RESUMO
The current article aimed to evaluate the in vitro mutagenicity of ten fried meat-based food extracts obtained from different catering companies from Navarra (Spain). A miniaturized 6-well version of the Ames test in Salmonella typhimurium TA98, and the in vitro micronucleus test (OECD TG 487) in TK6 cells were performed. None of the ten extracts of fried meat-based food induced gene mutations in S. typhimurium TA98 with or without metabolic activation, but five induced chromosomal aberrations after 24 h treatment of TK6 without metabolic activation. More studies are needed to check the biological relevance of these in vitro studies.
Assuntos
Culinária , Mutagênicos/química , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Linhagem Celular , Aberrações Cromossômicas/induzido quimicamente , Cromossomos Bacterianos/efeitos dos fármacos , Comércio , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Humanos , Carne/análise , Testes para Micronúcleos , Mutagênese , Testes de Mutagenicidade , Salmonella typhimurium/efeitos dos fármacosRESUMO
We have extensively characterized base substitution mutations in the 795 base pair (bp) long E. coli thyA gene to define as many of the base substitution mutational sites that inactivate the gene as possible. The resulting catalog of mutational sites constitutes a system with up to 5 times as many sites for monitoring each of the six base substitution mutations as the widely used rpoB/Rifr system. We have defined 75 sites for the G:C -> A:T transition, 68 sites for the G:C -> T:A transversion, 53 sites for the G:C -> C:G transversion, 49 sites for the A:T -> G:C transition, 39 sites for the A:T -> T:A transversion, and 59 sites for the A:T -> C:G transversion. The system is thus comprised of 343 base substitution mutations at 232 different base pairs, all of which can be sequenced with a single primer pair. This allows for the examination of mutational spectra using a more detailed probe of known mutations, while still allowing one to compare the number of repeated occurrences at specific sites. We have examined several mutagens and mutators with this system, and show its utility by looking at the spectrum of cisplatin, that has a single hotspot, underscoring the value of having as large an array of sites as possible at which one can monitor repeat occurrences. To test for regions of the gene that might be hotspots for a number of mutagens, or "hot" (mutaphilic) regions, we have looked at the ratio of mutations per set of an equal number of mutational sites throughout the gene. The resulting graphs suggest that there are "hot" regions at intervals, and this may reflect aspects of secondary structures, of the higher order structure of the chromosome, or perhaps the nucleoid structure of the chromosome plus histone-like protein complexes.
Assuntos
Cromossomos Bacterianos/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Genes Bacterianos , Mutagênicos/farmacologia , Mutação , 2-Aminopurina/farmacologia , 4-Nitroquinolina-1-Óxido/farmacologia , Azacitidina/farmacologia , Sequência de Bases , Bromodesoxiuridina/farmacologia , Cisplatino/farmacologia , Códon , Primers do DNA/genética , Primers do DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Metanossulfonato de Etila/farmacologia , Código Genético , Sequenciamento de Nucleotídeos em Larga Escala , MutagêneseRESUMO
Hydrogen peroxide (H2O2, HP) is a universal toxin that organisms deploy to kill competing or invading cells. Bactericidal action of H2O2 presents several questions. First, the lethal H2O2 concentrations in bacterial cultures are 1000x higher than, for example, those calculated for the phagosome. Second, H2O2-alone kills bacteria in cultures either by mode-one, via iron-mediated chromosomal damage, or by mode-two, via unknown targets, but the killing mode in phagosomes is unclear. Third, phagosomal H2O2 toxicity is enhanced by production of nitric oxide (NO), but in vitro studies disagree: some show NO synergy with H2O2 antimicrobial action, others instead report alleviation. To investigate this "NO paradox," we treated Escherichia coli with various concentrations of H2O2-alone or H2O2+NO, measuring survival and chromosome stability. We found that all NO concentrations make sublethal H2O2 treatments highly lethal, via triggering catastrophic chromosome fragmentation (mode-one killing). Yet, NO-alone is not lethal, potentiating H2O2 toxicity by blocking H2O2 scavenging in cultures. Catalases represent obvious targets of NO inhibition, and catalase-deficient mutants are indeed killed equally by H2O2-alone or H2O2+NO treatments, also showing similar levels of chromosome fragmentation. Interestingly, iron chelation blocks chromosome fragmentation in catalase-deficient mutants without blocking H2O2-alone lethality, indicating mode-two killing. In fact, mode-two killing of WT cells by much higher H2O2 concentrations is transiently alleviated by NO, reproducing the "NO paradox." We conclude that NO potentiates H2O2 toxicity by promoting mode-one killing (via catastrophic chromosome fragmentation) by otherwise static low H2O2 concentrations, while transiently suppressing mode-two killing by immediately lethal high H2O2 concentrations.
Assuntos
Catalase/antagonistas & inibidores , Cromossomos Bacterianos/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Óxido Nítrico/farmacologia , Animais , Catalase/metabolismo , Bovinos , Cromossomos Bacterianos/genética , Fragmentação do DNA/efeitos dos fármacos , Reparo do DNA , Sinergismo Farmacológico , Escherichia coli/genética , Testes de Sensibilidade Microbiana , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genéticaRESUMO
The heat-stable nucleoid-structuring (H-NS) protein is a global transcriptional regulator implicated in coordinating the expression of over 200 genes in Escherichia coli, including many involved in adaptation to osmotic stress. We have applied superresolved microscopy to quantify the intracellular and spatial reorganization of H-NS in response to a rapid osmotic shift. We found that H-NS showed growth phase-dependent relocalization in response to hyperosmotic shock. In stationary phase, H-NS detached from a tightly compacted bacterial chromosome and was excluded from the nucleoid volume over an extended period of time. This behavior was absent during rapid growth but was induced by exposing the osmotically stressed culture to a DNA gyrase inhibitor, coumermycin. This chromosomal compaction/H-NS exclusion phenomenon occurred in the presence of either potassium or sodium ions and was independent of the presence of stress-responsive sigma factor σS and of the H-NS paralog StpA.IMPORTANCE The heat-stable nucleoid-structuring (H-NS) protein coordinates the expression of over 200 genes in E. coli, with a large number involved in both bacterial virulence and drug resistance. We report on the novel observation of a dynamic compaction of the bacterial chromosome in response to exposure to high levels of salt. This stress response results in the detachment of H-NS proteins and their subsequent expulsion to the periphery of the cells. We found that this behavior is related to mechanical properties of the bacterial chromosome, in particular, to how tightly twisted and coiled is the chromosomal DNA. This behavior might act as a biomechanical response to stress that coordinates the expression of genes involved in adapting bacteria to a salty environment.
Assuntos
Cromossomos Bacterianos/efeitos dos fármacos , Proteínas de Escherichia coli/genética , Escherichia coli/efeitos dos fármacos , Proteínas de Fímbrias/genética , Regulação Bacteriana da Expressão Gênica , Pressão Osmótica , Cloreto de Potássio/farmacologia , Adaptação Fisiológica , Aminocumarinas/farmacologia , Cátions Monovalentes , Cromossomos Bacterianos/metabolismo , Cromossomos Bacterianos/ultraestrutura , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/ultraestrutura , Proteínas de Escherichia coli/metabolismo , Proteínas de Fímbrias/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Potássio/metabolismo , Transporte Proteico/efeitos dos fármacos , Fator sigma/genética , Fator sigma/metabolismo , Sódio/metabolismo , Inibidores da Topoisomerase II/farmacologia , Transcrição GênicaRESUMO
Spreading resistance to antibiotics and the emergence of multidrug-resistant strains have become frequent in many bacterial species, including mycobacteria, which are the causative agents of severe diseases and which have profound impacts on global health. Here, we used a system of microfluidics, fluorescence microscopy, and target-tagged fluorescent reporter strains of Mycobacterium smegmatis to perform real-time monitoring of replisome and chromosome dynamics following the addition of replication-altering drugs (novobiocin, nalidixic acid, and griselimycin) at the single-cell level. We found that novobiocin stalled replication forks and caused relaxation of the nucleoid and that nalidixic acid triggered rapid replisome collapse and compaction of the nucleoid, while griselimycin caused replisome instability, with the subsequent overinitiation of chromosome replication and overrelaxation of the nucleoid. In addition to study target-drug interactions, our system also enabled us to observe how the tested antibiotics affected the physiology of mycobacterial cells (i.e., growth, chromosome segregation, etc.).
Assuntos
Anti-Infecciosos/farmacologia , Replicação do DNA/efeitos dos fármacos , Mycobacterium smegmatis/efeitos dos fármacos , Mycobacterium smegmatis/genética , Inibidores da Síntese de Ácido Nucleico/farmacologia , Proteínas de Bactérias/genética , Segregação de Cromossomos/efeitos dos fármacos , Segregação de Cromossomos/genética , Cromossomos Bacterianos/efeitos dos fármacos , Cromossomos Bacterianos/genética , Avaliação de Medicamentos/métodos , Microfluídica/métodos , Microscopia de Fluorescência/métodos , Imagem com Lapso de Tempo/métodosRESUMO
In Escherichia coli, an increase in the frequency of chromosome replication is lethal. In order to identify compounds that affect chromosome replication, we screened for molecules capable of restoring the viability of hyper-replicating cells. We made use of two E. coli strains that over-initiate DNA replication by keeping the DnaA initiator protein in its active ATP bound state. While viable under anaerobic growth or when grown on poor media, these strains become inviable when grown in rich media. Extracts from actinomycetes strains were screened, leading to the identification of deferoxamine (DFO) as the active compound in one of them. We show that DFO does not affect chromosomal replication initiation and suggest that it was identified due to its ability to chelate cellular iron. This limits the formation of reactive oxygen species, reduce oxidative DNA damage and promote processivity of DNA replication. We argue that the benzazepine derivate (±)-6-Chloro-PB hydrobromide acts in a similar manner.
Assuntos
Replicação do DNA/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Quelantes de Ferro/farmacologia , Proteínas de Bactérias/metabolismo , Cromossomos Bacterianos/efeitos dos fármacos , Cromossomos Bacterianos/genética , Replicação do DNA/genética , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , Desferroxamina/farmacologia , Escherichia coli/genética , Escherichia coli/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Inibidores da Topoisomerase II/farmacologiaRESUMO
Bacteriophage-encoded genetic elements control bacterial biological functions. Enterohemorrhagic Escherichia coli (EHEC) strains harbor lambda-phages encoding the Shiga-toxin (Stx), which is expressed during the phage lytic cycle and associated with exacerbated disease. Phages also reside dormant within bacterial chromosomes through their lysogenic cycle, but how this impacts EHEC virulence remains unknown. We find that during lysogeny the phage transcription factor Cro activates the EHEC type III secretion system (T3SS). EHEC lambdoid phages are lysogenic under anaerobic conditions when Cro binds to and activates the promoters of T3SS genes. Interestingly, the Cro sequence varies among phages carried by different EHEC outbreak strains, and these changes affect Cro-dependent T3SS regulation. Additionally, infecting mice with the related pathogen C. rodentium harboring the bacteriophage cro from EHEC results in greater T3SS gene expression and enhanced virulence. Collectively, these findings reveal the role of phages in impacting EHEC virulence and their potential to affect outbreak strains.
Assuntos
Colífagos/metabolismo , Escherichia coli Êntero-Hemorrágica/efeitos dos fármacos , Escherichia coli Êntero-Hemorrágica/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Proteínas Repressoras/farmacologia , Proteínas Virais Reguladoras e Acessórias/farmacologia , Fatores de Virulência/genética , Animais , Cromossomos Bacterianos/efeitos dos fármacos , Citrobacter rodentium/patogenicidade , Colífagos/genética , Modelos Animais de Doenças , Infecções por Enterobacteriaceae/metabolismo , Infecções por Enterobacteriaceae/patologia , Escherichia coli Êntero-Hemorrágica/patogenicidade , Escherichia coli Êntero-Hemorrágica/virologia , Escherichia coli/genética , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/genética , Genes Bacterianos/efeitos dos fármacos , Células HeLa , Humanos , Lipídeos , Lisogenia , Camundongos , Camundongos Endogâmicos C3H , Proteínas Repressoras/genética , Toxina Shiga/genética , Fatores de Transcrição , Sistemas de Secreção Tipo III/efeitos dos fármacos , Sistemas de Secreção Tipo III/genética , Sistemas de Secreção Tipo III/metabolismo , Ensaio de Placa Viral , Proteínas Virais Reguladoras e Acessórias/genética , Virulência/efeitos dos fármacos , Virulência/genéticaRESUMO
Staphylococcus aureus isolates resistant to several antimicrobials have been gradually emerged since the beginning of the antibiotic era. Consequently, the first isolation of methicillin-resistant S. aureus occurred in 1960, which was described a few years later in Chile. Currently, S. aureus resistant to antistaphylococcal penicillins is endemic in Chilean hospitals and worldwide, being responsible for a high burden of morbidity and mortality. This resistance is mediated by the expression of a new transpeptidase, named PBP2a or PBP2', which possesses lower affinity for the ß-lactam antibiotics, allowing the synthesis of peptidoglycan even in presence of these antimicrobial agents. This new enzyme is encoded by the mecA gene, itself embedded in a chromosomal cassette displaying a genomic island structure, of which there are several types and subtypes. Methicillin resistance is mainly regulated by an induction mechanism activated in the presence of ß-lactams, through a membrane receptor and a repressor of the gene expression. Although mec-independent methicillin resistance mechanisms have been described, they are clearly infrequent.
Assuntos
Proteínas de Bactérias/genética , Estruturas Genéticas/genética , Staphylococcus aureus Resistente à Meticilina/genética , Proteínas de Ligação às Penicilinas/genética , Antibacterianos/química , Antibacterianos/farmacologia , Proteínas de Bactérias/efeitos dos fármacos , Cromossomos Bacterianos/efeitos dos fármacos , Genes Bacterianos/efeitos dos fármacos , Meticilina/química , Meticilina/farmacologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Estrutura Molecular , Proteínas de Ligação às Penicilinas/efeitos dos fármacosRESUMO
Resumen Desde el inicio de la era antimicrobiana se han ido seleccionando gradualmente cepas de Staphylococcus aureus resistentes a antimicrobianos de amplio uso clínico. Es así como en 1960 se describen en Inglaterra las primeras cepas resistentes a meticilina, y algunos años después son informadas en hospitales de Chile. Actualmente, S. aureus resistente a penicilinas antiestafilocóccicas es endémico en los hospitales de nuestro país y del mundo, siendo responsable de una alta morbimortalidad. La resistencia es mediada habitualmente por la síntesis de una nueva transpeptidasa, denominada PBP2a o PBP2' que posee menos afinidad por el β-lactámico, y es la que mantiene la síntesis de peptidoglicano en presencia del antimicrobiano. Esta nueva enzima se encuentra codificada en el gen mecA, a su vez inserto en un cassette cromosomal con estructura de isla genómica, de los cuales existen varios tipos y subtipos. La resistencia a meticilina se encuentra regulada, principalmente, por un mecanismo de inducción de la expresión del gen en presencia del β-lactámico, a través de un receptor de membrana y un represor de la expresión. Si bien se han descrito mecanismos generadores de resistencia a meticilina mec independientes, son categóricamente menos frecuentes.
Staphylococcus aureus isolates resistant to several antimicrobials have been gradually emerged since the beginning of the antibiotic era. Consequently, the first isolation of methicillin-resistant S. aureus occurred in 1960, which was described a few years later in Chile. Currently, S. aureus resistant to antistaphylococcal penicillins is endemic in Chilean hospitals and worldwide, being responsible for a high burden of morbidity and mortality. This resistance is mediated by the expression of a new transpeptidase, named PBP2a or PBP2', which possesses lower affinity for the β-lactam antibiotics, allowing the synthesis of peptidoglycan even in presence of these antimicrobial agents. This new enzyme is encoded by the mecA gene, itself embedded in a chromosomal cassette displaying a genomic island structure, of which there are several types and subtypes. Methicillin resistance is mainly regulated by an induction mechanism activated in the presence of β-lactams, through a membrane receptor and a repressor of the gene expression. Although mec-independent methicillin resistance mechanisms have been described, they are clearly infrequent.
Assuntos
Proteínas de Bactérias/genética , Estruturas Genéticas/genética , Proteínas de Ligação às Penicilinas/genética , Staphylococcus aureus Resistente à Meticilina/genética , Proteínas de Bactérias/efeitos dos fármacos , Estrutura Molecular , Cromossomos Bacterianos/efeitos dos fármacos , Proteínas de Ligação às Penicilinas/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Genes Bacterianos/efeitos dos fármacos , Meticilina/farmacologia , Meticilina/química , Antibacterianos/farmacologia , Antibacterianos/químicaRESUMO
Mushrooms have long been used worldwide for culinary and medicinal purposes because of their various nutrients and active constituents. The safety of mushrooms as a culinary ingredient requires validation. Although Mycoleptodonoides aitchisonii has long been used for culinary purposes in East Asia, it has not been authorized by a regulatory agency. In this study we conducted genotoxicity and single-treatment toxicity tests according to the guidelines of the Organisation for Economic Co-operation and Development. We performed genotoxicity tests (bacterial reverse mutation study, chromosome aberration test, and micronucleus test), single-treatment toxicity tests, and in vivo mammalian alkaline comet assay of M. aitchisonii water extract (WT). A single treatment with 5000 mg/kg M. aitchisonii WT showed no toxicity. M. aitchisonii WT induced bacterial reverse mutation and chromosome aberration but showed a negative result in the micronucleus test. Thus, an in vivo mammalian alkaline comet assay was performed; however, no genotoxicity was detected. Treatment with <5000 mg/kg M. aitchisonii WT is nontoxic and can be used for culinary purposes.
Assuntos
Basidiomycota/química , Mutagênicos/toxicidade , Animais , Cromossomos Bacterianos/efeitos dos fármacos , Ensaio Cometa , Cricetulus , Escherichia coli/efeitos dos fármacos , Feminino , Masculino , Camundongos , Camundongos Endogâmicos ICR , Testes de Sensibilidade Microbiana , Testes para Micronúcleos , Ratos , Ratos Sprague-Dawley , Salmonella typhimurium/efeitos dos fármacosRESUMO
Nutrient starvation usually halts cell growth rather than causing death. Thymine starvation is exceptional, because it kills cells rapidly. This phenomenon, called thymineless death (TLD), underlies the action of several antibacterial, antimalarial, anticancer, and immunomodulatory agents. Many explanations for TLD have been advanced, with recent efforts focused on recombination proteins and replication origin (oriC) degradation. Because current proposals account for only part of TLD and because reactive oxygen species (ROS) are implicated in bacterial death due to other forms of harsh stress, we investigated the possible involvement of ROS in TLD. Here, we show that thymine starvation leads to accumulation of both single-stranded DNA regions and intracellular ROS, and interference with either event protects bacteria from double-stranded DNA breakage and TLD. Elevated levels of single-stranded DNA were necessary but insufficient for TLD, whereas reduction of ROS to background levels largely abolished TLD. We conclude that ROS contribute to TLD by converting single-stranded DNA lesions into double-stranded DNA breaks. Participation of ROS in the terminal phases of TLD provides a specific example of how ROS contribute to stress-mediated bacterial self-destruction.
Assuntos
Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Viabilidade Microbiana , Espécies Reativas de Oxigênio/metabolismo , Espécies Reativas de Oxigênio/toxicidade , Inanição , Timina/metabolismo , Cromossomos Bacterianos/efeitos dos fármacos , Cromossomos Bacterianos/metabolismo , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , DNA de Cadeia Simples/efeitos dos fármacos , DNA de Cadeia Simples/metabolismo , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Modelos Genéticos , RNA Bacteriano/efeitos dos fármacos , Recombinação Genética , Origem de Replicação/efeitos dos fármacosRESUMO
Condensation studies of chromosomal DNA in E. coli with a tetranuclear ruthenium complex are carried out and images obtained with wide-field fluorescence microscopy. Remarkably different condensate morphologies resulted, depending upon the treatment protocol. The occurrence of condensed nucleoid spirals in live bacteria provides evidence for the transertion hypothesis.
Assuntos
Cromossomos Bacterianos , DNA Bacteriano , Escherichia coli/genética , Cromossomos Bacterianos/efeitos dos fármacos , DNA Bacteriano/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Microscopia de Fluorescência , Compostos Organometálicos/química , Compostos Organometálicos/farmacologia , Ribossomos/metabolismo , Rutênio/químicaRESUMO
While the action of many antimicrobial drugs is well understood at the molecular level, a systems-level physiological response to antibiotics remains largely unexplored. This work considers fluctuation dynamics of both the chromosome and cytosol in Escherichia coli, and their response to sublethal treatments of a clinically important antibiotic, rifampicin. We precisely quantify the changes in dynamics of chromosomal loci and cytosolic aggregates (a rheovirus nonstructural protein known as µNS-GFP), measuring short time-scale displacements across several hours of drug exposure. To achieve this we develop an empirical method correcting for photo-bleaching and loci size effects. This procedure allows us to characterize the dynamic response to rifampicin in different growth conditions, including a customised microfluidic device. We find that sub-lethal doses of rifampicin cause a small but consistent increase in motility of both the chromosomal loci and cytosolic aggregates. Chromosomal and cytosolic responses are consistent with each other and between different growth conditions.
Assuntos
Antibacterianos/farmacologia , Infecções por Escherichia coli/tratamento farmacológico , Infecções por Escherichia coli/microbiologia , Escherichia coli/efeitos dos fármacos , Rifampina/farmacologia , Cromossomos Bacterianos/efeitos dos fármacos , Cromossomos Bacterianos/genética , Escherichia coli/citologia , Escherichia coli/genética , Genoma Bacteriano/efeitos dos fármacos , HumanosRESUMO
Iron-dependent oxidative DNA damage in vivo by hydrogen peroxide (H2O2, HP) induces copious single-strand(ss)-breaks and base modifications. HP also causes infrequent double-strand DNA breaks, whose relationship to the cell killing is unclear. Since hydrogen peroxide only fragments chromosomes in growing cells, these double-strand breaks were thought to represent replication forks collapsed at direct or excision ss-breaks and to be fully reparable. We have recently reported that hydrogen peroxide kills Escherichia coli by inducing catastrophic chromosome fragmentation, while cyanide (CN) potentiates both the killing and fragmentation. Remarkably, the extreme density of CN+HP-induced chromosomal double-strand breaks makes involvement of replication forks unlikely. Here we show that this massive fragmentation is further amplified by inactivation of ss-break repair or base-excision repair, suggesting that unrepaired primary DNA lesions are directly converted into double-strand breaks. Indeed, blocking DNA replication lowers CN+HP-induced fragmentation only â¼2-fold, without affecting the survival. Once cyanide is removed, recombinational repair in E. coli can mend several double-strand breaks, but cannot mend â¼100 breaks spread over the entire chromosome. Therefore, double-strand breaks induced by oxidative damage happen at the sites of unrepaired primary one-strand DNA lesions, are independent of replication and are highly lethal, supporting the model of clustered ss-breaks at the sites of stable DNA-iron complexes.
Assuntos
Cromossomos Bacterianos/genética , Fragmentação do DNA/efeitos dos fármacos , Reparo do DNA , Peróxido de Hidrogênio/farmacologia , Cromossomos Bacterianos/efeitos dos fármacos , Cianetos/farmacologia , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Desoxirribonuclease I/metabolismo , Interações Medicamentosas , Escherichia coli/citologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genéticaRESUMO
The aim of this study was to investigate the potential usefulness of DNA fragmentation as a quick and simple procedure for detecting resistance to fluoroquinolones (FQ) in isogenic Escherichia coli strains harboring defined and multiple quinolone resistance mechanisms, including low-level quinolone resistance (LLQR) phenotypes. DNA fragmentation assay (Micromax(®)) was evaluated for detecting resistance to FQ in 71 isogenic strains of E. coli harboring specific quinolone resistance mechanisms frequently found in clinical isolates. These isogenic strains represent a consistent and reliable model of increasing minimum inhibitory concentrations (MICs) of ciprofloxacin (CIP), ranging from 0.004 to 16 mg/L. According to CLSI criteria, the assay correctly identified all CIP-resistant strains (MIC ≥4 mg/L). As regards susceptible strains, 96% of bacterial strains were correctly assigned as susceptible to CIP. Moreover, the procedure enabled LLQR phenotypes to be efficiently identified; this subset may show different levels of DNA damage depending on the strain, even with similar MIC. Interestingly, despite increasing the dose according to the MIC, a lower response to quinolones occurs in strains with higher MIC values. This is a simple, rapid, and reliable test for evaluating susceptibility to FQ of E. coli, including the detection of strains harboring LLQR mechanisms.
Assuntos
Antibacterianos/farmacologia , Cromossomos Bacterianos/efeitos dos fármacos , Ciprofloxacina/farmacologia , Fragmentação do DNA/efeitos dos fármacos , Farmacorresistência Bacteriana/genética , Escherichia coli/efeitos dos fármacos , Cromossomos Bacterianos/química , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Infecções por Escherichia coli/microbiologia , Genótipo , Humanos , Testes de Sensibilidade Microbiana , Fenótipo , Sensibilidade e EspecificidadeRESUMO
Copper homeostasis in bacteria is challenged by periodic elevation of copper levels in the environment, arising from both natural sources and human inputs. Several mechanisms have evolved to efflux copper from bacterial cells, including thecus(copper sensing copper efflux system), andpco(plasmid-borne copper resistance system) systems. The genes belonging to these two systems can be physically clustered in a Copper Homeostasis and Silver Resistance Island (CHASRI) on both plasmids and chromosomes in Enterobacteria. Increasing use of copper in agricultural and industrial applications raises questions about the role of human activity in the evolution of novel copper resistance mechanisms. Here we present evidence that CHASRI emerged and diversified in response to copper deposition across aerobic and anaerobic environments. An analysis of diversification rates and a molecular clock model suggest that CHASRI experienced repeated episodes of elevated diversification that could correspond to peaks in human copper production. Phylogenetic analyses suggest that CHASRI originated in a relative ofEnterobacter cloacaeas the ultimate product of sequential assembly of several pre-existing two-gene modules. Once assembled, CHASRI dispersed via horizontal gene transfer within Enterobacteriaceae and also to certain members of Shewanellaceae, where the originalpcomodule was replaced by a divergentpcohomolog. Analyses of copper stress mitigation suggest that CHASRI confers increased resistance aerobically, anaerobically, and during shifts between aerobic and anaerobic environments, which could explain its persistence in facultative anaerobes and emergent enteric pathogens.
Assuntos
Cobre/toxicidade , Enterobacteriaceae/genética , Metais Pesados/toxicidade , Filogenia , Cromossomos Bacterianos/efeitos dos fármacos , Cromossomos Bacterianos/genética , Cobre/metabolismo , Enterobacteriaceae/efeitos dos fármacos , Enterobacteriaceae/metabolismo , Transferência Genética Horizontal , Homeostase/genética , Humanos , Metais Pesados/metabolismo , Plasmídeos/genética , Shewanella/genéticaRESUMO
Hydrogen peroxide (HP) or cyanide (CN) are bacteriostatic at low-millimolar concentrations for growing Escherichia coli, whereas CN + HP mixture is strongly bactericidal. We show that this synergistic toxicity is associated with catastrophic chromosomal fragmentation. Since CN alone does not kill at any concentration, while HP alone kills at 20 mM, CN must potentiate HP poisoning. The CN + HP killing is blocked by iron chelators, suggesting Fenton's reaction. Indeed, we show that CN enhances plasmid DNA relaxation due to Fenton's reaction in vitro. However, mutants with elevated iron or HP pools are not acutely sensitive to HP-alone treatment, suggesting that, in addition, in vivoâ CN recruits iron from intracellular depots. We found that part of the CN-recruited iron pool is managed by ferritin and Dps: ferritin releases iron on cue from CN, while Dps sequesters it, quelling Fenton's reaction. We propose that disrupting intracellular iron trafficking is a common strategy employed by the immune system to kill microbes.
Assuntos
Cromossomos Bacterianos/genética , Cianetos/toxicidade , Fragmentação do DNA/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/toxicidade , Ferro/metabolismo , Cromossomos Bacterianos/efeitos dos fármacos , Cromossomos Bacterianos/metabolismo , Sinergismo Farmacológico , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismoRESUMO
Rates of infection with hospital-acquired Acinetobacter baumannii have exploded over the past decade due to our inability to limit persistence and effectively treat disease. A. baumannii quickly acquires antibiotic resistance, and its genome encodes mechanisms to tolerate biocides and desiccation, which enhance its persistence in hospital settings. With depleted antibiotic options, new methods to treat A. baumannii infections are desperately needed. A comprehensive understanding detailing A. baumannii cellular factors that contribute to its resiliency at genetic and mechanistic levels is vital to the development of new treatment options. Tools to rapidly dissect the A. baumannii genome will facilitate this goal by quickly advancing our understanding of A. baumannii gene-phenotype relationships. We describe here a recombination-mediated genetic engineering (recombineering) system for targeted genome editing of A. baumannii. We have demonstrated that this system can perform directed mutagenesis on wide-ranging genes and operons and is functional in various strains of A. baumannii, indicating its broad application. We utilized this system to investigate key gene-phenotype relationships in A. baumannii biology important to infection and persistence in hospitals, including oxidative stress protection, biocide resistance mechanisms, and biofilm formation. In addition, we have demonstrated that both the formation and movement of type IV pili play an important role in A. baumannii biofilm. Importance: Acinetobacter baumannii is the causative agent of hospital-acquired infections, including pneumonia and serious blood and wound infections. A. baumannii is an emerging pathogen and has become a threat to public health because it quickly develops antibiotic resistance, making treatment difficult or impossible. While the threat of A. baumannii is well recognized, our understanding of even its most basic biology lags behind. Analysis of A. baumannii cellular functions to identify potential targets for drug development has stalled due in part to laborious genetic techniques. Here we have pioneered a novel recombineering system that facilitates efficient genome editing in A. baumannii by single PCR products. This technology allows for rapid genome editing to quickly ascertain gene-phenotype relationships. To demonstrate the power of recombineering in dissecting A. baumannii biology, we use this system to establish key gene-phenotype relationships important to infection and persistence in hospitals, including oxidative stress protection, biocide resistance, and biofilm formation.
Assuntos
Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/genética , Cromossomos Bacterianos/genética , Genoma Bacteriano/genética , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Biofilmes/efeitos dos fármacos , Clorexidina/farmacologia , Cromossomos Bacterianos/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Estudos de Associação Genética , Testes de Sensibilidade MicrobianaRESUMO
The excessive and often unreasonable use of antibacterial drugs leads to rise of antibioticresistant strains. To overcome this problem, new antibiotics are searched and the new drug targets are investigated. The proteins involved in replication of bacterial chromosomes seem to be promising candidates for drug targets since they are involved in crucial life pathways and are structurally and/or functionally different from the eukaryotic homologues. Within last few years, a large number of newly developed methods allowed to search among thousands of molecules for the ones that specifically inhibit DNA synthesis in the prokaryotic cell. In this review, we present some of these methods.
Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Cromossomos Bacterianos/efeitos dos fármacos , Cromossomos Bacterianos/genética , Descoberta de Drogas/métodos , Replicação do DNA/efeitos dos fármacos , Desenho de Fármacos , Modelos MolecularesRESUMO
ProQ is a cytoplasmic protein with RNA chaperone activities that reside in FinO- and Hfq-like domains. Lesions at proQ decrease the level of the osmoregulatory glycine betaine transporter ProP. Lesions at proQ eliminated ProQ and Prc, the periplasmic protease encoded by the downstream gene prc. They dramatically slowed the growth of Escherichia coli populations and altered the morphologies of E. coli cells in high-salinity medium. ProQ and Prc deficiencies were associated with different phenotypes. ProQ-deficient bacteria were elongated unless glycine betaine was provided. High-salinity cultures of Prc-deficient bacteria included spherical cells with an enlarged periplasm and an eccentric nucleoid. The nucleoid-containing compartment was bounded by the cytoplasmic membrane and peptidoglycan. This phenotype was not evident in bacteria cultivated at low or moderate salinity, nor was it associated with murein lipoprotein (Lpp) deficiency, and it differed from those elicited by the MreB inhibitor A-22 or the FtsI inhibitor aztreonam at low or high salinity. It was suppressed by deletion of spr, which encodes one of three murein hydrolases that are redundantly essential for enlargement of the murein sacculus. Prc deficiency may alter bacterial morphology by impairing control of Spr activity at high salinity. ProQ and Prc deficiencies lowered the ProP activity of bacteria cultivated at moderate salinity by approximately 70% and 30%, respectively, but did not affect other osmoregulatory functions. The effects of ProQ and Prc deficiencies on ProP activity are indirect, reflecting their roles in the maintenance of cell structure.