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1.
Braz. j. biol ; 84: e254016, 2024. tab
Artículo en Inglés | LILACS, VETINDEX | ID: biblio-1364529

RESUMEN

The present study was conducted to isolate and characterize bacteria from water and soil sample taken from the Lahore Canal at different sites i.e. Mall Road, Mohlanwal and Khera site. Isolated bacterial strains were identified on the basis of morphological and biochemical tests. Identification was confirmed by culturing bacteria on selective media. Antibiotic resistance test was also performed to observe the resistance of bacteria against different antibiotics. Blood agar test was performed for identification of different pathogenic bacteria. The result revealed that water and soil samples of Lahore Canal Lahore from different sites were contaminated with Escherichia coli, Salmonella sp., Vibrio sp., Bacillus spp., Enterococcus sp. and Staphylococcus spp. Due to presence of these pathogens, this water is not suitable for any domestic and irrigation use. Study also revealed that water of the Lahore Canal is harmful for human health as it is contaminated with bacteria that can cause severe disease e.g., Escherichia coli can cause gastroenteritis, Bacillus spp. can cause nausea and vomiting, Enterococcus may infect urinary tract, Salmonella sp. is responsible for Bacteremia, Staphylococcus spp. can cause mild fever and Vibrio sp. can be the reason of cholera. Thus it is rendered unfit for any kind of human use even other than drinking like swimming, bathing, washing etc., until and unless some remedial measures are employed to eradicate pathogenic microorganisms by WASA and LWMS according to standards of WHO. Similarly, it is quite harmful, when and where ever it is used for irrigation without proper treatment.


O presente estudo foi realizado para isolar e caracterizar bactérias de amostras de água e solo retiradas do Canal Lahore, em Lahore, em diferentes locais, ou seja, Mall Road, Mohlanwal e Khera. As cepas bacterianas isoladas foram identificadas com base em testes morfológicos e bioquímicos. A identificação foi confirmada por cultura de bactérias em testes de meios seletivos. O teste de resistência aos antibióticos também foi realizado para observar a resistência das bactérias a diferentes antibióticos. Foi realizado o teste de ágar sangue para identificar diferentes bactérias patogênicas. O resultado revelou que amostras de água e solo do Canal Lahore, Lahore, de diferentes localidades estavam contaminadas com Escherichia coli, Salmonella sp., Vibrio sp., Bacillus spp., Enterococcus sp. e Staphylococcus spp. Por causa da presença desses patógenos, essa água não é adequada para qualquer uso doméstico e de irrigação. O estudo revelou que a água do Canal Lahore é prejudicial à saúde humana, pois está contaminada com bactérias que podem causar doenças graves, por exemplo: Escherichia coli pode ocasionar gastroenterite; Bacillus spp. pode causar náuseas e vômitos; Enterococcus sp. pode infectar o trato urinário; Salmonella sp. é responsável pela bacteremia; Staphylococcus spp. pode causar febre leve; e Vibrio sp. pode ser a razão da cólera. Assim, torna-se imprópria para uso humano, como natação, banho, lavagem etc., até que algumas medidas corretivas sejam empregadas para erradicar microrganismos patogênicos por WASA e LWMS de acordo com os padrões da OMS. Da mesma forma, é bastante prejudicial, quando usada para irrigação sem tratamento adequado.


Asunto(s)
Animales , Suelo , Staphylococcus , Vibrio , Farmacorresistencia Microbiana , Muestras de Agua , Enterococcus , Escherichia coli
2.
Braz. j. biol ; 84: e255755, 2024. tab, graf
Artículo en Inglés | LILACS, VETINDEX | ID: biblio-1355898

RESUMEN

Abstract The present study involves the chemical and bacteriological analysis of water from different sources i.e., bore, wells, bottle, and tap, from Peshawar, Mardan, Swat and Kohat districts of Khyber Pakhtunkhwa (KP) province, Pakistan. From each district, 50 water samples (10 samples from each source), regardless of urban and rural status, were collected from these sources and analysed for sulphates, nitrates, nitrites, chlorides, total soluble solids and coliforms (E. coli). Results indicated that majority of the water sources had unacceptable E. coli count i.e.> 34 CFU/100mL. E. coli positive samples were high in Mardan District, followed by Kohat, Swat and Peshawar district. Besides this, the some water sources were also chemically contaminated by different inorganic fertilizers (nitrates/nitrites of sodium, potassium) but under safe levels whereas agricultural and industrial wastes (chloride and sulphate compounds) were in unsafe range. Among all districts, the water quality was found comparatively more deteriorated in Kohat and Mardan districts than Peshawar and Swat districts. Such chemically and bacteriologically unfit water sources for drinking and can cause human health problems.


Resumo O presente estudo envolve a análise química e bacteriológica de água de diferentes fontes, ou seja, furo, poços, garrafa e torneira, dos distritos de Peshawar, Mardan, Swat e Kohat da província de Khyber Pakhtunkhwa (KP), Paquistão. De cada distrito, 50 amostras de água (10 amostras de cada fonte), independentemente do status urbano e rural, foram coletadas dessas fontes e analisadas para sulfatos, nitratos, nitritos, cloretos, sólidos solúveis totais e coliformes (E. coli). Os resultados indicaram que a maioria das fontes de água tinha uma contagem inaceitável de E. coli, ou seja, > 34 UFC / 100 mL. As amostras positivas para E. coli foram elevadas no distrito de Mardan, seguido por Kohat, Swat e distrito de Peshawar. Além disso, algumas fontes de água também foram contaminadas quimicamente por diferentes fertilizantes inorgânicos (nitratos/nitritos de sódio, potássio), mas em níveis seguros, enquanto os resíduos agrícolas e industriais (compostos de cloreto e sulfato) estavam em níveis inseguros. Entre todos os distritos, a qualidade da água foi considerada comparativamente mais deteriorada nos distritos de Kohat e Mardan do que nos distritos de Peshawar e Swat. Essas fontes de água química e bacteriologicamente impróprias para beber podem causar problemas à saúde humana.


Asunto(s)
Humanos , Agua Potable , Calidad del Agua , Pakistán , Escherichia coli
3.
Toxicon ; 223: 107012, 2023 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-36592762

RESUMEN

The methylotrophic yeast Pichia pastoris has been one of the most widely used organisms in recent years as an expression system for a wide variety of recombinant proteins with therapeutic potential. Its popularity as an alternative system to Escherichia coli is mainly due to the easy genetic manipulation and the ability to produce high levels of heterologous proteins, either intracellularly or extracellularly. Being a eukaryotic organism, P. pastoris carries out post-translational modifications that allow it to produce soluble and correctly folded recombinant proteins. This work, evaluated the expression capacity in P. pastoris of two single-chain variable fragments (scFvs) of human origin, 10FG2 and LR. These scFvs were previously obtained by directed evolution against scorpion venom toxins and are able to neutralize different toxins and venoms of Mexican species. The yield obtained in P. pastoris was higher than that obtained in bacterial periplasm (E. coli), and most importantly, biochemical and functional properties were not modified. These results confirm that P. pastoris yeast can be a good expression system for the production of antibody fragments of a new recombinant antivenom.


Asunto(s)
Escorpiones , Ponzoñas , Animales , Humanos , Escorpiones/química , Ponzoñas/metabolismo , Saccharomyces cerevisiae/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/química , Fragmentos de Inmunoglobulinas/genética , Fragmentos de Inmunoglobulinas/metabolismo
4.
Nature ; 613(7945): 783-789, 2023 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-36631609

RESUMEN

Efficient and accurate termination is required for gene transcription in all living organisms1,2. Cellular RNA polymerases in both bacteria and eukaryotes can terminate their transcription through a factor-independent termination pathway3,4-called intrinsic termination transcription in bacteria-in which RNA polymerase recognizes terminator sequences, stops nucleotide addition and releases nascent RNA spontaneously. Here we report a set of single-particle cryo-electron microscopy structures of Escherichia coli transcription intrinsic termination complexes representing key intermediate states of the event. The structures show how RNA polymerase pauses at terminator sequences, how the terminator RNA hairpin folds inside RNA polymerase, and how RNA polymerase rewinds the transcription bubble to release RNA and then DNA. These macromolecular snapshots define a structural mechanism for bacterial intrinsic termination and a pathway for RNA release and DNA collapse that is relevant for factor-independent termination by all RNA polymerases.


Asunto(s)
ARN Polimerasas Dirigidas por ADN , Transcripción Genética , Microscopía por Crioelectrón , ARN Polimerasas Dirigidas por ADN/metabolismo , ARN , ADN , Escherichia coli/genética , Escherichia coli/metabolismo , Bacterias/genética , Regiones Terminadoras Genéticas , ARN Bacteriano/genética
5.
J Med Chem ; 66(2): 1380-1425, 2023 Jan 26.
Artículo en Inglés | MEDLINE | ID: mdl-36634346

RESUMEN

We have developed compounds with a promising activity against Acinetobacter baumannii and Pseudomonas aeruginosa, which are both on the WHO priority list of antibiotic-resistant bacteria. Starting from DNA gyrase inhibitor 1, we identified compound 27, featuring a 10-fold improved aqueous solubility, a 10-fold improved inhibition of topoisomerase IV from A. baumannii and P. aeruginosa, a 10-fold decreased inhibition of human topoisomerase IIα, and no cross-resistance to novobiocin. Cocrystal structures of 1 in complex with Escherichia coli GyrB24 and (S)-27 in complex with A. baumannii GyrB23 and P. aeruginosa GyrB24 revealed their binding to the ATP-binding pocket of the GyrB subunit. In further optimization steps, solubility, plasma free fraction, and other ADME properties of 27 were improved by fine-tuning of lipophilicity. In particular, analogs of 27 with retained anti-Gram-negative activity and improved plasma free fraction were identified. The series was found to be nongenotoxic, nonmutagenic, devoid of mitochondrial toxicity, and possessed no ion channel liabilities.


Asunto(s)
Acinetobacter baumannii , Inhibidores de Topoisomerasa II , Humanos , Inhibidores de Topoisomerasa II/farmacología , Inhibidores de Topoisomerasa II/química , Pseudomonas aeruginosa/metabolismo , Acinetobacter baumannii/metabolismo , Antibacterianos/farmacología , Antibacterianos/química , Escherichia coli/metabolismo , Benzotiazoles , Pruebas de Sensibilidad Microbiana , Girasa de ADN/metabolismo
6.
Nat Commun ; 14(1): 59, 2023 Jan 04.
Artículo en Inglés | MEDLINE | ID: mdl-36599844

RESUMEN

The aromatic side-chains of phenylalanine, tyrosine, and tryptophan interact with their environments via both hydrophobic and electrostatic interactions. Determining the extent to which these contribute to protein function and stability is not possible with conventional mutagenesis. Serial fluorination of a given aromatic is a validated method in vitro and in silico to specifically alter electrostatic characteristics, but this approach is restricted to a select few experimental systems. Here, we report a group of pyrrolysine-based aminoacyl-tRNA synthetase/tRNA pairs (tRNA/RS pairs) that enable the site-specific encoding of a varied spectrum of fluorinated phenylalanine amino acids in E. coli and mammalian (HEK 293T) cells. By allowing the cross-kingdom expression of proteins bearing these unnatural amino acids at biochemical scale, these tools may potentially enable the study of biological mechanisms which utilize aromatic interactions in structural and cellular contexts.


Asunto(s)
Aminoacil-ARNt Sintetasas , Fenilalanina , Aminoácidos/metabolismo , Aminoacil-ARNt Sintetasas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Halogenación , Fenilalanina/metabolismo , ARN de Transferencia/metabolismo , Humanos , Células HEK293
7.
Gut Microbes ; 15(1): 2163838, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-36656595

RESUMEN

Conflicting evidence exists on the association between consumption of non-steroidal anti-inflammatory drugs (NSAIDs) and symptomatic worsening of inflammatory bowel disease (IBD). We hypothesized that the heterogeneous prevalence of pathobionts [e.g., adherent-invasive Escherichia coli (AIEC)], might explain this inconsistent NSAIDs/IBD correlation. Using IL10-/- mice, we found that NSAID aggravated colitis in AIEC-colonized animals. This was accompanied by activation of the NLRP3 inflammasome, Caspase-8, apoptosis, and pyroptosis, features not seen in mice exposed to AIEC or NSAID alone, revealing an AIEC/NSAID synergistic effect. Inhibition of NLRP3 or Caspase-8 activity ameliorated colitis, with reduction in NLRP3 inflammasome activation, cell death markers, activated T-cells and macrophages, improved histology, and increased abundance of Clostridium cluster XIVa species. Our findings provide new insights into how NSAIDs and an opportunistic gut-pathobiont can synergize to worsen IBD symptoms. Targeting the NLRP3 inflammasome or Caspase-8 could be a potential therapeutic strategy in IBD patients with gut inflammation, which is worsened by NSAIDs.


Asunto(s)
Antiinflamatorios no Esteroideos , Colitis , Microbioma Gastrointestinal , Enfermedades Inflamatorias del Intestino , Animales , Ratones , Antiinflamatorios no Esteroideos/efectos adversos , Caspasa 8/metabolismo , Colitis/inducido químicamente , Colitis/tratamiento farmacológico , Colitis/microbiología , Inflamasomas , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Enfermedades Inflamatorias del Intestino/microbiología , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Inhibidores de Caspasas/farmacología , Escherichia coli/patogenicidad
8.
PLoS One ; 18(1): e0280627, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-36662754

RESUMEN

The vital roles of diagnostic tools and vaccines are prominent in controlling COVID-19. Spike protein of the SARS CoV-2, specifically the epitopes in that protein, are the critical components of the vaccines and immunological diagnostic tools. Two epitopes in the spike protein, the S14P5 and S21P2, identified previously are of great interest because they are linear and elicit neutralizing antibodies. The present study formulated each epitope in the tandem-repeat structure to increase their immunogenicity and facilitate their production. The tandem repeats (TR) were expressed efficiently in E. coli, yielding 58 mg and 46 mg per liter culture for TR-S14P5 and TR-S212, respectively. ELISA using either one of the repeating epitopes can be used as a serological test to identify individuals infected by the SARS-CoV-2 virus. The area under curves (AUC), based on testing 157 serum samples from COVID-19 patients and 26 from COVID-19-free individuals, were 0.806 and 0.889 for TR-S14P5 and TR-S21P2-based ELISAs, respectively. For 100% diagnostic specificity, the sensitivity was only 70%. The low sensitivity supposedly resulted from some samples being from early infection prior to antibody conversion. Both recombinant epitopes were highly immunogenic in rabbits, and the immune sera recognized inactivated SARS CoV-2 virus in dot-blot assays. These antibodies should be useful as a reagent for detecting SARS-CoV-2 antigens. Furthermore, the TR-S14P5 and TR-S21P2, being conserved and denaturation-resistant, are envisaged to be ideal for intra-nasal vaccines, which are required to complement current COVID-19 to overcome rapidly mutated SARS CoV-2.


Asunto(s)
COVID-19 , Animales , Conejos , Epítopos , COVID-19/diagnóstico , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus , Escherichia coli , Anticuerpos Antivirales , Anticuerpos Neutralizantes
9.
Sci Adv ; 9(3): eadd8659, 2023 Jan 20.
Artículo en Inglés | MEDLINE | ID: mdl-36662863

RESUMEN

Braun's lipoprotein (Lpp) plays a major role in stabilizing the integrity of the cell envelope in Escherichia coli, as it provides a covalent cross-link between the outer membrane and the peptidoglycan layer. An important challenge in elucidating the physiological role of Lpp lies in attaining a detailed understanding of its distribution on the peptidoglycan layer. Here, using atomic force microscopy, we visualized Lpp directly on peptidoglycan sacculi. Lpp is homogeneously distributed over the outer surface of the sacculus at a high density. However, it is absent at the constriction site during cell division, revealing its role in the cell division process with Pal, another cell envelope-associated protein. Collectively, we have established a framework to elucidate the distribution of Lpp and other peptidoglycan-bound proteins via a direct imaging modality.


Asunto(s)
Escherichia coli , Peptidoglicano , Escherichia coli/metabolismo , Peptidoglicano/metabolismo , Proteínas de la Membrana Bacteriana Externa/metabolismo , Membrana Celular/metabolismo , Lipoproteínas/metabolismo
10.
J Wound Ostomy Continence Nurs ; 50(1): 26-30, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-36668986

RESUMEN

PURPOSE: Recent reports have noted an emergence of unusual organisms in microflora of pilonidal sinus (PNS); this study was undertaken to identify the primary microbial flora associated with infected primary PNS over a period of 1 year. DESIGN: A prospective multiple case series. SUBJECTS AND SETTING: A case series of 20 patients with primary PNS in a tertiary care center in Varanasi, India, was studied. The study was conducted at the Department of Microbiology and General Surgery, Institute of Medical Sciences, Varanasi, over a period of 1 year (September 2016 to July 2017). METHODS: Purulent exudate (pus) samples were collected from 20 patients with primary PNS from the discharging sinuses by aseptic methods. Samples were assessed for aerobic and anaerobic flora by conventional culture and molecular methods. Antimicrobial susceptibility testing was done for bacterial isolates. Bacterial diversity was compared with the demographic and clinical profile of the sinuses by multiple correspondence analysis. RESULTS: Of the total cases, 11 (55%) had purulent discharge, among which all showed polymicrobial flora. The ratio of aerobic to anaerobic organisms was 1:2 (16/32). Escherichia coli (E. coli, 4, 36.36%) and Enterococcus faecalis (E. faecalis, 4, 36.36%) were commonly isolated. Bifidobacterium was the most frequent anaerobe. Detailed molecular analysis revealed the presence of Kocuria flava as an unusual pathogen. On statistical analysis, factors like male gender, increased body mass index, absence of hair in sinus, presence of features of hirsutism, and absence of Fusobacteria were closely associated with one another in these PNS cases. CONCLUSIONS: The case series revealed the predominance of anaerobes in primarily infected PNS cases. Bifidobacterium spp and unusual pathogens like K. flava were among the emerging pathogens in infected PNS. Use of better molecular diagnostic facilities in addition to the conventional methods might enhance the verified diversity of microorganisms in such cases.


Asunto(s)
Seno Pilonidal , Humanos , Masculino , Escherichia coli , Estudios Prospectivos , India
11.
Environ Monit Assess ; 195(2): 316, 2023 Jan 21.
Artículo en Inglés | MEDLINE | ID: mdl-36670262

RESUMEN

The present study has been carried out to assess the quality of groundwater and surface water resources of Thettiyar watershed, Kerala, India. Sixty-six water samples were collected during pre-monsoon (April, 2019) and monsoon (July, 2019) season, and analyzed for pH, electrical conductivity (EC), total dissolved solids (TDS), total hardness and major cations and anions and microbiological parameters as well. According to the piper diagram's plot, Ca and Mg dominate over Na among the cations, and Cl is the most prevalent anion in groundwater throughout both the studied seasons. The hydrochemical analysis of water samples revealed that all the parameters are within the permissible limit except for pH. For microbiological analysis, the samples were tested for total coliform and fecal coliform. Most of the groundwater samples collected have higher total coliform and fecal coliform (E. coli) content than the recommended count by BIS (2012). The pre-monsoon surface water samples owned a maximum number of 3700 cfu/100 ml (TNTC-too numerous to count) of total coliform and 1400 cfu/100 ml (TNTC) of fecal coliform. In monsoon, the number of total coliform and fecal coliform has increased to 3800 cfu/100 ml and 1900 cfu/100 ml respectively. E. coli and total coliform are effectively correlated with each other in both seasons, in accordance with the statistical study. Domestic, sewage dump, and other household wastes are the main sources of bacterial contamination in the study area, which in turn nourishes contaminant organisms. According to the results, the government or municipality should implement an appropriate system for managing solid waste and should take all necessary measures to clean up the study area.


Asunto(s)
Agua Subterránea , Contaminantes Químicos del Agua , Calidad del Agua , Monitoreo del Ambiente/métodos , Sistemas de Información Geográfica , Escherichia coli , Contaminantes Químicos del Agua/análisis , Agua Subterránea/química , Aniones/análisis , Agua/análisis , Cationes/análisis , India
12.
BMC Vet Res ; 19(1): 17, 2023 Jan 21.
Artículo en Inglés | MEDLINE | ID: mdl-36670436

RESUMEN

BACKGROUND: Little data exist on the presence of resistant pathogens in day-old chicks imported into Benin. The occurrence of pathogenic bacteria was assessed in 180 one-day-old chicks imported from Belgium and received at the Cardinal Bernardin Gantin International Airport in Cotonou (Benin). The samples included swabbing the blisters of 180 chicks, followed by 18 pools of 10 swabs for bacterial isolation. Classic bacteriological methods based on Gram staining, culture on specific media and biochemical characterization were used. Antibacterial susceptibility screening to antibiotics was conducted using the Kirby-Bauer disc diffusion method, and the results were interpreted according to guidelines from the European Committee on Antimicrobial Susceptibility Testing (EUCAST). DNA extraction was performed by the heat treatment method. Resistance genes were screened by real-time PCR. RESULTS: We isolated 32 bacteria, including Escherichia coli (50%), Enterococcus spp. (28%), and coagulase-negative staphylococci (10%). The isolates were investigated for antibiotic resistance against antibiotics using the disk diffusion method and showed that in the Escherichia coli strains isolated, the highest rate of resistance was obtained against ciprofloxacin (81%), followed by trimethoprim + sulfamethoxazole (62%). Enterobacter cloacae was sensitive to all the antibiotics tested. Pseudomonas spp. resistant to amoxicillin and trimethoprim + sulfamethoxazole was noted. The SulII gene was found in all cloacal samples, while the SulI and blaTEM genes were present at 44.44% and 16.67%, respectively. CONCLUSION: This study confirms that imported day-old chicks can be a potential source of dissemination of resistant bacteria in poultry production. A system for immediate detection of resistant bacteria in chicks upon arrival in the country is thus needed.


Asunto(s)
Antibacterianos , Bacterias , Animales , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Bélgica , Benin , Pruebas de Sensibilidad Microbiana/veterinaria , Combinación Trimetoprim y Sulfametoxazol , Escherichia coli/genética
13.
Microbiome ; 11(1): 12, 2023 Jan 20.
Artículo en Inglés | MEDLINE | ID: mdl-36670449

RESUMEN

BACKGROUND: The plasmid-mediated resistance gene mcr-1 confers colistin resistance in Escherichia coli and paves the way for the evolution to pan-drug resistance. We investigated the impact of mcr-1 in gut colonization in the absence of antibiotics using isogenic E. coli strains transformed with a plasmid encoding or devoid of mcr-1. RESULTS: In gnotobiotic and conventional mice, mcr-1 significantly enhanced intestinal anchoring of E. coli but impaired their lethal effect. This improvement of intestinal fitness was associated with a downregulation of intestinal inflammatory markers and the preservation of intestinal microbiota composition. The mcr-1 gene mediated a cross-resistance to antimicrobial peptides secreted by the microbiota and intestinal epithelial cells (IECs), enhanced E. coli adhesion to IECs, and decreased the proinflammatory activity of both E. coli and its lipopolysaccharides. CONCLUSION: Overall, mcr-1 changed multiple facets of bacterial behaviour and appeared as a factor enhancing commensal lifestyle and persistence in the gut even in the absence of antibiotics. Video Abstract.


Asunto(s)
Infecciones por Escherichia coli , Proteínas de Escherichia coli , Animales , Ratones , Escherichia coli/genética , Simbiosis , Proteínas de Escherichia coli/genética , Farmacorresistencia Bacteriana/genética , Antibacterianos/farmacología , Infecciones por Escherichia coli/microbiología , Pruebas de Sensibilidad Microbiana
14.
Biomolecules ; 13(1)2023 Jan 16.
Artículo en Inglés | MEDLINE | ID: mdl-36671567

RESUMEN

Ragweed pollen is highly allergenic and elicits type I hypersensitivity reactions in the exposed populations. Amb a 11 is a recently discovered component of this pollen, and its biological role in allergy is still being researched. In our study, ragweed allergy patients were recruited prospectively over a three-year period; a comprehensive questionnaire was administered, and sera were collected and stored. The production of recombinant Amb a 11 was achieved in parallel with patients' recruitment. The gene coding for mature protein was inserted in E. coli and in Sf9 Spodoptera frugiperda cells. The recombinant allergens (designated eAmb a 11 and iAmb a 11) were tested for His-tag presence in Western blot. IgE reactivity was evaluated in 150 patients' sera for both recombinant allergen forms in ELISA, with 5 positive sera being tested further by hRBL (humanized rat basophilic leukemia) hexosaminidase release assay. Both allergen forms were proven to be IgE-reactive His-tagged proteins, with an extensive overlap of positive sera (92 toward the former recombinant allergen, 100 toward the latter) and an overall Amb a 11 sensitization prevalence estimated at 68.67%. The hRBL mediator release assay revealed a significant, slightly weaker effect of recombinant allergens when compared with nAmb a 1. Sensitization to this major allergen appears to be associated with more severe asthma symptoms (OR = 4.71, 95% CI = 1.81-12.21). In conclusion, recombinant Amb a 11 is a bona fide allergen, which is IgE-reactive and an inducer of hRBL degranulation. It is an important IgE-reactive component from ragweed pollen, with high IgE sensitization prevalence in the sample population and allergenicity of the recombinant allergen comparable to Amb a 1.


Asunto(s)
Asma , Hipersensibilidad , Ratas , Animales , Alérgenos/genética , Ambrosia/metabolismo , Proteínas de Plantas/metabolismo , Escherichia coli/metabolismo , Inmunoglobulina E , Proteínas Recombinantes/genética
15.
Biosensors (Basel) ; 13(1)2023 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-36671910

RESUMEN

The development of a convenient, sensitive, rapid and self-sterilizing biosensor for microbial detection is important for the prevention and control of foodborne diseases. Herein, we designed a surface-enhanced Raman scattering (SERS) sensing nanoplatform based on a capture-enrichment-enhancement strategy to detect bacteria. The gold-Azo@silver-cetyltrimethylammonium bromide (Au-Azo@Ag-CTAB) SERS nanotags were obtained by optimizing the synthesis process conditions. The results showed that the modification of CTAB enabled the nanotags to bind to different bacteria electrostatically. This SERS sensing nanoplatform was demonstrated to be fast (15 min), accurate and sensitive (limit of detection (LOD): 300 and 400 CFU/mL for E. coli and S. aureus, respectively). Of note, the excellent endogenous antibacterial activity of CTAB allowed the complete inactivation of bacteria after the assay process, thus effectively avoiding secondary contamination.


Asunto(s)
Nanopartículas del Metal , Escherichia coli , Cetrimonio , Staphylococcus aureus , Bacterias , Espectrometría Raman/métodos , Oro
16.
Cells ; 12(2)2023 Jan 14.
Artículo en Inglés | MEDLINE | ID: mdl-36672251

RESUMEN

Cell-cycle progression is regulated by numerous intricate endogenous mechanisms, among which intracellular forces and protein motors are central players. Although it seems unlikely that it is possible to speed up this molecular machinery by applying tiny external forces to the cell, we show that magnetic forcing of magnetosensitive bacteria reduces the duration of the mitotic phase. In such bacteria, the coupling of the cell cycle to the splitting of chains of biogenic magnetic nanoparticles (BMNs) provides a biological realization of such forcing. Using a static gradient magnetic field of a special spatial configuration, in probiotic bacteria E. coli Nissle 1917, we shortened the duration of the mitotic phase and thereby accelerated cell division. Thus, focused magnetic gradient forces exerted on the BMN chains allowed us to intervene in the processes of division and growth of bacteria. The proposed magnetic-based cell division regulation strategy can improve the efficiency of microbial cell factories and medical applications of magnetosensitive bacteria.


Asunto(s)
Escherichia coli , Campos Magnéticos , Escherichia coli/metabolismo , División Celular , Ciclo Celular
17.
Nucleic Acids Res ; 51(1): 365-379, 2023 01 11.
Artículo en Inglés | MEDLINE | ID: mdl-36594161

RESUMEN

RNase E is an endoribonuclease found in many bacteria, including important human pathogens. Within Escherichia coli, it has been shown to have a major role in both the maturation of all classes of RNA involved in translation and the initiation of mRNA degradation. Thus, knowledge of the major determinants of RNase E cleavage is central to our understanding and manipulation of bacterial gene expression. We show here that the binding of RNase E to structured RNA elements is crucial for the processing of tRNA, can activate catalysis and may be important in mRNA degradation. The recognition of structured elements by RNase E is mediated by a recently discovered groove that is distant from the domains associated with catalysis. The functioning of this groove is shown here to be essential for E. coli cell viability and may represent a key point of evolutionary divergence from the paralogous RNase G family, which we show lack amino acid residues conserved within the RNA-binding groove of members of the RNase E family. Overall, this work provides new insights into the recognition and cleavage of RNA by RNase E and provides further understanding of the basis of RNase E essentiality in E. coli.


Asunto(s)
Proteínas de Escherichia coli , Escherichia coli , Humanos , Escherichia coli/genética , Escherichia coli/metabolismo , Endorribonucleasas/metabolismo , ARN/genética , ARN/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Catálisis , ARN Bacteriano/metabolismo
18.
Comput Biol Chem ; 102: 107807, 2023 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-36587565

RESUMEN

The current study reports synthesis of 2-aminoquinolines and 1-aminoisoquinolines derivatives and their characterization. Further, in vitro studies were conducted to determine antimicrobial activities. Compound 3 h showed maximum activity against B. subtilis (IC50: 0.10±0.02 µM) and E. coli (IC50: 0.13±0.01 µM) whereas compound 3i showed higher antimicrobial activity against E. coli (IC50: 0.11±0.01) and C. viswanathii (IC50: 0.10±0.05 µM). Safety profiles of the most potent derivatives were evaluated utilizing cell viability assay using RAW 264.7 and HeLa cell lines and in vitro hemolytic assay was carried out freshly isolated RBC from healthy rat. Furthermore, in silico studies, like molecular docking, binding free energy calculations and ADME predictions were done to get the best lead candidates. Additionally, molecular dynamic simulation for 100 ns was performed to know stability of protein and ligand complex. The active compounds were found to be non-toxic and non-hemolytic and hold great promise to become newer antimicrobial agents.


Asunto(s)
Antiinfecciosos , Antineoplásicos , Humanos , Ratas , Animales , Simulación del Acoplamiento Molecular , Relación Estructura-Actividad , Antineoplásicos/química , Células HeLa , Aminoquinolinas , Escherichia coli , Antiinfecciosos/farmacología , Estructura Molecular
19.
Biotechnol Lett ; 45(2): 299-307, 2023 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-36592259

RESUMEN

OBJECTIVES: To find glycine oxidase genes that can be applied to the breeding of glyphosate resistant crops. RESULTS: The glycine oxidase (GO, EC 1.4.3.19) gene (GenBank No: KC831746) from Bacillus licheniformis (B. licheniformis) was chemically synthesized and transformed into glyphosate-sensitive Escherichia coli (E. coli). The GO gene was transformed into Arabidopsis and rice through Agrobacterium-mediated transformation. The test results confirmed that transgenic plants containing GO genes are more resistant to glyphosate than wild-type plants. On solid Murashige and Skoog (MS) (Murashige and Skoog1962 ) medium containing 200 µM glyphosate, transgenic Arabidopsis thaliana grew normally, while wild-type plants were stunted and root growth was restricted. In a solution containing 500 µM glyphosate, wild-type rice showed severe yellowing, while transgenic rice grew normally. In addition, when sprayed with 10 mM glyphosate solution, wild-type rice withered and died, while transgenic rice grew well. The function of GO gene in glyphosate resistance and the application value of GO gene in the cultivation of glyphosate-resistant crops is proved. CONCLUSIONS: The glycine oxidase gene from B. licheniformis enhances the resistance of E. coli, Arabidopsis and rice to glyphosate.


Asunto(s)
Arabidopsis , Bacillus licheniformis , Herbicidas , Escherichia coli/genética , Plantas Modificadas Genéticamente/genética , Bacillus licheniformis/genética , Arabidopsis/genética , Fitomejoramiento
20.
Environ Pollut ; 319: 120983, 2023 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-36596379

RESUMEN

Plastics have been proposed as vectors of bacteria as they act as a substrate for biofilms. In this study, we evaluated the abundance of faecal and marine bacteria and antibiotic resistance genes (ARGs) from biofilms adhered to marine plastics. Floating plastics and plastics from sediments were collected in coastal areas impacted by human faecal pollution in the northwestern Mediterranean Sea. Culture and/or molecular methods were used to quantify faecal indicators (E. coli, Enterococci and crAssphage), and the ARGs sulI, tetW and blaTEM and the 16S rRNA were detected by qPCR assays. Pseudomonas and Vibrio species and heterotrophic marine bacteria were also analysed via culture-based methods. Results showed that, plastic particles covered by bacterial biofilms, primarily consisted of marine bacteria including Vibrio spp. Some floating plastics had a low concentration of viable E. coli and Enterococci (42% and 67% of the plastics respectively). Considering the median area of the plastics, we detected an average of 68 cfu E. coli per item, while a higher concentration of E. coli was detected on individual plastic items, when compared with 100 ml of the surrounding water. Using qPCR, we quantified higher values of faecal indicators which included inactive and dead microorganisms, detecting up to 2.6 × 102 gc mm-2. The ARGs were detected in 67-88% of the floating plastics and in 29-57% of the sediment plastics with a concentration of up to 6.7 × 102 gc mm-2. Furthermore, enrichment of these genes was observed in biofilms compared with the surrounding water. These results show that floating plastics act as a conduit for both the attachment and transport of faecal microorganisms. In contrast, low presence of faecal indicators was detected in plastic from seafloor sediments. Therefore, although in low concentrations, faecal bacteria, and potential pathogens, were identified in marine plastics, further suggesting plastics act as a reservoir of pathogens and ARGs.


Asunto(s)
Escherichia coli , Vibrio , Humanos , Escherichia coli/genética , Plásticos , Antibacterianos , ARN Ribosómico 16S , Farmacorresistencia Microbiana/genética , Genes Bacterianos , Vibrio/genética , Enterococcus/genética , Biopelículas , Agua
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