RESUMO
Antibiotic resistance (AR) is a major public health concern. Antibiotic intermediates (AIs) used in the production of semisynthetic antibiotics have the same bioactive structure as parent antibiotics and synthetic antibiotic production wastewater usually contains high concentrations of residual AIs; however, the effects of AIs and their interactive effects with antibiotics on the emergence of AR are unknown. In this study, antibiotic-sensitive E. coli K12 was exposed to five types of ß-lactam AIs and their parent antibiotic ampicillin to analyze their impact on the evolution of multiple AR. The results indicated that AI 6-APA inhibits bacterial growth and stimulates the production of reactive oxygen species, as well as induces AR and antibiotic persistence like the parent antibiotic AMP. Combined exposure to 6-APA and AMP synergistically stimulated the induction of multiple AR and antibiotic persistence. The resistance mutation frequency increased up to 6.1 × 106-fold under combined exposure and the combination index reached 1326.5, indicating a strong synergy of 6-APA and AMP. Phenotypic and genotypic analyses revealed that these effects were associated with the overproduction of reactive oxygen species, enhanced stress response signatures, and activation of efflux pumps. These findings provide evidence and mechanistic insights into AR induction by AIs in antibiotic production wastewater.
Assuntos
Antibacterianos , Espécies Reativas de Oxigênio , Águas Residuárias , Antibacterianos/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Ampicilina/farmacologia , Sinergismo Farmacológico , Escherichia coli K12/efeitos dos fármacos , Escherichia coli K12/genética , Escherichia coli K12/crescimento & desenvolvimento , Escherichia coli K12/metabolismo , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , beta-Lactamas/farmacologiaRESUMO
2,4-dihydroxybutyric acid (DHB) and 2-keto-4-hydroxybutyrate (OHB) are non-natural molecules obtained through synthetic pathways from renewable carbon source. As they are structurally similar to lactate and pyruvate respectively, they could possibly interfere with the metabolic network of Escherichia coli. In fact, we showed that DHB can be easily oxidized by the membrane associated L and D-lactate dehydrogenases encoded by lldD, dld and ykgF into OHB, and the latter being cleaved into pyruvate and formaldehyde by several pyruvate-dependent aldolases, with YagE being the most effective. While formaldehyde was readily detoxified into formate, Escherichia coli K12 MG1655 strain failed to grow on DHB despite of the production of pyruvate. To find out the reason for this failure, we constructed a mutant strain whose growth was rendered dependent on DHB and subjected this strain to adaptive evolution. Genome sequencing of the adapted strain revealed an essential role for ygbI encoding a transcriptional repressor of the threonate operon in this DHB-dependent growth. This critical function was attributed to the derepression of ygbN encoding a putative threonate transporter, which was found to exclusively transport the D form of DHB. A subsequent laboratory evolution was carried out with E. coli K12 MG1655 deleted for ΔygbI to adapt for growth on DHB as sole carbon source. Remarkably, only two additional mutations were disclosed in the adapted strain, which were demonstrated by reverse engineering to be necessary and sufficient for robust growth on DHB. One mutation was in nanR encoding the transcription repressor of sialic acid metabolic genes, causing 140-fold increase in expression of nanA encoding N-acetyl neuraminic acid lyase, a pyruvate-dependent aldolase, and the other was in the promoter of dld leading to 14-fold increase in D-lactate dehydrogenase activity on DHB. Taken together, this work illustrates the importance of promiscuous enzymes in underground metabolism and moreover, in the frame of synthetic pathways aiming at producing non-natural products, these underground reactions could potentially penalize yield and title of these bio-based products.
Assuntos
Carbono , Proteínas de Escherichia coli , Escherichia coli , Carbono/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/crescimento & desenvolvimento , Redes e Vias Metabólicas , Óperon , Hidroxibutiratos/metabolismo , Regulação Bacteriana da Expressão Gênica , Ácido Pirúvico/metabolismo , Escherichia coli K12/genética , Escherichia coli K12/metabolismo , Escherichia coli K12/crescimento & desenvolvimento , Escherichia coli K12/enzimologia , Mutação , Formaldeído/metabolismo , Ácido Láctico/metabolismoRESUMO
Before the absorption in the intestine, glucose encounters gut bacteria, which may serve as a barrier against hyperglycemia by metabolizing glucose. In the present study, we compared the capacity of enterobacterial strains to lower glucose levels in an in vitro model of nutrient-induced bacterial growth. Two probiotic strains, Hafnia alvei HA4597 (H. alvei) and Escherichia coli (E. coli) Nissle 1917, as well as E. coli K12, were studied. To mimic bacterial growth in the gut, a planktonic culture was supplemented twice daily by the Luria Bertani milieu with or without 0.5% glucose. Repeated nutrient provision resulted in the incremental growth of bacteria. However, in the presence of glucose, the maximal growth of both strains of E. coli but not of H. alvei was inhibited. When glucose was added to the culture medium, a continuous decrease in its concentration was observed during each feeding phase. At its highest density, H. alvei displayed more efficient glucose consumption accompanied by a more pronounced downregulation of glucose transporters' expression than E. coli K12. Thus, the study reveals that the probiotic strain H. alvei HA4597 is more resilient to maintain its growth than E. coli in the presence of 0.5% glucose accompanied by more efficient glucose consumption. This experimental approach offers a new strategy for the identification of probiotics with increased glucose metabolizing capacities potentially useful for the prevention and co-treatment of type 2 diabetes.
Assuntos
Glucose , Probióticos , Glucose/metabolismo , Escherichia coli/metabolismo , Hafnia alvei/metabolismo , Escherichia coli K12/metabolismo , Escherichia coli K12/crescimento & desenvolvimentoRESUMO
Tolerance to human gastrointestinal stressors is crucial for probiotics to exhibit their health benefits; however, there is no standardised method for screening their stress tolerance. In this study, we proposed a novel method for screening probiotic candidates tolerant to human gastrointestinal stress-gastrointestinal tolerance assay and culture (GTA-C) method-using black polyethylene terephthalate (PET) non-woven fabric as a scaffold to modify the specialized cellulose film (SCF) method. The modified SCF method showed excellent pH-based diffusion of medium components, had minimal effect on the growth of Escherichia coli K12, and improved the visibility of the colonies. Analysis of kimchi samples cultured using the SCF and modified SCF methods revealed that the modified method diversified the cultured bacteria. GTA in a simulated human fasting state using the modified SCF method showed that acid stress significantly affected the growth of four bacteria used as probiotics and that tolerance to acid stress may be species-dependent. Screening of probiotics in kimchi samples resulted in the identification of lactic acid bacteria tolerant to human gastrointestinal stress during fasting. Our results indicate that the modified SCF method (GTA-C method) is useful for screening probiotics resistant to the gastrointestinal environment during fasting.
Assuntos
Trato Gastrointestinal , Probióticos , Estresse Fisiológico , Humanos , Trato Gastrointestinal/microbiologia , Concentração de Íons de Hidrogênio , Alimentos Fermentados/microbiologia , Celulose , Jejum , Escherichia coli K12/efeitos dos fármacos , Escherichia coli K12/crescimento & desenvolvimentoRESUMO
Genome-scale metabolic models (GEMs) are mathematical representations of metabolism that allow for in silico simulation of metabolic phenotypes and capabilities. A prerequisite for these predictions is an accurate representation of the biomolecular composition of the cell necessary for replication and growth, implemented in GEMs as the so-called biomass objective function (BOF). The BOF contains the metabolic precursors required for synthesis of the cellular macro- and micromolecular constituents (e.g. protein, RNA, DNA), and its composition is highly dependent on the particular organism, strain, and growth condition. Despite its critical role, the BOF is rarely constructed using specific measurements of the modeled organism, drawing the validity of this approach into question. Thus, there is a need to establish robust and reliable protocols for experimental condition-specific biomass determination. Here, we address this challenge by presenting a general pipeline for biomass quantification, evaluating its performance on Escherichia coli K-12 MG1655 sampled during balanced exponential growth under controlled conditions in a batch-fermentor set-up. We significantly improve both the coverage and molecular resolution compared to previously published workflows, quantifying 91.6% of the biomass. Our measurements display great correspondence with previously reported measurements, and we were also able to detect subtle characteristics specific to the particular E. coli strain. Using the modified E. coli GEM iML1515a, we compare the feasible flux ranges of our experimentally determined BOF with the original BOF, finding that the changes in BOF coefficients considerably affect the attainable fluxes at the genome-scale.
Assuntos
Escherichia coli K12/crescimento & desenvolvimento , Escherichia coli K12/genética , Biomassa , Simulação por Computador , Genoma Bacteriano/genética , Modelos BiológicosRESUMO
Deuterium is a natural low abundance stable hydrogen isotope that in high concentrations negatively affects growth of cells. Here, we have studied growth of Escherichia coli MG1655, a wild-type laboratory strain of E. coli K-12, in deuterated glycerol minimal medium. The growth rate and final biomass in deuterated medium is substantially reduced compared to cells grown in ordinary medium. By using a multi-generation adaptive laboratory evolution-based approach, we have isolated strains that show increased fitness in deuterium-based growth media. Whole-genome sequencing identified the genomic changes in the obtained strains and show that there are multiple routes to genetic adaptation to growth in deuterium-based media. By screening a collection of single-gene knockouts of nonessential genes, no specific gene was found to be essential for growth in deuterated minimal medium. Deuteration of proteins is of importance for NMR spectroscopy, neutron protein crystallography, neutron reflectometry, and small angle neutron scattering. The laboratory evolved strains, with substantially improved growth rate, were adapted for recombinant protein production by T7 RNA polymerase overexpression systems and shown to be suitable for efficient production of perdeuterated soluble and membrane proteins for structural biology applications.
Assuntos
Adaptação Fisiológica/genética , Deutério/metabolismo , Escherichia coli K12/metabolismo , Marcação por Isótopo/métodos , Nêutrons , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cristalografia/métodos , Meios de Cultura/química , Meios de Cultura/farmacologia , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli K12/efeitos dos fármacos , Escherichia coli K12/genética , Escherichia coli K12/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Genes Essenciais , Glicerol/metabolismo , Glicerol/farmacologia , Glicerol Quinase/genética , Glicerol Quinase/metabolismo , Mutação , Difração de Nêutrons , Canais de Potássio/genética , Canais de Potássio/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Seleção Genética , Fator sigma/genética , Fator sigma/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Sequenciamento Completo do GenomaRESUMO
Predicting the fate of individual cells among a microbial population (i.e., growth and gene expression) remains a challenge, especially when this population is exposed to very dynamic environmental conditions, such as those encountered during continuous cultivation. Indeed, the dynamic nature of a continuous cultivation process implies the potential diversification of the microbial population resulting in genotypic and phenotypic heterogeneity. The present work focused on the induction of the arabinose operon in Escherichia coli as a model system to study this diversification process in continuous cultivations. As a preliminary step, the green fluorescent protein (GFP) level triggered by an arabinose-inducible ParaBAD promoter was tracked by flow cytometry in chemostat cultivations with glucose-arabinose co-feeding. For a wide range of glucose-arabinose co-feeding concentrations in the chemostats, the simultaneous occurrence of GFP positive and negative subpopulation was observed. In the second set of experiments, continuous cultivation was performed by adding glucose continuously and arabinose based on the capability of individual cells to switch from low GFP to high GFP expression states, performed with a technology setup called segregostat. In the segregostat cultivation mode, on-line flow cytometry analysis was used for adjusting the arabinose/glucose transitions based on the phenotypic switching profiles of the microbial population. This strategy allowed finding an appropriate arabinose pulsing frequency, leading to prolonged maintenance of the induction level with a limited increase in the phenotypic diversity for more than 60 generations. The results suggest that the steady forcing of individual cells into a given phenotypic trajectory may not be the best strategy for controlling cell populations. Instead, allowing individual cells to switch periodically around a predefined threshold seems to be a more robust strategy leading to oscillations, but within a predictable cell population behavior range.
Assuntos
Escherichia coli K12 , Proteínas de Fluorescência Verde/biossíntese , Regiões Promotoras Genéticas , Arabinose/genética , Arabinose/metabolismo , Escherichia coli K12/genética , Escherichia coli K12/crescimento & desenvolvimento , Proteínas de Fluorescência Verde/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genéticaRESUMO
To identify the physiological factors that limit the growth of Escherichia coli K-12 strains synthesizing minimal lipopolysaccharide (LPS), we describe the first construction of strains devoid of the entire waa locus and concomitantly lacking all three acyltransferases (LpxL/LpxM/LpxP), synthesizing minimal lipid IVA derivatives with a restricted ability to grow at around 21 °C. Suppressors restoring growth up to 37 °C of Δ(gmhD-waaA) identified two independent single-amino-acid substitutions-P50S and R310S-in the LPS flippase MsbA. Interestingly, the cardiolipin synthase-encoding gene clsA was found to be essential for the growth of ΔlpxLMP, ΔlpxL, ΔwaaA, and Δ(gmhD-waaA) bacteria, with a conditional lethal phenotype of Δ(clsA lpxM), which could be overcome by suppressor mutations in MsbA. Suppressor mutations basS A20D or basR G53V, causing a constitutive incorporation of phosphoethanolamine (P-EtN) in the lipid A, could abolish the Ca++ sensitivity of Δ(waaC eptB), thereby compensating for P-EtN absence on the second Kdo. A single-amino-acid OppA S273G substitution is shown to overcome the synthetic lethality of Δ(waaC surA) bacteria, consistent with the chaperone-like function of the OppA oligopeptide-binding protein. Furthermore, overexpression of GcvB sRNA was found to repress the accumulation of LpxC and suppress the lethality of LapAB absence. Thus, this study identifies new and limiting factors in regulating LPS biosynthesis.
Assuntos
Escherichia coli K12/crescimento & desenvolvimento , Genes Essenciais , Lipopolissacarídeos/biossíntese , Lipopolissacarídeos/genética , Transportadores de Cassetes de Ligação de ATP/genética , Aciltransferases/genética , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Cardiolipinas/genética , Escherichia coli K12/genética , Escherichia coli K12/metabolismo , Proteínas de Escherichia coli/genética , Lipoproteínas/genética , Proteínas de Membrana/genética , Mutações Sintéticas Letais , Transferases (Outros Grupos de Fosfato Substituídos)/genéticaRESUMO
Bacterial genomes encode various multidrug efflux pumps (MDR) whose specific conditions for fitness advantage are unknown. We show that the efflux pump MdtEF-TolC, in Escherichia coli, confers a fitness advantage during exposure to extreme acid (pH 2). Our flow cytometry method revealed pH-dependent fitness trade-offs between bile acids (a major pump substrate) and salicylic acid, a membrane-permeant aromatic acid that induces a drug resistance regulon but depletes proton motive force (PMF). The PMF drives MdtEF-TolC and related pumps such as AcrAB-TolC. Deletion of mdtE (with loss of the pump MdtEF-TolC) increased the strain's relative fitness during growth with or without salicylate or bile acids. However, when the growth cycle included a 2-h incubation at pH 2 (below the pH growth range), MdtEF-TolC conferred a fitness advantage. The fitness advantage required bile salts but was decreased by the presence of salicylate, whose uptake is amplified by acid. For comparison, AcrAB-TolC, the primary efflux pump for bile acids, conferred a PMF-dependent fitness advantage with or without acid exposure in the growth cycle. A different MDR pump, EmrAB-TolC, conferred no selective benefit during growth in the presence of bile acids. Without bile acids, all three MDR pumps incurred a large fitness cost with salicylate when exposed at pH 2. These results are consistent with the increased uptake of salicylate at low pH. Overall, we showed that MdtEF-TolC is an MDR pump adapted for transient extreme-acid exposure and that low pH amplifies the salicylate-dependent fitness cost for drug pumps. IMPORTANCE Antibiotics and other drugs that reach the gut must pass through stomach acid. However, little is known of how extreme acid modulates the effect of drugs on gut bacteria. We find that extreme-acid exposure leads to a fitness advantage for a multidrug pump that otherwise incurs a fitness cost. At the same time, extreme acid amplifies the effect of salicylate selection against multidrug pumps. Thus, organic acids and stomach acid could play important roles in regulating multidrug resistance in the gut microbiome. Our flow cytometry assay provides a way to measure the fitness effects of extreme-acid exposure to various membrane-soluble organic acids, including plant-derived nutrients and pharmaceutical agents. Therapeutic acids might be devised to control the prevalence of multidrug pumps in environmental and host-associated habitats.
Assuntos
Proteínas de Transporte/metabolismo , Escherichia coli K12/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Ácidos/metabolismo , Proteínas de Transporte/genética , Escherichia coli K12/genética , Escherichia coli K12/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras/genéticaRESUMO
Sustaining a robust metabolic network requires a balanced and fully functioning proteome. In addition to amino acids, many enzymes require cofactors (coenzymes and engrafted prosthetic groups) to function properly. Extensively validated resource allocation models, such as genome-scale models of metabolism and gene expression (ME-models), have the ability to compute an optimal proteome composition underlying a metabolic phenotype, including the provision of all required cofactors. Here we apply the ME-model for Escherichia coli K-12 MG1655 to computationally examine how environmental conditions change the proteome and its accompanying cofactor usage. We found that: (1) The cofactor requirements computed by the ME-model mostly agree with the standard biomass objective function used in models of metabolism alone (M-models); (2) ME-model computations reveal non-intuitive variability in cofactor use under different growth conditions; (3) An analysis of ME-model predicted protein use in aerobic and anaerobic conditions suggests an enrichment in the use of peroxyl scavenging acids in the proteins used to sustain aerobic growth; (4) The ME-model could describe how limitation in key protein components affect the metabolic state of E. coli. Genome-scale models have thus reached a level of sophistication where they reveal intricate properties of functional proteomes and how they support different E. coli lifestyles.
Assuntos
Biologia Computacional/métodos , Escherichia coli K12/crescimento & desenvolvimento , Nutrientes/metabolismo , Proteoma , Escherichia coli K12/metabolismo , Modelos BiológicosRESUMO
Understanding individual responses to nutrition and medicine is of growing interest and importance. There is evidence that differences in bitter taste receptor (TAS2R) genes which give rise to two frequent haplotypes, TAS2R38-PAV (functional) and TAS2R38-AVI (non-functional), may impact inter-individual differences in health status. We here analyzed the relevance of the TAS2R38 receptor in the regulation of the human immune response using the TAS2R38 agonist allyl isothiocyanate (AITC) from Brassica plants. A differential response in calcium mobilization upon AITC treatment in leucocytes from healthy humans confirmed a relevance of TAS2R38 functionality, independent from cation channel TRPV1 or TRPA1 activation. We further identified a TAS2R38-dependence of MAPK and AKT signaling activity, bactericidal (toxicity against E. coli) and anti-inflammatory activity (TNF-alpha inhibition upon cell stimulation). These in vitro results were derived at relevant human plasma levels in the low micro molar range as shown here in a human intervention trial with AITC-containing food.
Assuntos
Fatores Imunológicos/farmacologia , Isotiocianatos/farmacologia , Leucócitos/efeitos dos fármacos , Receptores Acoplados a Proteínas G/agonistas , Imunidade Adaptativa/efeitos dos fármacos , Adulto , Sinalização do Cálcio , Células Cultivadas , Dieta , Escherichia coli K12/crescimento & desenvolvimento , Feminino , Humanos , Imunidade Inata/efeitos dos fármacos , Fatores Imunológicos/administração & dosagem , Fatores Imunológicos/farmacocinética , Isotiocianatos/administração & dosagem , Isotiocianatos/farmacocinética , Leucócitos/imunologia , Leucócitos/metabolismo , Masculino , Viabilidade Microbiana , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Polimorfismo de Nucleotídeo Único , Medicina de Precisão , Gravidez , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Effect of fermentation parameters such as C/N ratio, specific growth rate, phosphate limitation, and plasmid instability on enhancing isoprene production is the focus of the current study. Isoprene productivity in the recombinant Escherichia coli K12_MVA strain showed a bell-shaped relationship with specific growth rate in bioreactor studies with isoprene volumetric productivity peaking at 0.35/h. This behavior was depicted by a production inhibition kinetic model which envisaged a serious competition between the cellular growth, acetic acid production, and isoprene biosynthesis. The model equation derived showed a reasonable fit with the experimental values. Judicious control of the growth rates and acetate accumulation by optimizing C/N ratio, phosphate concentration, and intermittent feeding strategy resulted in maximizing the carbon flux towards isoprene. Plasmid instability caused by metabolic burden posed by the presence of dual plasmids on the bacteria was simulated using first-order degradation kinetics. The experimental plasmid loss trend was in accordance with the model simulated trend, where higher plasmid loss correlated with higher specific growth rates. Modulating the growth rate, acetate accumulation, and plasmid instability resulted in achieving maximum isoprene volumetric productivity of 1.125 g/l/h with 46.67% of carbon flux towards isoprene and a isoprene titre of 18 g/l in 16 h fermentation run.
Assuntos
Escherichia coli K12/crescimento & desenvolvimento , Hemiterpenos/biossíntese , Microrganismos Geneticamente Modificados/crescimento & desenvolvimento , Butadienos , Carbono/farmacologia , Escherichia coli K12/genética , Hemiterpenos/genética , Microrganismos Geneticamente Modificados/genética , Nitrogênio/farmacologiaRESUMO
E. coli is associated with high rates of infection and resistance to drugs not only in China but also the rest of the world. In addition, the number of E. coli biofilm infections continue to increase with time. Notably, biofilms are attractive targets for the prevention of infections caused by multidrug-resistant bacteria. Moreover, the pgaABCD-encoded Poly-ß-1,6-N-acetyl-d-glucosamine (PNAG) plays an important role in biofilm formation. Therefore, this study aimed to explore the specific effect of the (R)-(+)-pulegone (PU) on growth and biofilm formation in multi-drug resistant E. coli. The molecular mechanisms involved were also examined. The results showed that PU had significant antibacterial and antibiofilm formation activity against E. coli K1, with MIC and MBC values of 23.68 and 47.35 mg/mL, respectively. On the other hand, the maximum inhibition rate for biofilm formation in the bacterium was 52.36 % at 94.70 mg/mL of PU. qRT-PCR data showed that PU significantly down-regulated expression of the pgaABCD genes (P < 0.05). PU was also broadly effective against biofilm formation in MG1655 and MG1655/ΔpgaABCD, exhibiting the maximum inhibition rates were 98.23 % and 93.35 %, respectively. In addition, PU destroyed pre-formed mature biofilm in both MG1655 and MG1655/ΔpgaABCD about 95.03 % and 92.4 %, respectively. The study therefore verified that pgaA was a potential and key target for PU in E. coli although it was not the only one. Overall, the findings indicated that PU is a potential and novel inhibitor of drug resistance, This therefore gives insights on new ways of preventing and treating biofilm-associated infections in the food industry as well as in clinical practice.
Assuntos
Antibacterianos/farmacologia , Proteínas da Membrana Bacteriana Externa/genética , Biofilmes/efeitos dos fármacos , Monoterpenos Cicloexânicos/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Escherichia coli K12/efeitos dos fármacos , Proteínas de Escherichia coli/genética , Amidoidrolases/genética , Biofilmes/crescimento & desenvolvimento , Escherichia coli K12/genética , Escherichia coli K12/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica , Testes de Sensibilidade MicrobianaRESUMO
Growth curve measurements are commonly used in microbiology, while the use of microplate readers for such measurements provides better temporal resolution and higher throughput. However, evaluating bacterial growth with microplate readers has been hurdled by barriers such as multiple scattering. Here, we report our development of a method based on the time derivatives of the optical density (OD) and/or fluorescence (FL) of bacterial cultures to overcome these barriers. First, we illustrated our method using quantitative models and numerical simulations, which predicted the number of bacteria and the number of fluorescent proteins in time as well as their time derivatives. Then, we systematically investigated how the time derivatives depend on the parameters in the models/simulations, providing a framework for understanding the FL growth curves. In addition, as a demonstration, we applied our method to study the lag time elongation of bacteria subjected to treatment with silver (Ag+) ions and found that the results from our method corroborated well with that from growth curve fitting by the Gompertz model that has been commonly used in the literature. Furthermore, this method was applied to the growth of bacteria in the presence of silver nanoparticles (AgNPs) at various concentrations, where the OD curve measurements failed. We showed that our method allowed us to successfully extract the growth behavior of the bacteria from the FL measurements and understand how the growth was affected by the AgNPs.
Assuntos
Densitometria , Escherichia coli K12/crescimento & desenvolvimento , Nanopartículas Metálicas/química , Prata/químicaRESUMO
Under stressful conditions, Escherichia coli forms biofilm for survival by sensing a variety of environmental conditions. CsgD, the master regulator of biofilm formation, controls cell aggregation by directly regulating the synthesis of Curli fimbriae. In agreement of its regulatory role, as many as 14 transcription factors (TFs) have so far been identified to participate in regulation of the csgD promoter, each monitoring a specific environmental condition or factor. In order to identify the whole set of TFs involved in this typical multi-factor promoter, we performed in this study 'promoter-specific transcription-factor' (PS-TF) screening in vitro using a set of 198 purified TFs (145 TFs with known functions and 53 hitherto uncharacterized TFs). A total of 48 TFs with strong binding to the csgD promoter probe were identified, including 35 known TFs and 13 uncharacterized TFs, referred to as Y-TFs. As an attempt to search for novel regulators, in this study we first analysed a total of seven Y-TFs, including YbiH, YdcI, YhjC, YiaJ, YiaU, YjgJ and YjiR. After analysis of curli fimbriae formation, LacZ-reporter assay, Northern-blot analysis and biofilm formation assay, we identified at least two novel regulators, repressor YiaJ (renamed PlaR) and activator YhjC (renamed RcdB), of the csgD promoter.
Assuntos
Biofilmes/crescimento & desenvolvimento , Escherichia coli K12/crescimento & desenvolvimento , Escherichia coli K12/genética , Proteínas de Escherichia coli/genética , Regiões Promotoras Genéticas , Transativadores/genética , Fatores de Transcrição/metabolismo , Sítios de Ligação , Escherichia coli K12/metabolismo , Proteínas de Escherichia coli/metabolismo , Fímbrias Bacterianas/fisiologia , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Transativadores/metabolismo , Fatores de Transcrição/genéticaRESUMO
With the stagnancy of antibiotics development, polymyxins have become the last defense for treatment of multidrug-resistant (MDR) Gram-negative bacteria, whereas the effect of polymyxin monotherapy is limited by resistance. The objective of this study was to evaluate the effects of polymyxin B (PMNB)-vorinostat (SAHA) combination therapy against Gram-negative pathogens in vitro and in vivo. The antibacterial activities of PMNB and SAHA were evaluated by susceptibility testing. The synergistic effect was assessed by checkerboard tests and time-killing kinetics experiments. Cellular morphology studies and reactive oxygen species (ROS) assay were conducted to explore potential mechanisms. Also, Galleria mellonella models were made to evaluate the antibacterial effects in vivo. PMNB-SAHA had the synergistic effect against all tested isolates, reducing >2 log10 colony-forming units (CFU)/mL at 40 minutes, and showed more powerful antibacterial effects than PMNB alone in the 24-hour window. Cellular morphology study showed the change of membrane and disruption of integrity. ROS assay showed more oxidative stress in combination than PMNB or SAHA monotherapy. In animal models, PMNB-SAHA showed a higher survival rate than that of monotherapy. This study is the first to report the synergistic antibacterial effect of PMNB-SAHA therapy against MDR Gram-negative bacteria. Further clinical research is needed to confirm the results.
Assuntos
Antibacterianos/farmacologia , Infecções Bacterianas/tratamento farmacológico , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Polimixina B/farmacologia , Vorinostat/farmacologia , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/crescimento & desenvolvimento , Animais , Infecções Bacterianas/microbiologia , Infecções Bacterianas/mortalidade , Combinação de Medicamentos , Sinergismo Farmacológico , Enterococcus faecalis/efeitos dos fármacos , Enterococcus faecalis/crescimento & desenvolvimento , Escherichia coli K12/efeitos dos fármacos , Escherichia coli K12/crescimento & desenvolvimento , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/crescimento & desenvolvimento , Larva/efeitos dos fármacos , Larva/microbiologia , Longevidade/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Mariposas/efeitos dos fármacos , Mariposas/microbiologia , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/crescimento & desenvolvimento , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimentoRESUMO
Plantaricin BM-1 is a class IIa bacteriocin with a strong bactericidal effect on gram-positive bacteria. Although plantaricin BM-1 also inhibits the growth of some gram-negative bacteria, including Escherichia coli, the mechanism is not clear. In this study, we used tandem mass tag-based quantitative proteomics analysis to examine the inhibitory mechanism of plantaricin BM-1 against E. coli K12, and evaluated the morphological effects by electron microscopy. The results demonstrated that plantaricin BM-1 inhibits the growth of E. coli K12 by bacteriostatic action, mainly acting on the surface of the cell wall, leading to its collapse. Proteomic analysis identified 976 differentially expressed proteins (>1.2-fold change, p < 0.05) under treatment with plantaricin BM-1, including 490 up-regulated proteins and 486 down-regulated proteins. These proteins were mainly involved in peptidoglycan synthesis and energy metabolism pathways, including amino acid, glyoxylate and dicarboxylate, ABC transporter, and quorum-sensing pathways. Specifically, plantaricin BM-1 treatment significantly improved peptidoglycan synthesis and enhanced the tricarboxylic acid cycle in E. coli K12, and altered the expression of cell membrane proteins. These results provide new insight into the inhibition mechanism of class IIa bacteriocins on gram-negative bacteria, which can lay the foundation for its broader use as an alternative to conventional antibiotics.
Assuntos
Bacteriocinas/farmacologia , Escherichia coli K12/efeitos dos fármacos , Escherichia coli K12/metabolismo , Peptidoglicano/biossíntese , Proteômica , Escherichia coli K12/crescimento & desenvolvimento , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana/metabolismoRESUMO
Countering the rise of antibiotic-resistant pathogens requires improved understanding of how resistance emerges and spreads in individual species, which are often embedded in complex microbial communities such as the human gut microbiome. Interactions with other microorganisms in such communities might suppress growth and resistance evolution of individual species (e.g., via resource competition) but could also potentially accelerate resistance evolution via horizontal transfer of resistance genes. It remains unclear how these different effects balance out, partly because it is difficult to observe them directly. Here, we used a gut microcosm approach to quantify the effect of three human gut microbiome communities on growth and resistance evolution of a focal strain of Escherichia coli. We found the resident microbial communities not only suppressed growth and colonisation by focal E. coli but also prevented it from evolving antibiotic resistance upon exposure to a beta-lactam antibiotic. With samples from all three human donors, our focal E. coli strain only evolved antibiotic resistance in the absence of the resident microbial community, even though we found resistance genes, including a highly effective resistance plasmid, in resident microbial communities. We identified physical constraints on plasmid transfer that can explain why our focal strain failed to acquire some of these beneficial resistance genes, and we found some chromosomal resistance mutations were only beneficial in the absence of the resident microbiota. This suggests, depending on in situ gene transfer dynamics, interactions with resident microbiota can inhibit antibiotic-resistance evolution of individual species.
Assuntos
Farmacorresistência Bacteriana/fisiologia , Escherichia coli K12/efeitos dos fármacos , Microbioma Gastrointestinal/fisiologia , Ampicilina/farmacologia , Antibacterianos/farmacologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Escherichia coli K12/genética , Escherichia coli K12/crescimento & desenvolvimento , Escherichia coli K12/fisiologia , Proteínas de Escherichia coli/genética , Fezes/microbiologia , Microbioma Gastrointestinal/efeitos dos fármacos , Humanos , Mutação , PlasmídeosRESUMO
The method of pulsed laser processing with a nanosecond pulse duration was employed to obtain a nanotexture on the surface of copper alloys. The effect of the obtained micro- and nanotexture on the bactericidal properties of the surface upon its contact with suspensions containing of E. coli K12 C600 or K. pneumoniae 811 cells in a nutrient medium were studied. The evolution of cell morphology after on the nanotextured surface was analyzed using scanning electron microscopy, and changes in biological fluid during this contact were studied by mass spectrometry. It was shown that massive death of bacterial cells both in the suspension and on the nanotextured surface was determined by combined toxic effects of the hierarchically textured surface and high concentration of Cu2+ ions in the medium.
Assuntos
Ligas/farmacologia , Antibacterianos/farmacologia , Cobre/farmacologia , Escherichia coli K12/efeitos dos fármacos , Klebsiella pneumoniae/efeitos dos fármacos , Nanopartículas/toxicidade , Ligas/química , Ligas/efeitos da radiação , Antibacterianos/química , Antibacterianos/efeitos da radiação , Cobre/química , Cobre/efeitos da radiação , Escherichia coli K12/crescimento & desenvolvimento , Escherichia coli K12/ultraestrutura , Interações Hidrofóbicas e Hidrofílicas , Klebsiella pneumoniae/crescimento & desenvolvimento , Klebsiella pneumoniae/ultraestrutura , Lasers , Testes de Sensibilidade Microbiana , Microscopia Eletrônica de Varredura , Nanopartículas/química , Nanopartículas/efeitos da radiação , Propriedades de SuperfícieRESUMO
ABSTRACT: Unpasteurized liquid egg can be contaminated with pathogenic microorganisms and may cause foodborne outbreaks. Thus, it is essential to decontaminate the liquid egg to ensure food safety. Pulsed UV light is one of the emerging technologies for food decontamination in recent years. This static treatment system has been studied previously in our laboratory. However, continuous processing using a flow-through treatment system needs to be evaluated for potential commercial applications. Therefore, in this study, a flow-through treatment system of pulsed UV light was evaluated and optimized for inactivation of Escherichia coli K12NSR for liquid egg white decontamination. Treatment factors including flow rate (40 to 80 mL/min), number of passes (one to three passes), and distance from the sample to the pulsed UV light strobe (5 to 13 cm) were optimized using response surface methodology. This methodology suggested three passes with 40 mL/min flow rate and a 5-cm distance as the optimum conditions. The model was then validated for the maximum reduction of E. coli K12NSR, which was measured as 1.57 log CFU/mL at the optimal conditions. The energy doses of the pulsed UV light and temperature changes of the liquid egg white during the treatment were measured. Furthermore, several quality parameters were assessed at the optimum treatment conditions to determine the impact of the flow-through pulsed UV processing on the quality of liquid egg white. The results showed significant differences in pH, lipid oxidation, turbidity, and color between control and pulsed UV light-treated samples (P < 0.05). However, there was no significant difference in foaming ability or foam stability between pulsed UV light-treated samples and the control. Overall, this study demonstrated the potential of flow-through pulsed UV light to decontaminate liquid egg white, but further research is needed for optimal enhancement.