RESUMO
The dynamics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission are influenced by a variety of factors, including social restrictions and the emergence of distinct variants. In this study, we delve into the origins and dissemination of the Alpha, Delta, and Omicron-BA.1 variants of concern in Galicia, northwest Spain. For this, we leveraged genomic data collected by the EPICOVIGAL Consortium and from the GISAID database, along with mobility information from other Spanish regions and foreign countries. Our analysis indicates that initial introductions during the Alpha phase were predominantly from other Spanish regions and France. However, as the pandemic progressed, introductions from Portugal and the United States became increasingly significant. The number of detected introductions varied from 96 and 101 for Alpha and Delta to 39 for Omicron-BA.1. Most of these introductions left a low number of descendants (<10), suggesting a limited impact on the evolution of the pandemic in Galicia. Notably, Galicia's major coastal cities emerged as critical hubs for viral transmission, highlighting their role in sustaining and spreading the virus. This research emphasizes the critical role of regional connectivity in the spread of SARS-CoV-2 and offers essential insights for enhancing public health strategies and surveillance measures.
Assuntos
COVID-19 , SARS-CoV-2 , Espanha/epidemiologia , COVID-19/epidemiologia , COVID-19/transmissão , COVID-19/virologia , Humanos , SARS-CoV-2/genética , Genoma Viral , Filogenia , PandemiasRESUMO
BACKGROUND: The methodology described in previous literature for Monkeypox virus (MPXV) sequencing shows low efficiency when using metagenomic approaches. The aim of the present study was to evaluate a new fine-tuned method for extraction and enrichment of genomic MPXV DNA using clinical samples and to compare it to a non-enrichment metagenomic approach. RESULTS: A new procedure that allows sample enrichment in MPXV DNA, avoiding wasting the sequencing capacity in human DNA, was designed. This procedure consisted of host DNA depletion using a saponin/NaCl combination treatment and DNase, together with high g-force centrifugations. After typical quality control, samples using the enrichment method contained around 96% of reads not classified as human DNA, while the non-enrichment protocol showed around 5-10%. When reads not belonging to Orthopoxvirus were removed, enriched samples kept about 50% of the original read counts, while non-enriched ones kept only 2-7%. CONCLUSIONS: Results showed a very significant improvement in sequencing efficiency, increasing the number of reads belonging to MPXV, the depth of coverage and the trustworthiness of the consensus sequences. This, in turn, allows for more samples to be included in a single cartridge, reducing costs and time to diagnosis, which can be very important factors when dealing with a contagious disease.
Assuntos
Monkeypox virus , Mpox , Humanos , Monkeypox virus/genética , Mpox/diagnóstico , DNA Viral/genéticaRESUMO
Control of infections caused by carbapenem-resistant Klebsiella pneumoniae continues to be challenging. The success of this pathogen is favored by its ability to acquire antimicrobial resistance and to spread and persist in both the environment and in humans. The emergence of clinically important clones, such as sequence types 11, 15, 101, and 258, has been reported worldwide. However, the mechanisms promoting the dissemination of such high-risk clones are unknown. Unraveling the factors that play a role in the pathobiology and epidemicity of K. pneumoniae is therefore important for managing infections. To address this issue, we studied a carbapenem-resistant ST-15 K. pneumoniae isolate (Kp3380) that displayed a remarkable adherent phenotype with abundant pilus-like structures. Genome sequencing enabled us to identify a chaperone-usher pili system (Kpi) in Kp3380. Analysis of a large K. pneumoniae population from 32 European countries showed that the Kpi system is associated with the ST-15 clone. Phylogenetic analysis of the operon revealed that Kpi belongs to the little-characterized γ2-fimbrial clade. We demonstrate that Kpi contributes positively to the ability of K. pneumoniae to form biofilms and adhere to different host tissues. Moreover, the in vivo intestinal colonizing capacity of the Kpi-defective mutant was significantly reduced, as was its ability to infect Galleria mellonella The findings provide information about the pathobiology and epidemicity of Kpi+K. pneumoniae and indicate that the presence of Kpi may explain the success of the ST-15 clone. Disrupting bacterial adherence to the intestinal surface could potentially target gastrointestinal colonization.
Assuntos
Fímbrias Bacterianas/genética , Klebsiella pneumoniae/genética , Chaperonas Moleculares/genética , Células A549 , Animais , Antibacterianos , Aderência Bacteriana/efeitos dos fármacos , Aderência Bacteriana/genética , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Carbapenêmicos/farmacologia , Linhagem Celular , Modelos Animais de Doenças , Farmacorresistência Bacteriana Múltipla/genética , Células Epiteliais/microbiologia , Europa (Continente) , Feminino , Deleção de Genes , Genes Bacterianos/genética , Humanos , Infecções por Klebsiella , Klebsiella pneumoniae/citologia , Klebsiella pneumoniae/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Tipagem de Sequências Multilocus , Óperon , FilogeniaRESUMO
Pseudomonas aeruginosa is one of the leading causes of nosocomial pneumonia and its associated mortality. Moreover, extensively drug-resistant high-risk clones are globally widespread, presenting a major challenge to the healthcare systems. Despite this, no vaccine is available against this high-concerning pathogen. Here we tested immunogenicity and protective efficacy of an experimental live vaccine against P. aeruginosa pneumonia, consisting of an auxotrophic strain which lacks the key enzyme involved in D-glutamate biosynthesis, a structural component of the bacterial cell wall. As the amounts of free D-glutamate in vivo are trace substances in most cases, blockage of the cell wall synthesis occurs, compromising the growth of this strain, but not its immunogenic properties. Indeed, when delivered intranasally, this vaccine stimulated production of systemic and mucosal antibodies, induced effector memory, central memory and IL-17A-producing CD4+ T cells, and recruited neutrophils and mononuclear phagocytes into the airway mucosa. A significant improvement in mice survival after lung infection caused by ExoU-producing PAO1 and PA14 strains was observed. Nearly one third of the mice infected with the XDR high-risk clone ST235 were also protected. These findings highlight the potential of this vaccine for the control of acute pneumonia caused by this bacterial pathogen.
Assuntos
Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa/imunologia , Vacinas Atenuadas/imunologia , Administração Intranasal , Animais , Anticorpos Antibacterianos/imunologia , Feminino , Imunidade nas Mucosas , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pneumonia Bacteriana/imunologia , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/patogenicidade , Sistema Respiratório/imunologia , Infecções Respiratórias/imunologia , Vacinas Atenuadas/farmacologiaRESUMO
In the last decades, personalized medicine has been increasing its presence in different fields of medicine, including ophthalmology. A new factor that can help us direct medicine towards the challenge of personalized treatments is the microbiome. The gut microbiome plays an important role in controlling immune response, and dysbiosis has been associated with immune-mediated diseases such as non-infectious uveitis (NIU). In this review, we gather the published evidence, both in the pre-clinical and clinical studies, that support the possible role of intestinal dysbiosis in the pathogenesis of NIU, as well as the modulation of the gut microbiota as a new possible therapeutic target. We describe the different mechanisms that have been proposed to involve dysbiosis in the causality of NIU, as well as the potential pharmacological tools that could be used to modify the microbiome (dietary supplementation, antibiotics, fecal microbiota transplantation, immunomodulators, or biologic drugs) and, consequently, in the control of the NIU. Furthermore, there is increasing scientific evidence suggesting that the treatment with anti-TNF not only restores the composition of the gut microbiota but also that the study of the composition of the gut microbiome will help predict the response of each patient to anti-TNF treatment.
Assuntos
Microbioma Gastrointestinal , Microbiota , Uveíte , Disbiose , Transplante de Microbiota Fecal , Microbioma Gastrointestinal/fisiologia , Humanos , Microbiota/fisiologia , Inibidores do Fator de Necrose Tumoral , Uveíte/terapiaRESUMO
BACKGROUND: Infections caused by multidrug-resistant pathogens such as Acinetobacter baumannii constitute a major health problem worldwide. In this study we present a global in vivo transcriptomic analysis of A. baumannii isolated from the lungs of mice with pneumonia infection. METHODS: Mice were infected with A. baumannii ATCC 17978 and AbH12O-A2 strains and the total bacterial RNA were analyzed by RNA sequencing. Lists of differentially expressed genes were obtained and 14 of them were selected for gene deletion and further analysis. RESULTS: Transcriptomic analysis revealed a specific gene expression profile in A. baumannii during lung infection with upregulation of genes involved in iron acquisition and host invasion. Mutant strains lacking feoA, mtnN, yfgC, basB, hisF, oatA, and bfnL showed a significant loss of virulence in murine pneumonia. A decrease in biofilm formation, adherence to human epithelial cells, and growth rate was observed in selected mutants. CONCLUSIONS: This study provides an insight into A. baumannii gene expression profile during murine pneumonia infection. Data revealed that 7 in vivo upregulated genes were involved in virulence and could be considered new therapeutic targets.
Assuntos
Infecções por Acinetobacter , Acinetobacter baumannii , Pneumonia Bacteriana , Transcriptoma , Fatores de Virulência , Infecções por Acinetobacter/microbiologia , Acinetobacter baumannii/genética , Animais , Aderência Bacteriana , Células Cultivadas , Células Epiteliais/microbiologia , Humanos , Camundongos , Pneumonia Bacteriana/microbiologia , Fatores de Virulência/genéticaRESUMO
OBJECTIVES: Carbapenemases are the most important mechanism responsible for carbapenem resistance in Enterobacteriaceae. Among carbapenemases, OXA-48 presents unique challenges as it is resistant to ß-lactam inhibitors. Here, we test the capacity of the compound LN-1-255, a 6-alkylidene-2'-substituted penicillanic acid sulfone, to inhibit the activity of the carbapenemase OXA-48. METHODS: The OXA-48 gene was cloned and expressed in Klebsiella pneumoniae and Escherichia coli in order to obtain MICs in the presence of inhibitors (clavulanic acid, tazobactam and sulbactam) and LN-1-255. OXA-48 was purified and steady-state kinetics was performed with LN-1-255 and tazobactam. The covalent binding mode of LN-1-255 with OXA-48 was studied by docking assays. RESULTS: Both OXA-48-producing clinical and transformant strains displayed increased susceptibility to carbapenem antibiotics in the presence of 4 mg/L LN-1-255 (2-32-fold increased susceptibility) and 16 mg/L LN-1-255 (4-64-fold increased susceptibility). Kinetic assays demonstrated that LN-1-255 is able to inhibit OXA-48 with an acylation efficiency (k2/K) of 10â±â1â×â10(4) M(-1) s(-1) and a slow deacylation rate (koff) of 7â±â1â×â10(-4) s(-1). IC50 was 3 nM for LN-1-255 and 1.5 µM for tazobactam. Lastly, kcat/kinact was 500-fold lower for LN-1-255 than for tazobactam. CONCLUSIONS: In these studies, carbapenem antibiotics used in combination with LN-1-255 are effective against the carbapenemase OXA-48, an important emerging mechanism of antibiotic resistance. This provides an incentive for further investigations to maximize the efficacy of penicillin sulfone inhibition of class D plasmid-carried Enterobacteriaceae carbapenemases.
Assuntos
Óxidos S-Cíclicos/metabolismo , Escherichia coli/efeitos dos fármacos , Klebsiella pneumoniae/efeitos dos fármacos , Penicilinas/metabolismo , Sulbactam/metabolismo , Inibidores de beta-Lactamases/metabolismo , beta-Lactamases/metabolismo , Antibacterianos/farmacologia , Carbapenêmicos/farmacologia , Clonagem Molecular , Escherichia coli/enzimologia , Escherichia coli/genética , Expressão Gênica , Cinética , Klebsiella pneumoniae/enzimologia , Klebsiella pneumoniae/genética , Testes de Sensibilidade Microbiana , Ligação Proteica , beta-Lactamases/isolamento & purificaçãoRESUMO
The phosphoryl-transfer mechanism of shikimate kinase from Mycobacterium tuberculosis and Helicobacter pylori, which is an attractive target for antibiotic drug discovery, has been studied by 1D (1)H and (31)Pâ NMR spectroscopy. Metaphosphoric acid proved to be a good mimetic of the metaphosphate intermediate and facilitated the ready and rapid evaluation by NMR spectroscopic analysis of a dissociative mechanism. The required closed form of the active site for catalysis was achieved by the use of ADP (product) or two synthetic ADP analogues (AMPNP, AMPCP). Molecular dynamics simulation studies reported here also revealed that the essential arginine (Arg116/Arg117 in H. pylori and M. tuberculosis, respectively), which activates the γ-phosphate group of ATP for catalysis and triggers the release of the product for turnover, would also be involved in the stabilisation of the metaphosphate intermediate during catalysis. We believe that the studies reported here will be helpful for future structure-based design of inhibitors of this attractive target. The approach is also expected be useful for studies on the possible dissociative mechanism of other kinase enzymes.
Assuntos
Difosfato de Adenosina/química , Trifosfato de Adenosina/análogos & derivados , Helicobacter pylori/enzimologia , Mycobacterium tuberculosis/enzimologia , Ácidos Fosforosos/química , Fosfotransferases (Aceptor do Grupo Álcool)/química , Trifosfato de Adenosina/química , Sítios de Ligação , Catálise , Cristalografia por Raios X , Helicobacter pylori/química , Espectroscopia de Ressonância Magnética , Simulação de Dinâmica Molecular , Mycobacterium tuberculosis/química , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismoRESUMO
Two mechanisms of resistance to colistin have been described in Acinetobacter baumannii. One involves complete loss of lipopolysaccharide (LPS), resulting from mutations in lpxA, lpxC, or lpxD, and the second is associated with phosphoethanolamine addition to LPS, mediated through mutations in pmrAB. In order to assess the clinical impacts of both resistance mechanisms, A. baumannii ATCC 19606 and its isogenic derivatives, AL1851 ΔlpxA, AL1852 ΔlpxD, AL1842 ΔlpxC, and ATCC 19606 pmrB, were analyzed for in vitro growth rate, in vitro and in vivo competitive growth, infection of A549 respiratory alveolar epithelial cells, virulence in the Caenorhabditis elegans model, and virulence in a systemic mouse infection model. The in vitro growth rate of the lpx mutants was clearly diminished; furthermore, in vitro and in vivo competitive-growth experiments revealed a reduction in fitness for both mutant types. Infection of A549 cells with ATCC 19606 or the pmrB mutant resulted in greater loss of viability than with lpx mutants. Finally, the lpx mutants were highly attenuated in both the C. elegans and mouse infection models, while the pmrB mutant was attenuated only in the C. elegans model. In summary, while colistin resistance in A. baumannii confers a clear selective advantage in the presence of colistin treatment, it causes a noticeable cost in terms of overall fitness and virulence, with a more striking reduction associated with LPS loss than with phosphoethanolamine addition. Therefore, we hypothesize that colistin resistance mediated by changes in pmrAB will be more likely to arise in clinical settings in patients treated with colistin.
Assuntos
Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/patogenicidade , Colistina/farmacologia , Colistina/uso terapêutico , Animais , Linhagem Celular , Farmacorresistência Bacteriana , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , VirulênciaRESUMO
Wastewater treatment plants (WWTPs) are recognized as important niches of antibiotic-resistant bacteria that can be easily spread to the environment. In this study, we collected wastewater samples from the WWTP of A Coruña (NW Spain) from April 2020 to February 2022 to evaluate the presence of Gram-negative bacteria harboring carbapenemase genes. Bacteria isolated from wastewater were classified and their antimicrobial profiles were determined. In total, 252 Gram-negative bacteria carrying various carbapenemase genes were described. Whole-genome sequencing was conducted on 55 selected carbapenemase producing isolates using Oxford Nanopore technology. This study revealed the presence of a significant population of bacteria carrying carbapenemase genes in WWTP, which constitutes a public health problem due to their risk of dissemination to the environment. This emphasizes the usefulness of WWTP monitoring for combating antibiotic resistance. Data revealed the presence of different types of sequences harboring carbapenemase genes, such as blaKPC-2, blaGES-5, blaGES-6, blaIMP-11, blaIMP-28, blaOXA-24, blaOXA-48, blaOXA-58, blaOXA-217, and blaVIM-2. Importantly, the presence of the blaKPC-2 gene in wastewater, several months before any clinical case was detected in University Hospital of A Coruña, suggests that wastewater-based epidemiology can be used as an early warning system for the surveillance of antibiotic-resistant bacteria.
RESUMO
The incidence of colorectal cancer (CRC) has increased worldwide, and early diagnosis is crucial to reduce mortality rates. Therefore, new noninvasive biomarkers for CRC are required. Recent studies have revealed an imbalance in the oral and gut microbiomes of patients with CRC, as well as impaired gut vascular barrier function. In the present study, the microbiomes of saliva, crevicular fluid, feces, and non-neoplastic and tumor intestinal tissue samples of 93 CRC patients and 30 healthy individuals without digestive disorders (non-CRC) were analyzed by 16S rRNA metabarcoding procedures. The data revealed that Parvimonas, Fusobacterium, and Bacteroides fragilis were significantly over-represented in stool samples of CRC patients, whereas Faecalibacterium and Blautia were significantly over-abundant in the non-CRC group. Moreover, the tumor samples were enriched in well-known periodontal anaerobes, including Fusobacterium, Parvimonas, Peptostreptococcus, Porphyromonas, and Prevotella. Co-occurrence patterns of these oral microorganisms were observed in the subgingival pocket and in the tumor tissues of CRC patients, where they also correlated with other gut microbes, such as Hungatella. This study provides new evidence that oral pathobionts, normally located in subgingival pockets, can migrate to the colon and probably aggregate with aerobic bacteria, forming synergistic consortia. Furthermore, we suggest that the group composed of Fusobacterium, Parvimonas, Bacteroides, and Faecalibacterium could be used to design an excellent noninvasive fecal test for the early diagnosis of CRC. The combination of these four genera would significantly improve the reliability of a discriminatory test with respect to others that use a single species as a unique CRC biomarker.
Assuntos
Bacteroides , Biomarcadores Tumorais , Neoplasias Colorretais , Fezes , Fusobacterium , Humanos , Neoplasias Colorretais/microbiologia , Neoplasias Colorretais/diagnóstico , Fusobacterium/isolamento & purificação , Fusobacterium/genética , Masculino , Feminino , Bacteroides/isolamento & purificação , Bacteroides/genética , Pessoa de Meia-Idade , Fezes/microbiologia , Faecalibacterium/isolamento & purificação , Faecalibacterium/genética , Idoso , RNA Ribossômico 16S/genética , Microbioma Gastrointestinal/genética , Saliva/microbiologia , AdultoRESUMO
The dynamics of SARS-CoV-2 transmission are influenced by a variety of factors, including social restrictions and the emergence of distinct variants. In this study, we delve into the origins and dissemination of the Alpha, Delta, and Omicron variants of concern in Galicia, northwest Spain. For this, we leveraged genomic data collected by the EPICOVIGAL Consortium and from the GISAID database, along with mobility information from other Spanish regions and foreign countries. Our analysis indicates that initial introductions during the Alpha phase were predominantly from other Spanish regions and France. However, as the pandemic progressed, introductions from Portugal and the USA became increasingly significant. Notably, Galicia's major coastal cities emerged as critical hubs for viral transmission, highlighting their role in sustaining and spreading the virus. This research emphasizes the critical role of regional connectivity in the spread of SARS-CoV-2 and offers essential insights for enhancing public health strategies and surveillance measures.
RESUMO
Oral and intestinal samples from a cohort of 93 colorectal cancer (CRC) patients and 30 healthy controls (non-CRC) were collected for microbiome analysis. Saliva (28 non-CRC and 94 CRC), feces (30 non-CRC and 97 CRC), subgingival fluid (20 CRC), and tumor tissue samples (20 CRC) were used for 16S metabarcoding and/or RNA sequencing (RNAseq) approaches. A differential analysis of the abundance, performed with the ANCOM-BC package, adjusting the P-values by the Holm-Bonferroni method, revealed that Parvimonas was significantly over-represented in feces from CRC patients (P-value < 0.001) compared to healthy controls. A total of 11 Parvimonas micra isolates were obtained from the oral cavity and adenocarcinoma of CRC patients. Genome analysis identified a pair of isolates from the same patient that shared 99.2% identity, demonstrating that P. micra can translocate from the subgingival cavity to the gut. The data suggest that P. micra could migrate in a synergistic consortium with other periodontal bacteria. Metatranscriptomics confirmed that oral bacteria were more active in tumor than in non-neoplastic tissues. We suggest that P. micra could be considered as a CRC biomarker detected in non-invasive samples such as feces.
RESUMO
Wastewater-based epidemiology has been widely used as a cost-effective method for tracking the COVID-19 pandemic at the community level. Here we describe COVIDBENS, a wastewater surveillance program running from June 2020 to March 2022 in the wastewater treatment plant of Bens in A Coruña (Spain). The main goal of this work was to provide an effective early warning tool based in wastewater epidemiology to help in decision-making at both the social and public health levels. RT-qPCR procedures and Illumina sequencing were used to weekly monitor the viral load and to detect SARS-CoV-2 mutations in wastewater, respectively. In addition, own statistical models were applied to estimate the real number of infected people and the frequency of each emerging variant circulating in the community, which considerable improved the surveillance strategy. Our analysis detected 6 viral load waves in A Coruña with concentrations between 103 and 106 SARS-CoV-2 RNA copies/L. Our system was able to anticipate community outbreaks during the pandemic with 8-36 days in advance with respect to clinical reports and, to detect the emergence of new SARS-CoV-2 variants in A Coruña such as Alpha (B.1.1.7), Delta (B.1.617.2), and Omicron (B.1.1.529 and BA.2) in wastewater with 42, 30, and 27 days, respectively, before the health system did. Data generated here helped local authorities and health managers to give a faster and more efficient response to the pandemic situation, and also allowed important industrial companies to adapt their production to each situation. The wastewater-based epidemiology program developed in our metropolitan area of A Coruña (Spain) during the SARS-CoV-2 pandemic served as a powerful early warning system combining statistical models with mutations and viral load monitoring in wastewater over time.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , COVID-19/epidemiologia , Espanha/epidemiologia , Águas Residuárias , Pandemias , RNA Viral , Vigilância Epidemiológica Baseada em Águas Residuárias , Surtos de DoençasRESUMO
The development of a whole-cell vaccine from bacteria auxotrophic for D-amino acids present in the bacterial cell wall is considered a promising strategy for providing protection against bacterial infections. Here, we constructed a prototype vaccine, consisting of a glutamate racemase-deficient mutant, for preventing Klebsiella pneumoniae infections. The deletion mutant lacks the murI gene and requires exogenous addition of D-glutamate for growth. The results showed that the K. pneumoniae ΔmurI strain is attenuated and includes a favourable combination of antigens for inducing a robust immune response and conferring an adequate level of cross-protection against systemic infections caused by K. pneumoniae strains, including some hypervirulent serotypes with elevated production of capsule polysaccharide as well as multiresistant K. pneumoniae strains. The auxotroph also induced specific production of IL-17A and IFN-γ. The rapid elimination of the strain from the blood of mice without causing disease suggests a high level of safety for administration as a vaccine.
RESUMO
The quantification of the SARS-CoV-2 RNA load in wastewater has emerged as a useful tool to monitor COVID-19 outbreaks in the community. This approach was implemented in the metropolitan area of A Coruña (NW Spain), where wastewater from a treatment plant was analyzed to track the epidemic dynamics in a population of 369,098 inhabitants. Viral load detected in the wastewater and the epidemiological data from A Coruña health system served as main sources for statistical models developing. Regression models described here allowed us to estimate the number of infected people (R2 = 0.9), including symptomatic and asymptomatic individuals. These models have helped to understand the real magnitude of the epidemic in a population at any given time and have been used as an effective early warning tool for predicting outbreaks in A Coruña municipality. The methodology of the present work could be used to develop a similar wastewater-based epidemiological model to track the evolution of the COVID-19 epidemic anywhere in the world where centralized water-based sanitation systems exist.
Assuntos
COVID-19 , SARS-CoV-2 , Modelos Epidemiológicos , Humanos , RNA Viral , Espanha/epidemiologia , Carga Viral , Águas ResiduáriasRESUMO
Ethanol and endopolygalacturonase (endoPG) are simultaneously produced by the yeast Kluyveromyces marxianus CCEBI 2011. The aim of this study was to determine the optimal combination of seven environmental and nutritional variables, as well as the influence of each one, with respect to the fermentation process in yeast cultures in which sugarcane juice was the substrate. Simplex sequential optimization showed that after 15 runs the optimal conditions were: pH, 4.6; temperature, 31 ºC; total reducing sugars (TRS), 125 g/l; (NH(4))(2)SO(4), 2.48 g/l; (NH(4))(2)HPO(4), 2.73 g/l; CaCl(2), 0.33 g/l and MgSO(4)·7H(2)O, 0.54 g/l. Under these conditions, the ethanol concentration was 47.6 g/l and endoPG concentration was 9.8 U/ml, which represented increases of 22% and 10%, respectively, over the concentrations obtained under suboptimal conditions. Temperature and (NH(4))(2)SO(4) supplementation were the most significant factors influencing the co-production process.
Assuntos
Etanol/metabolismo , Kluyveromyces/metabolismo , Poligalacturonase/metabolismo , Saccharum/metabolismo , Meios de Cultura/química , Fermentação , Concentração de Íons de Hidrogênio , TemperaturaRESUMO
Acinetobacter baumannii has emerged in the last decade as an important nosocomial pathogen. To identify genes involved in the course of a pneumonia infection, gene expression profiles were obtained from A. baumannii ATCC 17978 grown in mouse infected lungs and in culture medium. Gene expression analysis allowed us to determine a gene, the A1S_0242 gene (feoA), over-expressed during the pneumonia infection. In the present work, we evaluate the role of this gene, involved in iron uptake. The inactivation of the A1S_0242 gene resulted in an increase susceptibility to oxidative stress and a decrease in biofilm formation, in adherence to A549 cells and in fitness. In addition, infection of G. mellonella and pneumonia in mice showed that the virulence of the Δ0242 mutant was significantly attenuated. Data presented in this work indicated that the A1S_0242 gene from A. baumannii ATCC 17978 strain plays a role in fitness, adhesion, biofilm formation, growth, and, definitively, in virulence. Taken together, these observations show the implication of the feoA gene plays in the pathogenesis of A. baumannii and highlight its value as a potential therapeutic target.
Assuntos
Infecções por Acinetobacter/fisiopatologia , Acinetobacter baumannii/patogenicidade , Proteínas de Transporte de Cátions/metabolismo , Pneumonia Bacteriana/fisiopatologia , Fatores de Virulência/metabolismo , Células A549 , Acinetobacter baumannii/genética , Animais , Aderência Bacteriana , Biofilmes/crescimento & desenvolvimento , Proteínas de Transporte de Cátions/genética , Modelos Animais de Doenças , Células Epiteliais/microbiologia , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Pulmão/microbiologia , Camundongos , Fatores de Virulência/genéticaRESUMO
Many strains of Acinetobacter baumannii have been described as being able to form biofilm. Small non-coding RNAs (sRNAs) control gene expression in many regulatory circuits in bacteria. The aim of the present work was to provide a global description of the sRNAs produced both by planktonic and biofilm-associated (sessile) cells of A. baumannii ATCC 17978, and to compare the corresponding gene expression profiles to identify sRNAs molecules associated to biofilm formation and virulence. sRNA was extracted from both planktonic and sessile cells and reverse transcribed. cDNA was subjected to 454-pyrosequencing using the GS-FLX Titanium chemistry. The global analysis of the small RNA transcriptome revealed different sRNA expression patterns in planktonic and biofilm associated cells, with some of the transcripts only expressed or repressed in sessile bacteria. A total of 255 sRNAs were detected, with 185 of them differentially expressed in the different types of cells. A total of 9 sRNAs were expressed only in biofilm cells, while the expression of other 21 coding regions were repressed only in biofilm cells. Strikingly, the expression level of the sRNA 13573 was 120 times higher in biofilms than in planktonic cells, an observation that prompted us to further investigate the biological role of this non-coding transcript. Analyses of an isogenic mutant and over-expressing strains revealed that the sRNA 13573 gene is involved in biofilm formation and attachment to A549 human alveolar epithelial cells. The present work serves as a basis for future studies examining the complex regulatory network that regulate biofilm biogenesis and attachment to eukaryotic cells in A. baumannii ATCC 17978.
Assuntos
Acinetobacter baumannii/genética , Biofilmes , Perfilação da Expressão Gênica , Pequeno RNA não Traduzido/genética , Células A549 , Acinetobacter baumannii/fisiologia , Linhagem Celular Tumoral , DNA Complementar/genética , Regulação Bacteriana da Expressão Gênica , Humanos , Microscopia Eletrônica de Varredura , RNA Bacteriano/genética , VirulênciaRESUMO
Genetic and functional studies showed that some components of the Acinetobacter baumannii ATCC 17978 A1S_0112-A1S_0119 gene cluster are critical for biofilm biogenesis and surface motility. Recently, our group has shown that the A1S_0114 gene was involved in biofilm formation, a process related with pathogenesis. Confirming our previous results, microscopy images revealed that the ATCC 17978 Δ0114 derivative lacking this gene was unable to form a mature biofilm structure. Therefore, other bacterial phenotypes were analyzed to determine the role of this gene in the pathogenicity of A. baumannii ATCC 17978. The interaction of the ATCC 17978 parental strain and the Δ0114 mutant with A549 human alveolar epithelial cells was quantified revealing that the A1S_0114 gene was necessary for proper attachment to A549 cells. This dependency correlates with the negative effect of the A1S_0114 deletion on the expression of genes coding for surface proteins and pili-assembly systems, which are known to play a role in adhesion. Three different experimental animal models, including vertebrate and invertebrate hosts, confirmed the role of the A1S_0114 gene in virulence. All of the experimental infection assays indicated that the virulence of the ATCC 17978 was significantly reduced when this gene was inactivated. Finally, we discovered that the A1S_0114 gene was involved in the production of a small lipopeptide-like compound herein referred to as acinetin 505 (Ac-505). Ac-505 was isolated from ATCC 17978 spent media and its chemical structure was interpreted by mass spectrometry. Overall, our observations provide novel information on the role of the A1S_0114 gene in A. baumannii's pathobiology and lay the foundation for future work to determine the mechanisms by which Ac-505, or possibly an Ac-505 precursor, could execute critical functions as a secondary metabolite.