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1.
J Microbiol ; 57(9): 781-794, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31452043

RESUMO

The phytopathogenic Burkholderia species B. glumae and B. plantarii are the causal agents of bacterial wilt, grain rot, and seedling blight, which threaten the rice industry globally. Toxoflavin and tropolone are produced by these phytopathogens and are considered the most hostile biohazards with a broad spectrum of target organisms. However, despite their nonspecific toxicity, the effects of toxoflavin and tropolone on bacteria remain unknown. RNA-seq based transcriptome analysis was employed to determine the genome-wide expression patterns under phytotoxin treatment. Expression of 2327 and 830 genes was differentially changed by toxoflavin and tropolone, respectively. Enriched biological pathways reflected the down-regulation of oxidative phosphorylation and ribosome function, beginning with the inhibition of membrane biosynthesis and nitrogen metabolism under oxidative stress or iron starvation. Conversely, several systems such as bacterial chemotaxis, flagellar assembly, biofilm formation, and sulfur/taurine transporters were highly expressed as countermeasures against the phytotoxins. In addition, our findings revealed that three hub genes commonly induced by both phytotoxins function as the siderophore enterobactin, an iron-chelator. Our study provides new insights into the effects of phytotoxins on bacteria for better understanding of the interactions between phytopathogens and other microorganisms. These data will also be applied as a valuable source in subsequent applications against phytotoxins, the major virulence factor.


Assuntos
Antibacterianos/toxicidade , Burkholderia/química , Proteínas de Escherichia coli/genética , Escherichia coli/efeitos dos fármacos , Doenças das Plantas/microbiologia , Pirimidinonas/toxicidade , Triazinas/toxicidade , Tropolona/toxicidade , Antibacterianos/metabolismo , Burkholderia/metabolismo , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Oryza/microbiologia , Pirimidinonas/metabolismo , Transcriptoma/efeitos dos fármacos , Triazinas/metabolismo , Tropolona/metabolismo
2.
J Med Microbiol ; 68(9): 1359-1366, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31364964

RESUMO

Methodology. Biochemical and molecular methods were used to identify 100 lactobacilli isolated from rectal swabs. Among these, L. paracasei ssp. paracasei LP5 and L. brevis LP9 showed significant antibacterial activity against S. agalactiae and L. monocytogenes. Accordingly, characterization of their bacteriocins, BacLP5 and BacLP9, was conducted to obtain information on their kinetic production, sensitivity to chemico-physical parameters and molecular weight. To investigate the possible use of the two Lactobacillus strains as probiotics, their gastrointestinal resistance, cellular adhesiveness and sensitivity to antibiotics were also studied.Results. The obtained data show that BacLP5 and BacLP9 most likely belong to class II bacteriocins and both have a molecular weight of approximately 3 kDa. The production of BacLP5 and BacLP9 started after 4 h (40 and 80 AU ml-1), respectively. Both of the Lactobacillus strains survived gastric and intestinal juices well and showed adhesive capability on HEp-2 cells.Conclusion. Due to their peculiar antimicrobial characteristics, L. paracasei ssp. paracasei LP5 and L. brevis LP9 are suitable for use in the treatment of vaginal disorders, through both oral and transvaginal administration.


Assuntos
Antibacterianos/metabolismo , Bacteriocinas/metabolismo , Lactobacillus brevis/metabolismo , Lactobacillus paracasei/metabolismo , Antibacterianos/química , Antibacterianos/isolamento & purificação , Aderência Bacteriana , Bacteriocinas/química , Bacteriocinas/isolamento & purificação , Ácidos e Sais Biliares/farmacologia , Linhagem Celular , Fenômenos Químicos , Suco Gástrico/metabolismo , Humanos , Lactobacillus brevis/classificação , Lactobacillus brevis/isolamento & purificação , Lactobacillus paracasei/classificação , Lactobacillus paracasei/isolamento & purificação , Listeria monocytogenes/efeitos dos fármacos , Listeria monocytogenes/crescimento & desenvolvimento , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Peso Molecular , Probióticos , Reto/microbiologia , Streptococcus agalactiae/efeitos dos fármacos , Streptococcus agalactiae/crescimento & desenvolvimento
3.
Phys Chem Chem Phys ; 21(35): 19192-19200, 2019 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-31436279

RESUMO

Despite advances, tuberculosis remains a significant infectious disease, whose mortality presents alarming numbers. Although it can be cured, the number of cases of antimicrobial resistant strains is increasing, requiring the use of less efficient second-line drugs. Capreomycin and streptomycin are part of this group, being antibiotics whose mechanism of action is the inhibition of protein synthesis when interacting with the tuberculosis bacterial ribosome. Their binding mechanisms are distinct: capreomycin is able to bind to both ribosomal (30S and 50S) subunits, whereas streptomycin binds only to the smaller one (30S). In this context, the biochemical characterization of these binding sites for a proper understanding of their complex interactions is of crucial importance to increase their efficacy. Through crystallographic data and computer simulations, in this work we calculated the interaction binding energies of capreomycin and streptomycin in complex with the tuberculosis bacterial ribosome subunits, by using density functional theory (DFT) within the molecular fractionation with conjugated caps (MFCC) approach. For capreomycin in the 30S (50S) subunit, we investigated the binding energies of 44 (30) residues presented within a pocket radius of 14 Å (30 Å). Regarding streptomycin, 60 nucleotide (25 amino acid) residues distributed up to 12.5 Å (15 Å) away from the drug in the 30S subunit (S12 protein) were taken into account. We also identify the contributions of hydrogen bonds and hydrophobic interactions in the drug-receptor complex, and the regions of the drugs that most contributed to the anchorages of them in their binding sites, as well as identify residues that are most associated with mutations.


Assuntos
Antibacterianos/química , Capreomicina/química , Metabolismo Energético , Mycobacterium tuberculosis/metabolismo , Subunidades Ribossômicas/química , Subunidades Ribossômicas/metabolismo , Estreptomicina/química , Antibacterianos/metabolismo , Antibacterianos/uso terapêutico , Capreomicina/metabolismo , Capreomicina/uso terapêutico , Simulação por Computador , Cristalização , Humanos , Mutação , Mycobacterium tuberculosis/química , Receptores de Droga/genética , Receptores de Droga/metabolismo , Estreptomicina/metabolismo , Estreptomicina/uso terapêutico , Tuberculose/tratamento farmacológico , Tuberculose/microbiologia
4.
Chem Pharm Bull (Tokyo) ; 67(8): 810-815, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31366830

RESUMO

Helicobacter pylori (H. pylori) infection is common and can result in gastric and duodenal ulcers, and in some cases, gastric lymphoma and cancer. Omeprazole (OMP)-in combination with clarithromycin (CLR), amoxicillin (AMX), tinidazole (TND), or metronidazole (MET)-is used in double or triple combination therapy for eradication of H. pylori. However, the roles of the drugs other than OMP are not clearly understood. Therefore, in the present study, we aimed to investigate any effects of these drugs on OMP metabolism by wild-type CYP2C19 using spectroscopy and enzyme kinetics. The dissociation constants (Kd) for CYP2C19 with OMP, CLR, AMX, TND, and MET were 8.6, 126, 156, 174, and 249 µM, respectively. The intrinsic clearance of OMP was determined to be 355 mL/min/µmol of CYP2C19. Metabolism of OMP was significantly inhibited by 69, 66, 28, and 40% in the presence of CLR, TND, AMX, and MET, respectively. Moreover, the combination of CLR and TND resulted in 76% inhibition of OMP metabolism, while the combination of AMX and MET resulted in 48% inhibition of OMP metabolism. Both combinations of drugs not only have antibacterial effects, but also enhance the effect of OMP by inhibiting its metabolism by CYP2C19. These results indicate that drug-drug interactions of co-administered drugs can cause complex effects, providing a basis for OMP dose adjustment when used in combination therapy for H. pylori eradication.


Assuntos
Antibacterianos/farmacologia , Citocromo P-450 CYP2C19/metabolismo , Infecções por Helicobacter/tratamento farmacológico , Helicobacter pylori/efeitos dos fármacos , Omeprazol/farmacologia , Amoxicilina/química , Amoxicilina/farmacologia , Antibacterianos/química , Antibacterianos/metabolismo , Cromatografia Líquida de Alta Pressão , Claritromicina/química , Claritromicina/farmacologia , Citocromo P-450 CYP2C19/química , Combinação de Medicamentos , Humanos , Metronidazol/química , Metronidazol/farmacologia , Testes de Sensibilidade Microbiana , Estrutura Molecular , Omeprazol/antagonistas & inibidores , Omeprazol/metabolismo , Tinidazol/química , Tinidazol/farmacologia
5.
Bioresour Technol ; 291: 121853, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31377510

RESUMO

The concentration of antibiotics in anaerobically digested swine wastewater (ADSW) usually gradually increases due to the addition of antibiotics in livestock feed. Lemna aequinoctialis was used to treatment synthetic ADSW contaminated by oxytetracycline (OTC) whose concentrations were 0.05, 0.25, 0.50 and 1.00 mg/L, and its influences on NH3-N and TP remove were investigated. The fresh weight, photosynthetic pigment and protein content of duckweed were also investigated. Results have shown that nutrient removal and duckweed growth followed the "dose-response" relationships, and 0.05 mg/L OTC could significantly promote the synthesis of photosynthetic pigments and proteins in duckweed. Meanwhile, the protein content gradually decreased during investigation. More important, the degradation products and possible degradation pathways of OTC were diagrammatized via liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS), and twelve intermediates were detected in the duckweed systems. This study can offer a novel view for phytoremediation of ADSW containing antibiotics by aquatic plants.


Assuntos
Antibacterianos/metabolismo , Araceae/metabolismo , Nutrientes , Oxitetraciclina/metabolismo , Águas Residuárias/química , Anaerobiose , Animais , Biodegradação Ambiental , Cromatografia Líquida , Suínos , Espectrometria de Massas em Tandem
6.
Chem Commun (Camb) ; 55(69): 10192-10213, 2019 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-31411602

RESUMO

Light is unsurpassed in its ability to modulate biological interactions. Since their discovery, chemists have been fascinated by photosensitive molecules capable of switching between isomeric forms, known as photoswitches. Photoswitchable peptides have been recognized for many years; however, their functional implementation in biological systems has only recently been achieved. Peptides are now acknowledged as excellent protein-protein interaction modulators and have been important in the emergence of photopharmacology. In this review, we briefly explain the different classes of photoswitches and summarize structural studies when they are incorporated into peptides. Importantly, we provide a detailed overview of the rapidly increasing number of examples, where biological modulation is driven by the structural changes. Furthermore, we discuss some of the remaining challenges faced in this field. These exciting proof-of-principle studies highlight the tremendous potential of photocontrollable peptides as optochemical tools for chemical biology and biomedicine.


Assuntos
Descoberta de Drogas , Peptídeos/química , Peptídeos/farmacologia , Sequência de Aminoácidos , Animais , Antibacterianos/química , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Morte Celular/efeitos dos fármacos , Descoberta de Drogas/métodos , Humanos , Isomerismo , Luz , Modelos Moleculares , Ácidos Nucleicos/metabolismo , Peptídeos/metabolismo , Processos Fotoquímicos , Mapas de Interação de Proteínas/efeitos dos fármacos
7.
J Agric Food Chem ; 67(35): 9705-9718, 2019 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-31393722

RESUMO

Antimicrobial resistance is among the most urgent global challenges facing sustainable animal production systems. The use of antibiotics as growth promoters and for infectious disease prevention in intensive animal-farming practices has translated into the selection and spread of antimicrobial resistance genes in an unprecedented fashion. Several multi-resistant bacterial strains have been isolated from food-producing animals, thus constituting an alarming food-safety issue. Many industrial byproducts with potential antimicrobial properties are currently being investigated to identify empirical and affordable solutions/alternatives that can potentially be used in feed for animals. Grape pomace is among such byproducts that gained the attention as a result of its low cost, abundance, and, most importantly, its bioactive and antibacterial properties. This review discusses the recently reported studies with regard to exploring the use of grape pomace (and its extracts) in animal production to control pathogens, along with the promotion of beneficial bacterial species in the gut to ultimately alleviate antibacterial resistance. The review further summarizes realistic expectations connected with grape pomace usage and lists the still-to-be-addressed concerns about its application in animal agriculture.


Assuntos
Ração Animal/análise , Antibacterianos/administração & dosagem , Infecções Bacterianas/veterinária , Extratos Vegetais/administração & dosagem , Vitis/química , Resíduos/análise , Animais , Antibacterianos/química , Antibacterianos/metabolismo , Infecções Bacterianas/tratamento farmacológico , Infecções Bacterianas/metabolismo , Infecções Bacterianas/microbiologia , Extratos Vegetais/química , Extratos Vegetais/metabolismo , Vitis/metabolismo
8.
J Agric Food Chem ; 67(35): 9749-9756, 2019 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-31415718

RESUMO

Bovine lactoferrin N-lobe plays an important key in the nonimmunological defense system. In this work, the most suitable promoter Pveg was selected and the fragment coding bovine lactoferrin N-lobe was optimized according to codon bias of Bacillus. The recombinant plasmid pMA0911-Pveg-mBLF-N was introduced into Baicillus subtilis 168 to create B. subtilis/pMA0911-Pveg-mBLF-N. The bovine lactoferrin N-lobe was highly expressed at 28 °C for 15 h. Its purified protein was obtained with 16.5 mg/L and a purity of 93.6% using ammonium sulfate precipitation, Ni-NTA, and molecular exclusion. About 200 ng/mL purified bovine lactoferrin N-lobe completely inhibited cell-growth of Escherichia coli JM109 (DE3), 70.3% of Pseudomonas aeruginosa CGMCC 1.6740, and 41.5% of Staphylococcus aureus CGMCC 1.282. To our knowledge, this is the first report about active expression, purification, and characterization of bovine lactoferrin N-lobe in safe bacterium B. subtilis, which opens an available application way in the biomedical and food industries.


Assuntos
Antibacterianos/farmacologia , Bacillus subtilis/genética , Códon/genética , Lactoferrina/genética , Regiões Promotoras Genéticas , Animais , Antibacterianos/metabolismo , Bacillus subtilis/metabolismo , Clonagem Molecular , Códon/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Expressão Gênica , Engenharia Genética , Lactoferrina/metabolismo , Lactoferrina/farmacologia , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/crescimento & desenvolvimento , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimento
9.
J Agric Food Chem ; 67(29): 8191-8196, 2019 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-31282662

RESUMO

Conversion of free fatty acids into monoacylglycerol gives rise to new structural properties, particularly amphipathic property. Therefore, monoacylglycerols are widely used in pharmaceutical and food industries and are also reported to facilitate better absorption into the human body. A functional fatty acid when transformed into a monoacylglycerol will possibly conserve both the original functionality and amphipathic property. The compound 7,10-dihydroxy-8(E)-octadecenoic acid (DOD) was generated from oleic acid by Pseudomonas aeruginosa PR3 and was known to contain antimicrobial activities against a broad range of food-borne and plant pathogenic bacteria. Here, we attempted to convert DOD into its monoacylglycerol form using lipase for producing an amphipathic antibacterial agent. Consequently, the monoacylglycerol of DOD (DOD-MAG) was successfully produced by coincubating DOD, glycerol, and lipase at 30 °C. The maximum conversion yield reached 70% after 12 h of incubation. Antibacterial activity of DOD-MAG was enhanced by 8 times from the original activity of DOD against food-borne bacteria.


Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Monoglicerídeos/química , Ácidos Oleicos/química , Ácidos Oleicos/farmacologia , Pseudomonas aeruginosa/química , Antibacterianos/metabolismo , Microbiologia de Alimentos , Ácidos Oleicos/metabolismo , Pseudomonas aeruginosa/metabolismo , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimento
10.
J Agric Food Chem ; 67(31): 8468-8475, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31310114

RESUMO

Fermentation of the fungal strain Skeletocutis sp. originating from Mount Elgon Natural Reserve in Kenya, followed by bioassay guided fractionation led to the isolation of 12 previously undescribed metabolites named skeletocutins A-L (1-5 and 7-13) together with the known tyromycin A (6). Their structures were assigned by NMR spectroscopy complemented by HR-ESIMS. Compounds 1-6 and 11-13 exhibited selective activities against Gram-positive bacteria, while compound 10 weakly inhibited the formation of biofilm of Staphylococcus aureus. The isolated metabolites were also evaluated for inhibition of L-leucine aminopeptidase, since tyromycin A had previously been reported to possess such activities but only showed weak effects. Furthermore, all compounds were tested for antiviral activity against Hepatitis C virus (HCV), and compound 6 moderately inhibited HCV infectivity with an IC50 of 6.6 µM.


Assuntos
Antibacterianos/farmacologia , Polyporales/química , Madeira/microbiologia , Antibacterianos/química , Antibacterianos/metabolismo , Antivirais/química , Antivirais/metabolismo , Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Quênia , Testes de Sensibilidade Microbiana , Estrutura Molecular , Polyporales/crescimento & desenvolvimento , Polyporales/isolamento & purificação , Polyporales/metabolismo
11.
Sci Total Environ ; 691: 80-92, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31319261

RESUMO

The emergence and dissemination of infections caused by carbapenem-resistant Klebsiella pneumoniae (CRKP) are of great concern worldwide, as there are limited options for their treatment. Thus, in this study, whole-genome sequencing (WGS) was applied to assess CRKP distribution and dissemination from hospital settings to the aquatic environment in order to identify the extent of the problem. Samples were collected from hospital wastewaters and receiving water bodies. Susceptible K. pneumoniae and CRKP were enumerated and isolated using standard methods. Seventeen CRKP were DNA-sequenced using an Illumina HiSeq X™ platform. De novo assembly and annotation were performed using SPAdes and RAST, respectively. The study analysed antibiotic resistance traits (antibiotic resistant genes, mobile genetic elements, and virulence genes) in CRKP isolates. Although influent of wastewater harboured the highest CRKP, wastewater treatment plants were efficient in reducing the threat. In terms of resistance per matrix, benthic sediment proved to harbour more CRKP (22.88%) versus susceptible K. pneumoniae, as revealed by their resistant quotient analysis, while effluent of wastewaters (4.21%) and water bodies (4.64%) had the lowest CRKP loads. The disseminating CRKP consisted of six sequence types (ST) - ST307 (n = 7), a novel ST3559 (n = 5), ST15 (n = 2), and one isolate of each of ST39, 152 and 298. All CRKP isolates harboured ß-lactams (blaCTX-M-15 and blaOXA-1), quinolone (oqxA and oqxB) and fosfomycin (fosA) resistance genes as well as virulence genes. This study highlights the dissemination of 'high' importance and novel ST CRKP from hospital wastewater to waterbodies. This is concerning, particularly in the African context where a sizable number of people still rely on direct water resources for household use, including drinking. Further research is needed to systematically track the occurrence and distribution of these bacteria so as to mitigate their threat.


Assuntos
Antibacterianos/metabolismo , Carbapenêmicos/metabolismo , Farmacorresistência Bacteriana/genética , Klebsiella pneumoniae/fisiologia , Biodegradação Ambiental , Monitoramento Ambiental , Sequenciamento Completo do Genoma
12.
Pol J Microbiol ; 68(2): 269-280, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31257793

RESUMO

Two glucose-limited realkalized fed-batch cultures of Lactococcus lactis CECT 539 were carried out in a diluted whey medium (DW) using two different feeding media. The cultures were fed a mixture of a 400 g/l concentrated lactose and a concentrated mussel processing waste (CMPW, 101.72 g glucose/l) medium (fermentation I) or a CMPW medium supplemented with glucose and KH2PO4 up to concentrations of 400 g glucose/l and 3.21 g total phosphorus/l, respectively (fermentation II). For an accurate description and a better understanding of the kinetics of both cultures, the growth and product formation by L. lactis CECT 539 were both modelled, for the first time, as a function of the amounts of glucose (G) added and the pH gradient (VpH) generated in every realkalization and feeding cycle, by using an empirical polynomial model. With this modeling procedure, the kinetics of biomass, viable cell counts, nisin, lactic acid, acetic acid and butane-2,3-diol production in both cultures were successfully described (R 2 values > 0.970) and interpreted for the first time. In addition, the optimum VpH and G values for each product were accurately calculated in the two realkalized fed-batch cultures. This approach appears to be useful for designing feeding strategies to enhance the productions of biomass, bacteriocin, and metabolites by the nisin-producing strain in wastes from the food industry.Two glucose-limited realkalized fed-batch cultures of Lactococcus lactis CECT 539 were carried out in a diluted whey medium (DW) using two different feeding media. The cultures were fed a mixture of a 400 g/l concentrated lactose and a concentrated mussel processing waste (CMPW, 101.72 g glucose/l) medium (fermentation I) or a CMPW medium supplemented with glucose and KH2PO4 up to concentrations of 400 g glucose/l and 3.21 g total phosphorus/l, respectively (fermentation II). For an accurate description and a better understanding of the kinetics of both cultures, the growth and product formation by L. lactis CECT 539 were both modelled, for the first time, as a function of the amounts of glucose (G) added and the pH gradient (VpH) generated in every realkalization and feeding cycle, by using an empirical polynomial model. With this modeling procedure, the kinetics of biomass, viable cell counts, nisin, lactic acid, acetic acid and butane-2,3-diol production in both cultures were successfully described (R 2 values > 0.970) and interpreted for the first time. In addition, the optimum VpH and G values for each product were accurately calculated in the two realkalized fed-batch cultures. This approach appears to be useful for designing feeding strategies to enhance the productions of biomass, bacteriocin, and metabolites by the nisin-producing strain in wastes from the food industry.


Assuntos
Antibacterianos/metabolismo , Bacteriocinas/metabolismo , Conservantes de Alimentos/metabolismo , Glucose/metabolismo , Lactococcus lactis/crescimento & desenvolvimento , Nisina/metabolismo , Probióticos/metabolismo , Ácido Acético/metabolismo , Biomassa , Fermentação/fisiologia , Concentração de Íons de Hidrogênio , Ácido Láctico/metabolismo
13.
Anal Bioanal Chem ; 411(20): 5209-5222, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31183522

RESUMO

The developed method was evaluated for the determination of 10 antibiotics belonging to four chemical classes (fluoroquinolones, sulfonamides, lincosamides, and metoxybenzylpyrimidines) and six of their metabolites in four vegetable matrices (lettuce, tomato, cauliflower, and broad beans). The reported method detection limits were sufficiently low (0.1-5.8 ng/g dry weight) to detect target compounds in vegetables under real agricultural practices. Absolute and relative recovery values ranged from 40 to 118% and from 70 to 118%, respectively, for all targeted compounds at the spike level of 100 ng/g dry weight. Regarding method precision, the highest relative standard deviation (RSD) was obtained for enrofloxacin in lettuce (20%), while for the rest of the compounds in all matrices, the RSD values were below 20% for the same spike level. Matrix effects, due to electrospray ionization, ranged from - 26 to 29% for 85% of all estimated values. In a field study, four of the 10 targeted antibiotics were detected in tested vegetables. For the first time, antibiotic metabolites were quantified in vegetables grown under real field conditions. More specifically, decarboxyl ofloxacin and TMP304 were detected in tomato fruits (1.5 ng/g dry weight) and lettuce leaves (21.0-23.1 ng/g dry weight), respectively. It is important to remark that the concentration of TMP304 was five times higher than that from the parental compound, emphasizing the importance of metabolite analysis in monitoring studies. Therefore, the method provided a robust, reliable, and simple-to-use tool that could prove useful for routine multiclass analysis of antibiotics and their metabolites in vegetable samples. Graphical abstract.


Assuntos
Antibacterianos/análise , Produtos Agrícolas/química , Verduras/química , Irrigação Agrícola , Antibacterianos/metabolismo , Cromatografia Líquida/métodos , Limite de Detecção , Reprodutibilidade dos Testes , Extração em Fase Sólida , Espectrometria de Massas por Ionização por Electrospray
14.
Prep Biochem Biotechnol ; 49(8): 800-806, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31156029

RESUMO

In this study, azurin, a bacteriocin with anticancer property, was produced by food-grade Lactococcus lactis using the Nisin Controlled Gene Expression (NICE) System. In addition, the antibacterial and cytotoxic properties of recombinant azurin in the culture supernatant were also investigated. Azurin gene from Pseudomonas aeruginosa was cloned into the pNZ8149 vector and the resulting recombinant DNA was transformed into food grade L. lactis NZ3900. The expression of azurin protein was induced by the optimum concentration of nisin for 3 h. Inhibition zones for Escherichia coli and Bacillus cereus were observed at 5.0 and 10 mg/mL concentrations of lyophilized supernatants containing azurin, but no inhibition zone at azurin-free lyophilized supernatants. When HUVEC, HT29, HCT116, and MCF7 cell lines were treated with lyophilized culture supernatants with azurin or without azurin, cell viability decreased with increasing concentrations of the supernatant. Furthermore, the supernatants containing azurin showed more anti-proliferative effect than the azurin-free supernatants. This work provides a practicable method to produce recombinant azurin in the food grade L. lactis strain. As a result, the recombinant L. lactis strain, producing azurin, can be used in the investigation of food biopreservatives and in the development of a therapeutic probiotic.


Assuntos
Azurina/genética , Clonagem Molecular/métodos , Lactococcus lactis/genética , Pseudomonas aeruginosa/genética , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Azurina/farmacologia , Bacillus cereus/efeitos dos fármacos , Linhagem Celular , Escherichia coli/efeitos dos fármacos , Amplificação de Genes , Humanos , Plasmídeos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Transformação Genética
15.
J Agric Food Chem ; 67(28): 8029-8034, 2019 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-31246026

RESUMO

A special levan-type exopolysaccharide (EPS) from Bacillus amyloliquefaciens JN4 with antiadhesive activity against enterotoxigenic Escherichia coli (ETEC) was purified and identified. Chemical analysis indicated that EPS-JN4 with a low molecular weight of 8 kDa is composed of fructose and glucose with a molar ratio of 46.1:1. Structural analysis clarified that EPS-JN4 contains a main chain of ß-(2,6)-linked Fruf residues and intensive branches of a single 2-linked Fruf at every six residues. Furthermore, the superior antiadhesive activity of EPS-JN4 against ETEC showed its potential usage as an antiadhesive agent for diarrhea prevention. EPS-JN4 is a specific type of levan family, for its small molecular size and intensive branches. The results expand the knowledge on structural types of levan and illustrate its potential as an antiadhesive agent for diarrhea prevention, which will be conducive to elucidate the relation between structure and function.


Assuntos
Antibacterianos/farmacologia , Bacillus amyloliquefaciens/química , Aderência Bacteriana/efeitos dos fármacos , Escherichia coli Enterotoxigênica/efeitos dos fármacos , Frutanos/farmacologia , Polissacarídeos Bacterianos/farmacologia , Antibacterianos/química , Antibacterianos/metabolismo , Bacillus amyloliquefaciens/metabolismo , Escherichia coli Enterotoxigênica/fisiologia , Frutanos/química , Frutanos/metabolismo , Espectroscopia de Ressonância Magnética , Peso Molecular , Polissacarídeos Bacterianos/química , Polissacarídeos Bacterianos/metabolismo
16.
J Med Microbiol ; 68(7): 1021-1032, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31188094

RESUMO

INTRODUCTION: The spread of carbapenem-resistant Acinetobacter baumannii has led to a worldwide healthcare problem. Carbapenem resistance in A. baumannii is mainly mediated by the acquisition of the carbapenem-hydrolyzing oxacillinase OXA-23. The phenotypic detection of carbapenem-producing A. baumannii is challenging and time-consuming. Hence, there is an unmet medical need for reliable and rapid diagnostic tools to detect OXA-23-producing Acinetobacter isolates to enable successful patient management. AIM: Development of an immunochromatographic lateral flow test (ICT) for the rapid and reliable detection of OXA-23-producing carbapenem-resistant Acinetobacter isolates. METHODOLOGY: For the development of an antibody-based ICT, we generated anti-OXA-23 monoclonal antibodies (MoAbs) and screened them sequentially for their ability to bind native OXA-23. Selected OXA-23-specific MoAbs were tested in different combinations for their capacity to capture and detect OXA-23His6 by sandwich enzyme-linked immunosorbent assay (ELISA) and ICT. A well-characterized collection of carbapenem-resistant Acinetobacter isolates with defined carbapenem resistance mechanisms were used to evaluate the specificity of the final OXA-23 ICT prototype. RESULTS: The antibody pairs best suited for the sandwich ELISA format did not match the best pairs in the ICT format selected during the development process of the final prototype OXA-23 ICT. This prototype was able to differentiate between OXA-23 subfamily-mediated carbapenem resistance and carbapenem-resistant Acinetobacter isolates overexpressing other OXAs with 100  % specificity and a turnaround time of 20 min from culture plate to result. CONCLUSION: With this rapid detection assay one can save 12-48 h of diagnostic time, which could help avoid inappropriate use of carbapenems and enable earlier intervention to control the transmission of OXA-23-producing carbapenem-resistant Acinetobacter isolates to other patients and healthcare workers.


Assuntos
Acinetobacter baumannii/isolamento & purificação , Antibacterianos/farmacologia , Carbapenêmicos/farmacologia , Farmacorresistência Bacteriana Múltipla , Imunoensaio/métodos , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/enzimologia , Acinetobacter baumannii/imunologia , Animais , Antibacterianos/metabolismo , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Proteínas de Bactérias , Carbapenêmicos/metabolismo , Clonagem Molecular , Feminino , Regulação Bacteriana da Expressão Gênica , Camundongos , Testes de Sensibilidade Microbiana , beta-Lactamases/genética , beta-Lactamases/imunologia , beta-Lactamases/metabolismo
17.
Nat Commun ; 10(1): 2613, 2019 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-31197182

RESUMO

Kistamicin is a divergent member of the glycopeptide antibiotics, a structurally complex class of important, clinically relevant antibiotics often used as the last resort against resistant bacteria. The extensively crosslinked structure of these antibiotics that is essential for their activity makes their chemical synthesis highly challenging and limits their production to bacterial fermentation. Kistamicin contains three crosslinks, including an unusual 15-membered A-O-B ring, despite the presence of only two Cytochrome P450 Oxy enzymes thought to catalyse formation of such crosslinks within the biosynthetic gene cluster. In this study, we characterise the kistamicin cyclisation pathway, showing that the two Oxy enzymes are responsible for these crosslinks within kistamicin and that they function through interactions with the X-domain, unique to glycopeptide antibiotic biosynthesis. We also show that the kistamicin OxyC enzyme is a promiscuous biocatalyst, able to install multiple crosslinks into peptides containing phenolic amino acids.


Assuntos
Actinobacteria/metabolismo , Antibacterianos/metabolismo , Vias Biossintéticas/genética , Glicopeptídeos/biossíntese , Peptídeos/metabolismo , Actinobacteria/genética , Antibacterianos/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biocatálise , Ciclização/genética , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Glicopeptídeos/química , Família Multigênica , Peptídeos/química
18.
World J Microbiol Biotechnol ; 35(6): 92, 2019 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-31187317

RESUMO

Polyketides and peptides obtained from actinobacteria are important therapeutic compounds which include front line antibiotics and anticancer drugs. Many screening programs are directed towards isolation of bioactive compounds from these organisms but the chances of finding novel antimicrobial leads among common actinobacteria are fast dwindling. As a result, the focus has shifted to the members of less exploited genera of rare actinobacteria. Three isolates, MMS8, MMS16 and KCR3 found to be potent polyketide and peptide producers were identified by 16S rRNA gene sequencing and their sequences deposited in the GenBank under the accession numbers MG407702, MG372012 and MG430204 respectively. MMS8 identified as Micromonospora auratinigra, yielded one potent compound determined to be chloroanthraquinone with an minimum inhibitory concentration (MIC) of 8 µg/ml against Bacillus subtilis and an IC50 value of 10 µg/ml and 4 µg/ml against HeLa and IMR cell lines respectively. This is the first report of the production of chloroanthraquinone by M. auratinigra. MMS16, identified as a member of the family Micromonosporaceae, yielded a potent compound MMS16B analyzed to be a novel bafilomycin analogue. The MIC of the compound was found to be 7 µg/ml against B.subtilis and IC50 value against HeLa and IMR was observed to be 9 µg/ml and 14 µg/ml respectively. MMS16B was also found to exhibit anti-quorum sensing (AQS) activity at sublethal concentrations. KCR3 identified as Kocuria kristinae yielded a novel antimicrobial peptide with antibacterial, antifungal and AQS activity. To the best of our knowledge, no antimicrobial activity has ever been reported from K. kristinae.


Assuntos
Actinobacteria/metabolismo , Peptídeos/metabolismo , Policetídeos/metabolismo , Actinobacteria/genética , Actinobacteria/isolamento & purificação , Animais , Antibacterianos/metabolismo , Anti-Infecciosos/metabolismo , Anti-Infecciosos/farmacologia , Antifúngicos/metabolismo , Antifúngicos/farmacologia , Antineoplásicos/metabolismo , Bacillus subtilis/efeitos dos fármacos , Linhagem Celular , Testes de Sensibilidade Microbiana , Micromonospora/genética , Micromonospora/isolamento & purificação , Micromonospora/metabolismo , Peptídeos/isolamento & purificação , Peptídeos/farmacologia , Policetídeos/isolamento & purificação , Policetídeos/farmacologia , RNA Ribossômico 16S/genética
19.
Ann Agric Environ Med ; 26(2): 203-209, 2019 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-31232046

RESUMO

The antibiotic resistance in many pathogenic bacteria has become a major clinical problem, therefore, the necessity arises to search for new therapeutic strategies. The most promising solution lies in bacteriophages, phage endolysins and antimicrobial peptides. The aim of this study is to review the possibilities for the common use of bacteriophages, phage endolysins and antimicrobial peptides, both in the form of combined therapies and new strategies for the production of peptide drugs. Bacteriophages are viruses that specifically infect and destroy pathogenic bacteria by penetration into bacterial cells, causing metabolism disorders and, consequently, cell lysis. Phage-encoded endolysins are bacteriolytic proteins produced at the end of the phage lytic cycle that destroy elements of bacterial cell wall and enable the release of phage progeny from host cells. Antimicrobial peptides (AMPs) constitute an element of the innate immunity of living organisms and are characterized by the activity against a broad spectrum of bacteria. In the literature, there are only a few reports on the direct interaction of bacteriophages, phage endolysins and antimicrobial peptides against pathogenic bacteria. In each of them, a synergistic effect was observed, and Phage-encoded antimicrobial peptides as a specific group of AMPs have were also discussed. Phage-display technique was also reviewed in terms of its applications to produce and deliver biologically active peptides. The literature data also suggest that bacteriophages, phage endolysins and antimicrobial peptides can be used in combined therapy, thus negating many of the limitations resulting from their specificity as a single antimicrobial agent.


Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Bactérias/efeitos dos fármacos , Infecções Bacterianas/tratamento farmacológico , Bacteriófagos/química , Endopeptidases/farmacologia , Animais , Antibacterianos/química , Antibacterianos/metabolismo , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/metabolismo , Bactérias/virologia , Infecções Bacterianas/microbiologia , Bacteriófagos/enzimologia , Bacteriófagos/genética , Bacteriófagos/fisiologia , Desenho de Drogas , Endopeptidases/química , Endopeptidases/metabolismo , Humanos
20.
J Microbiol ; 57(8): 704-710, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31187416

RESUMO

KatA is the major catalase required for hydrogen peroxide (H2O2) resistance and acute virulence in Pseudomonas aeruginosa PA14, whose transcription is governed by its dual promoters (katAp1 and katAp2). Here, we observed that KatA was not required for acute virulence in another wild type P. aeruginosa strain, PAO1, but that PAO1 exhibited higher KatA expression than PA14 did. This was in a good agreement with the observation that PAO1 was more resistant than PA14 to H2O2 as well as to the antibiotic peptide, polymyxin B (PMB), supposed to involve reactive oxygen species (ROS) for its antibacterial activity. The higher KatA expression in PAO1 than in PA14 was attributed to both katAp1 and katAp2 transcripts, as assessed by S1 nuclease mapping. In addition, it was confirmed that the PMB resistance is attributed to both katAp1 and katAp2 in a complementary manner in PA14 and PAO1, by exploiting the promoter mutants for each -10 box (p1m, p2m, and p1p2m). These results provide an evidence that the two widely used P. aeruginosa strains display different virulence mechanisms associated with OxyR and Anr, which need to be further characterized for better understanding of the critical virulence pathways that may differ in various P. aeruginosa strains.


Assuntos
Proteínas de Bactérias/genética , Catalase/genética , Regulação Bacteriana da Expressão Gênica , Peróxido de Hidrogênio/metabolismo , Polimixina B/metabolismo , Pseudomonas aeruginosa/enzimologia , Antibacterianos/metabolismo , Regiões Promotoras Genéticas , Pseudomonas aeruginosa/patogenicidade , Virulência
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