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2.
J Infect Dis ; 214(2): 173-81, 2016 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-26908752

RESUMO

BACKGROUND: Patients with recurrent Clostridium difficile infection (CDI) have a ≥60% risk of relapse, as conventional therapies do not address the underlying gastrointestinal dysbiosis. This exploratory study evaluated the safety and efficacy of bacterial spores for preventing recurrent CDI. METHODS: Stool specimens from healthy donors were treated with ethanol to eliminate pathogens. The resulting spores were fractionated and encapsulated for oral delivery as SER-109. Following their response to standard-of-care antibiotics, patients in cohort 1 were treated with SER-109 on 2 consecutive days (geometric mean dose, 1.7 × 10(9) spores), and those in cohort 2 were treated on 1 day (geometric mean dose, 1.1 × 10(8) spores). The primary efficacy end point was absence of C. difficile-positive diarrhea during an 8-week follow-up period. Microbiome alterations were assessed. RESULTS: Thirty patients (median age, 66.5 years; 67% female) were enrolled, and 26 (86.7%) met the primary efficacy end point. Three patients with early, self-limiting C. difficile-positive diarrhea did not require antibiotics and tested negative for C. difficile at 8 weeks; thus, 96.7% (29 of 30) achieved clinical resolution. In parallel, gut microbiota rapidly diversified, with durable engraftment of spores and no outgrowth of non-spore-forming bacteria found after SER-109 treatment. Adverse events included mild diarrhea, abdominal pain, and nausea. CONCLUSIONS: SER-109 successfully prevented CDI and had a favorable safety profile, supporting a novel microbiome-based intervention as a potential therapy for recurrent CDI.


Assuntos
Terapia Biológica/métodos , Clostridioides difficile/crescimento & desenvolvimento , Infecções por Clostridium/prevenção & controle , Microbioma Gastrointestinal , Trato Gastrointestinal/microbiologia , Prevenção Secundária/métodos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Terapia Biológica/efeitos adversos , Diarreia/prevenção & controle , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem
3.
Nature ; 524(7563): 59-64, 2015 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-26222023

RESUMO

Multidrug tolerance is largely responsible for chronic infections and caused by a small population of dormant cells called persisters. Selection for survival in the presence of antibiotics produced the first genetic link to multidrug tolerance: a mutant in the Escherichia coli hipA locus. HipA encodes a serine-protein kinase, the multidrug tolerance activity of which is neutralized by binding to the transcriptional regulator HipB and hipBA promoter. The physiological role of HipA in multidrug tolerance, however, has been unclear. Here we show that wild-type HipA contributes to persister formation and that high-persister hipA mutants cause multidrug tolerance in urinary tract infections. Perplexingly, high-persister mutations map to the N-subdomain-1 of HipA far from its active site. Structures of higher-order HipA-HipB-promoter complexes reveal HipA forms dimers in these assemblies via N-subdomain-1 interactions that occlude their active sites. High-persistence mutations, therefore, diminish HipA-HipA dimerization, thereby unleashing HipA to effect multidrug tolerance. Thus, our studies reveal the mechanistic basis of heritable, clinically relevant antibiotic tolerance.


Assuntos
Antibacterianos/farmacologia , Proteínas de Ligação a DNA/metabolismo , Farmacorresistência Bacteriana Múltipla/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Regiões Promotoras Genéticas/genética , Domínio Catalítico , Cristalografia por Raios X , Proteínas de Ligação a DNA/genética , Regulação para Baixo/genética , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Tolerância a Medicamentos/genética , Escherichia coli/genética , Escherichia coli/patogenicidade , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/genética , Humanos , Modelos Moleculares , Mutação/genética , Óperon/genética , Fenótipo , Multimerização Proteica , Estrutura Terciária de Proteína/genética , Transcrição Gênica/genética , Bexiga Urinária/microbiologia , Bexiga Urinária/patologia , Infecções Urinárias/tratamento farmacológico , Infecções Urinárias/microbiologia
4.
Genetics ; 195(4): 1265-76, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24077306

RESUMO

Bacteria exposed to bactericidal fluoroquinolone (FQ) antibiotics can survive without becoming genetically resistant. Survival of these phenotypically resistant cells, commonly called "persisters," depends on the SOS gene network. We have examined mutants in all known SOS-regulated genes to identify functions essential for tolerance in Escherichia coli. The absence of DinG and UvrD helicases and the Holliday junction processing enzymes RuvA and RuvB leads to a decrease in survival. Analysis of the respective mutants indicates that, in addition to repair of double-strand breaks, tolerance depends on the repair of collapsed replication forks and stalled transcription complexes. Mutation in recF results in increased survival, which identifies RecAF recombination as a poisoning mechanism not previously linked to FQ lethality. DinG acts upstream of SOS promoting its induction, whereas RuvAB participates in repair only. UvrD directly promotes all repair processes initiated by FQ-induced damage and prevents RecAF-dependent misrepair, making it one of the crucial SOS functions required for tolerance.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana/genética , Escherichia coli/genética , Fluoroquinolonas/farmacologia , Inibidores da Síntese de Ácido Nucleico/farmacologia , Resposta SOS em Genética , Quebras de DNA de Cadeia Dupla , DNA Helicases/genética , DNA Helicases/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo
5.
Antimicrob Agents Chemother ; 56(9): 4922-6, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22777047

RESUMO

Persisters are dormant phenotypic variants of regular cells that are tolerant to antibiotics and play an important role in recalcitrance of chronic infections to therapy. Persisters can be produced stochastically in a population untreated with antibiotics. At the same time, a deterministic component of persister formation has also been documented in a population of cells with DNA damaged by fluoroquinolone treatment. Expression of the SOS response under these conditions induces formation of persisters by increasing expression of the TisB toxin. This suggests that other stress responses may also contribute to persister formation. Of particular interest is oxidative stress that pathogens encounter during infection. Activated macrophages produce reactive oxygen and nitrogen species which induce the SoxRS and OxyR regulons. Genes controlled by these regulons deactivate the oxidants and promote repair. We examined the ability of oxidative stress induced by paraquat (PQ) to affect persister formation. Preincubation of cells with PQ produced a dramatic increase in the number of persisters surviving challenge with fluoroquinolone antibiotics. PQ did not affect killing by kanamycin or ampicillin. Persisters in a culture treated with PQ that survived a challenge with a fluoroquinolone were also highly tolerant to other antibiotics. PQ induces SoxRS, which in turn induces expression of the AcrAB-TolC multidrug-resistant (MDR) pump. Fluoroquinolones are extruded by this MDR pump, and the effect of PQ on antibiotic tolerance was largely abolished in a mutant that was defective in the pump. It appears that PQ, acting through AcrAB-TolC, reduces the concentration of fluoroquinolones in the cells. This allows a larger fraction of cells to become persisters in the presence of a fluoroquinolone. Analysis of a lexA3 mutant indeed showed a dependence of persister induction under these conditions on SOS. These findings show that induction of a classical resistance mechanism, MDR efflux, by oxidative stress leads to an increase in multidrug-tolerant persister cells.


Assuntos
Farmacorresistência Bacteriana Múltipla/genética , Tolerância a Medicamentos/genética , Escherichia coli/genética , Estresse Oxidativo/efeitos dos fármacos , Paraquat/farmacologia , Ampicilina/farmacologia , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fluoroquinolonas/farmacologia , Canamicina/farmacologia , Testes de Sensibilidade Microbiana , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Resposta SOS em Genética/efeitos dos fármacos , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ativação Transcricional/efeitos dos fármacos
6.
PLoS One ; 7(6): e39185, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22720069

RESUMO

Bacterial populations produce antibiotic-tolerant persister cells. A number of recent studies point to the involvement of toxin/antitoxin (TA) modules in persister formation. hipBA is a type II TA module that codes for the HipB antitoxin and the HipA toxin. HipA is an EF-Tu kinase, which causes protein synthesis inhibition and dormancy upon phosphorylation of its substrate. Antitoxins are labile proteins that are degraded by one of the cytosolic ATP-dependent proteases. We followed the rate of HipB degradation in different protease deficient strains and found that HipB was stabilized in a lon(-) background. These findings were confirmed in an in vitro degradation assay, showing that Lon is the main protease responsible for HipB proteolysis. Moreover, we demonstrated that degradation of HipB is dependent on the presence of an unstructured carboxy-terminal stretch of HipB that encompasses the last 16 amino acid residues. Further, substitution of the conserved carboxy-terminal tryptophan of HipB to alanine or even the complete removal of this 16 residue fragment did not alter the affinity of HipB for hipBA operator DNA or for HipA indicating that the major role of this region of HipB is to control HipB degradation and hence HipA-mediated persistence.


Assuntos
Antitoxinas/metabolismo , Toxinas Bacterianas/metabolismo , Escherichia coli/metabolismo , Sequência de Bases , Primers do DNA , Proteólise
7.
PLoS Biol ; 8(2): e1000317, 2010 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-20186264

RESUMO

Bacteria induce stress responses that protect the cell from lethal factors such as DNA-damaging agents. Bacterial populations also form persisters, dormant cells that are highly tolerant to antibiotics and play an important role in recalcitrance of biofilm infections. Stress response and dormancy appear to represent alternative strategies of cell survival. The mechanism of persister formation is unknown, but isolated persisters show increased levels of toxin/antitoxin (TA) transcripts. We have found previously that one or more components of the SOS response induce persister formation after exposure to a DNA-damaging antibiotic. The SOS response induces several TA genes in Escherichia coli. Here, we show that a knockout of a particular SOS-TA locus, tisAB/istR, had a sharply decreased level of persisters tolerant to ciprofloxacin, an antibiotic that causes DNA damage. Step-wise administration of ciprofloxacin induced persister formation in a tisAB-dependent manner, and cells producing TisB toxin were tolerant to multiple antibiotics. TisB is a membrane peptide that was shown to decrease proton motive force and ATP levels, consistent with its role in forming dormant cells. These results suggest that a DNA damage-induced toxin controls production of multidrug tolerant cells and thus provide a model of persister formation.


Assuntos
Anti-Infecciosos/farmacologia , Toxinas Bacterianas/metabolismo , Ciprofloxacina/farmacologia , Proteínas de Escherichia coli/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Toxinas Bacterianas/genética , Escherichia coli/fisiologia , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Resposta SOS em Genética/efeitos dos fármacos , Resposta SOS em Genética/genética , Resposta SOS em Genética/fisiologia
8.
PLoS Genet ; 5(12): e1000760, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20011100

RESUMO

Bacteria can survive antibiotic treatment without acquiring heritable antibiotic resistance. We investigated persistence to the fluoroquinolone ciprofloxacin in Escherichia coli. Our data show that a majority of persisters to ciprofloxacin were formed upon exposure to the antibiotic, in a manner dependent on the SOS gene network. These findings reveal an active and inducible mechanism of persister formation mediated by the SOS response, challenging the prevailing view that persisters are pre-existing and formed purely by stochastic means. SOS-induced persistence is a novel mechanism by which cells can counteract DNA damage and promote survival to fluoroquinolones. This unique survival mechanism may be an important factor influencing the outcome of antibiotic therapy in vivo.


Assuntos
Antibacterianos/farmacologia , Ciprofloxacina/farmacologia , Escherichia coli/efeitos dos fármacos , Resposta SOS em Genética , Resistência Microbiana a Medicamentos/genética , Escherichia coli/genética , Processos Estocásticos
9.
Antimicrob Agents Chemother ; 52(8): 2718-26, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18519731

RESUMO

Bacterial populations produce a small number of persister cells that exhibit multidrug tolerance. Persister cells are largely responsible for the antibiotic recalcitrance of biofilm infections. The mechanism of persister cell formation largely remains unknown due to the challenges in identifying persister genes. We screened an ordered comprehensive library of 3,985 Escherichia coli knockout strains to identify mutants with altered antibiotic tolerance. Stationary-state cultures in 96-well plates were exposed to ofloxacin at a concentration which allows only tolerant persister cells to survive. The persister cell level of each culture was determined. A total of 150 mutants with decreased persistence were identified in the initial screen, and subsequent validation confirmed that neither the growth rate nor the ofloxacin MIC was affected for 10 of them. The genes affected in these strains were dnaJ and dnaK (chaperones), apaH (diadenosine tetraphosphatase), surA (peptidyl-prolyl cis-trans isomerase), fis and hns (global regulators), hnr (response regulator of RpoS), dksA (transcriptional regulator of rRNA transcription), ygfA (5-formyl-tetrahydrofolate cyclo-ligase), and yigB (flavin mononucleotide [FMN] phosphatase). The prominent presence of global regulators among these strains pointed to the likely redundancy of persister cell formation mechanisms: the elimination of a regulator controlling several redundant persister genes would be expected to produce a phenotype. This observation is consistent with previous findings for a possible role of redundant genes such as toxin/antitoxin modules in persister cell formation. ygfA and yigB were of special interest. The mammalian homolog of YgfA (methenyltetrahydrofolate synthetase) catalyzes the conversion of 5-formyl-tetrahydrofolate (THF) into the rapidly degraded 5,10-methenyl-THF, depleting the folate pool. The YigB protein is a phosphatase of FMN which would deplete the pool of this cofactor. Stochastic overexpression of these genes could lead to dormancy and, hence, tolerance by depleting the folate and FMN pools, respectively. Consistent with this scenario, the overexpression of both genes produced increased tolerance to ofloxacin.


Assuntos
Antibacterianos/farmacologia , Proteínas de Escherichia coli/metabolismo , Escherichia coli/efeitos dos fármacos , Nucleotídeos/metabolismo , Farmacorresistência Bacteriana Múltipla , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Mutação , Ofloxacino/farmacologia
10.
Science ; 319(5869): 1533-6, 2008 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-18339941

RESUMO

Conjugation allows bacteria to acquire genes for antibiotic resistance, novel virulence attributes, and alternative metabolic pathways. Using a fluorescent protein fusion, SeqA-YFP, we have visualized this process in real time and in single cells of Escherichia coli. We found that the F pilus mediates DNA transfer at considerable cell-to-cell distances. Integration of transferred DNA by recombination occurred in up to 96% of recipients; in the remaining cells, the transferred DNA was fully degraded by the RecBCD helicase/nuclease. The acquired integrated DNA was tracked through successive replication rounds and was found to occasionally split and segregate with different chromosomes, leading to the inheritance of different gene clusters within the cell lineage. The incidence of DNA splitting corresponds to about one crossover per cell generation.


Assuntos
Conjugação Genética , DNA Bacteriano/genética , Escherichia coli/genética , Transferência Genética Horizontal , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/metabolismo , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Exodesoxirribonuclease V/metabolismo , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência , Pili Sexual/fisiologia , Proteínas Recombinantes de Fusão/metabolismo , Recombinação Genética
11.
J Bacteriol ; 188(14): 5136-44, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16816185

RESUMO

Bacterial populations produce dormant persister cells that are resistant to killing by all antibiotics currently in use, a phenomenon known as multidrug tolerance (MDT). Persisters are phenotypic variants of the wild type and are largely responsible for MDT of biofilms and stationary populations. We recently showed that a hipBA toxin/antitoxin locus is part of the MDT mechanism in Escherichia coli. In an effort to find additional MDT genes, an E. coli expression library was selected for increased survival to ampicillin. A clone with increased persister production was isolated and was found to overexpress the gene for the conserved aerobic sn-glycerol-3-phosphate dehydrogenase GlpD. The GlpD overexpression strain showed increased tolerance to ampicillin and ofloxacin, while a strain with glpD deleted had a decreased level of persisters in the stationary state. This suggests that GlpD is a component of the MDT mechanism. Further genetic studies of mutants affected in pathways involved in sn-glycerol-3-phosphate metabolism have led to the identification of two additional multidrug tolerance loci, glpABC, the anaerobic sn-glycerol-3-phosphate dehydrogenase, and plsB, an sn-glycerol-3-phosphate acyltransferase.


Assuntos
Acetiltransferases/genética , Escherichia coli/genética , Glicerol-3-Fosfato Desidrogenase (NAD+)/genética , Acetiltransferases/metabolismo , Antibacterianos/farmacologia , Sequência de Bases , Primers do DNA , Tolerância a Medicamentos , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Genótipo , Glicerol-3-Fosfato Desidrogenase (NAD+)/metabolismo , Plasmídeos , Proteínas Recombinantes/metabolismo
12.
J Bacteriol ; 184(11): 2898-905, 2002 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12003929

RESUMO

During prolonged incubation in stationary phase Escherichia coli undergoes starvation-induced differentiation, resulting in highly resistant cells. In rich medium with high amino acid content further incubation of cultures at high cell density leads to the generation of a population of cells no longer able to form colonies. The viability loss is due to some component of spent medium, active at high pH and high cell density, and can be prevented either by keeping the pH close to neutrality, by washing off the nonsalt components of the medium, or by keeping the saturating cell density low. Exposure to short-chain n-alcohols within a specific time window in stationary phase also prevents viability loss, in an rpoS-dependent fashion. The development of stress resistance, a hallmark of stationary-phase cells, is affected following alcohol treatment, as is the response to extracellular factors in spent medium. Alcohols seem to block cells in an early phase of starvation-induced differentiation, most likely by interfering with processes important for regulation of sigma(s) such as cell density signals and sensing the nutrient content of the medium.


Assuntos
Álcoois/farmacologia , Escherichia coli/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Adaptação Fisiológica/efeitos dos fármacos , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/metabolismo , Diferenciação Celular , Sobrevivência Celular , Meios de Cultura , Escherichia coli/metabolismo , Escherichia coli/fisiologia , Concentração de Íons de Hidrogênio , Mutação , Fator sigma/metabolismo
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