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Salmonella enterica is a globally disseminated pathogen that is the cause of over 100 million infections per year. The resulting diseases are dependent upon host susceptibility and the infecting serovar. As S. enterica serovar Typhimurium induces a typhoid-like disease in mice, this model has been used extensively to illuminate various aspects of Salmonella infection and host responses. Due to the severity of infection in this model, researchers often use strains of mice resistant to infection or attenuated Salmonella. Despite decades of research, many aspects of Salmonella infection and fundamental biology remain poorly understood. Here, we use a transposon insertion sequencing technique to interrogate the essential genomes of widely used isogenic wild-type and attenuated S. Typhimurium strains. We reveal differential essential pathways between strains in vitro and provide a direct link between iron starvation, DNA synthesis, and bacterial membrane integrity.IMPORTANCESalmonella enterica is an important clinical pathogen that causes a high number of deaths and is increasingly resistant to antibiotics. Importantly, S. enterica is used widely as a model to understand host responses to infection. Understanding how Salmonella survives in vivo is important for the design of new vaccines to combat this pathogen. Live attenuated vaccines have been used clinically for decades. A widely used mutation, aroA, is thought to attenuate Salmonella by restricting the ability of the bacterium to access particular amino acids. Here we show that this mutation limits the ability of Salmonella to acquire iron. These observations have implications for the interpretation of many previous studies and for the use of aroA in vaccine development.
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Salmonella enterica serovar Dublin (S. Dublin) is an important zoonotic pathogen with high invasiveness. In the prevention and control of the Salmonella epidemic, the live attenuated vaccine plays a very important role. To prevent and control the epidemic of S. Dublin in cattle farms, the development of more effective vaccines is necessary. In this study, we constructed two gene deletion mutants, Sdu189ΔspiC and Sdu189ΔspiCΔaroA, with the parental strain S. Dublin Sdu189. The immunogenicity and protective efficacy were evaluated in the mice model. First, both mutant strains were much less virulent than the parental strain, as determined by the 50% lethal dose (LD50) for specific pathogen-free (SPF) 6-week-old female BALB/c mice. Second, the specific IgG antibody level and the expression level of cytokine TNF-α, IFN-γ, IL-4, and IL-18 were increased significantly in the vaccinated mice compared to the control group. In addition, the deletion strains were cleared rapidly from organs of immunized mice within 14 d after immunization, while the parental strain could still be detected in the spleen and liver after 21 d of infection. Compared with the parental strain infected group, no obvious lesions were detected in the liver, spleen, and cecum of the deletion strain vaccinated groups of mice. Immunization with Sdu189ΔspiC and Sdu189ΔspiCΔaroA both provided 100% protection against subsequent challenges with the wild-type Sdu189 strain. These results demonstrated that these two deletion strains showed the potential as live attenuated vaccines against S. Dublin infection. The present study established a foundation for screening a suitable live attenuated Salmonella vaccine.
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Dermonecrotic toxin (DNT) is an important bacterial virulence factor produced by the zoonotic pathogens Bordetella bronchiseptica and Pasteurella multocida. This study aims to explore the possibility of expressing different fragments of P. multocida toxin (PMT) in the chromosome of attenuated B. bronchiseptica to generate single-component mucosal vaccine candidates. To achieve this, a 954-bp fragment (basepairs 301 â¼ 1254) of the B. bronchiseptica aroA gene was replaced with an N-terminal, 930-bp fragment (basepairs 1-930; PMTN) or a C-terminal, 900-bp fragment (base pairs 2959 â¼ 3858; PMTC) of the PMT encoding gene toxA. The resulting strains, denoted as Bb-PMTN or Bb-PMTC, expressed PMTN and PMTC, as evidenced by ELISA using polyclonal against full-length of PMT. Phenotypical analyses revealed that Bb-PMTN and Bb-PMTC grew much slower than wild type strains in tryptic soy broth. These strains also displayed significantly decreased 161-fold-virulence compared to the wildtype strains in mouse models. Intranasal immunization of Bb-PMTN and Bb-PMTC in mice induced high levels of antibodies against B. bronchiseptica and PMT, as well as IFN-γ and IL-10 in mouse sera, and most importantly, high titers of sIgA in mouse lungs. Vaccination with these two engineering strains provided 100% protection of mice against lethal challenge with B. bronchiseptica and 80%â¼100% protection against lethal challenge with PMT, with Bb-PMTN exhibiting 1.25-fold greater immunogenic efficacy over Bb-PMTC. This study highlights the use of B. bronchiseptica attenuated strains as live mucosal vectors to deliver heterologous antigens.
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Toxinas Bacterianas , Infecciones por Bordetella , Bordetella bronchiseptica , Infecciones por Pasteurella , Pasteurella multocida , Animales , Proteínas Bacterianas , Toxinas Bacterianas/genética , Infecciones por Bordetella/prevención & control , Bordetella bronchiseptica/genética , Ratones , Infecciones por Pasteurella/prevención & control , Pasteurella multocida/genética , Vacunas AtenuadasRESUMEN
Vaccination remains the most effective approach for prevention and control of infectious diseases in aquaculture. Edwardsiella piscicida is a causative agent of edwardsiellosis leading to mass mortality in a variety of fish species, leading to huge economic losses in the aquaculture industry. In this study, we have deleted the aroA and phoP genes in E. piscicida and investigated the phenotype, degrees of attenuation, immunogenicity, and ability to confer immune protection in zebrafish host. Our vaccine strain χ16028 with genotype ΔaroA11 ΔphoP12, showed significantly reduced growth, motility, biofilm formation and intracellular replication compared to the wild-type strain J118. In this regard, χ16028 exhibited retarded colonization and attenuation phenotype in zebrafish. Studies showed that χ16028 induced TLR4 and TLR5 mediated NF-kB pathway and upregulated cytokine gene expression i.e., TNF-α, IL-1ß, IL-6, IL-8 and type-I IFN in zebrafish. Zebrafish immunized by intracoelomic injection (i.c.) with χ16028 showed systemic and mucosal IgM responses and protection against the wild-type E. piscicida i.c. injection challenge. However, the protection was only 25% in zebrafish following i.c. challenge. We speculate that our vaccine strain might be very attenuated; a booster dose may trigger better immune response and increase the percentage of survival to a more significant level.
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Infecciones por Enterobacteriaceae , Enfermedades de los Peces , Animales , Edwardsiella , Infecciones por Enterobacteriaceae/prevención & control , Infecciones por Enterobacteriaceae/veterinaria , Enfermedades de los Peces/prevención & control , Virulencia , Pez CebraRESUMEN
BACKGROUND: Glyphosate is a non-selective systemic herbicide with a broad spectrum of weed control that inhibits a key enzyme, 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase, in the shikimate pathway. OBJECTIVES: Isolation and analysis of the epsps (aroA) gene responsible for glyphosate-tolerance in bacteria from Roundup- contaminated soils was the aim of this study. MATERIALS AND METHODS: Sampling was done from the soil of the gardens which were heavily contaminated by Roundup herbicide and then bacterial screening was performed in the presence of high concentrations of glyphosate. The genus of bacterium was identified via molecular methods such as 16S rRNA sequencing. The aroA gene of this bacterium (aroA HA-09) was isolated using the primers designed-upon specific regions of aroA genes available in NCBI GenBank database. The PCR product was cloned, sequenced and subcloned into pET28a as an expression vector and transferred into E. coli strain BL21(DE3). The cells were inoculated in liquid M9 minimal medium containing IPTG and different concentrations of glyphosate. RESULTS: The genus of bacterium was identified as Pseudomonas sp. strain HA-09. The isolated aroA HA-09 gene from this bacterium was approximately 2.2 kb in size. Bioassay of E. coli expressing this gene showed high tolerance to glyphosate (up to 300 mM). CONCLUSION: The aroA HA-09 gene could be considered as a novel and efficient candidate for development of glyphosate-tolerant crop plants.
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Bordetella bronchiseptica is a leading cause of swine respiratory disorders which depict a great threat to well-flourished porcine industry. Vaccination remains an effective way for the prevention of B. bronchiseptica infections, as live B. bronchiseptica vaccines possess many advantages compared to inactivated vaccines and/or sub-unit vaccines, however, their safety is not up to the mark. In present study, we constructed marker-free aroA/bscN double deleted B. bronchiseptica QH09 through two-step homologous recombination strategy. Our data showed that QH09 attenuated virulence to mice compared with the parent aroA deleted B. bronchiseptica QH0814. We also found that QH09 meets the vaccine safety standards, upon challenge in piglets, did not cause any visible clinical signs or lesions on organs. Finally, we demonstrated that vaccination of QH09 activated the systemic as well as the mucosal immunity in pigs and provided protection against lethal bacterial challenge. These findings suggest that the aroA/bscN double deleted B. bronchiseptica QH09 may be an effective vaccine candidate, with safety assurance of animals against B. bronchiseptica infections.
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Poultry vaccine programs are important for control of Salmonella infections. Although there are vaccines for Salmonella Enteritidis, Salmonella Typhimurium and Salmonella Typhi, there are no vaccines for Salmonella Infantis which has an increased rate in the world. In this study, it was aimed to generate aroA gene deleted mutant bacteria for the constitution of S. Infantis vaccine prototype and the in vitro characterisation of this bacterium. S. Infantis auxotrophic mutant which has a block at any step of chorismate pathway has been constituted for the first time in the world and it was determined that this bacterium gets susceptibility against some antibiotics and antimicrobial substances. It was also observed that the adhesion and invasion rate of mutant strain tenfold decreased in comparison with the field strain in cell culture assay. It is understood from the in vitro evaluation of this mutant strain that it can be used as a vaccine candidate in further vaccine development studies.
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3-Fosfoshikimato 1-Carboxiviniltransferasa/genética , Enfermedades de las Aves de Corral/prevención & control , Infecciones por Salmonella/prevención & control , Salmonella/genética , Salmonella/inmunología , Vacunas Sintéticas/inmunología , Ácido 4-Aminobenzoico/metabolismo , Animales , Células CACO-2 , Línea Celular Tumoral , Humanos , Aves de Corral/microbiología , Infecciones por Salmonella/inmunología , Infecciones por Salmonella/microbiología , Vacunas Tifoides-Paratifoides/inmunología , Vacunas Atenuadas/inmunologíaRESUMEN
The emergence of multidrug-resistant Klebsiella pneumoniae has rendered a large array of infections difficult to treat. In a high-throughput genetic screen of factors required for K. pneumoniae survival in the lung, amino acid biosynthesis genes were critical for infection in both immunosuppressed and wild-type (WT) mice. The limited pool of amino acids in the lung did not change during infection and was insufficient for K. pneumoniae to overcome attenuating mutations in aroA, hisA, leuA, leuB, serA, serB, trpE, and tyrA in WT and immunosuppressed mice. Deletion of aroA, which encodes 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase class I, resulted in the most severe attenuation. Treatment with the EPSP synthase-specific competitive inhibitor glyphosate decreased K. pneumoniae growth in the lungs. K. pneumoniae expressing two previously identified glyphosate-resistant mutations in EPSP synthase had significant colonization defects in lung infection. Selection and characterization of six spontaneously glyphosate-resistant mutants in K. pneumoniae yielded no mutations in aroA Strikingly, glyphosate treatment of mice lowered the bacterial burden of two of three spontaneous glyphosate-resistant mutants and further lowered the burden of the less-attenuated EPSP synthase catalytic mutant. Of 39 clinical isolate strains, 9 were resistant to glyphosate at levels comparable to those of selected resistant strains, and none appeared to be more highly resistant. These findings demonstrate amino acid biosynthetic pathways essential for K. pneumoniae infection are promising novel therapeutic targets.
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Aminoácidos/metabolismo , Klebsiella pneumoniae/metabolismo , Klebsiella pneumoniae/patogenicidad , Pulmón/microbiología , Animales , Antibacterianos/uso terapéutico , Proteínas Bacterianas/genética , Vías Biosintéticas/efectos de los fármacos , Vías Biosintéticas/genética , Farmacorresistencia Bacteriana Múltiple , Femenino , Glicina/análogos & derivados , Glicina/uso terapéutico , Huésped Inmunocomprometido , Klebsiella pneumoniae/efectos de los fármacos , Ratones , GlifosatoRESUMEN
Vaccination of chicks with Salmonella (S.) Typhimurium aroA deletion mutants has previously been shown to inhibit intestinal colonization of wild-type S. Typhimurium strains. In Australia, Bioproperties VaxSafe™ STM1 strain is the only licensed and commercially available S. Typhimurium vaccine. This vaccine is a live attenuated aroA deletion mutant. Currently, it is recommended that the first dose of the STM1 vaccine is administered through coarse spray. It is unclear whether this mode of administration effectively permits intestinal colonization. Furthermore, it is not known whether the STM1 strain prevents or inhibits Salmonella colonization of chicks following this first dose. This study investigated both in vitro and in vivo colonization parameters. Invasiveness was assessed using an in vitro invasion assay into sections of ileum and caecum collected from day-old chicks. The S. Typhimurium definitive types (DT) 9 and 44 exhibited the greatest invasion into both intestinal segments. STM1 was invasive but was significantly less so than both isolates of S. Typhimurium. In dual and triple infections, no competitive microbial interactions between STM1 and wild-type Salmonella were observed. In vivo colonization inhibition was also tested. Vaccinated and nonvaccinated day-old chicks were challenged with S. Typhimurium DT9. Both STM1 and S. Typhimurium DT9 were found in spleen, liver, ileum, caecum and caecal contents from day 2 postinfection. No significant exclusion effect was observed in vaccinated and challenged chicks.
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Pollos/microbiología , Intestinos/microbiología , Enfermedades de las Aves de Corral/prevención & control , Vacunas contra la Salmonella/inmunología , Salmonella typhimurium , Animales , Portador Sano , Embrión de Pollo , Femenino , Masculino , Enfermedades de las Aves de Corral/microbiología , Salmonella typhimurium/genética , Salmonella typhimurium/inmunologíaRESUMEN
OBJECTIVE: To discover and isolate a glyphosate-resistant gene from a microorganism through gene mining. RESULTS: The full aroM gene from Acremonium sp. (named aroMA.sp.) was cloned using rapid amplification of cDNA ends. The transcriptional expression level of each domain increased significantly after glyphosate treatment in the aroMA.sp. complex and reached its maximum at 48 h. The aroA domain of the aroMA.sp. (named aroA A.sp.) was expressed in Escherichia coli BL21 (DE3) and the product was purified through Ni-NTA affinity chromatography. Furthermore, 45 KDa was indicated by SDS-PAGE and its enzyme activity was optimal at 30 °C and PH 7.0. The Ki/Km value of aroAA.sp. was 0.106, and the E. coli BL21 harboring aroAA.sp. could grow in the M9 minimal medium with 100 mM glyphosate. CONCLUSION: The aroAA.sp. from the aroMA.sp. complex had high enzyme activity and glyphosate resistance. Therefore, this research offers a new strategy for improving glyphosate resistance using the aroA domain of the aroM complex in the fungi.
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3-Fosfoshikimato 1-Carboxiviniltransferasa/química , 3-Fosfoshikimato 1-Carboxiviniltransferasa/genética , Acremonium/enzimología , Resistencia a los Herbicidas , 3-Fosfoshikimato 1-Carboxiviniltransferasa/metabolismo , Acremonium/genética , Clonación Molecular , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Glicina/análogos & derivados , Glicina/farmacología , Dominios Proteicos , Regulación hacia Arriba , GlifosatoRESUMEN
Glyphosate is a widely used herbicide that inhibits the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS)-encoding aroA gene in the shikimate pathway. The discovery and cloning of the aroA gene with high resistance is central to breeding a transgenic glyphosate-resistant plant. A novel aroAPantoea gene from Pantoea G-1 was previously isolated and cloned. The aroA Pantoea enzyme was defined as a new class I EPSPS with glyphosate resistance. The aroA Pantoea gene was introduced into tobacco through Agrobacteriummediated transformation. The transgenic tobacco plants were confirmed by PCR, RT-PCR, and Southern blot. The analysis of glyphosate resistance also showed that the transgenic tobacco plants could survive at 15 mM glyphosate; the glyphosate resistance level of the transgenic plants is higher than the agricultural application level recommended by most manufacturers. Overall, this study shows that aroAPantoea can be used as a candidate gene for the development of genetically modified crops.
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BACKGROUND: Glyphosate is a herbicide that acts by inhibition of the enzyme, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), involved in the catalysis of an essential step in the biosynthesis of aromatic amino acids. The objective of this study was the isolation of glyphosate-resistant bacterial strains and subsequent characterization of the gene(s) encoding glyphosate resistance in these isolates. Using an enrichment culture technique, a glyphosate-resistant bacterium, Ochrobactrum intermedium Sq20 was isolated from glyphosate-contaminated indigenous soil and characterized. RESULTS: An open reading frame (ORF) comprising of 1353 bp potentially encoding aroAO. intermediumSq20 was amplified from O. intermedium Sq20. It showed 97% homology with aroA genes from other Ochrobactrum spp. Physicochemical characterization revealed that aroAO. intermediumSq20 encodes a polypeptide of 450 amino acids with a calculated molecular mass of 48.9782 kDa and an isoelectric point of 5.21. Secondary structure prediction of AroAO. intermediumSq20 demonstrated a high percentage of random coils and α helices. Methodical optimization and validation of the protein structure helped to build a reliable protein model indicating the presence of 91.8% amino acid residues in most favoured regions. In addition, strain Sq20 was found to be capable of complete degradation of glyphosate at 500 mg L-1 initial concentration as the sole carbon and energy source within 4 days. CONCLUSION: A glyphosate-resistant bacterial strain O. intermedium Sq20 was discovered. Sequence analysis and structure modelling demonstrated that AroAO. intermediumSq20 closely resembles class II EPSPS and possesses high glyphosate resistance. This provides a good foundation for functional analysis of experimentally derived crystal structures. The cloning and characterization of AroAO. intermediumSq20 will further help in understanding its role at the molecular level and its potential use in the production of glyphosate-resistant transgenic crops. © 2017 Society of Chemical Industry.
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3-Fosfoshikimato 1-Carboxiviniltransferasa/genética , Proteínas Bacterianas/genética , Glicina/análogos & derivados , Resistencia a los Herbicidas/genética , Herbicidas/farmacología , Ochrobactrum/genética , 3-Fosfoshikimato 1-Carboxiviniltransferasa/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Glicina/farmacología , Ochrobactrum/efectos de los fármacos , Ochrobactrum/metabolismo , Filogenia , Alineación de Secuencia , GlifosatoRESUMEN
Salmonellosis ranks among the major diseases of commercial poultry, and its presence in poultry flocks is responsible for economic losses and risks related to public health. Vaccines are an important tool within integrated programmes to control salmonellosis. The purpose of this study was to assess cross-protection provided by the Poulvac® ST vaccine in the control of Salmonella Heidelberg in experimentally challenged 3- and 21-day-old birds. Eighty birds were identified and separated into four treatments (T1: vaccinated and challenged at 3 days of age, T2: unvaccinated and challenged at 3 days of age, T3: vaccinated and challenged at 21 days of age, and T4: unvaccinated and challenged at 21 days of age). The inoculum was produced from a Brazilian field strain of SH. At the end of the experiment, caecum and liver/spleen samples were collected for quantitative and qualitative analysis of SH, respectively. Analysis of the liver/spleen showed that Poulvac® ST significantly (P ≤ 0.05) reduced the percentage of SH positivity in the group challenged at 3 days of age, while in the group challenged at 21 days this difference was almost considered significant (P = 0.1818). On the other hand, there was no statistically significant difference in SH count in the caecum (CFU/g) in the group challenged at 3 days, but for the group challenged at 21 days the SH counts were significantly (P ≤ 0.05) lower in the vaccinated group when compared to the positive control.
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Pollos/inmunología , Enfermedades Transmitidas por los Alimentos/prevención & control , Enfermedades de las Aves de Corral/prevención & control , Salmonelosis Animal/prevención & control , Vacunas contra la Salmonella/inmunología , Salmonella enterica/inmunología , Animales , Pollos/microbiología , Protección Cruzada , Enfermedades Transmitidas por los Alimentos/microbiología , Humanos , Intestinos/inmunología , Intestinos/microbiología , Enfermedades de las Aves de Corral/microbiología , Salmonelosis Animal/microbiología , Vacunas contra la Salmonella/genética , Salmonella enterica/genética , Vacunas Atenuadas/genética , Vacunas Atenuadas/inmunología , Vísceras/inmunología , Vísceras/microbiologíaRESUMEN
Microbially-mediated arsenic (As) transformation in soils affects As speciation and plant uptake. However, little is known about the impacts of As on bacterial communities and their functional genes in the rhizosphere of As-hyperaccumulator Pteris vittata. In this study, arsenite (AsIII) oxidase genes (aroA-like) and arsenate (AsV) reductase genes (arsC) were amplified from three soils, which were amended with 50mgkg-1 As and/or 1.5% phosphate rock (PR) and grew P. vittata for 90 d. The aroA-like genes in the rhizosphere were 50 times more abundant than arsC genes, consistent with the dominance of AsV in soils. According to functional gene alignment, most bacteria belonged to α-, ß- and γ-Proteobacteria. Moreover, aroA-like genes showed a higher biodiversity than arsC genes based on clone library analysis and could be grouped into nine clusters based on terminal restriction fragment length polymorphism (T-RFLP) analysis. Besides, AsV amendment elevated aroA-like gene diversity, but decreased arsC gene diversity. Redundancy analysis indicated that soil pH, available Ca and P, and AsV concentration were key factors driving diverse compositions in aroA-like gene community. This work identified new opportunities to screen for As-oxidizing and/or -reducing bacteria to aid phytoremediation of As-contaminated soils.
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Arseniato Reductasas/genética , Arsénico/toxicidad , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Oxidorreductasas/genética , Fosfatos/toxicidad , Pteris/microbiología , Contaminantes del Suelo/toxicidad , Arsénico/metabolismo , Biodiversidad , Gammaproteobacteria/efectos de los fármacos , Gammaproteobacteria/enzimología , Genes Bacterianos , Fosfatos/metabolismo , Pteris/metabolismo , Rizosfera , Contaminantes del Suelo/metabolismoRESUMEN
The innate immune receptors, NOD1 and NOD2, are key regulators of intestinal homeostasis. NOD2 deficiency is linked to increased risk for Crohn's disease, a type of inflammatory bowel disease characterized by chronic inflammatory pathology and dysbiosis within resident microbial communities. However, the relationship between NOD protein-regulated immune functions and dysbiosis remains unclear. We hypothesized that the relationship between NOD1 or NOD2 deficiency and altered community structure during chronic disease may arise via NOD-dependent impairment of community resilience over time. Using the Salmonella ΔaroA model of chronic colitis with littermate mice to control for environmental influences on the microbiota, we show that NOD proteins exert a relatively minor impact on the chronic inflammatory environment and do not significantly contribute to bacterial abundance or community resilience following infection. Rather, temporal shifts in relative abundance of targeted bacterial groups correlated with inflammatory phenotype driven by presence of the pathogen and the ensuing complex immune response.
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Microbioma Gastrointestinal , Inmunidad Innata , Proteína Adaptadora de Señalización NOD1/metabolismo , Proteína Adaptadora de Señalización NOD2/metabolismo , Salmonelosis Animal/inmunología , Animales , Colitis/inmunología , Modelos Animales de Enfermedad , Disbiosis , Ratones Endogámicos C57BLRESUMEN
BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterial pathogen frequently isolated in both hospital and community environments. Methicillin-resistant Staphylococcus aureus is considered a major nosocomial pathogen that causes severe morbidity and mortality. OBJECTIVES: The main objective of this study was to determine the genotypes of MRSA strains isolated from the nares of hospitalized and community patients in Kermanshah Hospital, western Iran, by PCR-restriction fragment length polymorphism (PCR-RFLP). MATERIALS AND METHODS: Of 1387 patients, 1217 patients were screened for more than 48 hours after admission in hospital wards and 170 patients were screened in the hemodialysis unit of Kermanshah Hospital, which is the largest hospital in western Iran. S. aureus was identified by standard biochemical tests, including colonial morphology, production of coagulase, and DNase and the API20 Staph test. Methicillin-resistant Staphylococcus aureus was identified by the Oxacillin strip test. RESULTS: In total, 258 S. aureus isolates were recovered from 1387 samples, of which 96 isolates were MRSA, 82 were hospital acquired, and 14 were community acquired. Digestion of the aro A gene revealed only one distinctive RFLP pattern in the 258 isolates. CONCLUSIONS: Methicillin-resistant Staphylococcus aureus is an increasingly common cause of nosocomial infections. Our results are in agreement with those of other studies reporting that a few specialized clones are responsible for most cases of MRSA nasal carriage. In this study, MRSA strains isolated from different wards of hospital were closely related when analyzed by coagulase gene typing. Identifying patients colonized with MRSA during hospitalization and rapidly typing them with these methods may facilitate detection of outbreaks and prevention of the spread of organisms in hospitals.
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The food safety of stacked trait genetically modified (GM) maize GH5112E-117C containing insect-resistance gene Cry1Ah and glyphosate-resistant gene G2-aroA was evaluated in comparison to non-GM Hi-II maize fed to Sprague-Dawley rats during a 90-day subchronic feeding study. Three different dietary concentrations (12.5, 25 and 50 %, w/w) of the GM maize were used or its corresponding non-GM maize. No biologically significant differences in the animals' clinical signs, body weights, food consumption, hematology, clinical chemistry, organ weights and histopathology were found between the stacked trait GM maize groups, and the non-GM maize groups. The results of the 90-day subchronic feeding study demonstrated that the stacked trait GM maize GH5112E-117C is as safe as the conventional non-GM maize Hi-II.
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Alimentación Animal , Inocuidad de los Alimentos , Plantas Modificadas Genéticamente/genética , Zea mays/genética , Alimentación Animal/análisis , Animales , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/genética , Proteínas Bacterianas/toxicidad , Análisis Químico de la Sangre , Endotoxinas/genética , Endotoxinas/toxicidad , Femenino , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/toxicidad , Masculino , Tamaño de los Órganos , Ratas Sprague-Dawley , Pruebas de Toxicidad Subcrónica , Aumento de PesoRESUMEN
In this study, an aroA-deletion avian pathogenic Escherichia coli (APEC) mutant (strain DE17ΔaroA) and aroA and luxS double deletion APEC mutant (strain DE17ΔluxSΔaroA) were constructed from the APEC DE17 strain. The results showed that as compared to DE17ΔaroA, the virulence of DE17ΔluxSΔaroA was further attenuated by 200- and 31.7-fold, respectively, in ducklings based on the 50% lethal dose. The adherence and invasion abilities of DE17ΔluxSΔaroA and DE17ΔaroA were reduced by 36.5%/42.5% and 25.8%/29.3%, respectively, as compared to the wild-type strain DE17 (p < 0.05 and 0.01, respectively). Furthermore, in vivo studies showed that the bacterial loads of DE17ΔluxSΔaroA were reduced by 8400- and 11,333-fold in the spleen and blood of infected birds, respectively, while those of DE17ΔaroA were reduced by 743- and 1000-fold, respectively, as compared to the wild-type strain DE17. Histopathological analysis showed both that the mutants were associated with reduced pathological changes in the liver, spleen, and kidney of ducklings, and changes in DE17ΔluxSΔaroA-infected ducklings were reduced to a greater degree than those infected with DE17ΔaroA. Real-time polymerase chain reaction analysis further demonstrated that the mRNA levels of virulence-related genes (i.e., tsh, ompA, vat, iucD, pfs, fyuA, and fimC) were significantly decreased in DE17ΔaroA, especially in DE17ΔluxSΔaroA, as compared to DE17 (p < 0.05). In addition, the deletion of aroA or the double deletion of aroA and luxS reduced bacterial motility. To evaluate the potential use of DE17ΔluxSΔaroA as a vaccine candidate, 50 7-day-old ducklings were divided randomly into five groups of ten each for the experiment. The results showed that the ducklings immunized with inactivated DE17, DE17ΔluxS, DE17ΔaroA, and DE17ΔluxSΔaroA were 70.0%, 70.0%, 70.0, and 80.0% protected, respectively, after challenge with strain APEC DE17. The results of this study suggest that the double deletion of luxS and aroA attenuated APEC pathogenicity and DE17ΔluxSΔaroA was more appropriate for development of a future vaccine against avian colibacillosis than DE17ΔaroA.
Asunto(s)
3-Fosfoshikimato 1-Carboxiviniltransferasa/genética , Proteínas Bacterianas/genética , Liasas de Carbono-Azufre/genética , Escherichia coli/patogenicidad , Eliminación de Gen , Factores de Virulencia/genética , 3-Fosfoshikimato 1-Carboxiviniltransferasa/deficiencia , Estructuras Animales/microbiología , Estructuras Animales/patología , Animales , Animales Recién Nacidos , Adhesión Bacteriana , Carga Bacteriana , Liasas de Carbono-Azufre/deficiencia , Patos , Escherichia coli/genética , Infecciones por Escherichia coli/microbiología , Infecciones por Escherichia coli/patología , Infecciones por Escherichia coli/veterinaria , Vacunas contra Escherichia coli/administración & dosificación , Vacunas contra Escherichia coli/inmunología , Histocitoquímica , Enfermedades de las Aves de Corral/microbiología , Enfermedades de las Aves de Corral/patología , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/inmunología , VirulenciaRESUMEN
The 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) is a key enzyme in the aromatic amino acid biosynthetic pathway in microorganisms and plants, which catalyzes the formation of 5-enolpyruvylshikimate-3-phosphate (EPSP) from shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP). In this study, a novel AroA-encoding gene was identified from the deep sea bacterium Alcanivorax sp. L27 through screening the genomic library and termed as AroAA.sp. A phylogenetic analysis revealed that AroAA.sp (1317 bp and 438 amino acids) is a class II AroA. This enzyme exhibited considerable activity between pH 5.5 and pH 8.0 and notable activity at low temperatures. The KM for PEP and IC50 [glyphosate] values (the concentration of glyphosate that inhibited enzyme activity by 50%) of AroAA.sp were 78 µM and 1.5 mM, respectively. Furthermore, site-directed mutagenesis revealed that the G100A mutant had a 30-fold increase in the IC50 [glyphosate] value; while the L105P mutant showed only 20% catalytic activity compared to wild-type AroAA.sp. The specific activity of the wild-type AroAA.sp, the G100A mutant and the L105P mutant were 7.78 U/mg, 7.26 U/mg and 1.76 U/mg, respectively. This is the first report showing that the G100A mutant of AroA displays considerably improved glyphosate resistance and demonstrates that Leu105 is essential for the enzyme's activity.
Asunto(s)
3-Fosfoshikimato 1-Carboxiviniltransferasa/aislamiento & purificación , Alcanivoraceae/enzimología , Proteínas Bacterianas/aislamiento & purificación , 3-Fosfoshikimato 1-Carboxiviniltransferasa/antagonistas & inhibidores , 3-Fosfoshikimato 1-Carboxiviniltransferasa/genética , 3-Fosfoshikimato 1-Carboxiviniltransferasa/metabolismo , Agrobacterium tumefaciens/enzimología , Agrobacterium tumefaciens/genética , Alcanivoraceae/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Clonación Molecular , Frío , Secuencia de Consenso , Escherichia coli/enzimología , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Biblioteca de Genes , Genes Bacterianos , Vectores Genéticos/genética , Glicina/análogos & derivados , Glicina/farmacología , Concentración de Iones de Hidrógeno , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Filogenia , Conformación Proteica , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Ácido Shikímico/análogos & derivados , Ácido Shikímico/metabolismo , GlifosatoRESUMEN
Rivers in northern Chile have arsenic (As) concentrations at levels that are toxic for humans and other organisms. Microorganism-mediated redox reactions have a crucial role in the As cycle; the microbial oxidation of As (As(III) to As(V)) is a critical transformation because it favors the immobilization of As in the solid phase. We studied the role of microbial As oxidation for controlling the mobility of As in the extreme environment found in the Chilean Altiplano (i.e., > 4000 meters above sea level (masl) and < 310 mm annual rainfall), which are conditions that have rarely been studied. Our model system was the upper Azufre River sub-basin, where the natural attenuation of As from hydrothermal discharge (pH 4-6) was observed. As(III) was actively oxidized by a microbial consortium, leading to a significant decrease in the dissolved As concentrations and a corresponding increase in the sediment's As concentration downstream of the hydrothermal source. In-situ oxidation experiments demonstrated that the As oxidation required biological activity, and microbiological molecular analysis confirmed the presence of As(III)-oxidizing groups (aroA-like genes) in the system. In addition, the pH measurements and solid phase analysis strongly suggested that the As removal mechanism involved adsorption or coprecipitation with Fe-oxyhydroxides. Taken together, these results indicate that the microorganism-mediated As oxidation contributed to the attenuation of As concentrations and the stabilization of As in the solid phase, therefore controlling the amount of As transported downstream. This study is the first to demonstrate the microbial oxidation of As in Altiplano basins and its relevance in the immobilization of As.