RESUMEN
Antibiotic-resistant Enterobacterales pose a major threat to healthcare systems worldwide, necessitating the development of novel strategies to fight such hard-to-kill bacteria. One potential approach is to develop molecules that force bacteria to hyper-activate prodrug antibiotics, thus rendering them more effective. In the present work, we aimed to obtain proof-of-concept data to support that small molecules targeting transcriptional regulators can potentiate the antibiotic activity of the prodrug metronidazole (MTZ) against Escherichia coli under aerobic conditions. By screening a chemical library of small molecules, a series of structurally related molecules were identified that had little inherent antibiotic activity but showed substantial activity in combination with ineffective concentrations of MTZ. Transcriptome analyses, functional genetics, thermal shift assays, and electrophoretic mobility shift assays were then used to demonstrate that these MTZ boosters target the transcriptional repressor MarR, resulting in the upregulation of the marRAB operon and its downstream MarA regulon. The associated upregulation of the flavin-containing nitroreductase, NfsA, was then shown to be critical for the booster-mediated potentiation of MTZ antibiotic activity. Transcriptomic studies, biochemical assays, and electron paramagnetic resonance measurements were then used to show that under aerobic conditions, NfsA catalyzed 1-electron reduction of MTZ to the MTZ radical anion which in turn induced lethal DNA damage in E. coli. This work reports the first example of prodrug boosting in Enterobacterales by transcriptional modulators and highlights that MTZ antibiotic activity can be chemically induced under anaerobic growth conditions.
Asunto(s)
Antibacterianos , Proteínas de Escherichia coli , Escherichia coli , Metronidazol , Nitrorreductasas , Proteínas Represoras , Nitrorreductasas/metabolismo , Nitrorreductasas/genética , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Escherichia coli/genética , Metronidazol/farmacología , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Antibacterianos/farmacología , Antibacterianos/química , Aerobiosis , Proteínas Represoras/metabolismo , Proteínas Represoras/genética , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Bibliotecas de Moléculas Pequeñas/químicaRESUMEN
Antimicrobial resistance is a global problem, rendering conventional treatments less effective and requiring innovative strategies to combat this growing threat. The tripartite AcrAB-TolC efflux pump is the dominant constitutive system by which Enterobacterales like Escherichia coli and Klebsiella pneumoniae extrude antibiotics. Here, we describe the medicinal chemistry development and drug-like properties of BDM91288, a pyridylpiperazine-based AcrB efflux pump inhibitor. In vitro evaluation of BDM91288 confirmed it to potentiate the activity of a panel of antibiotics against K. pneumoniae as well as revert clinically relevant antibiotic resistance mediated by acrAB-tolC overexpression. Using cryo-EM, BDM91288 binding to the transmembrane region of K. pneumoniae AcrB was confirmed, further validating the mechanism of action of this inhibitor. Finally, proof of concept studies demonstrated that oral administration of BDM91288 significantly potentiated the in vivo efficacy of levofloxacin treatment in a murine model of K. pneumoniae lung infection.
Asunto(s)
Antibacterianos , Proteínas de Escherichia coli , Animales , Ratones , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/farmacología , Klebsiella pneumoniae/metabolismo , Escherichia coli , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/farmacologíaRESUMEN
Objectives: In Acinetobacter baumannii, multidrug efflux pumps belonging to the resistance-nodulation-division (RND) superfamily result in decreased antibiotic susceptibility. Improving the activity of current antibiotics via efflux pump inhibitors (EPIs) represents an attractive alternative approach to control this bacterium. Pyridylpiperazines (PyrPips) are a new class of EPIs that can effectively inhibit the Escherichia coli RND efflux pump AcrAB-TolC and boost the activity of several antibiotics. Here we have evaluated and characterized whether the PyrPip chemical family is also able to boost antibiotic activity through inhibition of the RND efflux pumps in A. baumannii. Methods: Comparative structural modelling and docking, structure-activity relationship studies alongside molecular genetic approaches were deployed to improve, characterize and validate PyrPips' target. Results: We showed that two enhanced PyrPip EPIs are capable of rescuing the activity of different classes of antibiotics in A. baumannii. By expressing A. baumannii main efflux pumps (AdeB, AdeG and AdeJ) individually in E. coli recombinant strains, we could gain further insights about the EPIs' capacity to act upon each pump. Finally, we showed that PyrPip EPIs are mostly acting through AdeJ inhibition via interactions with two key charged residues, namely E959 and E963. Conclusions: Our work demonstrates that PyrPip EPIs are capable of inhibiting RND efflux pumps of A. baumannii, and thus may present a promising chemical scaffold for further development.
RESUMEN
Multidrug-resistant Escherichia coli is a continuously growing worldwide public health problem, in which the well-known AcrAB-TolC tripartite RND efflux pump is a critical driver. We have previously described pyridylpiperazines as a novel class of allosteric inhibitors of E. coli AcrB which bind to a unique site in the protein transmembrane domain, allowing for the potentiation of antibiotic activity. Here, we show a rational optimization of pyridylpiperazines by modifying three specific derivatization points of the pyridine core to improve the potency and the pharmacokinetic properties of this chemical series. In particular, this work found that the introduction of a primary amine to the pyridine through ester (29, BDM91270) or oxadiazole (44, BDM91514) based linkers allowed for analogues with improved antibiotic boosting potency through AcrB inhibition. In vitro studies, using genetically engineered mutants, showed that this improvement in potency is mediated through novel interactions with distal acidic residues of the AcrB binding pocket. Of the two leads, compound 44 was found to have favorable physico-chemical properties and suitable plasma and microsomal stability. Together, this work expands the current structure-activity relationship data on pyridylpiperazine efflux pump inhibitors, and provides a promising step towards future in vivo proof of concept of pyridylpiperazines as antibiotic potentiators.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli , Escherichia coli/metabolismo , Antibacterianos/química , Piridinas/farmacología , Piridinas/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Proteínas Portadoras/metabolismoRESUMEN
Efflux transporters of the RND family confer resistance to multiple antibiotics in Gram-negative bacteria. Here, we identify and chemically optimize pyridylpiperazine-based compounds that potentiate antibiotic activity in E. coli through inhibition of its primary RND transporter, AcrAB-TolC. Characterisation of resistant E. coli mutants and structural biology analyses indicate that the compounds bind to a unique site on the transmembrane domain of the AcrB L protomer, lined by key catalytic residues involved in proton relay. Molecular dynamics simulations suggest that the inhibitors access this binding pocket from the cytoplasm via a channel exclusively present in the AcrB L protomer. Thus, our work unveils a class of allosteric efflux-pump inhibitors that likely act by preventing the functional catalytic cycle of the RND pump.
Asunto(s)
Antibacterianos/farmacología , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de Escherichia coli/química , Escherichia coli/efectos de los fármacos , Lipoproteínas/química , Proteínas de Transporte de Membrana/química , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/química , Piperazinas/farmacología , Piridinas/farmacología , Regulación Alostérica/efectos de los fármacos , Sitio Alostérico , Antibacterianos/química , Proteínas de la Membrana Bacteriana Externa/antagonistas & inhibidores , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Transporte Biológico/efectos de los fármacos , Cristalografía por Rayos X , Farmacorresistencia Bacteriana Múltiple , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/antagonistas & inhibidores , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expresión Génica , Lipoproteínas/antagonistas & inhibidores , Lipoproteínas/genética , Lipoproteínas/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Simulación de Dinámica Molecular , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/antagonistas & inhibidores , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Mutación , Oligopéptidos/química , Oligopéptidos/farmacología , Oxacilina/química , Oxacilina/farmacología , Piperazinas/síntesis química , Regiones Promotoras Genéticas , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Piridinas/síntesis química , Relación Estructura-ActividadRESUMEN
The adherent-invasive Escherichia coli (AIEC) pathotype has been implicated in the pathogenesis of inflammatory bowel diseases in general and in Crohn's disease (CD) in particular. AIEC strains are primarily characterized by their ability to adhere to and invade intestinal epithelial cells. However, the genetic and phenotypic features of AIEC isolates vary greatly as a function of the strain's clonality, host factors, and the gut microenvironment. It is thus essential to identify the determinants of AIEC pathogenicity and understand their role in intestinal epithelial barrier dysfunction and inflammation. We reasoned that soil nematode Caenorhabditis elegans (a simple but powerful model of host-bacterium interactions) could be used to study the virulence of AIEC vs. non- AIEC E. coli strains. Indeed, we found that the colonization of C. elegans (strain N2) by E. coli impacted survival in a strain-specific manner. Moreover, the AIEC strains' ability to invade cells in vitro was linked to the median lifespan in C. elegans (strain PX627). However, neither the E. coli intrinsic invasiveness (i.e., the fact for an individual strain to be characterized as invasive or not) nor AIEC's virulence levels (i.e., the intensity of invasion, established in % from the infectious inoculum) in intestinal epithelial cells was correlated with C. elegans' lifespan in the killing assay. Nevertheless, AIEC longevity of C. elegans might be a relevant model for screening anti-adhesion drugs and anti-invasive probiotics.
RESUMEN
To thrive, vector-borne pathogens must survive in the vector's gut. How these pathogens successfully exploit this environment in time and space has not been extensively characterized. Using Yersinia pestis (the plague bacillus) and its flea vector, we developed a bioluminescence-based approach and employed it to investigate the mechanisms of pathogenesis at an unprecedented level of detail. Remarkably, lipoylation of metabolic enzymes, via the biosynthesis and salvage of lipoate, increases the Y. pestis transmission rate by fleas. Interestingly, the salvage pathway's lipoate/octanoate ligase LplA enhances the first step in lipoate biosynthesis during foregut colonization but not during midgut colonization. Lastly, Y. pestis primarily uses lipoate provided by digestive proteolysis (presumably as lipoyl peptides) rather than free lipoate in blood, which is quickly depleted by the vector. Thus, spatial and temporal factors dictate the bacterium's lipoylation strategies during an infection, and replenishment of lipoate by digestive proteolysis in the vector might constitute an Achilles' heel that is exploited by pathogens.
Asunto(s)
Peste , Siphonaptera , Yersinia pestis , Animales , Biopelículas , Insectos Vectores , Yersinia pestis/genéticaRESUMEN
In flea-borne plague, blockage of the flea's foregut by Yersinia pestis hastens transmission to the mammalian host. Based on microscopy observations, we first suggest that flea blockage results from primary infection of the foregut and not from midgut colonization. In this model, flea infection is characterized by the recurrent production of a mass that fills the lumen of the proventriculus and encompasses a large number of Y. pestis. This recurrence phase ends when the proventricular cast is hard enough to block blood ingestion. We further showed that ymt (known to be essential for flea infection) is crucial for cast production, whereas the hmsHFRS operon (known to be essential for the formation of the biofilm that blocks the gut) is needed for cast consolidation. By screening a library of mutants (each lacking a locus previously known to be upregulated in the flea gut) for biofilm formation, we found that rpiA is important for flea blockage but not for colonization of the midgut. This locus may initially be required to resist toxic compounds within the proventricular cast. However, once the bacterium has adapted to the flea, rpiA helps to form the biofilm that consolidates the proventricular cast. Lastly, we used genetic techniques to demonstrate that ribose-5-phosphate isomerase activity (due to the recent gain of a second copy of rpiA (y2892)) accentuated blockage but not midgut colonization. It is noteworthy that rpiA is an ancestral gene, hmsHFRS and rpiA2 were acquired by the recent ancestor of Y. pestis, and ymt was acquired by Y. pestis itself. Our present results (i) highlight the physiopathological and molecular mechanisms leading to flea blockage, (ii) show that the role of a gene like rpiA changes in space and in time during an infection, and (iii) emphasize that evolution is a gradual process punctuated by sudden jumps.
Asunto(s)
Insectos Vectores/microbiología , Peste/transmisión , Siphonaptera/microbiología , Yersinia pestis/fisiología , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biopelículas , Sistema Digestivo/microbiología , Femenino , Humanos , Insectos Vectores/fisiología , Masculino , Ratones , Operón , Peste/microbiología , Siphonaptera/fisiología , Yersinia pestis/genéticaRESUMEN
The escalating burden of antibiotic drug resistance necessitates research into novel classes of antibiotics and their mechanism of action. Pyrrolomycins are a family of potent natural product antibiotics with nanomolar activity against Gram-positive bacteria, yet with an elusive mechanism of action. In this work, we dissect the apparent Gram-positive specific activity of pyrrolomycins and show that Gram-negative bacteria are equally sensitive to pyrrolomycins when drug efflux transporters are removed and that albumin in medium plays a large role in pyrrolomycin activity. The selection of resistant mutants allowed for the characterization and validation of a number of mechanisms of resistance to pyrrolomycins in both Staphylococcus aureus and an Escherichia coli ΔtolC mutant, all of which appear to affect compound penetration rather than being target associated. Imaging of the impact of pyrrolomycin on the E. coli ΔtolC mutant using scanning electron microscopy showed blebbing of the bacterial cell wall often at the site of bacterial division. Using potentiometric probes and an electrophysiological technique with an artificial bilayer lipid membrane, it was demonstrated that pyrrolomycins C and D are very potent membrane-depolarizing agents, an order of magnitude more active than conventional carbonyl cyanide m-chlorophenylhydrazone (CCCP), specifically disturbing the proton gradient and uncoupling oxidative phosphorylation via protonophoric action. This work clearly unveils the until-now-elusive mechanism of action of pyrrolomycins and explains their antibiotic activity as well as mechanisms of innate and acquired drug resistance in bacteria.
Asunto(s)
Antibacterianos/química , Antibacterianos/farmacología , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Escherichia coli/ultraestructura , Pruebas de Sensibilidad Microbiana , Microscopía Electrónica de Rastreo , Pirroles/química , Pirroles/farmacología , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/metabolismo , Staphylococcus aureus/ultraestructura , Relación Estructura-ActividadRESUMEN
The mechanisms involved in the virulence of Yersinia pestis, the plague pathogen, are not fully understood. In previous research, we found that a Y. pestis mutant lacking the HicB3 (YPO3369) putative orphan antitoxin was attenuated for virulence in a murine model of bubonic plague. Toxin-antitoxin systems (TASs) are widespread in prokaryotes. Most bacterial species possess many TASs of several types. In type II TASs, the toxin protein is bound and neutralized by its cognate antitoxin protein in the cytoplasm. Here we identify the hicA3 gene encoding the toxin neutralized by HicB3 and show that HicA3-HicB3 constitutes a new functional type II TAS in Y. pestis. Using biochemical and mutagenesis-based approaches, we demonstrate that the HicA3 toxin is an RNase with a catalytic histidine residue. HicB3 has two functions: it sequesters and neutralizes HicA3 by blocking its active site, and it represses transcription of the hicA3B3 operon. Gel shift assays and reporter fusion experiments indicate that the HicB3 antitoxin binds to two operators in the hicA3B3 promoter region. We solved the X-ray structures of HicB3 and the HicA3-HicB3 complex; thus, we present the first crystal structure of a TA complex from the HicAB family. HicB3 forms a tetramer that can bind two HicA3 toxin molecules. HicA3 is monomeric and folds as a double-stranded-RNA-binding domain. The HicB3 N-terminal domain occludes the HicA3 active site, whereas its C-terminal domain folds as a ribbon-helix-helix DNA-binding motif.
Asunto(s)
Antitoxinas/metabolismo , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Yersinia pestis/metabolismo , Animales , Antitoxinas/genética , Proteínas Bacterianas/genética , Toxinas Bacterianas/genética , Composición de Base , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Peste/microbiología , Regiones Promotoras Genéticas , Conformación Proteica , Virulencia , Yersinia pestis/genética , Yersinia pestis/patogenicidadRESUMEN
Serratia marcescens is an important nosocomial pathogen that can cause an array of infections, most notably of the urinary tract and bloodstream. Naturally, it is found in many environmental niches, and is capable of infecting plants and animals. The emergence and spread of multidrug-resistant strains producing extended-spectrum or metallo beta-lactamases now pose a threat to public health worldwide. Here we report the complete genome sequences of two carefully selected S. marcescens strains, a multidrug-resistant clinical isolate (strain SM39) and an insect isolate (strain Db11). Our comparative analyses reveal the core genome of S. marcescens and define the potential metabolic capacity, virulence, and multidrug resistance of this species. We show a remarkable intraspecies genetic diversity, both at the sequence level and with regards genome flexibility, which may reflect the diversity of niches inhabited by members of this species. A broader analysis with other Serratia species identifies a set of approximately 3,000 genes that characterize the genus. Within this apparent genetic diversity, we identified many genes implicated in the high virulence potential and antibiotic resistance of SM39, including the metallo beta-lactamase and multiple other drug resistance determinants carried on plasmid pSMC1. We further show that pSMC1 is most closely related to plasmids circulating in Pseudomonas species. Our data will provide a valuable basis for future studies on S. marcescens and new insights into the genetic mechanisms that underlie the emergence of pathogens highly resistant to multiple antimicrobial agents.
Asunto(s)
Genoma Bacteriano , Infecciones por Serratia/microbiología , Serratia marcescens/genética , Animales , Antibacterianos/farmacología , Resistencia a Múltiples Medicamentos , Evolución Molecular , Variación Genética , Humanos , Insectos/microbiología , Infecciones por Serratia/tratamiento farmacológico , Serratia marcescens/efectos de los fármacos , Serratia marcescens/aislamiento & purificación , Serratia marcescens/patogenicidadRESUMEN
Plague is transmitted by fleas or contaminated aerosols. To successfully produce disease, the causal agent (Yersinia pestis) must rapidly sense and respond to rapid variations in its environment. Here, we investigated the role of 2-component regulatory systems (2CSs) in plague because the latter are known to be key players in bacterial adaptation to environmental change. Along with the previously studied PhoP-PhoQ system, OmpR-EnvZ was the only one of Y. pestis' 23 other 2CSs required for production of bubonic, septicemic, and pneumonic plague. In vitro, OmpR-EnvZ was needed to counter serum complement and leukocytes but was not required for the secretion of antiphagocyte exotoxins. In vivo, Y. pestis lacking OmpR-EnvZ did not induce an early immune response in the skin and was fully virulent in neutropenic mice. We conclude that, throughout the course of Y. pestis infection, OmpR-EnvZ is required to counter toxic effectors secreted by polymorphonuclear leukocytes in the tissues.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/fisiología , Peste/microbiología , Yersinia pestis/fisiología , Animales , Proteínas del Sistema Complemento/inmunología , Femenino , Inmunidad Innata , Macrófagos/microbiología , Ratones , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Yersinia pestis/inmunología , Yersinia pestis/patogenicidadRESUMEN
Bubonic plague (a fatal, flea-transmitted disease) remains an international public health concern. Although our understanding of the pathogenesis of bubonic plague has improved significantly over the last few decades, researchers have still not been able to define the complete set of Y. pestis genes needed for disease or to characterize the mechanisms that enable infection. Here, we generated a library of Y. pestis mutants, each lacking one or more of the genes previously identified as being up-regulated in vivo. We then screened the library for attenuated virulence in rodent models of bubonic plague. Importantly, we tested mutants both individually and using a novel, "per-pool" screening method that we have developed. Our data showed that in addition to genes involved in physiological adaptation and resistance to the stress generated by the host, several previously uncharacterized genes are required for virulence. One of these genes (ympt1.66c, which encodes a putative helicase) has been acquired by horizontal gene transfer. Deletion of ympt1.66c reduced Y. pestis' ability to spread to the lymph nodes draining the dermal inoculation site--probably because loss of this gene decreased the bacteria's ability to survive inside macrophages. Our results suggest that (i) intracellular survival during the early stage of infection is important for plague and (ii) horizontal gene transfer was crucial in the acquisition of this ability.
Asunto(s)
Interacciones Huésped-Parásitos/genética , Peste/genética , Yersinia pestis/genética , Yersinia pestis/patogenicidad , Animales , Modelos Animales de Enfermedad , Femenino , Humanos , Macrófagos/microbiología , Ratas , VirulenciaRESUMEN
BACKGROUND: Yersinia pestis (the plague bacillus) and its ancestor, Yersinia pseudotuberculosis (which causes self-limited bowel disease), encode putative homologues of the periplasmic lysozyme inhibitor Ivy and the membrane-bound lysozyme inhibitor MliC. The involvement of both inhibitors in virulence remains subject to debate. METHODS: Mutants lacking ivy and/or mliC were generated. We evaluated the mutants' ability to counter lysozyme, grow in serum, and/or counter leukocytes; to produce disease in wild-type, neutropenic, or lysozyme-deficient rodents; and to induce host inflammation. RESULTS: MliC was not required for lysozyme resistance and the development of plague. Deletion of ivy decreased Y. pestis' ability to counter lysozyme and polymorphonuclear neutrophils, but it did not affect the bacterium's ability to grow in serum or resist macrophages. Y. pestis lacking Ivy had attenuated virulence, unless animals were neutropenic or lysozyme deficient. The Ivy mutant induced inflammation to a degree similar to that of the parental strain. Last, Y. pseudotuberculosis did not require Ivy to counter lysozyme and for virulence. CONCLUSIONS: Ivy is required to counter lysozyme during infection, but its role as a virulence factor is species dependent. Our study also shows that a gene that is not necessary for the virulence of an ancestral bacterium may become essential in the emergence of a new pathogen.
Asunto(s)
Proteínas Bacterianas/genética , Inmunidad Innata , Muramidasa/antagonistas & inhibidores , Factores de Virulencia/genética , Yersinia pestis/patogenicidad , Animales , Proteínas Bacterianas/metabolismo , Sangre/inmunología , Sangre/microbiología , Bovinos , Línea Celular , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Evolución Molecular , Femenino , Eliminación de Gen , Humanos , Macrófagos/inmunología , Masculino , Ratones , Ratones Noqueados , Muramidasa/metabolismo , Neutrófilos/metabolismo , Neutrófilos/microbiología , Periplasma/química , Fagocitos/metabolismo , Fagocitos/microbiología , Peste/inmunología , Peste/microbiología , Peste/patología , Ratas , Ratas Endogámicas BN , Albúmina Sérica Bovina/química , Virulencia , Factores de Virulencia/metabolismo , Yersinia pestis/genética , Yersinia pseudotuberculosis/genética , Yersinia pseudotuberculosis/patogenicidad , Infecciones por Yersinia pseudotuberculosis/inmunología , Infecciones por Yersinia pseudotuberculosis/microbiología , Infecciones por Yersinia pseudotuberculosis/patologíaRESUMEN
Potential benefits of combination antibiotic therapy for the treatment of plague have never been evaluated. We compared the efficacy of a ciprofloxacin (CIN) and gentamicin (GEN) combination therapy with that of each antibiotic administered alone (i) against Yersinia pestis in vitro and (ii) in a mouse model of bubonic plague in which animals were intravenously injected with antibiotics for five days, starting at two different times after infection (44 h and 56 h). In vitro, the CIN+GEN combination was synergistic at 0.5x the individual drugs' MICs and indifferent at 1x- or 2x MIC. In vivo, the survival rate for mice treated with CIN+GEN was similar to that observed with CIN alone and slightly higher than that observed for GEN alone 100, 100 and 85%, respectively when treatment was started 44 h post challenge. 100% of survivors were recorded in the CIN+GEN group vs 86 and 83% in the CIN and GEN groups, respectively when treatment was delayed to 56 h post-challenge. However, these differences were not statistically significant. Five days after the end of treatment, Y. pestis were observed in lymph nodes draining the inoculation site (but not in the spleen) in surviving mice in each of the three groups. The median lymph node log(10) CFU recovered from persistently infected lymph nodes was significantly higher with GEN than with CIN (5.8 vs. 3.2, p = 0.04) or CIN+GEN (5.8 vs. 2.8, p = 0.01). Taken as the whole, our data show that CIN+GEN combination is as effective as CIN alone but, regimens containing CIN are more effective to eradicate Y. pestis from the draining lymph node than the recommended GEN monotherapy. Moreover, draining lymph nodes may serve as a reservoir for the continued release of Y. pestis into the blood - even after five days of intravenous antibiotic treatment.
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Ciprofloxacina/uso terapéutico , Gentamicinas/uso terapéutico , Peste/tratamiento farmacológico , Peste/microbiología , Animales , Antiinfecciosos/sangre , Antiinfecciosos/farmacología , Carga Bacteriana/efectos de los fármacos , Ciprofloxacina/sangre , Ciprofloxacina/farmacología , Quimioterapia Combinada , Femenino , Gentamicinas/sangre , Gentamicinas/farmacología , Ganglios Linfáticos/efectos de los fármacos , Ganglios Linfáticos/microbiología , Ganglios Linfáticos/patología , Ratones , Pruebas de Sensibilidad Microbiana , Análisis de Supervivencia , Factores de Tiempo , Resultado del Tratamiento , Yersinia pestis/efectos de los fármacosRESUMEN
Among Yersinia spp., Y. enterocolitica is the species most frequently isolated from infected aneurysms. This report describes the first case of postaneurysmal prosthetic vascular infection due to a superantigen-negative Yersinia pseudotuberculosis strain, showing a potential affinity of this species for endovascular tissue.
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Aneurisma/cirugía , Infecciones Relacionadas con Prótesis/diagnóstico , Infecciones por Yersinia pseudotuberculosis/diagnóstico , Yersinia pseudotuberculosis/aislamiento & purificación , Proteínas Bacterianas/genética , ADN Bacteriano/genética , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Infecciones Relacionadas con Prótesis/microbiología , Factores de Virulencia/genética , Yersinia pseudotuberculosis/química , Yersinia pseudotuberculosis/metabolismo , Infecciones por Yersinia pseudotuberculosis/microbiologíaRESUMEN
Serratia marcescens is an entomopathogenic bacterium that opportunistically infects a wide range of hosts, including humans. In a model of septic injury, if directly introduced into the body cavity of Drosophila, this pathogen is insensitive to the host's systemic immune response and kills flies in a day. We find that S. marcescens resistance to the Drosophila immune deficiency (imd)-mediated humoral response requires the bacterial lipopolysaccharide O-antigen. If ingested by Drosophila, bacteria cross the gut and penetrate the body cavity. During this passage, the bacteria can be observed within the cells of the intestinal epithelium. In such an oral infection model, the flies succumb to infection only after 6 days. We demonstrate that two complementary host defense mechanisms act together against such food-borne infection: an antimicrobial response in the intestine that is regulated by the imd pathway and phagocytosis by hemocytes of bacteria that have escaped into the hemolymph. Interestingly, bacteria present in the hemolymph elicit a systemic immune response only when phagocytosis is blocked. Our observations support a model wherein peptidoglycan fragments released during bacterial growth activate the imd pathway and do not back a proposed role for phagocytosis in the immune activation of the fat body. Thanks to the genetic tools available in both host and pathogen, the molecular dissection of the interactions between S. marcescens and Drosophila will provide a useful paradigm for deciphering intestinal pathogenesis.
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Modelos Animales de Enfermedad , Drosophila/microbiología , Interacciones Huésped-Patógeno/fisiología , Intestinos/microbiología , Infecciones por Serratia/fisiopatología , Serratia marcescens/patogenicidad , Animales , Drosophila/inmunología , Hemolinfa/microbiología , Inmunohistoquímica , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Infecciones por Serratia/inmunología , Serratia marcescens/inmunologíaRESUMEN
The nematode Caenorhabditis elegans is present in soils and composts, where it can encounter a variety of microorganisms. Some bacteria in these rich environments are innocuous food sources for C. elegans, whereas others are pathogens. Under laboratory conditions, C. elegans will avoid certain pathogens, such as Serratia marcescens, by exiting a bacterial lawn a few hours after entering it. By combining bacterial genetics and nematode genetics, we show that C. elegans specifically avoids certain strains of Serratia based on their production of the cyclic lipodepsipentapeptide serrawettin W2. Lawn-avoidance behavior is chiefly mediated by the two AWB chemosensory neurons, probably through G protein-coupled chemoreceptors, and also involves the nematode Toll-like receptor gene tol-1. Purified serrawettin W2, added to an Escherichia coli lawn, can directly elicit lawn avoidance in an AWB-dependent fashion, as can another chemical detected by AWB. These findings represent an insight into chemical recognition between these two soil organisms and reveal sensory mechanisms for pathogen recognition in C. elegans.
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Reacción de Prevención/efectos de los fármacos , Caenorhabditis elegans/microbiología , Caenorhabditis elegans/fisiología , Lipoproteínas/farmacología , Péptidos Cíclicos/farmacología , Serratia marcescens/química , Animales , Productos Biológicos/metabolismo , Productos Biológicos/farmacología , Proteínas de Caenorhabditis elegans/fisiología , Lipoproteínas/metabolismo , Proteínas del Tejido Nervioso/fisiología , Péptidos Cíclicos/metabolismoRESUMEN
A bacteriophage (phiIF3) capable of mediating generalized transduction in Serratia marcescens strain Db11 has been isolated and characterized. The genome of this Serratia strain has recently been sequenced and is likely to become the reference strain for S. marcescens researchers. phiIF3 is most likely a virulent phage, which can transduce markers at frequencies of 10(-6) transductants per p.f.u. It has a lipopolysaccharide receptor and was determined to have a latent period of 50 min and a burst size of approximately 100 phages. The phage DNA was resistant to digestion with restriction enzymes. Electron microscopy showed phiIF3 to be a member of the family Myoviridae. This is the first report of a generalized transducing phage able to infect Db11 and this phage will be a valuable tool for functional genomic analysis of the pathogen host.
Asunto(s)
Bacteriófagos/genética , Serratia marcescens/genética , Serratia marcescens/virología , Transducción Genética , Bacteriófagos/aislamiento & purificación , Bacteriófagos/fisiología , Bacteriófagos/ultraestructura , ADN Bacteriano/genética , Genoma Bacteriano , Humanos , Lisogenia , Myoviridae/genética , Myoviridae/aislamiento & purificación , Myoviridae/fisiología , Myoviridae/ultraestructura , Serratia marcescens/crecimiento & desarrolloRESUMEN
OBJECTIVES: AcrAB-TolC is the major tripartite multidrug efflux pump in Enterobacter aerogenes while EefABC is a cryptic efflux system. This study was conducted to identify and characterize E. aerogenes mutants producing the EefABC efflux pump. METHODS: Four spontaneous chloramphenicol-resistant (CMR) mutants were isolated. The expression level of the eefABC promoter and the production of the EefA and B proteins were analysed in the mutants. Antibiotic susceptibilities were compared for wild-type and mutant strains. Efflux activity was investigated using an efflux pump inhibitor. RESULTS: The activation of the eefABC promoter was detected in four CMR mutants. These mutants showed increased resistance to erythromycin and ticarcillin, but not to fluoroquinolones, ketolides and detergents. Two additional efflux proteins were detected in the mutants. The CMR mutants bear no mutation in hns, which encodes a repressor of eefABC. No alteration of porin expression, a phenotype observed in marA or ramA multidrug-resistant mutants, was detected in the mutants. CONCLUSIONS: These observations suggest that eefABC activation can occur in vitro independently of the H-NS, MarA or RamA global regulators.