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1.
Microbiol Spectr ; 9(1): e0102321, 2021 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-34431721

RESUMO

The production of capsular polysaccharides by Klebsiella pneumoniae protects the bacterial cell from harmful environmental factors such as antimicrobial compounds and infection by bacteriophages (phages). To bypass this protective barrier, some phages encode polysaccharide-degrading enzymes referred to as depolymerases to provide access to cell surface receptors. Here, we characterized the phage RAD2, which infects K. pneumoniae strains that produce the widespread, hypervirulence-associated K2-type capsular polysaccharide. Using transposon-directed insertion sequencing, we have shown that the production of capsule is an absolute requirement for efficient RAD2 infection by serving as a first-stage receptor. We have identified the depolymerase responsible for recognition and degradation of the capsule, determined that the depolymerase forms globular appendages on the phage virion tail tip, and present the cryo-electron microscopy structure of the RAD2 capsule depolymerase at 2.7-Å resolution. A putative active site for the enzyme was identified, comprising clustered negatively charged residues that could facilitate the hydrolysis of target polysaccharides. Enzymatic assays coupled with mass spectrometric analyses of digested oligosaccharide products provided further mechanistic insight into the hydrolase activity of the enzyme, which, when incubated with K. pneumoniae, removes the capsule and sensitizes the cells to serum-induced killing. Overall, these findings expand our understanding of how phages target the Klebsiella capsule for infection, providing a framework for the use of depolymerases as antivirulence agents against this medically important pathogen. IMPORTANCE Klebsiella pneumoniae is a medically important pathogen that produces a thick protective capsule that is essential for pathogenicity. Phages are natural predators of bacteria, and many encode diverse "capsule depolymerases" which specifically degrade the capsule of their hosts, an exploitable trait for potential therapies. We have determined the first structure of a depolymerase that targets the clinically relevant K2 capsule and have identified its putative active site, providing hints to its mechanism of action. We also show that Klebsiella cells treated with a recombinant form of the depolymerase are stripped of capsule, inhibiting their ability to grow in the presence of serum, demonstrating the anti-infective potential of these robust and readily producible enzymes against encapsulated bacterial pathogens such as K. pneumoniae.


Assuntos
Cápsulas Bacterianas/virologia , Bacteriófagos/enzimologia , Klebsiella pneumoniae/virologia , Polissacarídeo-Liases/metabolismo , Proteínas Virais/metabolismo , Cápsulas Bacterianas/metabolismo , Cápsulas Bacterianas/ultraestrutura , Bacteriófagos/genética , Bacteriófagos/fisiologia , Microscopia Crioeletrônica , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/metabolismo , Klebsiella pneumoniae/ultraestrutura , Polissacarídeo-Liases/genética , Proteínas Virais/genética
2.
Protein Sci ; 30(5): 1035-1043, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33763934

RESUMO

Bacterial microcompartments (BMCs) are bacterial organelles involved in enzymatic processes, such as carbon fixation, choline, ethanolamine and propanediol degradation, and others. Formed of a semi-permeable protein shell and an enzymatic core, they can enhance enzyme performance and protect the cell from harmful intermediates. With the ability to encapsulate non-native enzymes, BMCs show high potential for applied use. For this goal, a detailed look into shell form variability is significant to predict shell adaptability. Here we present four novel 3D cryo-EM maps of recombinant Klebsiella pneumoniae GRM2 BMC shell particles with the resolution in range of 9 to 22 Å and nine novel 2D classes corresponding to discrete BMC shell forms. These structures reveal icosahedral, elongated, oblate, multi-layered and polyhedral traits of BMCs, indicating considerable variation in size and form as well as adaptability during shell formation processes.


Assuntos
Proteínas de Bactérias/química , Klebsiella pneumoniae/química , Klebsiella pneumoniae/ultraestrutura , Proteínas de Bactérias/metabolismo , Microscopia Crioeletrônica , Klebsiella pneumoniae/metabolismo
3.
Nat Biomed Eng ; 5(6): 613-623, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33707779

RESUMO

The de novo design of antimicrobial therapeutics involves the exploration of a vast chemical repertoire to find compounds with broad-spectrum potency and low toxicity. Here, we report an efficient computational method for the generation of antimicrobials with desired attributes. The method leverages guidance from classifiers trained on an informative latent space of molecules modelled using a deep generative autoencoder, and screens the generated molecules using deep-learning classifiers as well as physicochemical features derived from high-throughput molecular dynamics simulations. Within 48 days, we identified, synthesized and experimentally tested 20 candidate antimicrobial peptides, of which two displayed high potency against diverse Gram-positive and Gram-negative pathogens (including multidrug-resistant Klebsiella pneumoniae) and a low propensity to induce drug resistance in Escherichia coli. Both peptides have low toxicity, as validated in vitro and in mice. We also show using live-cell confocal imaging that the bactericidal mode of action of the peptides involves the formation of membrane pores. The combination of deep learning and molecular dynamics may accelerate the discovery of potent and selective broad-spectrum antimicrobials.


Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Aprendizado Profundo , Desenho de Fármacos , Descoberta de Drogas/métodos , Farmacorresistência Bacteriana/efeitos dos fármacos , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/crescimento & desenvolvimento , Acinetobacter baumannii/ultraestrutura , Sequência de Aminoácidos , Animais , Antibacterianos/síntese química , Peptídeos Catiônicos Antimicrobianos/síntese química , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/ultraestrutura , Feminino , Infecções por Klebsiella/tratamento farmacológico , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/crescimento & desenvolvimento , Klebsiella pneumoniae/ultraestrutura , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Simulação de Dinâmica Molecular , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/ultraestrutura , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus aureus/ultraestrutura , Relação Estrutura-Atividade
4.
Microb Pathog ; 143: 104120, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32169488

RESUMO

Rapid dissemination of carbapenem resistant Enterobacteriaceae (CRE) is considered to be a global issue. Quercetin is a well known antimicrobial agent. Hence, it would be important to investigate bactericidal and synergistic interaction of quercetin with meropenem and elucidate effects of quercetin alone and quercetin-meropenem combination on blaNDM, blaVIM,acrB, ompC, ompF, ompk35 and ompk36 expressions, cellular morphology and cell-wall/membrane integrity. MIC, Time-kill and Baclight assays were performed to determine antibacterial/bactericidal activity of quercetin. Synergism with meropenem was evaluated by checkerboard assay followed by dose-response, isobologram analysis and FIC index, combination index calculation. Effects of meropenem, quercetin and their combinations on blaNDM, blaVIM,acrB, ompC, ompF, ompk35 and ompk36 expressions were evaluated by qRT-PCR. SEM was performed to evaluate effects of aforesaid combinations on cellular morphology. Quercetin alone exhibited at least four-fold reduced MIC value (16-256 µg/mL) than that of meropenem against CRE. It exhibited synergism with meropenem against 89.25% CRE. Again, only 128 µg/mL quercetin killed upto 99.95% bacteria within 4-6 h of dosing, which increased further to 99.99% in MIC combination of meropenem-quercetin. Thus, effective bactericidal activity of quercetin-meropenem combination might have been achieved through alteration of blaVIM, ompC expression and cellular morphology of bacteria. Quercetin exhibited bactericidal and synergistic activity with meropenem.


Assuntos
Antibacterianos/farmacologia , Enterobacteriáceas Resistentes a Carbapenêmicos/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Klebsiella pneumoniae/efeitos dos fármacos , Meropeném/farmacologia , Quercetina/farmacologia , Antibacterianos/administração & dosagem , Sinergismo Farmacológico , Escherichia coli/ultraestrutura , Klebsiella pneumoniae/ultraestrutura , Meropeném/administração & dosagem , Testes de Sensibilidade Microbiana , Microscopia Eletrônica de Varredura , Quercetina/administração & dosagem
5.
Bull Exp Biol Med ; 168(4): 488-491, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32146628

RESUMO

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 Superfície
6.
Int J Mol Sci ; 21(2)2020 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-31936552

RESUMO

The emergence of multi-drug-resistant bacteria represents a major public-health threat. Phages constitute a promising alternative to chemical antibiotics due to their high host specificity, abundance in nature, and evolvability. However, phage host specificity means that highly diverse bacterial species are particularly difficult to target for phage therapy. This is the case of Klebsiella pneumoniae, which presents a hypervariable extracellular matrix capsule exhibiting dozens of variants. Here, we report four novel phages infecting K. pneumoniae capsular type K22 which were isolated from environmental samples in Valencia, Spain. Full genome sequencing showed that these phages belong to the Podoviridae family and encode putative depolymerases that allow digestion of specific K22 K. pneumoniae capsules. Our results confirm the capsular type-specificity of K. pneumoniae phages, as indicated by their narrow infectivity in a panel of K. pneumoniae clinical isolates. Nonetheless, this work represents a step forward in the characterization of phage diversity, which may culminate in the future use of large panels of phages for typing and/or for combating multi-drug-resistant K. pneumoniae.


Assuntos
Bacteriófagos/isolamento & purificação , Klebsiella pneumoniae/virologia , Bacteriófagos/genética , Bacteriófagos/ultraestrutura , Genoma Viral , Especificidade de Hospedeiro , Humanos , Klebsiella pneumoniae/ultraestrutura , Funções Verossimilhança , Filogenia , Domínios Proteicos , Espanha , Proteínas Virais/química
7.
Mol Microbiol ; 113(5): 889-905, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31912541

RESUMO

Hypervirulent Klebsiella pneumoniae (hvKP) causes Klebsiella-induced liver abscess. Capsule is important for the pathogenesis of Klebsiella in systemic infection, but its role in gut colonisation is not well understood. By generating ΔwcaJ, Δwza and Δwzy capsule-null mutants in a prototypical K1 hypervirulent isolate, we show that inactivation of wza (capsule exportase) and wzy (capsule polymerase) confer cell envelope defects in addition to capsule loss, making them susceptible to bile salts and detergent stress. Bile salt resistance is restored when the initial glycosyltransferase wcaJ was inactivated together with wzy, indicating that build-up of capsule intermediates contribute to cell envelope defects. Mouse gut colonisation competition assays show that the capsule and its regulator RmpA were not required for hvKP to persist in the gut, although initial colonisation was decreased in the mutants. Both ΔrmpA and ΔwcaJ mutants gradually outcompeted the wild type in the gut, whereas Δwza and Δwzy mutants were less fit than wild type. Together, our results advise caution in using the right capsule-null mutant for determination of capsule's role in bacterial pathogenesis. With the use of ΔwcaJ mutant, we found that although the capsule is important for bacterial survival outside the gut environment, it imposes a fitness cost in the gut.


Assuntos
Cápsulas Bacterianas/genética , Proteínas de Bactérias/genética , Klebsiella pneumoniae/fisiologia , Klebsiella pneumoniae/patogenicidade , Virulência/genética , Animais , Aderência Bacteriana , Cápsulas Bacterianas/metabolismo , Proteínas de Bactérias/metabolismo , DNA Bacteriano , Feminino , Regulação Bacteriana da Expressão Gênica , Humanos , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Fagocitose , Células RAW 264.7 , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
8.
Nat Commun ; 11(1): 388, 2020 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-31959751

RESUMO

Bacterial microcompartments (BMCs) are prokaryotic organelles consisting of a protein shell and an encapsulated enzymatic core. BMCs are involved in several biochemical processes, such as choline, glycerol and ethanolamine degradation and carbon fixation. Since non-native enzymes can also be encapsulated in BMCs, an improved understanding of BMC shell assembly and encapsulation processes could be useful for synthetic biology applications. Here we report the isolation and recombinant expression of BMC structural genes from the Klebsiella pneumoniae GRM2 locus, the investigation of mechanisms behind encapsulation of the core enzymes, and the characterization of shell particles by cryo-EM. We conclude that the enzymatic core is encapsulated in a hierarchical manner and that the CutC choline lyase may play a secondary role as an adaptor protein. We also present a cryo-EM structure of a pT = 4 quasi-symmetric icosahedral shell particle at 3.3 Å resolution, and demonstrate variability among the minor shell forms.


Assuntos
Proteínas de Bactérias/metabolismo , Klebsiella pneumoniae/citologia , Liases/metabolismo , Organelas/ultraestrutura , Proteínas de Bactérias/genética , Colina/metabolismo , Microscopia Crioeletrônica , Loci Gênicos , Klebsiella pneumoniae/enzimologia , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/ultraestrutura , Liases/genética , Organelas/enzimologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Biologia Sintética
9.
Oral Dis ; 26(3): 670-676, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31869492

RESUMO

OBJECTIVES: To evaluate clinical and pathologically cases of respiratory scleroma diagnosed in a 30-year period in Guatemala. MATERIAL AND METHODS: Fifty-one cases of respiratory scleroma diagnosed from 1988 to 2018 in a single pathology service in Guatemala were confirmed using Warthin-Starry staining. Immunohistochemical reactions against CD68, LCA, CD20, CD3, and CD138 were performed to illustrate the inflammatory infiltrate. Scanning electron microscopy (SEM) was performed to illustrate bacteria morphology. RESULTS: All 51 cases affected patients from poor areas of Guatemala, particularly women (66.7%), with a mean age of 31 years (range 7-66 years). Nose was affected in most cases (96.1%). Other sites involved included pharynx, larynx, palate, maxillary sinuses, and upper lip. Depending on the stage, the disease manifested as ulcerations, nasal deformities, or laryngeal stenosis. Nasal obstruction, epistaxis, dysphonia, fetid discharge, and pain were the main symptoms. Mikulicz cells (CD68+) in a plasma cell-rich inflammatory background (CD138+, CD20+, CD3+/-) were the typical microscopic presentation. In SEM, each macrophagic vacuole contained few to dozens of Klebsiella rhinoscleromatis diplobacilli. Treatment consisted of long-term trimethoprim and sulfamethoxazole, with adequate control of disease. CONCLUSION: Respiratory scleroma is a rare infectious disease affecting the upper respiratory tract, in poor regions of the world, including Guatemala.


Assuntos
Doenças Respiratórias/diagnóstico , Doenças Respiratórias/microbiologia , Rinoscleroma/diagnóstico , Rinoscleroma/microbiologia , Adolescente , Adulto , Idoso , Criança , Feminino , Guatemala , Humanos , Klebsiella pneumoniae/isolamento & purificação , Klebsiella pneumoniae/ultraestrutura , Macrófagos/microbiologia , Microscopia Eletrônica de Varredura , Pessoa de Meia-Idade , Obstrução Nasal , Doenças Respiratórias/patologia , Rinoscleroma/patologia , Adulto Jovem
10.
ACS Nano ; 13(12): 14377-14387, 2019 12 24.
Artigo em Inglês | MEDLINE | ID: mdl-31815423

RESUMO

Multidrug resistance in pathogenic bacteria is an increasing problem in patient care and public health. Molecular nanomachines (MNMs) have the ability to open cell membranes using nanomechanical action. We hypothesized that MNMs could be used as antibacterial agents by drilling into bacterial cell walls and increasing susceptibility of drug-resistant bacteria to recently ineffective antibiotics. We exposed extensively drug-resistant Klebsiella pneumoniae to light-activated MNMs and found that MNMs increase the susceptibility to Meropenem. MNMs with Meropenem can effectively kill K. pneumoniae that are considered Meropenem-resistant. We examined the mechanisms of MNM action using permeability assays and transmission electron microscopy, finding that MNMs disrupt the cell wall of extensively drug-resistant K. pneumoniae, exposing the bacteria to Meropenem. These observations suggest that MNMs could be used to make conventional antibiotics more efficacious against multi-drug-resistant pathogens.


Assuntos
Parede Celular/efeitos dos fármacos , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Klebsiella pneumoniae/efeitos dos fármacos , Meropeném/farmacologia , Nanopartículas/toxicidade , Animais , Antibacterianos/farmacologia , Morte Celular/efeitos dos fármacos , Linhagem Celular , Klebsiella pneumoniae/efeitos da radiação , Klebsiella pneumoniae/ultraestrutura , Luz , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Meropeném/química , Camundongos , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Movimento
11.
Nat Commun ; 10(1): 5437, 2019 11 28.
Artigo em Inglês | MEDLINE | ID: mdl-31780649

RESUMO

Bacterial type II secretion systems (T2SSs) translocate virulence factors, toxins and enzymes across the cell outer membrane. Here we use negative stain and cryo-electron microscopy to reveal the core architecture of an assembled T2SS from the pathogen Klebsiella pneumoniae. We show that 7 proteins form a ~2.4 MDa complex that spans the cell envelope. The outer membrane complex includes the secretin PulD, with all domains modelled, and the pilotin PulS. The inner membrane assembly platform components PulC, PulE, PulL, PulM and PulN have a relative stoichiometric ratio of 2:1:1:1:1. The PulE ATPase, PulL and PulM combine to form a flexible hexameric hub. Symmetry mismatch between the outer membrane complex and assembly platform is overcome by PulC linkers spanning the periplasm, with PulC HR domains binding independently at the secretin base. Our results show that the T2SS has a highly dynamic modular architecture, with implication for pseudo-pilus assembly and substrate loading.


Assuntos
Klebsiella pneumoniae/ultraestrutura , Sistemas de Secreção Tipo II/ultraestrutura , Proteínas da Membrana Bacteriana Externa/ultraestrutura , Proteínas de Bactérias/ultraestrutura , Microscopia Crioeletrônica , Proteínas de Membrana/ultraestrutura , Microscopia Eletrônica , Coloração Negativa
12.
Anal Bioanal Chem ; 411(27): 7315-7325, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31637462

RESUMO

Biofilms are communities of bacteria living embedded in a highly hydrated matrix composed of polysaccharides, proteins, and extracellular DNA. This life style confers numerous advantages to bacteria including protection against external threats. However, they also contribute to increase bacterial resistance against antimicrobials, an issue particularly relevant in dangerous infections. Due to the complexity of the matrix, few information is present in the literature on details of its architecture including the spatial distribution of the macromolecular components which might give hints on the way the biofilm scaffold is built up by bacteria. In this study, we investigated the possibility to combine well-established microbiological procedures with advanced microscopies to get information on composition and distribution of the macromolecular components of biofilm matrices. To this, confocal microscopy, diffraction-limited infrared (IR) spectral imaging, and atomic force microscopy (AFM) were used to explore biofilm produced by a clinical strain of Klebsiella pneumoniae. IR imaging permitted to have clues on how the biofilm grows and spreads on surfaces, and the local distribution of the components within it. Through the analysis of the pure component spectra, it was possible to assess the chemical and structural composition of the saccaridic matrix, confirming the data obtained by NMR. It was also possible to follow the time course of biofilm from 6 up to 48 h when the biofilm grew into a 3-dimensional multi-layered structure, characteristic of colonies of bacteria linked together by a complex matrix. In addition, nanoFTIR and AFM investigations allowed the estimation of biofilm growth in the vertical direction and the morphological analysis of bacterial colonies at different time points and the evaluation of the chemical composition at the nanoscale.


Assuntos
Biofilmes/crescimento & desenvolvimento , Matriz Extracelular de Substâncias Poliméricas/metabolismo , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/fisiologia , Matriz Extracelular de Substâncias Poliméricas/química , Matriz Extracelular de Substâncias Poliméricas/ultraestrutura , Humanos , Klebsiella pneumoniae/química , Klebsiella pneumoniae/ultraestrutura , Microscopia de Força Atômica , Microscopia Confocal , Espectrofotometria Infravermelho
13.
Artigo em Inglês | MEDLINE | ID: mdl-31481437

RESUMO

We compared the efficacies of meropenem alone and in combination with colistin against two strains of extended-spectrum-ß-lactamase-producing Klebsiella pneumoniae, using an in vitro pharmacodynamic model that mimicked two different biofilm conditions. Meropenem monotherapy achieved remarkable efficacy (even a bactericidal effect) under all conditions, whereas colistin was almost inactive and resistance emerged. The addition of colistin to meropenem produced no relevant benefits, in contrast to experiences with other microorganisms.


Assuntos
Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Colistina/farmacologia , Klebsiella pneumoniae/efeitos dos fármacos , Meropeném/farmacologia , beta-Lactamases/metabolismo , Antibacterianos/administração & dosagem , Colistina/administração & dosagem , Farmacorresistência Bacteriana Múltipla , Quimioterapia Combinada , Klebsiella pneumoniae/enzimologia , Klebsiella pneumoniae/ultraestrutura , Meropeném/administração & dosagem , Microscopia Eletrônica de Varredura , Resistência beta-Lactâmica
14.
Microb Drug Resist ; 25(5): 631-638, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30614757

RESUMO

Benzalkonium chloride (BAC) is widely used as a disinfectant and preservative. This study investigated the effect on antimicrobial susceptibility and the cellular changes that occurred after exposure of Klebsiella pneumoniae clinical isolates to sublethal concentrations of BAC. Minimum inhibitory concentration and minimum bactericidal concentration of BAC were determined for the collected 50 K. pneumoniae clinical isolates by broth microdilution method, and the tested isolates were adapted to increasing sublethal concentrations of BAC. The effect of adaptation on MICs of the tested 16 antimicrobial agents, the cell ultrastructure, efflux, and membrane depolarization of the tested isolates were examined. Interestingly, most K. pneumoniae isolates that adapted to BAC showed increased antimicrobial resistance, various morphological and structural changes, increased membrane depolarization, and enhanced efflux activity. The findings of this study suggest that the extensive use of BAC at sublethal concentrations could contribute to the emergence of antibiotic resistance in K. pneumoniae clinical isolates that might complicate the therapy of infections caused by this pathogen. In conclusion, the hazard associated with the prolonged exposure to sublethal concentrations of BAC represents a public health risk and therefore it should be a focus in both hospital and community sanitation practices.


Assuntos
Antibacterianos/farmacologia , Anti-Infecciosos Locais/farmacologia , Compostos de Benzalcônio/farmacologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Genes MDR/efeitos dos fármacos , Klebsiella pneumoniae/efeitos dos fármacos , Aminoglicosídeos/farmacologia , Carbapenêmicos/farmacologia , Cefalosporinas/farmacologia , Farmacorresistência Bacteriana/genética , Fluoroquinolonas/farmacologia , Expressão Gênica , Humanos , Infecções por Klebsiella/tratamento farmacológico , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/isolamento & purificação , Klebsiella pneumoniae/ultraestrutura , Macrolídeos/farmacologia , Proteínas de Membrana Transportadoras/agonistas , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Testes de Sensibilidade Microbiana , Proteínas Associadas à Resistência a Múltiplos Medicamentos/agonistas , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Nitrobenzenos/farmacologia , Penicilinas/farmacologia , Tetraciclinas/farmacologia
15.
Microb Pathog ; 125: 325-335, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30243551

RESUMO

The aim of the current study is to identify bioactive compound from marine endophytic actinomycetes (MEA) isolated from Gulf of Mannar region, Southeast coast of India. Among the isolated actinomycetes, strain GRG 4 exhibited excellent ability to inhibit isolated colistin resistant (CR) Pseudomonas aeruginosa (P. aeruginosa) and Klebsiella pneumoniae (K. pneumoniae), which is a emerging threat to the world. The strain was identified as Streptomyces coeruleorubidus GRG 4 (KY457708), based on morphological, biochemical, phenotypic and genotypic characters. The bioactive metabolites present in the methanolic extract were partially purified by TLC and preparative HPLC. The active HPLC fraction 2 showed 15, 20 mm zone of inhibition against both CR P. aeruginosa and K. pneumoniae respectively. Analytical HPLC and FT-IR results of fraction 2 showed with carbonyl group. Both GC-MS and LC-MS results confirmed that the fraction 2 contained chemical constituents of Bis (2-Ethylhexyl) Phthalate (BEP). The compromised structure with loosely integrated and ruptured cell wall of BEP treated CR bacteria were observed by confocal laser scanning microscope (CLSM) and scanning electron microscope (SEM) at 75 µg/mL of minimum inhibitory concentration (MIC) dose. Further, cytotoxic effect of BEP against A549 human lung cancer cells revealed complete inhibition by cell proliferation and apoptosis was observed at 100 µg/mL in 24 h treatment. In addition, irreversible ROS dependent oxidative damage was clearly observed at the IC50 concentration of BEP. The toxicity of BEP was also studied against Vibrio fischeri (V. fischeri) and found to be highly toxic after 15 and 30 min of treatment. Based on the results it could be concluded that the identified compound BEP is a potent inhibitor for CR bacteria and A549 lung cancer cells.


Assuntos
Antibacterianos/farmacologia , Antineoplásicos/farmacologia , Produtos Biológicos/farmacologia , Streptomyces/química , Células A549 , Aliivibrio fischeri/efeitos dos fármacos , Antibacterianos/isolamento & purificação , Antineoplásicos/isolamento & purificação , Organismos Aquáticos/química , Produtos Biológicos/isolamento & purificação , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Cromatografia em Camada Fina , Endófitos/química , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/fisiologia , Humanos , Índia , Concentração Inibidora 50 , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/ultraestrutura , Espectrometria de Massas , Testes de Sensibilidade Microbiana , Microscopia Confocal , Microscopia Eletrônica de Varredura , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/ultraestrutura , Streptomyces/isolamento & purificação
16.
Acta Biomater ; 78: 78-88, 2018 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-30031912

RESUMO

Klebsiella pneumoniae (K. pneumoniae) is one of the most common pathogens in hospital-acquired infections. It is often resistant to multiple antibiotics (including carbapenems), and can cause severe pneumonia. In search of effective antimicrobials, we recently developed polyionenes that were demonstrated to be potent against a broad-spectrum of microbes in vitro. In this study, polyionenes containing rigid amide bonds were synthesized to treat multidrug-resistant (MDR) K. pneumoniae lung infection. The polyionene exhibited broad-spectrum activity against clinically-isolated MDR bacteria with low minimum inhibitory concentrations (MICs). It also demonstrated stronger antimicrobial activity against 20 clinical strains of K. pneumoniae and more rapid killing kinetics than imipenem and other commonly used antibiotics. Multiple treatments with imipenem and gentamycin led to drug resistance in K. pneumoniae, while repeated use of the polymer did not cause resistance development due to its membrane-disruption antimicrobial mechanism. Additionally, the polymer showed potent anti-biofilm activity. In a MDR K. pneumoniae lung infection mouse model, the polymer demonstrated lower effective dose than imipenem with negligible systemic toxicity. The polymer treatment significantly alleviated lung injury, markedly reduced K. pneumoniae counts in the blood and major organs, and decreased mortality. Given its potent in vivo antimicrobial activity, negligible toxicity and ability of mitigating resistance development, the polyionene may be used to treat MDR K. pneumoniae lung infection. STATEMENT OF SIGNIFICANCE: Klebsiella pneumoniae (K. pneumoniae) is one of the most common pathogens in hospital-acquired infections, is often resistant to multiple antibiotics including carbapenems and can cause severe pneumonia. In this study, we report synthesis of antimicrobial polymers (polyionenes) and their use as antimicrobial agents for treatment of K. pneumoniae-caused pneumonia. The polymers have broad spectrum antibacterial activity against clinically isolated MDR bacteria, and eliminate MDR K. pneumoniae more effectively and rapidly than clinically used antibiotics. The polymer treatment also provides higher survival rate and faster bacterial removal from the major organs and the blood than the antibiotics. Repeated use of the polymer does not lead to resistance development. More importantly, at the therapeutic dose, the polymer treatment does not cause acute toxicity. Given its in vivo efficacy and negligible toxicity, the polymer is a promising candidate for the treatment of MDR K. pneumoniae-caused pneumonia.


Assuntos
Antibacterianos/uso terapêutico , Farmacorresistência Bacteriana Múltipla , Infecções por Klebsiella/tratamento farmacológico , Klebsiella pneumoniae/fisiologia , Pneumonia/tratamento farmacológico , Polímeros/uso terapêutico , Animais , Antibacterianos/farmacologia , Antibacterianos/toxicidade , Biofilmes/efeitos dos fármacos , Modelos Animais de Doenças , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Feminino , Hemólise/efeitos dos fármacos , Rim/efeitos dos fármacos , Rim/fisiopatologia , Cinética , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/isolamento & purificação , Klebsiella pneumoniae/ultraestrutura , Fígado/efeitos dos fármacos , Fígado/fisiopatologia , Camundongos Endogâmicos ICR , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Pneumonia/microbiologia , Pneumonia/patologia , Polímeros/síntese química , Polímeros/toxicidade , Ratos , Testes de Toxicidade
17.
Nanotechnology ; 29(36): 365705, 2018 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-29889049

RESUMO

High-resolution single-cell imaging in their native or near-native state has received considerable interest for decades. In this research, we present an innovative approach that can be employed to study both morphological and nano-mechanical properties of hydrated single bacterial cells. The proposed strategy is to encapsulate wet cells with monolayer graphene with a newly developed water membrane approach, followed by imaging with both electron microscopy (EM) and atomic force microscopy (AFM). A computational framework was developed to provide additional insights, with the detailed nanoindentation process on graphene modelled based on the finite element method. The model was first validated by calibration with polymer materials of known properties, and the contribution of graphene was then studied and corrected to determine the actual moduli of the encapsulated hydrated sample. Application of the proposed approach was performed on hydrated bacterial cells (Klebsiella pneumoniae) to correlate the structural and mechanical information. EM and energy-dispersive x-ray spectroscopy imaging confirmed that the cells in their near-native stage can be studied inside the miniaturised environment enabled with graphene encapsulation. The actual moduli of the encapsulated hydrated cells were determined based on the developed computational model in parallel, with results comparable with those acquired with wet AFM. It is expected that the successful establishment of controlled graphene encapsulation offers a new route for probing liquid/live cells with scanning probe microscopy, as well as correlative imaging of hydrated samples for both biological and material sciences.


Assuntos
Grafite/química , Klebsiella pneumoniae/citologia , Nanopartículas/química , Simulação por Computador , Análise de Elementos Finitos , Klebsiella pneumoniae/ultraestrutura , Microscopia de Força Atômica , Nanopartículas/ultraestrutura
18.
J Biosci ; 42(4): 623-636, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29229880

RESUMO

In the last decade the detection of the resistance of bacteria to antibiotics treatment, developed by different kind of bacteria, is becoming a huge problem. We hereby present a different approach to the current problem of detection of bacteria resistance to antibiotics. Our aims were to use the atomic force microscopy (AFM) to investigate bacteria morphological changes in response to antibiotics treatment and explore the possibility of reducing the time required to obtain information on their resistance. In particular, we studied Klebsiella pneumoniae bacteria provided by the Lavagna Hospital ASL4 Liguria (Italy), where there are cases linked with antibiotics resistance of the Klebsiella pneumoniae. By comparing AFM images of bacteria strains treated with different antibiotics is possible to identify unambiguously the Klebsiella pneumoniae strains resistant to antibiotics. In fact, the analysis of the AFM images of the antibiotic-sensitive bacteria shows clearly the presence of morphological alterations in the cell wall. While in the case of the antibiotic-resistant bacteria morphological alterations are not present. This approach is based on an easy and potentially rapid AFM analysis.


Assuntos
Antibacterianos/farmacologia , Parede Celular/ultraestrutura , Farmacorresistência Bacteriana Múltipla , Klebsiella pneumoniae/ultraestrutura , Microscopia de Força Atômica/estatística & dados numéricos , Ceftazidima/farmacologia , Parede Celular/efeitos dos fármacos , Colistina/farmacologia , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/crescimento & desenvolvimento , Meropeném , Testes de Sensibilidade Microbiana , Tienamicinas/farmacologia
19.
Molecules ; 22(11)2017 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-29113046

RESUMO

Combinatory therapies have been commonly applied in the clinical setting to tackle multi-drug resistant bacterial infections and these have frequently proven to be effective. Specifically, combinatory therapies resulting in synergistic interactions between antibiotics and adjuvant have been the main focus due to their effectiveness, sidelining the effects of additivity, which also lowers the minimal effective dosage of either antimicrobial agent. Thus, this study was undertaken to look at the effects of additivity between essential oils and antibiotic, via the use of cinnamon bark essential oil (CBO) and meropenem as a model for additivity. Comparisons between synergistic and additive interaction of CBO were performed in terms of the ability of CBO to disrupt bacterial membrane, via zeta potential measurement, outer membrane permeability assay and scanning electron microscopy. It has been found that the additivity interaction between CBO and meropenem showed similar membrane disruption ability when compared to those synergistic combinations which was previously reported. Hence, results based on our studies strongly suggest that additive interaction acts on a par with synergistic interaction. Therefore, further investigation in additive interaction between antibiotics and adjuvant should be performed for a more in depth understanding of the mechanism and the impacts of such interaction.


Assuntos
Permeabilidade da Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Infecções por Klebsiella/tratamento farmacológico , Klebsiella pneumoniae/metabolismo , Óleos Voláteis/farmacologia , Tienamicinas/agonistas , Tienamicinas/farmacologia , Membrana Celular/ultraestrutura , Sinergismo Farmacológico , Quimioterapia Combinada/métodos , Infecções por Klebsiella/metabolismo , Klebsiella pneumoniae/ultraestrutura , Meropeném , Óleos Voláteis/química , Tienamicinas/química
20.
Enzyme Microb Technol ; 97: 114-121, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28010768

RESUMO

Silver nanoparticles are powerful antimicrobial agents. Here, the synthesis of silver chloride nanoparticles (AgCl-NPs) was consistently evidenced from a commercially valuable microalgae species, Chlorella vulgaris. Incubation of C. vulgaris conditioned medium with AgNO3 resulted in a medium color change to yellow/brown (with UV-vis absorbance at 415nm), indicative of silver nanoparticle formation. Energy-dispersive X-ray spectroscopy (EDS) of purified nanoparticles confirmed the presence of both silver and chlorine atoms, and X-ray diffraction (XRD) showed the typical pattern of cubic crystalline AgCl-NPs. Transmission electron microscopy (TEM) showed that most particles (65%) were spherical, with average diameter of 9.8±5.7nm. Fourier transform infrared spectroscopy (FTIR) of purified nanoparticle fractions suggested that proteins are the main molecular entities involved in AgCl-NP formation and stabilization. AgCl-NPs (from 10µg/mL) decreased by 98% the growth of Gram-positive Staphylococcus aureus and Gram-negative Klebsiella pneumoniae bacterial pathogens, and had a dose-dependent effect on cell viability, which was measured by automated image-based high content screening (HCS). Ultrastructural analysis of treated bacteria by TEM revealed the abnormal arrangement of the chromosomal DNA. Our findings strongly indicated that the AgCl-NPs from C. vulgaris conditioned medium is a promising 'green' alternative for biomedical application as antimicrobials.


Assuntos
Anti-Infecciosos/metabolismo , Anti-Infecciosos/farmacologia , Nanopartículas Metálicas/química , Compostos de Prata/química , Chlorella vulgaris/metabolismo , Química Verde , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/ultraestrutura , Nanopartículas Metálicas/ultraestrutura , Microalgas/metabolismo , Nanotecnologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/ultraestrutura
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