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1.
Int J Nanomedicine ; 15: 4275-4288, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32606677

RESUMO

Purpose: Selenium nanoparticles (Se NPs) are promising antibacterial agents to tackle the growing problem of antimicrobial resistance. The aim of this study was to fabricate Se NPs with a net positive charge to enhance their antibacterial efficacy. Methods: Se NPs were coated with a positively charged protein - recombinant spider silk protein eADF4(κ16) - to give them a net positive surface charge. Their cytotoxicity and antibacterial activity were investigated, with negatively charged polyvinyl alcohol coated Se NPs as a control. Besides, these eADF4(κ16)-coated Se NPs were immobilized on the spider silk films, and the antibacterial activity of these films was investigated. Results: Compared to the negatively charged polyvinyl alcohol coated Se NPs, the positively charged eADF4(κ16)-coated Se NPs demonstrated a much higher bactericidal efficacy against the Gram-negative bacteria E. coli, with a minimum bactericidal concentration (MBC) approximately 50 times lower than that of negatively charged Se NPs. Cytotoxicity testing showed that the eADF4(κ16)-coated Se NPs are safe to both Balb/3T3 mouse embryo fibroblasts and HaCaT human skin keratinocytes up to 31 µg/mL, which is much higher than the MBC of these particles against E. coli (8 ± 1 µg/mL). In addition, antibacterial coatings were created by immobilising the eADF4(κ16)-coated Se NPs on positively charged spider silk films and these were shown to retain good bactericidal efficacy and overcome the issue of low particle stability in culture broth. It was found that these Se NPs needed to be released from the film surface in order to exert their antibacterial effects and this release can be regulated by the surface charge of the film, such as the change of the spider silk protein used. Conclusion: Overall, eADF4(κ16)-coated Se NPs are promising new antibacterial agents against life-threatening bacteria.


Assuntos
Antibacterianos/farmacologia , Nanopartículas/química , Proteínas Recombinantes/farmacologia , Selênio/farmacologia , Seda/farmacologia , Células 3T3 , Animais , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Contagem de Colônia Microbiana , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Humanos , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Nanopartículas/ultraestrutura , Tamanho da Partícula
2.
PLoS One ; 15(7): e0235633, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32628709

RESUMO

The antibacterial efficacy of the tetracycline antibiotics has been greatly reduced by the development of resistance, hence a decline in their clinical use. The hok/sok locus is a type I toxin/antitoxin plasmid stability element, often associated with multi-drug resistance plasmids, especially ESBL-encoding plasmids. It enhances host cell survivability and pathogenicity in stressful growth conditions, and increases bacterial tolerance to ß-lactam antibiotics. The hok/sok locus forms dsRNA by RNA:RNA interactions between the toxin encoding mRNA and antitoxin non-coding RNA, and doxycycline has been reported to bind dsRNA structures and inhibit their cleavage/processing by the dsRNase, RNase III. This study investigated the antibacterial activities of doxycycline in hok/sok host bacteria cells, the effects on hok/sok-induced changes in growth and the mechanism(s) involved. Diverse strains of E. coli were transformed with hok/sok plasmids and assessed for doxycycline susceptibility and growth changes. The results show that the hok/sok locus increases bacterial susceptibility to doxycycline, which is more apparent in strains with more pronounced hok/sok-induced growth effects. The increased doxycycline susceptibility occurs despite ß-lactam resistance imparted by hok/sok. Doxycycline was found to induce bacterial death in a manner phenotypically characteristic of Hok toxin expression, suggesting that it inhibits the toxin/antitoxin dsRNA degradation, leading to Hok toxin expression and cell death. In this way, doxycycline could counteract the multi-drug resistance plasmid maintenance/propagation, persistence and pathogenicity mechanisms associated with the hok/sok locus, which could potentially help in efforts to mitigate the rise of antimicrobial resistance.


Assuntos
Antibacterianos/farmacologia , Toxinas Bacterianas/genética , Doxiciclina/farmacologia , Proteínas de Escherichia coli/genética , Escherichia coli/efeitos dos fármacos , RNA Bacteriano/genética , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Plasmídeos/genética , Plasmídeos/metabolismo , RNA Bacteriano/metabolismo , RNA de Cadeia Dupla/metabolismo
3.
Nature ; 584(7821): 470-474, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32669712

RESUMO

The rate of cell growth is crucial for bacterial fitness and drives the allocation of bacterial resources, affecting, for example, the expression levels of proteins dedicated to metabolism and biosynthesis1,2. It is unclear, however, what ultimately determines growth rates in different environmental conditions. Moreover, increasing evidence suggests that other objectives are also important3-7, such as the rate of physiological adaptation to changing environments8,9. A common challenge for cells is that these objectives cannot be independently optimized, and maximizing one often reduces another. Many such trade-offs have indeed been hypothesized on the basis of qualitative correlative studies8-11. Here we report a trade-off between steady-state growth rate and physiological adaptability in Escherichia coli, observed when a growing culture is abruptly shifted from a preferred carbon source such as glucose to fermentation products such as acetate. These metabolic transitions, common for enteric bacteria, are often accompanied by multi-hour lags before growth resumes. Metabolomic analysis reveals that long lags result from the depletion of key metabolites that follows the sudden reversal in the central carbon flux owing to the imposed nutrient shifts. A model of sequential flux limitation not only explains the observed trade-off between growth and adaptability, but also allows quantitative predictions regarding the universal occurrence of such tradeoffs, based on the opposing enzyme requirements of glycolysis versus gluconeogenesis. We validate these predictions experimentally for many different nutrient shifts in E. coli, as well as for other respiro-fermentative microorganisms, including Bacillus subtilis and Saccharomyces cerevisiae.


Assuntos
Adaptação Fisiológica , Meio Ambiente , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Acetatos/metabolismo , Bacillus subtilis/citologia , Bacillus subtilis/crescimento & desenvolvimento , Bacillus subtilis/metabolismo , Divisão Celular , Escherichia coli/enzimologia , Escherichia coli/genética , Fermentação , Gluconeogênese , Glucose/metabolismo , Glicólise , Metabolômica , Modelos Biológicos , Mutação , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo
4.
PLoS One ; 15(7): e0235740, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32678859

RESUMO

This study evaluated the larvicidal activity of Origanum majorana Linnaeus essential oil, identified the chemical composition, evaluated the antimicrobial, cytotoxic and antioxidant potential. The larvicidal activity was evaluated against larvae of the third stage of Aedes aegypti Linaeus, whereas the chemical composition was identified by gas chromatography coupled to mass spectrometer, the antimicrobial activity was carried out against the bacteria Pseudomonas aeruginosa, Escherichia coli and Staphylococcus auereus, the antioxidant activity was evaluated from of 2.2-diphenyl-1-picryl-hydrazila sequestration and Artemia salina Leach cytotoxicity. Regarding to the results, the larvicidal activity showed that O. majorana L. essential oil caused high mortality in A. aegypti L. larvae. In the chromatographic analysis, the main component found in O. majorana L. essential oil was pulegone (57.05%), followed by the other components verbenone (16.92%), trans-p-menthan-2-one (8.57%), iso-menthone (5.58%), piperitone (2.83%), 3-octanol (2.35%) and isopulegol (1.47%). The antimicrobial activity showed that E. coli and P. aeruginosa bacteria were more sensitive to oil than S. aureus, which was resistant at all concentrations. Essential oil did not present antioxidant activity, but it has high cytotoxic activity against A. salina L.


Assuntos
Aedes/efeitos dos fármacos , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Inseticidas/farmacologia , Larva/efeitos dos fármacos , Óleos Voláteis/farmacologia , Origanum/química , Animais , Antibacterianos/química , Antioxidantes/farmacologia , Bactérias/crescimento & desenvolvimento , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Folhas de Planta/química , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/crescimento & desenvolvimento , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimento
5.
Phys Rev Lett ; 125(2): 028103, 2020 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-32701325

RESUMO

Bacterial ribosomes are composed of one-third protein and two-thirds RNA by mass. The predominance of RNA is often attributed to a primordial RNA world, but why exactly two-thirds remains a long-standing mystery. Here we present a quantitative analysis, based on the kinetics of ribosome self-replication, demonstrating that the 1∶2 protein-to-RNA mass ratio uniquely maximizes cellular growth rates in E. coli. A previously unrecognized growth law, and an invariant of bacterial growth, also follow from our analysis. The growth law reveals that the ratio between the number of ribosomes and the number of polymerases making ribosomal RNA is proportional to the cellular doubling time. The invariant is conserved across growth conditions and specifies how key microscopic parameters in the cell, such as transcription and translation rates, are coupled to cellular physiology. Quantitative predictions from the growth law and invariant are shown to be in excellent agreement with E. coli data despite having no fitting parameters. Our analysis can be readily extended to other bacteria once data become available.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Modelos Biológicos , RNA Bacteriano/metabolismo , RNA Ribossômico/metabolismo , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Escherichia coli/genética , Ribossomos/genética
6.
Proc Natl Acad Sci U S A ; 117(31): 18540-18549, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32675239

RESUMO

Once described as mere "bags of enzymes," bacterial cells are in fact highly organized, with many macromolecules exhibiting nonuniform localization patterns. Yet the physical and biochemical mechanisms that govern this spatial heterogeneity remain largely unknown. Here, we identify liquid-liquid phase separation (LLPS) as a mechanism for organizing clusters of RNA polymerase (RNAP) in Escherichia coli Using fluorescence imaging, we show that RNAP quickly transitions from a dispersed to clustered localization pattern as cells enter log phase in nutrient-rich media. RNAP clusters are sensitive to hexanediol, a chemical that dissolves liquid-like compartments in eukaryotic cells. In addition, we find that the transcription antitermination factor NusA forms droplets in vitro and in vivo, suggesting that it may nucleate RNAP clusters. Finally, we use single-molecule tracking to characterize the dynamics of cluster components. Our results indicate that RNAP and NusA molecules move inside clusters, with mobilities faster than a DNA locus but slower than bulk diffusion through the nucleoid. We conclude that RNAP clusters are biomolecular condensates that assemble through LLPS. This work provides direct evidence for LLPS in bacteria and demonstrates that this process can serve as a mechanism for intracellular organization in prokaryotes and eukaryotes alike.


Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/enzimologia , Nucléolo Celular/genética , Nucléolo Celular/metabolismo , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/genética , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Imagem Individual de Molécula , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/metabolismo
7.
Mol Cell ; 79(2): 280-292.e8, 2020 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-32533919

RESUMO

Toxin-antitoxin (TA) systems are ubiquitous genetic elements in bacterial genomes, but their functions are controversial. Although they are frequently postulated to regulate cell growth following stress, few null phenotypes for TA systems have been reported. Here, we show that TA transcript levels can increase substantially in response to stress, but toxin is not liberated. We find that the growth of an Escherichia coli strain lacking ten TA systems encoding endoribonuclease toxins is not affected following exposure to six stresses that each trigger TA transcription. Additionally, using RNA sequencing, we find no evidence of mRNA cleavage following stress. Stress-induced transcription arises from antitoxin degradation and relief of transcriptional autoregulation. Importantly, although free antitoxin is readily degraded in vivo, antitoxin bound to toxin is protected from proteolysis, preventing release of active toxin. Thus, transcription is not a reliable marker of TA activity, and TA systems do not strongly promote survival following individual stresses.


Assuntos
Toxinas Bacterianas/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Estresse Fisiológico , Sistemas Toxina-Antitoxina , Transcrição Genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/crescimento & desenvolvimento , Plasmídeos/genética , Proteólise , RNA Bacteriano/metabolismo , RNA-Seq , Sistemas Toxina-Antitoxina/genética
8.
PLoS One ; 15(5): e0233518, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32433662

RESUMO

The emergence of multidrug-resistant Escherichia coli has become a great challenge in treating nosocomial infections. The polymyxin antibiotic colistin is used as a 'last-line' therapy for such strains, but resistance to colistin is increasingly emerging all over the world. In this study, we investigated lipopolysaccharides (LPS) of colistin-resistant isolates and examined mutations in lpx genes in strains not harbouring mcr genes. We examined 351 clinical E. coli isolates with 38 showing reduced susceptibility to colistin. These isolates were collected from different clinical specimens including blood, urine, and wounds, but no stool. After confirmation of the isolates via a BD Phoenix-100 system (Becton Dickinson, USA), we performed antimicrobial susceptibility tests to characterize the resistance pattern of these isolates to different classes of antibiotics, using the disk diffusion test. The Minimum Inhibitory Concentration (MIC) of colistin was determined using E-test strips. The presence of mobile colistin resistance (mcr-1 and mcr-2) genes was tested for all isolates. LPS (including lipid A) were extracted from all isolates and associated lpx genes analyzed by PCR and sequencing. Among the 38 clinical E. coli isolates with reduced susceptibility to colistin, 52% were resistant to colistin. The MICs of colistin ranged from 0.5 µg/ml to ˃256 µg/ml. Within the 20 colistin-resistant strains, six isolates carried the mcr-1 gene, but not mcr-2. Heterologous expression of the mcr-1 gene in susceptible E. coli DH5α increased the MIC of colistin by eight-fold. The remaining 14 isolates, were negative for both mcr genes. Six isolates were further negative for LPS production and five showed rough LPS phenotypes. Here we present evidence that loss of LPS or lipid A-deficiency can lead to colistin-resistance in clinical E. coli isolates not harbouring mcr genes.


Assuntos
Colistina/farmacologia , Farmacorresistência Bacteriana Múltipla , Proteínas de Escherichia coli , Escherichia coli , Deleção de Genes , Lipídeo A , Proteínas de Membrana , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/isolamento & purificação , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Lipídeo A/genética , Lipídeo A/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo
9.
Ecotoxicol Environ Saf ; 199: 110750, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32446103

RESUMO

Both antibiotics and surfactants commonly exist in natural environment and have generated great concerns due to their biological influence on the ecosystem. A major concern lies in the capacity of antibiotics to induce bacterial filaments formation, which has potential health risks. However, their joint effect is not clear so far. Here, we studied the joint effect of cephalexin (Cex), a typical antibiotic, and differently charged surfactants on the formation of E. coli filaments. Three kinds of surfactants characterized by different charges were used: cationic surfactant (CTAB), anionic surfactant (SDS) and nonionic surfactant (Tween). Data showed that Cex alone caused the formation of E. coli filaments, elongating their maximum profile from ca. 2 µm (a single E. coli cell) to tens of micrometers (an E. coli filament). A joint use of surfactants with Cex could produce even longer E. coli filaments, elongating the maximum length of the bacteria to larger than 100 µm. The capacity order of different surfactants under their optimum concentrations to produce elongated E. coli filaments was Tween > SDS > CTAB. The E. coli filaments were characterized with a normal DNA distribution and a good cell membrane integrity. We measured the stiffness of bacterial cell wall by atomic force microscopy and correlated the elongation capacity of the E. coli filaments to the stiffness of cell wall. Zeta potential measurement indicated that inserting into or being bound to the cell surface in a large quantity was tested not to be the major way that surfactants interacted with bacteria.


Assuntos
Antibacterianos/toxicidade , Cefalexina/toxicidade , Poluentes Ambientais/toxicidade , Escherichia coli/efeitos dos fármacos , Polissorbatos/toxicidade , Tensoativos/toxicidade , Parede Celular/efeitos dos fármacos , Parede Celular/ultraestrutura , Sinergismo Farmacológico , Ecossistema , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/ultraestrutura
10.
Int J Food Microbiol ; 326: 108650, 2020 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-32402916

RESUMO

Use of carbon dots (CDs) in combination with aqueous chitosan solution to extend shelf life and improve stability of soy milk was investigated. Soy milk samples with chitosan solution (0.00%, 0.08%, 0.12%, 0.16% and 0.20%) and banana-based CDs (4%, 6% and 8%) were prepared and stored at room temperature (25-30 °C) for shelf life evaluation. Soy milk with 0.16% chitosan solution exhibited improved stability as evident by increased viscosity, stability coefficient, zeta potential and decreased centrifugation rate compared with soy milk without chitosan. The suitable amount of carbon dots could effectively inhibit the growth of Escherichia coli, Staphylococcus aureus and Bacillus subtilis. Soy milk with 0.16% chitosan and 8% CDs exhibited longer shelf life and significantly lower total bacterial count after storage at room temperature for up to 4 days. Electronic nose-based flavor characteristics of all treated soy milk samples were not far from that of the control sample.


Assuntos
Bacillus subtilis/crescimento & desenvolvimento , Quitosana/farmacologia , Escherichia coli/crescimento & desenvolvimento , Armazenamento de Alimentos/estatística & dados numéricos , Leite de Soja/metabolismo , Staphylococcus aureus/crescimento & desenvolvimento , Bacillus subtilis/efeitos dos fármacos , Carbono/farmacologia , Escherichia coli/efeitos dos fármacos , Contaminação de Alimentos/análise , Pontos Quânticos , Staphylococcus aureus/efeitos dos fármacos , Paladar , Água/farmacologia
11.
J Food Sci ; 85(4): 947-955, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32237089

RESUMO

Edible packaging films have been widely studied because of its safety, green, and effective characteristics. In this paper, chitosan (CH) edible films containing hexahydro-ß-acids (HBA) were prepared, and its physical and mechanical properties, bioactivity, and their impact on the shelf life of pork were investigated. The infrared spectra indicated that the molecular interaction between CH and HBA was observed. Scanning electron microscopy was used to analyze the surface morphology of the film, and light transmittance analysis displayed that the addition of HBA enhanced the film's UV blocking performance. Compared to the CH film, the tensile strength of CH-HBA film increased to 29.19 ± 0.45 MPa, and the scavenging activity of 2,2-diphenyl-1-picrylhydrazyl (DPPH) reached 1.40 ± 0.01 mg rutin/cm2 of the film. The antibacterial activity of the CH-HBA film on Escherichia coli (44825) and Staphylococcus aureus (26001) showed that the CH-HBA film is a feasible antibacterial package. Furthermore, compared to pork packaged in CH and polyethylene films, fresh pork packaged with CH-HBA films displayed prolongation of shelf life due to reduction in microbial proliferation, thiobarbituric values, pH, and total volatile base nitrogen contents during storage at 4 °C for 16 days. The freshness of pork was prolonged by 7-8 days when the dosage of HBA was increased to 0.3% from 0.1% (w/v). These results revealed that the CH-HBA film can effectively extend the shelf life of pork. PRACTICAL APPLICATION: This study effectively prolonged the shelf life of pork. A chitosan-edible film combined with hexahydro-ß-acids has a potential application value in replacing traditional packaged fresh meat.


Assuntos
Antibacterianos/farmacologia , Quitosana/farmacologia , Embalagem de Alimentos/instrumentação , Carne Vermelha/análise , Ácidos/análise , Ácidos/farmacologia , Animais , Antibacterianos/análise , Quitosana/análise , Filmes Comestíveis , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Embalagem de Alimentos/métodos , Armazenamento de Alimentos , Carne Vermelha/microbiologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimento , Suínos , Resistência à Tração
12.
Mol Cell ; 78(4): 670-682.e8, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32343944

RESUMO

Biomolecular condensates play a key role in organizing RNAs and proteins into membraneless organelles. Bacterial RNP-bodies (BR-bodies) are a biomolecular condensate containing the RNA degradosome mRNA decay machinery, but the biochemical function of such organization remains poorly defined. Here, we define the RNA substrates of BR-bodies through enrichment of the bodies followed by RNA sequencing (RNA-seq). We find that long, poorly translated mRNAs, small RNAs, and antisense RNAs are the main substrates, while rRNA, tRNA, and other conserved non-coding RNAs (ncRNAs) are excluded from these bodies. BR-bodies stimulate the mRNA decay rate of enriched mRNAs, helping to reshape the cellular mRNA pool. We also observe that BR-body formation promotes complete mRNA decay, avoiding the buildup of toxic endo-cleaved mRNA decay intermediates. The combined selective permeability of BR-bodies for both enzymes and substrates together with the stimulation of the sub-steps of mRNA decay provide an effective organization strategy for bacterial mRNA decay.


Assuntos
Caulobacter crescentus/metabolismo , Endorribonucleases/metabolismo , Escherichia coli/metabolismo , Complexos Multienzimáticos/metabolismo , Organelas/metabolismo , Polirribonucleotídeo Nucleotidiltransferase/metabolismo , RNA Helicases/metabolismo , Estabilidade de RNA , RNA Mensageiro/metabolismo , Caulobacter crescentus/genética , Caulobacter crescentus/crescimento & desenvolvimento , Endorribonucleases/genética , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Humanos , Complexos Multienzimáticos/genética , Organelas/genética , Polirribonucleotídeo Nucleotidiltransferase/genética , RNA Helicases/genética , RNA Antissenso/genética , RNA Antissenso/metabolismo , RNA Mensageiro/genética , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , RNA não Traduzido/genética , RNA não Traduzido/metabolismo
13.
PLoS One ; 15(3): e0230489, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32226038

RESUMO

Surveillance of antimicrobial resistance (AMR) enables monitoring of trends in AMR prevalence. WHO recommends laboratory-based surveillance to obtain actionable AMR data at local or national level. However, laboratory-based surveillance may lead to overestimation of the prevalence of AMR due to bias. The objective of this study is to assess the difference in resistance prevalence between laboratory-based and population-based surveillance (PBS) among uropathogens in Indonesia. We included all urine samples submitted to the laboratory growing Escherichia coli and Klebsiella pneumoniae in the laboratory-based surveillance. Population-based surveillance data were collected in a cross-sectional survey of AMR in E. coli and K. pneumoniae isolated from urine samples among consecutive patients with symptoms of UTI, attending outpatient clinics and hospital wards. Data were collected between 1 April 2014 until 31 May 2015. The difference in percentage resistance (95% confidence intervals) between laboratory- and population-based surveillance was calculated for relevant antibiotics. A difference larger than +/- 5 percent points was defined as a biased result, precluding laboratory-based surveillance for guiding empirical treatment. We observed high prevalence of AMR ranging between 63.1% (piperacillin-tazobactam) and 85% (ceftriaxone) in laboratory-based surveillance and 41.3% (piperacillin-tazobactam) and 74.2% (ceftriaxone) in population-based surveillance, except for amikacin and meropenem (5.7%/9.8%; 10.8%/5.9%; [laboratory-/population-based surveillance], respectively). Laboratory-based surveillance yielded significantly higher AMR prevalence estimates than population-based surveillance. This difference was much larger when comparing surveillance data from outpatients than from inpatients. All point estimates of the difference between the two surveillance systems were larger than 5 percent points, except for amikacin and meropenem. Laboratory-based AMR surveillance of uropathogens, is not adequate to guide empirical treatment for community-based settings in Indonesia.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana , Infecções por Escherichia coli , Escherichia coli , Infecções por Klebsiella , Klebsiella pneumoniae , Infecções Urinárias , Estudos Transversais , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/isolamento & purificação , Infecções por Escherichia coli/tratamento farmacológico , Infecções por Escherichia coli/epidemiologia , Infecções por Escherichia coli/microbiologia , Feminino , Humanos , Indonésia , Infecções por Klebsiella/tratamento farmacológico , Infecções por Klebsiella/epidemiologia , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/crescimento & desenvolvimento , Klebsiella pneumoniae/isolamento & purificação , Masculino , Testes de Sensibilidade Microbiana , Infecções Urinárias/tratamento farmacológico , Infecções Urinárias/epidemiologia , Infecções Urinárias/microbiologia
14.
Proc Natl Acad Sci U S A ; 117(19): 10271-10277, 2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32341159

RESUMO

Viomycin, an antibiotic that has been used to fight tuberculosis infections, is believed to block the translocation step of protein synthesis by inhibiting ribosomal subunit dissociation and trapping the ribosome in an intermediate state of intersubunit rotation. The mechanism by which viomycin stabilizes this state remains unexplained. To address this, we have determined cryo-EM and X-ray crystal structures of Escherichia coli 70S ribosome complexes trapped in a rotated state by viomycin. The 3.8-Å resolution cryo-EM structure reveals a ribosome trapped in the hybrid state with 8.6° intersubunit rotation and 5.3° rotation of the 30S subunit head domain, bearing a single P/E state transfer RNA (tRNA). We identify five different binding sites for viomycin, four of which have not been previously described. To resolve the details of their binding interactions, we solved the 3.1-Å crystal structure of a viomycin-bound ribosome complex, revealing that all five viomycins bind to ribosomal RNA. One of these (Vio1) corresponds to the single viomycin that was previously identified in a complex with a nonrotated classical-state ribosome. Three of the newly observed binding sites (Vio3, Vio4, and Vio5) are clustered at intersubunit bridges, consistent with the ability of viomycin to inhibit subunit dissociation. We propose that one or more of these same three viomycins induce intersubunit rotation by selectively binding the rotated state of the ribosome at dynamic elements of 16S and 23S rRNA, thus, blocking conformational changes associated with molecular movements that are required for translocation.


Assuntos
Escherichia coli/metabolismo , Biossíntese de Proteínas , RNA Ribossômico/metabolismo , Ribossomos/metabolismo , Viomicina/farmacologia , Antibacterianos/farmacologia , Cristalografia por Raios X , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Modelos Moleculares , Conformação Molecular , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Ribossômico/genética , RNA de Transferência/química , RNA de Transferência/metabolismo , Proteínas Ribossômicas/metabolismo , Ribossomos/química
15.
World J Microbiol Biotechnol ; 36(4): 59, 2020 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-32236784

RESUMO

An endogenous homoethanol pathway (glucose/1.2 xylose => 2 pyruvate => 2 ethanol) was previously engineered in Escherichia coli SZ410 via eliminating acid-producing pathways and anaerobic expression of the pyruvate dehydrogenase complex (aceEF-lpd operon). This ethanologenic derivative was subsequently engineered through adaptive evolution and partial deletion of the RNase G, resulting in an improved strain of E. coli RM10 for ethanol production using C6 and C5 sugars. Nevertheless, compared to the ethanol tolerance and/or ethanol titer achieved by industrial yeast, further incremental improvement of RM10 was needed for ethanol production using cellulosic biomass derived C6 and C5 sugars. In this study, the role of aldB gene (encoding for acetaldehyde dehydrogenase, AldB, which oxidizes acetaldehyde to acetic acid) was evaluated for ethanol/acetaldehyde tolerance and xylose fermentation by RM10. Deletion of aldB gene decreased ethanol tolerance, fermentative cell growth and ethanol production from xylose; while overexpression of aldB gene improved fermentative cell growth, and increased ethanol production from xylose. The improvement is likely attributed to preventing acetaldehyde accumulation (a toxic intermediate of homoethanol pathway) via AldB catalyzed oxidation.


Assuntos
Aldeído Oxirredutases/metabolismo , Escherichia coli/crescimento & desenvolvimento , Etanol/metabolismo , Xilose/metabolismo , Aldeído Oxirredutases/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Endorribonucleases/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fermentação , Deleção de Genes
16.
J Food Sci ; 85(5): 1523-1535, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32282078

RESUMO

Bacteriocins are defined as ribosomally synthesized antibacterial peptides/proteins that either kill or inhibit the growth of other bacteria. In the present study, the physicochemical properties, mode of action, and potential use in food preservation of a novel bacteriocin BM1122 from Lactobacillus crustorum MN047 were studied. It exhibited a broad inhibitory spectrum against selected Gram-positive and Gram-negative bacteria. Kinetic curves revealed efficient time-dependent bactericidal activity. Moreover, BM1122 possessed low hemolytic activity and good thermal stability between 60 and 120 °C. It was resistant to a wide range of pH (2 to 11) and proteinases. The scanning and transmission electron microscopy showed that BM1122 led to plasmolysis of Staphylococcus aureus and pore formation in Escherichia coli. Flow cytometric analysis demonstrated that BM1122 destroyed cell membrane integrity. Additionally, BM1122 could also inhibit biofilm formation and disturb the normal cell cycles of S. aureus and E. coli. Finally, BM1122 may enhance the inhibition of S. aureus and E. coli on beef meat stored at 4 °C for a duration of 10 days. These findings indicated that BM1122 had the potential for use as a natural preservative in the food industry. PRACTICAL APPLICATION: Fresh raw meats are highly perishable products. Bacteriocin BM1122 with a broad antibacterial spectrum can inhibit the growth of microorganisms in beef meat during refrigerated storage.


Assuntos
Antibacterianos/farmacologia , Bacteriocinas/farmacologia , Lactobacillus/química , Animais , Antibacterianos/química , Antibacterianos/metabolismo , Bacteriocinas/química , Bacteriocinas/metabolismo , Bovinos , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Conservação de Alimentos , Lactobacillus/metabolismo , Carne Vermelha/microbiologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimento
17.
Proc Natl Acad Sci U S A ; 117(12): 6811-6821, 2020 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-32156726

RESUMO

Emerging evidence suggests the Pseudomonas aeruginosa accessory genome is enriched with uncharacterized virulence genes. Identification and characterization of such genes may reveal novel pathogenic mechanisms used by particularly virulent isolates. Here, we utilized a mouse bacteremia model to quantify the virulence of 100 individual P. aeruginosa bloodstream isolates and performed whole-genome sequencing to identify accessory genomic elements correlated with increased bacterial virulence. From this work, we identified a specific contact-dependent growth inhibition (CDI) system enriched among highly virulent P. aeruginosa isolates. CDI systems contain a large exoprotein (CdiA) with a C-terminal toxin (CT) domain that can vary between different isolates within a species. Prior work has revealed that delivery of a CdiA-CT domain upon direct cell-to-cell contact can inhibit replication of a susceptible target bacterium. Aside from mediating interbacterial competition, we observed our virulence-associated CdiA-CT domain to promote toxicity against mammalian cells in culture and lethality during mouse bacteremia. Structural and functional studies revealed this CdiA-CT domain to have in vitro tRNase activity, and mutations that abrogated this tRNAse activity in vitro also attenuated virulence. Furthermore, CdiA contributed to virulence in mice even in the absence of contact-dependent signaling. Overall, our findings indicate that this P. aeruginosa CDI system functions as both an interbacterial inhibition system and a bacterial virulence factor against a mammalian host. These findings provide an impetus for continued studies into the complex role of CDI systems in P. aeruginosa pathogenesis.


Assuntos
Proteínas de Bactérias/metabolismo , Inibição de Contato/genética , Escherichia coli/crescimento & desenvolvimento , Genômica/métodos , Pseudomonas aeruginosa/crescimento & desenvolvimento , Fatores de Virulência/metabolismo , Virulência , Animais , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Escherichia coli/metabolismo , Infecções por Escherichia coli/microbiologia , Feminino , Genoma Bacteriano , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/isolamento & purificação , Pseudomonas aeruginosa/metabolismo , Transdução de Sinais , Fatores de Virulência/genética
18.
Proc Natl Acad Sci U S A ; 117(12): 6777-6783, 2020 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-32152098

RESUMO

Tol-Pal is a multiprotein system present in the envelope of Gram-negative bacteria. Inactivation of this widely conserved machinery compromises the outer membrane (OM) layer of these organisms, resulting in hypersensitivity to many antibiotics. Mutants in the tol-pal locus fail to complete division and form cell chains. This phenotype along with the localization of Tol-Pal components to the cytokinetic ring in Escherichia coli has led to the proposal that the primary function of the system is to promote OM constriction during division. Accordingly, a poorly constricted OM is believed to link the cell chains formed upon Tol-Pal inactivation. However, we show here that cell chains of E. coli tol-pal mutants are connected by an incompletely processed peptidoglycan (PG) layer. Genetic suppressors of this defect were isolated and found to overproduce OM lipoproteins capable of cleaving the glycan strands of PG. Among the factors promoting cell separation in mutant cells was a protein of previously unknown function (YddW), which we have identified as a divisome-localized glycosyl hydrolase that cleaves peptide-free PG glycans. Overall, our results indicate that the cell chaining defect of Tol-Pal mutants cannot simply be interpreted as a defect in OM constriction. Rather, the complex also appears to be required for the activity of several OM-localized enzymes with cell wall remodeling activity. Thus, the Tol-Pal system may play a more general role in coordinating OM invagination with PG remodeling at the division site than previously appreciated.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Divisão Celular , Parede Celular/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Lipoproteínas/metabolismo , Peptidoglicano/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Membrana Celular/metabolismo , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Lipoproteínas/genética , Ligação Proteica
19.
Nat Commun ; 11(1): 1496, 2020 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-32198415

RESUMO

The ability to grow at moderate acidic conditions (pH 4.0-5.0) is important to Escherichia coli colonization of the host's intestine. Several regulatory systems are known to control acid resistance in E. coli, enabling the bacteria to survive under acidic conditions without growth. Here, we characterize an acid-tolerance response (ATR) system and its regulatory circuit, required for E. coli exponential growth at pH 4.2. A two-component system CpxRA directly senses acidification through protonation of CpxA periplasmic histidine residues, and upregulates the fabA and fabB genes, leading to increased production of unsaturated fatty acids. Changes in lipid composition decrease membrane fluidity, F0F1-ATPase activity, and improve intracellular pH homeostasis. The ATR system is important for E. coli survival in the mouse intestine and for production of higher level of 3-hydroxypropionate during fermentation. Furthermore, this ATR system appears to be conserved in other Gram-negative bacteria.


Assuntos
Tolerância a Medicamentos/fisiologia , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/genética , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/metabolismo , Animais , Proteínas de Bactérias/metabolismo , Sequência de Bases , Sítios de Ligação , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Ácido Graxo Sintase Tipo II/genética , Ácido Graxo Sintase Tipo II/metabolismo , Ácidos Graxos Insaturados/metabolismo , Feminino , Fermentação , Regulação Bacteriana da Expressão Gênica , Homeostase , Hidroliases/genética , Hidroliases/metabolismo , Concentração de Íons de Hidrogênio , Intestinos/microbiologia , Ácido Láctico/análogos & derivados , Ácido Láctico/metabolismo , Fluidez de Membrana , Lipídeos de Membrana , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Quinases/metabolismo , Transcrição Genética
20.
J Food Sci ; 85(4): 1193-1202, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32144762

RESUMO

In this study, starch-based films incorporating metal oxide (MO2 ) nanoparticles (NPs) of TiO2 and SiO2 (at a concentration of 1 to 4 wt. %) were produced by solution casting method. In order to exhibit antimicrobial properties, MO2 NPs were modified by synthesizing silver (Ag) ions over the NPs using cationic adsorption method. Ag ions were then reduced to metallic Ag by sodium borohydride solution. Scanning electron microscopy showed a smooth surface for the pure starch film. Incorporating MO2 @Ag NPs in the films increased surface roughness with agglomerated NPs within starch matrix. Energy dispersive X-ray analysis exhibited a uniform dispersion of Ag-loaded MO2 NPs, which increases surface contact between these NPs and the biopolymer matrix leading to improved physical and mechanical properties of the resulting films. With increasing in the NPs concentrations, the tensile strength and elongation at break % of the films increased and decreased, respectively. Incorporating MO2 @Ag NPs into starch matrix decreased solubility in water and water vapor permeability of the obtained films, and significantly inhibited the growth of Escherichia coli and Staphylococcus aureus. The most antibacterial effect was obtained for the films containing higher weight concentrations of Ag-loaded SiO2 -NPs.


Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Dióxido de Silício/química , Prata/química , Prata/farmacologia , Titânio/química , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Nanocompostos/química , Nanopartículas/química , Permeabilidade , Solubilidade , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimento , Resistência à Tração
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