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1.
Carbohydr Polym ; 255: 117484, 2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33436244

RESUMO

Wound dressing composed of chitosan, based crosslinked gelatin/ polyvinyl pyrrolidone, embedded silver nanoparticles were fabricated using solution casting method. The membrane was characterized by FTIR, SEM and TGA. Glutaraldehyde (0.5 %) was used for the crosslinking of membrane components and associated with 7-folds boosted mechanical performance, 28 % more hydrolytic stability, 3-folds thickness reduction and morphological roughness. Silver nanoparticles were characterized by UV-vis, XRD and TEM for an average size of 9.9 nm. The membrane with higher concentration of silver nanoparticles showed maximum antibacterial activity against human pathogenic bacteria; and the measured inhibition zones ranged from 1.5 to 3 cm. The activity of the particles ranged from severe to complete reduction in Penicillin, Erythromycin and Macrolide family's resistance genes expression such as ß-Lactamase, mecA and erm. This developed membrane can serve as promising and cost-effective system against severe diabetic and burn wound infections.


Assuntos
Antibacterianos/farmacologia , Bandagens , Quitosana/química , Citrullus colocynthis/química , Gelatina/química , Povidona/química , Prata/farmacologia , Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Farmacorresistência Bacteriana Múltipla/genética , Eritromicina/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Humanos , Macrolídeos/farmacologia , Nanopartículas Metálicas/química , Nanopartículas Metálicas/ultraestrutura , Metiltransferases/genética , Metiltransferases/metabolismo , Testes de Sensibilidade Microbiana , Proteínas de Ligação às Penicilinas/genética , Proteínas de Ligação às Penicilinas/metabolismo , Penicilinas/farmacologia , Cultura Primária de Células , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/crescimento & desenvolvimento , Salmonella typhi/efeitos dos fármacos , Salmonella typhi/crescimento & desenvolvimento , Prata/química , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimento , beta-Lactamases/genética , beta-Lactamases/metabolismo
2.
Proc Natl Acad Sci U S A ; 118(1)2021 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-33443205

RESUMO

The type 6 secretion system (T6SS) is a dynamic organelle encoded by many gram-negative bacteria that can be used to kill competing bacterial prey species in densely occupied niches. Some predatory species, such as Vibrio cholerae, use their T6SS in an untargeted fashion while in contrast, Pseudomonas aeruginosa assembles and fires its T6SS apparatus only after detecting initial attacks by other bacterial prey cells; this targeted attack strategy has been termed the T6SS tit-for-tat response. Molecules that interact with the P. aeruginosa outer membrane such as polymyxin B can also trigger assembly of T6SS organelles via a signal transduction pathway that involves protein phosphorylation. Recent work suggests that a phospholipase T6SS effector (TseL) of V. cholerae can induce T6SS dynamic activity in P. aeruginosa when delivered to or expressed in the periplasmic space of this organism. Here, we report that inhibiting expression of essential genes involved in outer membrane biogenesis can also trigger T6SS activation in P. aeruginosa Specifically, we developed a CRISPR interference (CRISPRi) system to knock down expression of bamA, tolB, and lptD and found that these knockdowns activated T6SS activity. This increase in T6SS activity was dependent on the same signal transduction pathway that was previously shown to be required for the tit-for-tat response. We conclude that outer membrane perturbation can be sensed by P. aeruginosa to activate the T6SS even when the disruption is generated by aberrant cell envelope biogenesis.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Sistemas CRISPR-Cas , Membrana Celular/metabolismo , Genes Essenciais/fisiologia , Proteínas Periplásmicas/metabolismo , Pseudomonas aeruginosa/genética , Sistemas de Secreção Tipo VI/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Membrana Celular/genética , Membrana Celular/patologia , Sobrevivência Celular/genética , Técnicas de Silenciamento de Genes , Inativação Gênica , Genes Essenciais/genética , Genótipo , Proteínas Periplásmicas/genética , Fenótipo , Pseudomonas aeruginosa/citologia , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/metabolismo , RNA-Seq , Transdução de Sinais/genética , Estresse Fisiológico , Vibrio cholerae/genética , Vibrio cholerae/crescimento & desenvolvimento
3.
Int J Biol Macromol ; 173: 99-108, 2021 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-33460660

RESUMO

The present investigation reports an in-vitro study using combination of laccase and an enhancer capable of inhibiting the growth of pathogenic microorganisms, preventing biofilm formation, and whitening teeth. Laccase-cinnamic acid system remarkably inhibited the growth of Aggregatibacter actinomycetemcomitans, Candida albicans, S. aureus, and Streptococcus mutans whilst showed no significant effects on Gram-negative bacteria. Data presented that cinnamic acid (10 mM) with laccase (0.125 U ml-1) led to a maximum decrease of about 90%, in S. mutans biofilm formation. The confocal laser scanning microscopy showed considerable detachment of S. mutans cells from glass substratum. The combined laccase-cinnamic acid system could remove teeth discoloration caused by coffee. SEM of the teeth surface exhibited no damages such as surface cracking or fracture. Liquid chromatography-tandem mass spectrometry (LC-MS) and cyclic voltammetry (CV) studies showed that laccase can catalyze the one-electron oxidation of cinnamic acid to the respective radical. This radical can then undergo several fates, including recombination with another radical to form a dimeric species, dismutation of the radical back to cinnamic acid or decarboxylation to give various reduced oxygen species. Therefore, the redox potential values of phenolic monomers/oligomers are related with their biological activities.


Assuntos
Aggregatibacter actinomycetemcomitans/efeitos dos fármacos , Antibacterianos/farmacologia , Cinamatos/farmacologia , Proteínas Fúngicas/farmacologia , Lacase/farmacologia , Aggregatibacter actinomycetemcomitans/crescimento & desenvolvimento , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Ácidos Cafeicos/farmacologia , Candida albicans/efeitos dos fármacos , Candida albicans/crescimento & desenvolvimento , Catecóis/farmacologia , Sinergismo Farmacológico , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Proteínas Fúngicas/isolamento & purificação , Ácido Gálico/farmacologia , Hidroquinonas/farmacologia , Lacase/isolamento & purificação , Lactobacillus/efeitos dos fármacos , Lactobacillus/crescimento & desenvolvimento , Testes de Sensibilidade Microbiana , Oxirredução , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/crescimento & desenvolvimento , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimento , Streptococcus mutans/efeitos dos fármacos , Streptococcus mutans/crescimento & desenvolvimento , Clareadores Dentários/farmacologia
4.
Mol Cell ; 81(3): 571-583.e6, 2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33412111

RESUMO

The arms race between bacteria and phages has led to the evolution of diverse anti-phage defenses, several of which are controlled by quorum-sensing pathways. In this work, we characterize a quorum-sensing anti-activator protein, Aqs1, found in Pseudomonas phage DMS3. We show that Aqs1 inhibits LasR, the master regulator of quorum sensing, and present the crystal structure of the Aqs1-LasR complex. The 69-residue Aqs1 protein also inhibits PilB, the type IV pilus assembly ATPase protein, which blocks superinfection by phages that require the pilus for infection. This study highlights the remarkable ability of small phage proteins to bind multiple host proteins and disrupt key biological pathways. As quorum sensing influences various anti-phage defenses, Aqs1 provides a mechanism by which infecting phages might simultaneously dampen multiple defenses. Because quorum-sensing systems are broadly distributed across bacteria, this mechanism of phage counter-defense may play an important role in phage-host evolutionary dynamics.


Assuntos
Proteínas de Bactérias/metabolismo , Bacteriófagos/metabolismo , Pseudomonas aeruginosa/metabolismo , Percepção de Quorum , Transativadores/metabolismo , Proteínas Virais/metabolismo , Proteínas de Bactérias/genética , Bacteriófagos/genética , Bacteriófagos/patogenicidade , Fímbrias Bacterianas/metabolismo , Interações Hospedeiro-Patógeno , Oxirredutases/genética , Oxirredutases/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/crescimento & desenvolvimento , Piocianina/metabolismo , Transativadores/genética , Proteínas Virais/genética
5.
Biochim Biophys Acta Biomembr ; 1863(1): 183482, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-33002450

RESUMO

BACKGROUND: Pseudomonas aeruginosa is a bacterium able to induce serious pulmonary infections in cystic fibrosis (CF) patients. This bacterium is very often antibiotic resistant, partly because of its membrane impermeability, which is linked to the membrane lipid composition. This work aims to study the membrane phospholipids of P. aeruginosa grown in CF sputum-like media. METHODS: Three media were used: Mueller Hilton broth (MHB), synthetic cystic fibrosis medium (SCFM) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) complemented SCFM (SCFM-PC). Lipids were extracted and LC-MS/MS analyses were performed. Growth curves, atomic force microscopy images and minimal inhibitory concentration determination were performed in order to compare the growth and potentially link lipid modifications to antibiotic resistance. RESULTS: Semi-quantification showed phospholipid quantity variation depending on the growth medium. Phosphatidylcholines were detected in traces in SCFM. MS/MS experiments showed an increase of phospholipids derived from DOPC in SCFM-PC. We observed no influence of the medium on the bacterial growth and a minor influence on the bacterial shape. MIC values were generally higher in SCFM and SCFM-PC than in MHB. CONCLUSIONS: We defined a CF sputum-like media which can be used for the membrane lipid extraction of P. aeruginosa. We also showed that the growth medium does have an influence on its membrane lipid composition and antibiotic resistance, especially for SCFM-PC in which P. aeruginosa uses DOPC, in order to make its own membrane. GENERAL SIGNIFICANCE: Our results show that considerable caution must be taken when choosing a medium for lipid identification and antibiotic testing -especially for phospholipids-enriched media.


Assuntos
Membrana Celular/metabolismo , Fibrose Cística/microbiologia , Fosfolipídeos/metabolismo , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/crescimento & desenvolvimento , Escarro/microbiologia , Meios de Cultura , Fibrose Cística/metabolismo , Humanos , Infecções por Pseudomonas/metabolismo
6.
Int J Biol Macromol ; 171: 44-58, 2021 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-33373634

RESUMO

Fatty acids-assisted superparamagnetic maghemite (γ-Fe2O3) NPs was biologically synthesized using extract of polyherbal drug Liv52 (L52E). The NPs were characterized by UV-vis spectroscopy, FT-IR, SEM, TEM, EDX, XRD and VSM. The major biological molecules present in L52E analysed by GC-MS were saturated fatty acids (palmitic acid 21.95%; stearic acid 13.99%; myristic acid 1.14%), monounsaturated fatty acid (oleic acid 18.43%), polyunsaturated fatty acid (linoleic acid 20.45%), and aromatic phenol (cardanol monoene 11.92%) that could imply in bio-fabrication and stabilization of γ-Fe2O3 NPs. The FT-IR spectra revealed involvement of carboxylic group of fatty acids, amide group of proteins and hydroxyl group of phenolic compounds that acts as reducing and capping agents. The synthesized NPs were used to investigate their antimicrobial, antibiofilm activity against P. aeruginosa, MRSA and C. albicans and anticancer activity on colon cancer cells (HCT-116) for biomedical applications. Further, molecular docking study was performed to explore the interaction of Fe2O3 NPs with major cell wall components i.e., peptidoglycan and mannoproteins. The docking studies revealed that Fe2O3 interacted efficiently with peptidoglycan and mannoproteins and Fe2O3 get accommodated into catalytic cleft of mannoprotein. Due to magnetic property, the biological activity of γ-Fe2O3 can be further enhanced by applying external magnetic field alone or in amalgamation with other therapeutics drugs.


Assuntos
Anti-Infecciosos/farmacologia , Antineoplásicos/farmacologia , Glicoproteínas de Membrana/farmacologia , Peptidoglicano/farmacologia , Anti-Infecciosos/química , Antineoplásicos/química , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Candida albicans/efeitos dos fármacos , Candida albicans/crescimento & desenvolvimento , Sobrevivência Celular/efeitos dos fármacos , Células HCT116 , Humanos , Ácido Linoleico/química , Glicoproteínas de Membrana/química , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/crescimento & desenvolvimento , Viabilidade Microbiana/efeitos dos fármacos , Simulação de Acoplamento Molecular , Ácido Mirístico/química , Ácido Oleico/química , Ácido Palmítico/química , Peptidoglicano/química , Fenóis/química , Estrutura Secundária de Proteína , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/crescimento & desenvolvimento , Ácidos Esteáricos/química
7.
PLoS Pathog ; 16(12): e1008893, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33326490

RESUMO

Bacterial bloodstream infections (BSI) are a major health concern and can cause up to 40% mortality. Pseudomonas aeruginosa BSI is often of nosocomial origin and is associated with a particularly poor prognosis. The mechanism of bacterial persistence in blood is still largely unknown. Here, we analyzed the behavior of a cohort of clinical and laboratory Pseudomonas aeruginosa strains in human blood. In this specific environment, complement was the main defensive mechanism, acting either by direct bacterial lysis or by opsonophagocytosis, which required recognition by immune cells. We found highly variable survival rates for different strains in blood, whatever their origin, serotype, or the nature of their secreted toxins (ExoS, ExoU or ExlA) and despite their detection by immune cells. We identified and characterized a complement-tolerant subpopulation of bacterial cells that we named "evaders". Evaders shared some features with bacterial persisters, which tolerate antibiotic treatment. Notably, in bi-phasic killing curves, the evaders represented 0.1-0.001% of the initial bacterial load and displayed transient tolerance. However, the evaders are not dormant and require active metabolism to persist in blood. We detected the evaders for five other major human pathogens: Acinetobacter baumannii, Burkholderia multivorans, enteroaggregative Escherichia coli, Klebsiella pneumoniae, and Yersinia enterocolitica. Thus, the evaders could allow the pathogen to persist within the bloodstream, and may be the cause of fatal bacteremia or dissemination, in particular in the absence of effective antibiotic treatments.


Assuntos
Infecções Bacterianas/sangue , Infecções Bacterianas/imunologia , Ativação do Complemento/imunologia , Acinetobacter baumannii/crescimento & desenvolvimento , Acinetobacter baumannii/patogenicidade , Bacteriemia/sangue , Bacteriemia/imunologia , Bacteriemia/microbiologia , Bactérias , Burkholderia/crescimento & desenvolvimento , Burkholderia/patogenicidade , Proteínas do Sistema Complemento/imunologia , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/patogenicidade , Humanos , Klebsiella pneumoniae/crescimento & desenvolvimento , Klebsiella pneumoniae/patogenicidade , Testes de Sensibilidade Microbiana , Infecções por Pseudomonas/sangue , Infecções por Pseudomonas/imunologia , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/patogenicidade , Yersinia enterocolitica/crescimento & desenvolvimento , Yersinia enterocolitica/patogenicidade
8.
Proc Natl Acad Sci U S A ; 117(52): 33519-33529, 2020 12 29.
Artigo em Inglês | MEDLINE | ID: mdl-33318204

RESUMO

Pseudomonas aeruginosa causes severe multidrug-resistant infections that often lead to bacteremia and sepsis. Physiologically relevant conditions can increase the susceptibility of pathogens to antibiotics, such as azithromycin (AZM). When compared to minimal-inhibitory concentrations (MICs) in laboratory media, AZM had a 16-fold lower MIC in tissue culture medium with 5% Mueller Hinton broth (MHB) and a 64-fold lower MIC in this tissue culture medium with 20% human serum. AZM also demonstrated increased synergy in combination with synthetic host-defense peptides DJK-5 and IDR-1018 under host-like conditions and in a murine abscess model. To mechanistically study the altered effects of AZM under physiologically relevant conditions, global transcriptional analysis was performed on P. aeruginosa with and without effective concentrations of AZM. This revealed that the arn operon, mediating arabinosaminylation of lipopolysaccharides and related regulatory systems, was down-regulated in host-like media when compared to MHB. Inactivation of genes within the arn operon led to increased susceptibility of P. aeruginosa to AZM and great increases in synergy between AZM and other antimicrobial agents, indicating that dysregulation of the arn operon might explain increased AZM uptake and synergy in host-like media. Furthermore, genes involved in central and energy metabolism and ribosome biogenesis were dysregulated more in physiologically relevant conditions treated with AZM, likely due to general changes in cell physiology as a result of the increased effectiveness of AZM in these conditions. These data suggest that, in addition to the arn operon, there are multiple factors in host-like environments that are responsible for observed changes in susceptibility.


Assuntos
Azitromicina/farmacologia , Meios de Cultura/farmacologia , Pseudomonas aeruginosa/crescimento & desenvolvimento , Antibacterianos/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Sinergismo Farmacológico , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Humanos , Macrolídeos/farmacologia , Testes de Sensibilidade Microbiana , Óperon/genética , Peptídeos/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/genética , Soro
9.
PLoS Biol ; 18(8): e3000805, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32810152

RESUMO

Antibiotics are losing efficacy due to the rapid evolution and spread of resistance. Treatments targeting bacterial virulence factors have been considered as alternatives because they target virulence instead of pathogen viability, and should therefore exert weaker selection for resistance than conventional antibiotics. However, antivirulence treatments rarely clear infections, which compromises their clinical applications. Here, we explore the potential of combining antivirulence drugs with antibiotics against the opportunistic human pathogen Pseudomonas aeruginosa. We combined two antivirulence compounds (gallium, a siderophore quencher, and furanone C-30, a quorum sensing [QS] inhibitor) together with four clinically relevant antibiotics (ciprofloxacin, colistin, meropenem, tobramycin) in 9×9 drug concentration matrices. We found that drug-interaction patterns were concentration dependent, with promising levels of synergies occurring at intermediate drug concentrations for certain drug pairs. We then tested whether antivirulence compounds are potent adjuvants, especially when treating antibiotic resistant (AtbR) clones. We found that the addition of antivirulence compounds to antibiotics could restore growth inhibition for most AtbR clones, and even abrogate or reverse selection for resistance in five drug combination cases. Molecular analyses suggest that selection against resistant clones occurs when resistance mechanisms involve restoration of protein synthesis, but not when efflux pumps are up-regulated. Altogether, our work provides a first systematic analysis of antivirulence-antibiotic combinatorial treatments and suggests that such combinations have the potential to be both effective in treating infections and in limiting the spread of antibiotic resistance.


Assuntos
Antibacterianos/farmacologia , Ciprofloxacino/farmacologia , Colistina/farmacologia , Furanos/farmacologia , Gálio/farmacologia , Meropeném/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Tobramicina/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Combinação de Medicamentos , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Sinergismo Farmacológico , Humanos , Testes de Sensibilidade Microbiana , Biossíntese de Proteínas/efeitos dos fármacos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/metabolismo , Percepção de Quorum/efeitos dos fármacos , Virulência
10.
Arch Microbiol ; 202(10): 2825-2840, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32747998

RESUMO

M18 strain of Streptococcus salivarius is a bacterial replacement probiotic that has been suggested for use in the oral cavity. Here, we have shown that S. salivarius M18 cell-free supernatant reduced the growth of the two most common human pathogens Pseudomonas aeruginosa and Klebsiella pneumonia and sensitized the pathogenic bacteria to antibiotic. Besides, the supernatant inhibited biofilm formation of P. aeruginosa drastically. For pinpointing the biomolecular changes that occurred in P. aeruginosa incubated with the probiotic supernatant, attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy was used. Unsupervised learning algorithms, principal component analysis (PCA) and hierarchical cluster analysis (HCA), and intensity analyses of individual spectral bands exhibited comprehensive alterations in the polysaccharide and lipid contents and compositions of P. aeruginosa cultivated with S. salivarius M18 cell-free supernatant. These results indicate that S. salivarius M18 has the potential for the prevention or alleviation of different pathogen-induced infections along with the infections of oral pathogens.


Assuntos
Antibiose/fisiologia , Klebsiella pneumoniae/crescimento & desenvolvimento , Probióticos/farmacologia , Pseudomonas aeruginosa/crescimento & desenvolvimento , Streptococcus salivarius/química , Biofilmes/crescimento & desenvolvimento , Humanos , Klebsiella pneumoniae/patogenicidade , Boca/microbiologia , Análise de Componente Principal , Pseudomonas aeruginosa/patogenicidade , Espectroscopia de Infravermelho com Transformada de Fourier
11.
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
12.
Proc Natl Acad Sci U S A ; 117(32): 19455-19464, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32703812

RESUMO

A better understanding of how antibiotic exposure impacts the evolution of resistance in bacterial populations is crucial for designing more sustainable treatment strategies. The conventional approach to this question is to measure the range of concentrations over which resistant strain(s) are selectively favored over a sensitive strain. Here, we instead investigate how antibiotic concentration impacts the initial establishment of resistance from single cells, mimicking the clonal expansion of a resistant lineage following mutation or horizontal gene transfer. Using two Pseudomonas aeruginosa strains carrying resistance plasmids, we show that single resistant cells have <5% probability of detectable outgrowth at antibiotic concentrations as low as one-eighth of the resistant strain's minimum inhibitory concentration (MIC). This low probability of establishment is due to detrimental effects of antibiotics on resistant cells, coupled with the inherently stochastic nature of cell division and death on the single-cell level, which leads to loss of many nascent resistant lineages. Our findings suggest that moderate doses of antibiotics, well below the MIC of resistant strains, may effectively restrict de novo emergence of resistance even though they cannot clear already-large resistant populations.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Pseudomonas aeruginosa/efeitos dos fármacos , Relação Dose-Resposta a Droga , Farmacorresistência Bacteriana/genética , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Modelos Teóricos , Plasmídeos/genética , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/crescimento & desenvolvimento , Análise de Célula Única , Processos Estocásticos
13.
J Vis Exp ; (160)2020 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-32597860

RESUMO

fDrug research for the treatment of lung infections is progressing towards predictive in vitro models of high complexity. The multifaceted presence of bacteria in lung models can re-adapt epithelial arrangement, while immune cells coordinate an inflammatory response against the bacteria in the microenvironment. While in vivo models have been the choice for testing new anti-infectives in the context of cystic fibrosis, they still do not accurately mimic the in vivo conditions of such diseases in humans and the treatment outcomes. Complex in vitro models of the infected airways based on human cells (bronchial epithelial and macrophages) and relevant pathogens could bridge this gap and facilitate the translation of new anti-infectives into the clinic. For such purposes, a co-culture model of the human cystic fibrosis bronchial epithelial cell line CFBE41o- and THP-1 monocyte-derived macrophages has been established, mimicking an infection of the human bronchial mucosa by P. aeruginosa at air-liquid interface (ALI) conditions. This model is set up in seven days, and the following parameters are simultaneously assessed: epithelial barrier integrity, macrophage transmigration, bacterial survival, and inflammation. The present protocol describes a robust and reproducible system for evaluating drug efficacy and host responses that could be relevant for discovering new anti-infectives and optimizing their aerosol delivery to the lungs.


Assuntos
Ar , Anti-Infecciosos/farmacologia , Brônquios/patologia , Técnicas de Cocultura , Células Epiteliais/microbiologia , Macrófagos/microbiologia , Pseudomonas aeruginosa/fisiologia , Morte Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Contagem de Colônia Microbiana , Citocinas/metabolismo , Impedância Elétrica , Células Epiteliais/efeitos dos fármacos , Proteínas de Fluorescência Verde/metabolismo , Humanos , Cinética , L-Lactato Desidrogenase/metabolismo , Macrófagos/efeitos dos fármacos , Permeabilidade , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/crescimento & desenvolvimento , Células THP-1 , Tobramicina/farmacologia
14.
PLoS One ; 15(5): e0234013, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32470039

RESUMO

The healthy cornea is remarkably resistant to infection, quickly clearing deliberately inoculated bacteria such as Pseudomonas aeruginosa and Staphylococcus aureus. Contrasting with the adjacent conjunctiva and other body surfaces, it also lacks a resident viable bacterial microbiome. Corneal resistance to microbes depends on intrinsic defenses involving tear fluid and the corneal epithelium. Dry eye, an ocular surface disease associated with discomfort and inflammation, can alter tear fluid composition and volume, and impact epithelial integrity. We previously showed that experimentally-induced dry eye (EDE) in mice does not increase corneal susceptibility to P. aeruginosa infection. Here, we explored if EDE alters corneal resistance to bacterial colonization. EDE was established in mice using scopolamine injections and dehumidified air-flow, and verified by phenol-red thread testing after 5 and 10 days. As expected, EDE corneas showed increased fluorescein staining versus controls consistent with compromised epithelial barrier function. Confocal imaging using mT/mG knock-in mice with red-fluorescent membranes revealed no other obvious morphological differences between EDE corneas and controls for epithelium, stroma, and endothelium. EDE corneas were imaged ex vivo and compared to controls after alkyne-functionalized D-alanine labeling of metabolically-active colonizing bacteria, or by FISH using a universal 16S rRNA gene probe. Both methods revealed very few viable bacteria on EDE corneas after 5 or 10 days (median of 0, upper quartile of ≤ 1 bacteria per field of view for each group [9-12 eyes per group]) similar to control corneas. Furthermore, there was no obvious difference in abundance of conjunctival bacteria, which included previously reported filamentous forms. Thus, despite reduced tear flow and apparent compromise to corneal barrier function (fluorescein staining), EDE murine corneas continue to resist bacterial colonization and maintain the absence of a resident viable bacterial microbiome.


Assuntos
Córnea/microbiologia , Síndromes do Olho Seco/microbiologia , Infecções Oculares Bacterianas/microbiologia , Pseudomonas aeruginosa/crescimento & desenvolvimento , Animais , Contagem de Colônia Microbiana , Modelos Animais de Doenças , Feminino , Camundongos Endogâmicos C57BL
15.
PLoS One ; 15(4): e0231965, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32324772

RESUMO

Pseudomonas aeruginosa is a Gram-negative γ-proteobacterium that forms part of the normal human microbiota and it is also an opportunistic pathogen, responsible for 30% of all nosocomial urinary tract infections. P. aeruginosa carries a highly branched respiratory chain that allows the colonization of many environments, such as the urinary tract, catheters and other medical devices. P. aeruginosa respiratory chain contains three different NADH dehydrogenases (complex I, NQR and NDH-2), whose physiologic roles have not been elucidated, and up to five terminal oxidases: three cytochrome c oxidases (COx), a cytochrome bo3 oxidase (CYO) and a cyanide-insensitive cytochrome bd-like oxidase (CIO). In this work, we studied the composition of the respiratory chain of P. aeruginosa cells cultured in Luria Broth (LB) and modified artificial urine media (mAUM), to understand the metabolic adaptations of this microorganism to the growth in urine. Our results show that the COx oxidases play major roles in mAUM, while P. aeruginosa relies on CYO when growing in LB medium. Moreover, our data demonstrate that the proton-pumping NQR complex is the main NADH dehydrogenase in both LB and mAUM. This enzyme is resistant to HQNO, an inhibitory molecule produced by P. aeruginosa, and may provide an advantage against the natural antibacterial agents produced by this organism. This work offers a clear picture of the composition of this pathogen's aerobic respiratory chain and the main roles that NQR and terminal oxidases play in urine, which is essential to understand its physiology and could be used to develop new antibiotics against this notorious multidrug-resistant microorganism.


Assuntos
Materiais Biomiméticos , Meios de Cultura , Oxirredutases/metabolismo , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/metabolismo , Urina , Aerobiose , Transporte de Elétrons , NADH Desidrogenase/metabolismo , Quinonas/metabolismo
16.
Biofouling ; 36(2): 234-244, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32321306

RESUMO

This study evaluated adhesion and biofilm formation by Candida albicans, Pseudomonas aeruginosa and Staphylococcus epidermidis on surfaces of titanium (Ti) and titanium coated with F18 Bioactive Glass (BGF18). Biofilms were grown and the areas coated with biofilm were determined after 2, 4 and 8 h. Microscopy techniques were applied in order to visualize the structure of the mature biofilm and the extracellular matrix. On the BGF18 specimens, there was less biofilm formation by C. albicans and S. epidermidis after incubation for 8 h. For P. aeruginosa biofilm, a reduction was observed after incubation for 4 h, and it remained reduced after 8 h on BGF18 specimens. All biofilm matrices seemed to be thicker on BGF18 surface than on titanium surfaces. BGF18 showed significant anti-biofilm activity in comparison with Ti in the initial periods of biofilm formation; however, there was extensive biofilm after incubation for 48 h.


Assuntos
Materiais Biocompatíveis/química , Biofilmes/crescimento & desenvolvimento , Vidro/química , Próteses e Implantes/microbiologia , Titânio/química , Candida albicans/crescimento & desenvolvimento , Pseudomonas aeruginosa/crescimento & desenvolvimento , Staphylococcus epidermidis/crescimento & desenvolvimento , Propriedades de Superfície
18.
Sci Rep ; 10(1): 4325, 2020 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-32152407

RESUMO

Aerosol inhalation is a promising strategy for the delivery of antibiotic agents. The efficacy of antibiotic treatment by aerosol inhalation is reduced by the formation of microbial biofilms in the respiratory system and excessive airway mucus build-up. Various approaches have been taken in order to overcome this barrier. In this in vitro study, we used hypertonic saline (7%, by weight), a low cost Food and Drug Administration-approved reagent, as an aerosol carrier to study its effects with the antibiotic, gentamicin, on the most common respiratory opportunistic pathogen, Pseudomonas aeruginosa, present in the mucus. The results indicated that the hypertonic saline aerosol containing gentamicin, a low cost antibiotic, significantly eliminated biofilm growth by ~3-fold, compared to the regular saline aerosol containing gentamicin. In addition to enhancing the penetration efficiency of drug molecules by 70%, bacterial motility also decreased (~50%) after treatment with aerosolised hypertonic saline. In conclusion, our results demonstrate that hypertonic saline can significantly enhance the efficacy of antibiotic aerosols, which may contribute to the current use of inhaled therapeutic compounds.


Assuntos
Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Gentamicinas/farmacologia , Muco/efeitos dos fármacos , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/efeitos dos fármacos , Solução Salina Hipertônica/farmacologia , Biofilmes/crescimento & desenvolvimento , Humanos , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/crescimento & desenvolvimento
19.
Klin Lab Diagn ; 65(1): 37-41, 2020.
Artigo em Russo | MEDLINE | ID: mdl-32155005

RESUMO

Due to the spreading highly resistant strains among clinically significant P. aeruginosa clones, it becomes necessary to prescribe antibiotics not only taking into account the knowledge of sensitivity spectrum of a particular isolate but the data of microorganism biofilm activity as well. To study the dependence of biofilm-forming ability on the sensitivity to antibacterial preparations of P. aeruginosa clinical strains, isolated from patients with chronic osteomyelitis. 36 patients above 18 with chronic osteomyelitis of long tubular bones who were treated in the center of purulent osteology took part in the experiment. Object of the study - material isolated from wounds, fistulas, as well as from inflammatory foci. The sensitivity of isolated microorganisms to 10 antibiotics was analyzed: Piperacillin/Tazobactam, Imipenem, Meropenem, Aztreonam, Amikacin, Ciprofloxacin, Ceftriaxone, Ceftazidime, Cefotaxime, Cefepime. High- and medium-adhesive strains accounted for 86,1 % among P. aeruginosa clinical isolates, obtained from the wounds of patients with chronic osteomyelitis of long tubular bones. Highly adhesive strains are resistant to a wide range of antibacterial preparations used clinically. Penicillins were the most effective preparations when analyzing antibioticograms obtained for highly adhesive strains, for medium adhesive strains - penicillins, aminoglycosides and carbapenems, for low adhesive ones - aminoglycosides, penicillins, carbapenems, monobactams, quinolones. P. aeruginosa multi-resistance is a serious problem in the treatment of patients with chronic osteomyelitis. Spreading antibiotic-resistant strains of P. aeruginosa is associated with the presence of bacteria in the biofilm. Since adhesion is the first step in the biofilm formation, it is important to identify strains having high adhesive ability timely.


Assuntos
Antibacterianos/farmacologia , Biofilmes , Osteomielite/microbiologia , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/crescimento & desenvolvimento , Carbapenêmicos , Farmacorresistência Bacteriana Múltipla , Humanos , Testes de Sensibilidade Microbiana
20.
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
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