgG antibodies (median level of 13,460, IQR (2557-29,930) versus median level of 1190, IQR (347-4964) compared to the homologous group. Accordingly, the percentage of subjects with a percentage of neutralizing antibodies > 90% was significantly higher in the mix-and-match group (90.0%) versus the homologous (60.6%). Interestingly, no severe events were reported within 30 days after the second dose of vaccination in both groups. (4) Conclusions: Our data showed the superiority of the mix-and-match CoronaVac/BNT162b2 vaccination compared to the CoronaVac/CoronaVac regimen in terms of immunogenicity, thus constituting a proof-of-concept study supporting the use of inactivated vaccines in a mix-and-match strategy while ensuring good immunogenicity and safety.
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To investigate the persistence risk of Campylobacter spp. in poultry farms, and to study the virulence and antimicrobial resistance characteristics in the recovered strains, we collected 362 samples from breeding hen flocks, before and after disinfection. The virulence factors were investigated by targeting the genes; flaA, cadF, racR, virB11, pldA, dnaJ, cdtA, cdtB, cdtC, ciaB, wlaN, cgtB, and ceuE by PCR. Antimicrobial susceptibility was tested and genes encoding antibiotic resistance were investigated by PCR and MAMA-PCR. Among the analyzed samples, 167 (46.13%) were positive for Campylobacter. They were detected in 38.7% (38/98) and 3% (3/98) of environment samples before and after disinfection, respectively, and in 126 (75.9%) out of 166 feces samples. In total, 78 C. jejuni and 89 C. coli isolates were identified and further studied. All isolates were resistant to macrolids, tetracycline, quinolones, and chloramphenicol. However, lower rates were observed for beta-lactams [ampicillin (62.87%), amoxicillin-clavulanic acid (47.3%)] and gentamicin (0.6%). The tet(O) and the cmeB genes were detected in 90% of resistant isolates. The blaOXA-61 gene and the specific mutations in the 23S rRNA were detected in 87% and 73.5% of isolates, respectively. The A2075G and the Thr-86-Ile mutations were detected in 85% and 73.5% of macrolide and quinolone-resistant isolates, respectively. All isolates carried the flaA, cadF, CiaB, cdtA, cdtB, and cdtC genes. The virB11, pldA, and racR genes were frequent in both C. jejuni (89%, 89%, and 90%, respectively) and C. coli (89%, 84%, and 90%). Our findings highlight the high occurrence of Campylobacter strains exhibiting antimicrobial resistance with potential virulence traits in the avian environment. Thus, the improvement of biosecurity measures in poultry farms is essential to control bacterial infection persistence and to prevent the spread of virulent and resistant strains.
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Globally, Campylobacter is a significant contributor to gastroenteritis. Efficient pathogens are qualified by their virulence power, resistance to antibiotics and epidemic spread. However, the correlation between antimicrobial resistance (AR) and the pathogenicity power of pathogens is complex and poorly understood. In this study, we aimed to investigate genes encoding virulence and AR mechanisms in 177 Campylobacter isolates collected from layer hens and eggs in Tunisia and to assess associations between AR and virulence characteristics. Virulotyping was determined by searching 13 virulence genes and AR-encoding genes were investigated by PCR and MAMA-PCR. The following genes were detected in C. jejuni and C. coli isolates: tet(O) (100%/100%), blaOXA-61 (18.82%/6.25%), and cmeB (100%/100%). All quinolone-resistant isolates harbored the Thr-86-Ile substitution in GyrA. Both the A2074C and A2075G mutations in 23S rRNA were found in all erythromycin-resistant isolates; however, the erm(B) gene was detected in 48.38% and 64.15% of the C. jejuni and C. coli isolates, respectively. The machine learning algorithm Random Forest was used to determine the association of virulence genes with AR phenotypes. This analysis showed that C. jejuni virulotypes with gene clusters encompassing the racR, ceuE, virB11, and pldA genes were strongly associated with the majority of phenotypic resistance. Our findings showed high rates of AR and virulence genes among poultry Campylobacter, which is a cause of concern to human health. In addition, the correlations of specific virulence genes with AR phenotypes were established by statistical analysis.
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Antibiotic resistance in foodborne pathogens is an emergent global health concern. The objectives of this study were to assess antimicrobial resistance (AMR) in Campylobacter isolates from chicken carcasses and to investigate the AMR molecular mechanisms as well as the presence of virulence determinants. The study was performed on 257 samples collected from abattoirs and retail shops in northeastern Tunisia. Forty-eight Campylobacter isolates were recovered and identified as C. jejuni (n = 33) and C. coli (n = 15). Antibiotic resistance was tested against eight antibiotics and high resistance rates were observed against tetracycline (100%), erythromycin (97.9%), ciprofloxacin (73%), nalidixic acid (85.4%), ampicillin (83.3%), amoxicillin/clavulanic acid (22.9%), chloramphenicol (75%), and gentamicin (27.1%). All isolates were multidrug-resistant, and 22 resistance patterns were found. All isolates were screened for AMR genes (tet(O), tet(A), tet(B), tet(L), cmeB, ermB, blaOXA-61, and aphA-3), and for point mutations in gyrA (C257T substitution) and 23SrRNA (A2075G/A2074C) genes. All screened AMR genes, as well as the C257T and the A2075G mutations, were detected. The virulence genotypes were also determined, and all isolates carried the motility (flaA) and invasion (cadF) genes. Most of them also harbored the cdtA, cdtB, and cdtC genes, encoding the Campylobacter toxin. The screening of the cgtB and the wlaN genes, involved in Guillain-Barré Syndrome expression, revealed the presence of the cgtB in 21.2% of C. jejuni strains, whereas none of them carried the wlaN gene. Our findings highlight the emergence of Campylobacter strains simultaneously harboring several virulence and AMR determinants, which emphasizes the risk of transmission of MDR strains to humans via the food chain. Hence, controlling the dissemination of foodborne pathogens "from the farm to the fork" as well as restricting the use of antimicrobials in husbandry are mandatory to prevent the risk for consumers and to mitigate the dissemination of MDR pathogens.
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Despite the importance of eggs in the human diet, and unlike other products, for which food safety risks are widely investigated, information on the occurrence of Campylobacter and antimicrobial resistance in eggs and layer hen flocks is lacking in Tunisia. This study was conducted to determine the occurrence of Campylobacter and the antimicrobial resistance in layer hens and on eggshells. Thus, 366 cloacal swabs and 86 eggshell smear samples were collected from five layer hen farms in the North-East of Tunisia. The occurrence of Campylobacter infection, and the antimicrobial resistance rates and patterns, were analyzed. The occurrence rates of Campylobacter infection in laying hens and eggshells were 42.3% and 25.6%, respectively, with a predominance of C. jejuni (68.4%, 81.9%), followed by C. coli (31.6%, 18.2%). The antimicrobial susceptibility testing revealed high resistance rates against macrolides, tetracycline, quinolones, ß-lactams, and chloramphenicol, with percentages ranging from 35.5% to 100%. All isolates were multidrug resistant (MDR) and five resistance patterns were observed. These results emphasized the risk to consumer health and the need to establish a surveillance strategy to control and prevent the emergence and the spread of resistant strains of Campylobacter in poultry and humans.
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BACKGROUND: Thermo-tolerant Campylobacter species are the major cause of foodborne diseases worldwide. This study aimed to evaluate the prevalence of virulence genes and antibiotic resistance determinants in Campylobacter jejuni and Campylobacter coli isolates, and to investigate the relationship between these two traits. METHODS: A total of 132 Campylobacter isolates from poultry were tested for the presence of 13 virulence genes; flaA, cadF, racR, virB11, pldA, dnaJ, cdtA, cdtB, cdtC, ciaB, wlaN, cgtB and ceuE. The mechanisms underlying antibiotic resistance phenotypes were also studied by PCR and MAMA-PCR. RESULTS: PCR results revealed the presence of antimicrobial resistance genes in C. jejuni and C. coli as follows: cmeB (80% and 100%), tet(O) (100% and 80%), and the blaOXA-61 (81% and 93%), respectively. None of these strains harbored the aphA-3 gene. The Thr-86-Ile mutation associated with resistance to quinolones was found in 90% of C. jejuni and 80% of C. coli isolates. While the A2075G and A2074C mutations linked to the erythromycin resistance were detected in 100% of both species. Virulence genes were prevalent and ranged from 40 to 100%. A positive relationship was revealed between cadF, racR, and ciaB genes and resistance to ampicillin, amoxicillin/clavulanic acid, chloramphenicol, and nalidixic acid, in C. jejuni. However, no association was observed for C. coli isolated strains. CONCLUSION: This study provides for the first time an overview of antibiotic resistance mechanisms and pathogenic profiles of Campylobacter isolates, which emphasizes the potential risk for consumer health.
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Infecciones por Campylobacter , Campylobacter coli , Campylobacter jejuni , Campylobacter , Animales , Antibacterianos/farmacología , Campylobacter/genética , Campylobacter coli/genética , Infecciones por Campylobacter/veterinaria , Infecciones por Campylobacter/epidemiología , Campylobacter jejuni/genética , Pollos , Farmacorresistencia Microbiana , Túnez , Virulencia/genéticaRESUMEN
BACKGROUND: Current decision about when to operate abdominal aortic aneurysms (AAAs) is based only on the maximum aneurysm diameter (MAD). However, small aneurysms still rupture and we can observe very large AAA without any symptom. A simple morphologic analysis could be a tool to assess the risk of rupture. The main objective of this study was to assess the relevance of ratios between MAD and healthy aorta on computed tomography (CT) as a risk factor of AAA rupture. The secondary objective was to evaluate CT signs as risk factors of AAA rupture. METHODS: Retrospective observational bicentric study comparing CT scans of a ruptured AAA group and a control group treated electively was conducted. Appariement 1:1 based on MAD was applied. Ratios between healthy aorta diameters at several levels, celiac trunk (CTR), superior mesenteric artery (SMA), highest renal artery (RA), and the MAD were calculated. The presence of blebs, crescent signs, ruptures of calcifications of the aneurysm sack, and draped aorta were notified. RESULTS: From 2010 to 2016, 38 ruptured AAA and 38 controls were included. Ratios were superior in the rupture group, respectively: MAD/CTR [2.77 (±0.5) versus 2.58 (±0.4) P < 0.095], MAD/SMA [2.92 (±0.7) versus 2.74 (±0.5) P < 0.194], and MAD/RA [3.02 (±0.70) versus 2.76 (±0.5) P < 0.054] but not significatively. Receiver operating characteristic curve analysis demonstrated optimal threshold to detect rupture at 2.8 for the ratio MAD/CTR (area under the curve (AUC) 0.593, sensitivity 47.4%, specificity 78.9%), at 3.3 for the ratio MAD/SMA (AUC 0.564, sensitivity 31.6%, specificity 92.1%), and at 3.3 for the ratio MAD/RA (AUC 0.591, sensitivity 31.6%, specificity 94.7%). Bivariate analysis for rupture risk factor showed significance for the three ratios (MAD/CTR > 2.8 [OR = 11 (1.42; 85.20) P < 0.0217], MAD/SMA > 3.3 [OR = 10 (1.28; 78.12) P < 0.0281], and MAD/RA >3.3 [OR = 11.00 (1.42; 85.20) P < 0.0217]). One scannographic sign was more present in the rupture group: crescent sign 36.8% versus 5.3%, P = 0.0007, as well in bivariate analysis [OR = 7 (1.59; 30.80) P < 0.0326]. CONCLUSIONS: In our experience, specific ratios when they exceed calculated threshold, seem to be more prone to rupture. We could consider that these measures, easy to apply in clinical practice, would be complementary keys for rupture risk individual assessment.
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Aorta Abdominal/diagnóstico por imagen , Aneurisma de la Aorta Abdominal/diagnóstico por imagen , Rotura de la Aorta/etiología , Aortografía , Angiografía por Tomografía Computarizada , Anciano , Anciano de 80 o más Años , Puntos Anatómicos de Referencia , Aneurisma de la Aorta Abdominal/complicaciones , Rotura de la Aorta/diagnóstico por imagen , Femenino , Francia , Humanos , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Estudios Retrospectivos , Medición de Riesgo , Factores de RiesgoAsunto(s)
Arterias/anomalías , Estenosis Carotídea/complicaciones , Infarto Cerebral/etiología , Lengua/irrigación sanguínea , Estenosis Carotídea/diagnóstico por imagen , Estenosis Carotídea/cirugía , Angiografía Cerebral , Infarto Cerebral/diagnóstico por imagen , Angiografía por Tomografía Computarizada , Endarterectomía Carotidea , Femenino , Humanos , Persona de Mediana Edad , Resultado del TratamientoRESUMEN
The aim of the current study is to assess the prevalence of Campylobacter infection in broiler chickens, raised in intensive production conditions, and to evaluate the antimicrobial susceptibility of recovered Campylobacter isolates. A total of 590 cloacal swab samples were taken from 13 broiler chicken flocks in the North East of Tunisia. All samples were tested for the presence of thermophilic Campylobacter by culture and PCR, targeting the mapA and ceuE genes, respectively. Susceptibility to antimicrobial drugs was tested against 8 antibiotics. Prevalence of Campylobacter infection, relationship with geographic origins and seasons, antimicrobial resistance rates and patterns were analyzed. Total prevalence of Campylobacter infection in broiler flocks was in the range of 22.4%, with a predominance of C. jejuni (68.9%), followed by C. coli (31.1%). Positive association was highlighted between the infection level and the season (P < 0.001), but no link was emphasized considering the geographic origin. Antimicrobial susceptibility testing revealed very high resistance rates detected against macrolide, tetracycline, quinolones, and chloramphenicol, ranging from 88.6% to 100%. Lower resistance prevalence was noticed for ß-lactams (47% and 61.4%) and gentamicin (12.9%). 17 R-type patterns were observed, and a common pattern was found in 30.3% of isolates. This study provides updates and novel data on the prevalence and the AMR of broiler campylobacters in Tunisia, revealing the occurrence of high resistance to several antibiotics and emphasizing the requirement of better surveillance and careful regulation of antimicrobials use.
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Antibacterianos/farmacología , Campylobacter/aislamiento & purificación , Pollos/microbiología , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Animales , Campylobacter/efectos de los fármacos , Campylobacter/genética , Infecciones por Campylobacter/tratamiento farmacológico , Infecciones por Campylobacter/microbiología , Farmacorresistencia Bacteriana Múltiple/genética , Microbiología de Alimentos/métodos , Genes Bacterianos/genética , Pruebas de Sensibilidad Microbiana/métodos , Enfermedades de las Aves de Corral/tratamiento farmacológico , Enfermedades de las Aves de Corral/microbiología , Prevalencia , TúnezRESUMEN
Diseases caused by food-borne pathogens constitute a major burden to consumers, food business operators, and national governments. Bacterial and viral pathogens are the major biotic factors influencing food safety. A vast array of culture dependent analytical methods and protocols have been developed. Recently, nucleic acid-based methods have begun to replace or complement culture-based methods for routine use in food control laboratories. Basic advantages provided by nucleic acid-based technologies are faster speed and more information, such as sub-species identification, antibiotic resistance, and food microbiology. In particular, PCR and alternative methods have been developed to a stage that provides good speed, sensitivity, specificity, and reproducibility with minimized risk of carryover contamination. This review briefly summarizes currently available and developing molecular technologies that may be candidates for involvement in microbiological molecular diagnostic methods in the next decade.
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The positive single-stranded RNA genome of the Coxsackievirus B3 (CVB3) contains a 5' untranslated region (UTR) which hosts the internal ribosome entry site (IRES) element that governs cap-independent translation initiation and a polyadenylated 3' UTR which is required for stimulating the IRES activity. Viral RNA genomes could circularize to regulate initiation of translation and RNA synthesis at 5' and 3' ends. Interactions could either take place by direct RNA-RNA contacts, through cellular protein bridges mediating RNA circularization or both. Accordingly, we aimed to assess the nature of molecular interactions between these two regions and to evaluate cellular factors required for mRNA 3' end-mediated stimulation of CVB3 IRES-driven translation. By gel shift assays, we have showed that combining, in vitro, 5' and 3' UTR fragments had no discernible effect on the structures of RNAs, arguing against the presence of specific canonical RNA-RNA cyclization sequences between these two regions. Competitive UV crosslinking assays using BHK-21 cell extract showed common cellular proteins eIF3b, PTB, and La binding to both 5'- and 3' end RNAs. PCBP 1-2 and PABP were shown to bind, respectively, to 5' and 3' UTR probes. Taking together, these data suggest that CVB3 5'-3' end bridging occurs through 5' UTR-protein-protein-3' UTR interactions and not through RNA-RNA direct contact. The dual involvement of the 3' and 5' UTRs in controlling viral translation and RNA synthesis highlights the relevance of these regions in the infectious virus life cycle, making them suitable candidates for targeted CVB3 antiviral therapy.
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Regiones no Traducidas 3' , Regiones no Traducidas 5' , Enterovirus Humano B/fisiología , Interacciones Huésped-Patógeno , Biosíntesis de Proteínas , Proteínas/metabolismo , ARN Viral/metabolismo , Sitios Internos de Entrada al Ribosoma , Unión Proteica , ARN/metabolismo , ARN CircularRESUMEN
Coxsackievirus B3 (CVB3) causes viral myocarditis, and can ultimately result in dilated cardiomyopathy. There is no vaccine available for clinical use. In the present work, we assessed whether the Sabin3-like mutant of CVB3 could induce a protective immunity against virulent CVB3 Nancy and CVB4 E2 strains in mice by both oral and intraperitoneal (IP) routes. Serum samples, taken from mice inoculated with Sabin3-like, were assayed in vitro for their anti-CVB3 neutralizing activity. CVB3 Sabin3-like was highly attenuated in vivo and was able to induce an anti-CVB3 activity of the serum. However, at 4 days post-CVB3 challenge, significant increased titers of CVB3 neutralizing antibodies were detectable in the sera of immunized mice over the next 6 days. Non-immunized mice challenged with CVB3 Nancy had no anti-CVB3 activity in their sera until 10 days post-infection. CVB3 Nancy induced higher viral titers than did the mutant strain. There was no variation of the neutralizing activity of serum taken from mice immunized with CVB3 Sabin3-like and challenged with CVB4 E2, compared to non-immunized mice. Despite the fact that CVB3 and CVB4 are closely related viruses, virus-neutralizing activity clearly distinguish between these viruses. A variable and limited amount of pancreatic inflammation was seen in some mice 10 days after Sabin3-like inoculation by IP route, whereas there was no evidence of pancreatic damage in mice inoculated by oral route. All immunized mice were protected from myocarditis and pancreatitis at 8 days post-challenge with CVB3 or CVB4 E2. These findings strongly suggest that the mutant strain could be considered a candidate for an attenuated CVB3 vaccine.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Infecciones por Coxsackievirus/inmunología , Infecciones por Coxsackievirus/virología , Enterovirus Humano B/genética , Enterovirus Humano B/inmunología , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Niño , Chlorocebus aethiops , Infecciones por Coxsackievirus/prevención & control , Femenino , Humanos , Inflamación/patología , Ratones , Mutación , Miocardio/patología , Pruebas de Neutralización , Páncreas/patología , Células VeroRESUMEN
Internal ribosome entry site (IRES) elements fold into highly organized conserved secondary and probably tertiary structures that guide the ribosome to an internal site of the RNA at the IRES 3'end. The composition of the cellular proteome is under the control of multiple processes, one of the most important being translation initiation. In each poliovirus Sabin vaccine strain, a single point mutation in the IRES secondary-structure domain V is a major determinant of neurovirulence and translation attenuation. Here we are extrapolating poliovirus findings to a genomic related virus named coxsackievirus B3 CVB3); a causative agent of viral myocarditis. We have previously reported that Sabin3-like mutation (U473 â C) introduced in the domain V sequence of the CVB3 IRES led to a defective mutant with a serious reduction in translation efficiency and ribosomal initiation complex assembly, besides an impaired RNA-protein binding pattern. With the aim to identify proteins interacting with both CVB3 wild-type and Sabin3-like domain V RNAs and to assess the effect of the Sabin3-like mutation on these potential interactions, we have used a proteomic approach. This procedure allowed the identification of three RNA-binding proteins interacting with the domain V: eIF4G (p220), eIF3b (p116) and eIF4B (p80). Moreover, we report that this single-nucleotide exchange impairs the interaction pattern and the binding affinity of these standard translation initiation factors within the IRES domain V of the mutant strain. Taken together, these data indicate how this decisive Sabin3-like mutation mediates viral translation attenuation; playing a key role in the understanding of the cardiovirulence attenuation within this construct. Hence, these data provide further evidence for the crucial role of RNA structure for the IRES activity, and reinforce the idea of a distribution of function between the different IRES structural domains. VIRTUAL SLIDE: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/6160165131045880.
Asunto(s)
Regiones no Traducidas 5' , Enterovirus Humano B/metabolismo , Factor 3 de Iniciación Eucariótica/metabolismo , Factor 4G Eucariótico de Iniciación/metabolismo , Factores Eucarióticos de Iniciación/metabolismo , Miocarditis/virología , ARN Viral/metabolismo , Ribosomas/metabolismo , Animales , Sitios de Unión , Clonación Molecular , Cricetinae , Enterovirus Humano B/genética , Enterovirus Humano B/patogenicidad , Factor 3 de Iniciación Eucariótica/química , Factor 4G Eucariótico de Iniciación/química , Factores Eucarióticos de Iniciación/química , Células HeLa , Humanos , Peso Molecular , Mutagénesis Sitio-Dirigida , Mutación , Conformación de Ácido Nucleico , Iniciación de la Cadena Peptídica Traduccional , Proteómica/métodos , ARN Viral/química , ARN Viral/genética , VirulenciaRESUMEN
Internal ribosome entry site (IRES) elements are highly structured RNA sequences that function to recruit ribosomes for the initiation of translation. In contrast to the canonical cap-binding, the mechanism of IRES-mediated translation initiation is still poorly understood. Translation initiation of the coxsackievirus B3 (CVB3), a causative agent of viral myocarditis, has been shown to be mediated by a highly ordered structure of the 5' untranslated region (5'UTR), which harbors an IRES. Taking into account that efficient initiation of mRNA translation depends on temporally and spatially orchestrated sequence of RNA-protein and RNA-RNA interactions, and that, at present, little is known about these interactions, we aimed to describe recent advances in our understanding of molecular structures and biochemical functions of the translation initiation process. Thus, this review will explore the IRES elements as important RNA structures and the significance of these structures in providing an alternative mechanism of translation initiation of the CVB3 RNA. Since translation initiation is the first intracellular step during the CVB3 infection cycle, the IRES region provides an ideal target for antiviral therapies. Interestingly, the 5' and 3'UTRs represent promising candidates for the study of CVB3 cardiovirulence and provide new insights for developing live-attenuated vaccines.