RESUMO
The crystal violet microtiter plate biofilm assay is often used to compare the amount of biofilm formed by a mutant versus wild-type or a compound-treated biofilm versus the non-treatment control. In many of these studies the amount of biofilm is assessed only at one single time point. However, if the dynamics of biofilm development of the mutant (or compound-treated biofilm) is different than that of the wild-type (or non-treatment control), then biofilm quantification at a single time point may give misleading results. To overcome this shortcoming of the common biofilm quantification technique, we recommend to use a serial dilution-based crystal violet microtiter plate biofilm assay for easy assessment of the dynamics of biofilm development and dispersal. We demonstrate that the dilution-resolved crystal violet assay displays the dynamics of Pseudomonas aeruginosa biofilm development and dispersal as efficient as a time-resolved crystal violet assay. In addition, focusing on mutants of different parts of the c-di-GMP signaling system in P. aeruginosa, we provide an example illustrating the need to assess biofilm dynamics instead of quantifying biofilm biomass at a single time point.
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This paper presents the work performed to transition a lab-scale synthesis (1 g) to a large-scale (400 g) synthesis of the 3-5-diamino-1H-Pyrazole Disperazol, a new pharmaceutical for treatment of antibiotic-resistant Pseudomonas aeruginosa biofilm infections. The potentially hazardous diazotisation step in the lab-scale synthesis was transformed to a safe and easy-to-handle flow chemistry step. Additionally, the paper presents an OSHA-recommended safety assessment of active compound E, as performed by Fauske and Associates, LLC, Burr Ridge, IL, USA.
Assuntos
Pseudomonas aeruginosa , Pirazóis , Pirazóis/química , Pirazóis/farmacologia , Pirazóis/síntese química , Pseudomonas aeruginosa/efeitos dos fármacos , Antibacterianos/farmacologia , Antibacterianos/síntese química , Antibacterianos/química , Biofilmes/efeitos dos fármacos , Humanos , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/microbiologia , Medição de RiscoRESUMO
Persistent urinary tract infections (UTIs) in hospitalized patients constitute an important medical problem. It is estimated that 75% of nosocomial UTIs are associated with urinary tract catheters with P. aeruginosa being a species that forms biofilms on these catheters. These infections are highly resistant to standard-of-care antibiotics, and the effects of the host immune defenses, which allows for development of persistent infections. With antibiotics losing their efficacy, new treatment options against resilient infections, such as catheter-associated urinary tract infections (CAUTIs), are critically needed. Central to our anti-biofilm approach is the manipulation of the c-di-GMP signaling pathway in P. aeruginosa to switch bacteria from the protective biofilm to the unprotected planktonic mode of life. We recently identified a compound (H6-335-P1), that stimulates the c-di-GMP degrading activity of the P. aeruginosa BifA protein which plummets the intracellular c-di-GMP content and induces dispersal of P. aeruginosa biofilm bacteria into the planktonic state. In the present study, we formulated H6-335-P1 as a hydrochloride salt (Disperazol), which is water-soluble and facilitates delivery via injection or oral administration. Disperazol can work as a monotherapy, but we observed a 100-fold improvement in efficacy when treating murine P. aeruginosa CAUTIs with a Disperazol/ciprofloxacin combination. Biologically active Disperazol reached the bladder 30 min after oral administration. Our study provides proof of concept that Disperazol can be used in combination with a relevant antibiotic for effective treatment of CAUTIs.
RESUMO
The cell-to-cell communication system quorum sensing (QS), used by various pathogenic bacteria to synchronize gene expression and increase host invasion potentials, is studied as a potential target for persistent infection control. To search for novel molecules targeting the QS system in the Gram-negative opportunistic pathogen Pseudomonas aeruginosa, a chemical library consisting of 3,280 small compounds from LifeArc was screened. A series of 10 conjugated phenones that have not previously been reported to target bacteria were identified as inhibitors of QS in P. aeruginosa. Two lead compounds (ethylthio enynone and propylthio enynone) were re-synthesized for verification of activity and further elucidation of the mode of action. The isomeric pure Z-ethylthio enynone was used for RNA sequencing, revealing a strong inhibitor of QS-regulated genes, and the QS-regulated virulence factors rhamnolipid and pyocyanin were significantly decreased by treatment with the compounds. A transposon mutagenesis screen performed in a newly constructed lasB-gfp monitor strain identified the target of Z-ethylthio enynone in P. aeruginosa to be the MexEF-OprN efflux pump, which was further established using defined mex knockout mutants. Our data indicate that the QS inhibitory capabilities of Z-ethylthio enynone were caused by the drainage of intracellular signal molecules as a response to chemical-induced stimulation of the MexEF-oprN efflux pump, thereby inhibiting the autogenerated positive feedback and its enhanced signal-molecule synthesis.
Assuntos
Pseudomonas aeruginosa , Percepção de Quorum , Pseudomonas aeruginosa/genética , Percepção de Quorum/genética , Fatores de Virulência/genética , Proteínas de Bactérias/genéticaRESUMO
In the Pseudomonas aeruginosa type strain PA14, 40 genes are known to encode for diguanylate cyclases (DGCs) and/or phosphodiesterases (PDEs), which modulate the intracellular pool of the nucleotide second messenger c-di-GMP. While in general, high levels of c-di-GMP drive the switch from highly motile phenotypes towards a sessile lifestyle, the different c-di-GMP modulating enzymes are responsible for smaller and in parts nonoverlapping phenotypes. In this study, we sought to utilize previously recorded P. aeruginosa gene expression datasets on 414 clinical isolates to uncover transcriptional changes as a result of a high expression of genes encoding DGCs. This approach might provide a unique opportunity to bypass the problem that for many c-di-GMP modulating enzymes it is not known under which conditions their expression is activated. However, while we demonstrate that the selection of subgroups of clinical isolates with high versus low expression of sigma factor encoding genes served the identification of their downstream regulons, we were unable to confirm the predicted DGC regulons, because the high c-di-GMP associated phenotypes were rapidly lost in the clinical isolates,. Further studies are needed to determine the specific mechanisms underlying the loss of cyclase activity upon prolonged cultivation of clinical P. aeruginosa isolates.
RESUMO
The bacterium Pseudomonas aeruginosa is involved in chronic infections of cystic fibrosis lungs and chronic wounds. In these infections the bacteria are present as aggregates suspended in host secretions. During the course of the infections there is a selection for mutants that overproduce exopolysaccharides, suggesting that the exopolysaccharides play a role in the persistence and antibiotic tolerance of the aggregated bacteria. Here, we investigated the role of individual P. aeruginosa exopolysaccharides in aggregate-associated antibiotic tolerance. We employed an aggregate-based antibiotic tolerance assay on a set of P. aeruginosa strains that were genetically engineered to over-produce a single, none, or all of the three exopolysaccharides Pel, Psl, and alginate. The antibiotic tolerance assays were conducted with the clinically relevant antibiotics tobramycin, ciprofloxacin and meropenem. Our study suggests that alginate plays a role in the tolerance of P. aeruginosa aggregates toward tobramycin and meropenem, but not ciprofloxacin. However, contrary to previous studies we did not observe a role for Psl or Pel in the tolerance of P. aeruginosa aggregates toward tobramycin, ciprofloxacin, and meropenem.
RESUMO
Efficient colonization of mucosal surfaces is essential for opportunistic pathogens like Pseudomonas aeruginosa, but how bacteria collectively and individually adapt to optimize adherence, virulence and dispersal is largely unclear. Here we identified a stochastic genetic switch, hecR-hecE, which is expressed bimodally and generates functionally distinct bacterial subpopulations to balance P. aeruginosa growth and dispersal on surfaces. HecE inhibits the phosphodiesterase BifA and stimulates the diguanylate cyclase WspR to increase c-di-GMP second messenger levels and promote surface colonization in a subpopulation of cells; low-level HecE-expressing cells disperse. The fraction of HecE+ cells is tuned by different stress factors and determines the balance between biofilm formation and long-range cell dispersal of surface-grown communities. We also demonstrate that the HecE pathway represents a druggable target to effectively counter P. aeruginosa surface colonization. Exposing such binary states opens up new ways to control mucosal infections by a major human pathogen.
Assuntos
Aderência Bacteriana , Pseudomonas aeruginosa , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/fisiologia , BiofilmesRESUMO
Overproduction of the exopolysaccharide alginate contributes to the pathogenicity and antibiotic tolerance of Pseudomonas aeruginosa in chronic infections. The second messenger, c-di-GMP, is a positive regulator of the production of various biofilm matrix components and is known to regulate alginate synthesis at the posttranslational level in P. aeruginosa. We provide evidence that c-di-GMP also regulates transcription of the alginate operon in P. aeruginosa. Previous work has shown that transcription of the alginate operon is regulated by nine different proteins, AmrZ, AlgP, IHFα, IHFß, CysB, Vfr, AlgR, AlgB, and AlgQ, and we investigated if some of these proteins function as a c-di-GMP effector. We found that deletion of algP, algQ, IHFα, and IHFß had only a marginal effect on the transcription of the alginate operon. Deletion of vfr and cysB led to decreased transcription of the alginate operon, and the dependence of the c-di-GMP level was less pronounced, indicating that Vfr and CysB could be partially required for c-di-GMP-mediated regulation of alginate operon transcription. Our experiments indicated that the AmrZ, AlgR, and AlgB proteins are absolutely required for transcription of the alginate operon. However, differential radial capillary action of ligand assay (DRaCALA) and site-directed mutagenesis indicated that c-di-GMP does not bind to any of the AmrZ, AlgR, and AlgB proteins. IMPORTANCE The proliferation of alginate-overproducing P. aeruginosa variants in the lungs of cystic fibrosis patients often leads to chronic infection. The alginate functions as a biofilm matrix that protects the bacteria against host immune defenses and antibiotic treatment. Knowledge about the regulation of alginate synthesis is important in order to identify drug targets for the development of medicine against chronic P. aeruginosa infections. We provide evidence that c-di-GMP positively regulates transcription of the alginate operon in P. aeruginosa. Moreover, we revisited the role of the known alginate regulators, AmrZ, AlgP, IHFα, IHFß, CysB, Vfr, AlgR, AlgB, and AlgQ, and found that their effect on transcription of the alginate operon is highly varied. Deletion of algP, algQ, IHFα, or IHFß only had a marginal effect on transcription of the alginate operon, whereas deletion of vfr or cysB led to decreased transcription and deletion of amrZ, algR, or algB abrogated transcription.
Assuntos
Regulação Bacteriana da Expressão Gênica , Pseudomonas aeruginosa , Alginatos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , Humanos , Óperon , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismoRESUMO
Microbial biofilms are involved in a number of infections that cannot be cured, as microbes in biofilms resist host immune defenses and antibiotic therapies. With no strict biofilm-antibiotic in the current pipelines, there is an unmet need for drug candidates that enable the current antibiotics to eradicate bacteria in biofilms. We used high-throughput screening to identify chemical compounds that reduce the intracellular c-di-GMP content in Pseudomonas aeruginosa. This led to the identification of a small molecule that efficiently depletes P. aeruginosa for c-di-GMP, inhibits biofilm formation, and disperses established biofilm. A combination of our lead compound with standard of care antibiotics showed improved eradication of an implant-associated infection established in mice. Genetic analyses provided evidence that the anti-biofilm compound stimulates the activity of the c-di-GMP phosphodiesterase BifA in P. aeruginosa. Our work constitutes a proof of concept for c-di-GMP phosphodiesterase-activating drugs administered in combination with antibiotics as a viable treatment strategy for otherwise recalcitrant infections.
Assuntos
Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , GMP Cíclico/análogos & derivados , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/fisiologia , Transdução de Sinais/efeitos dos fármacos , Animais , Antibacterianos/química , Cromatografia Líquida de Alta Pressão , GMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Descoberta de Drogas , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Sequenciamento de Nucleotídeos em Larga Escala , Camundongos , Espectrometria de Massas em Tandem , TranscriptomaRESUMO
A decade of research has shown that the molecule c-di-GMP functions as a central second messenger in many bacteria. A high level of c-di-GMP is associated with biofilm formation, whereas a low level of c-di-GMP is associated with a planktonic single-cell bacterial lifestyle. c-di-GMP is formed by diguanylate cyclases and is degraded by specific phosphodiesterases. We previously presented evidence that the ectopic expression of the Escherichia coli phosphodiesterase YhjH in Pseudomonas aeruginosa results in biofilm dispersal. More recently, however, evidence has been presented that the induction of native c-di-GMP phosphodiesterases does not lead to a dispersal of P. aeruginosa biofilms. The latter result may discourage attempts to use c-di-GMP signaling as a target for the development of antibiofilm drugs. However, here, we demonstrate that the induction of the P. aeruginosa c-di-GMP phosphodiesterases PA2133 and BifA indeed results in the dispersal of P. aeruginosa biofilms in both a microtiter tray biofilm assay and a flow cell biofilm system.
Assuntos
Proteínas de Escherichia coli , Pseudomonas aeruginosa , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biofilmes , GMP Cíclico/análogos & derivados , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismoRESUMO
Bacteria have evolved distinct molecular mechanisms as a defense against oxidative stress. The foremost regulator of the oxidative stress response has been found to be OxyR. However, the molecular details of regulation upstream of OxyR remain largely unknown and need further investigation. Here, we characterize an oxidative stress and antibiotic tolerance regulator, OsaR (PA0056), produced by Pseudomonas aeruginosa Knocking out of osaR increased bacterial tolerance to aminoglycoside and ß-lactam antibiotics, as well as to hydrogen peroxide. Expression of the oxyR regulon genes oxyR, katAB, and ahpBCF was increased in the osaR mutant. However, the OsaR protein does not regulate the oxyR regulon genes through direct binding to their promoters. PA0055, osaR, PA0057, and dsbM are in the same gene cluster, and we provide evidence that expression of those genes involved in oxidant tolerance is controlled by the binding of OsaR to the intergenic region between osaR and PA0057, which contain two divergent promoters. The gene cluster is also regulated by PA0055 via an indirect effect. We further discovered that OsaR formed intramolecular disulfide bonds when exposed to oxidative stress, resulting in a change of its DNA binding affinity. Taken together, our results indicate that OsaR is inactivated by oxidative stress and plays a role in the tolerance of P. aeruginosa to aminoglycoside and ß-lactam antibiotics.
Assuntos
Pseudomonas aeruginosa , Regulon , Aminoglicosídeos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Peróxido de Hidrogênio/metabolismo , Oxirredução , Estresse Oxidativo , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Regulon/genética , TransativadoresRESUMO
Microbial biofilms are the cause of persistent infections associated with various medical implants and distinct body sites such as the urinary tract, lungs, and wounds. Compared with their free living counterparts, bacteria in biofilms display a highly increased resistance to immune system activities and antibiotic treatment. Therefore, biofilm infections are difficult or impossible to treat with our current armory of antibiotics. The challenges associated with biofilm infections have urged researchers to pursue a better understanding of the molecular mechanisms that are involved in the formation and dispersal of biofilms, and this has led to the identification of several steps that could be targeted in order to eradicate these challenging infections. Here we describe mechanisms that are involved in the regulation of biofilm development in Pseudomonas aeruginosa, Escherichia coli, and Acinetobacter baumannii, and provide examples of chemical compounds that have been developed to specifically inhibit these processes. These compounds include (i) pilicides and curlicides which inhibit the initial steps of biofilm formation by E. coli; (ii) compounds that interfere with c-di-GMP signaling in P. aeruginosa and E. coli; and (iii) compounds that inhibit quorum-sensing in P. aeruginosa and A. baumannii. In cases where compound series have a defined molecular target, we focus on elucidating structure activity relationship (SAR) trends within the particular compound series.
RESUMO
The human pathogen Pseudomonas aeruginosa can cause both acute infections and chronic biofilm-based infections. Expression of acute virulence factors is positively regulated by cAMP, whereas biofilm formation is positively regulated by c-di-GMP. We provide evidence that increased levels of cAMP, caused by either a lack of degradation or increased production, inhibit P. aeruginosa biofilm formation. cAMP-mediated inhibition of P. aeruginosa biofilm formation required Vfr, and involved a reduction of the level of c-di-GMP, as well as reduced production of biofilm matrix components. A mutant screen and characterization of defined knockout mutants suggested that a subset of c-di-GMP-degrading phosphodiesterases is involved in cAMP-Vfr-mediated biofilm inhibition in P. aeruginosa.
Assuntos
Biofilmes/crescimento & desenvolvimento , AMP Cíclico/metabolismo , GMP Cíclico/análogos & derivados , Pseudomonas aeruginosa/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteína Receptora de AMP Cíclico/genética , Proteína Receptora de AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Matriz Extracelular de Substâncias Poliméricas/metabolismo , Mutação , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/metabolismoRESUMO
Bacteria in the biofilm mode of growth cause numerous problematic infections due to their resistance to antimicrobials and the immune system. Because conventional antimicrobial compounds cannot efficiently eradicate biofilm infections, we urgently need new efficient anti-biofilm drugs. The secondary messenger c-di-GMP is a positive regulator of biofilm formation in many clinically relevant bacteria, and it is assumed that drugs that lower the intracellular level of c-di-GMP will force biofilm bacteria into a more treatable planktonic lifestyle. We describe a protocol for high-throughput screening of chemical libraries for compounds that lower the c-di-GMP level in bacteria, and potentially can serve as lead compounds in the development of novel biofilm dismantling drugs.
Assuntos
Bactérias/efeitos dos fármacos , Bactérias/metabolismo , GMP Cíclico/análogos & derivados , Descoberta de Drogas/métodos , Ensaios de Triagem em Larga Escala , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Antibacterianos/farmacologia , Bactérias/crescimento & desenvolvimento , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , GMP Cíclico/metabolismo , Bibliotecas de Moléculas Pequenas , Fluxo de TrabalhoRESUMO
Biofilm infections caused by Pseudomonas aeruginosa are frequently treated with ciprofloxacin (CIP); however, resistance rapidly develops. One of the primary resistance mechanisms is the overexpression of the MexCD-OprJ pump due to a mutation in nfxB, encoding the transcriptional repressor of this pump. The aim of this study was to investigate the effect of subinhibitory concentrations of CIP on the occurrence of nfxB mutants in the wild-type PAO1 flow cell biofilm model. For this purpose, we constructed fluorescent reporter strains (PAO1 background) with an mCherry tag for constitutive red fluorescence and chromosomal transcriptional fusion between the P mexCD promoter and gfp leading to green fluorescence upon mutation of nfxB We observed a rapid development of nfxB mutants by live confocal laser scanning microscopy (CLSM) imaging of the flow cell biofilm (reaching 80 to 90% of the whole population) when treated with 1/10 minimal biofilm inhibitory concentration of CIP for 24 h and 96 h. Based on the observed developmental stages, we propose that nfxB mutants emerged de novo in the biofilm during CIP treatment from filamentous cells, which might have arisen due to the stress responses induced by CIP. Identical nfxB mutations were found in fluorescent colonies from the same flow cell biofilm, especially in 24-h biofilms, suggesting selection and clonal expansion of the mutants during biofilm growth. Our findings point at the significant role of high-enough antibiotic dosages or appropriate combination therapy to avoid the emergence of resistant mutants in biofilms.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Ciprofloxacina/farmacologia , Proteínas de Ligação a DNA/genética , Farmacorresistência Bacteriana/genética , Regulação Bacteriana da Expressão Gênica , Mutação , Pseudomonas aeruginosa/genética , Fatores de Transcrição/genética , Proteínas de Bactérias/metabolismo , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Proteínas de Ligação a DNA/metabolismo , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Testes de Sensibilidade Microbiana , Microscopia Confocal , Microscopia de Fluorescência , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Reologia , Seleção Genética , Imagem com Lapso de Tempo , Fatores de Transcrição/metabolismo , Proteína Vermelha FluorescenteRESUMO
Current antibiotic treatments are insufficient in eradicating bacterial biofilms, which represent the primary cause of chronic bacterial infections. Thus, there is an urgent need for new strategies to eradicate biofilm infections. The second messenger c-di-GMP is a positive regulator of biofilm formation in many clinically relevant bacteria. It is hypothesized that drugs lowering the intracellular level of c-di-GMP will force biofilm bacteria into a more treatable planktonic lifestyle. To identify compounds capable of lowering c-di-GMP levels in Pseudomonas aeruginosa, we screened 5000 compounds for their potential c-di-GMP-lowering effect using a recently developed c-di-GMP biosensor strain. Our screen identified the anti-cancerous drug doxorubicin as a potent c-di-GMP inhibitor. In addition, the drug decreased the transcription of many biofilm-related genes. However, despite its effect on the c-di-GMP content in P. aeruginosa, doxorubicin was unable to inhibit biofilm formation or disperse established biofilms. On the contrary, the drug was found to promote P. aeruginosa biofilm formation, possibly through release of extracellular DNA from a subpopulation of killed bacteria. Our findings emphasize that lowering of the c-di-GMP content in bacteria might not be sufficient to mediate biofilm inhibition or dispersal.
Assuntos
Antineoplásicos/farmacologia , Biofilmes/efeitos dos fármacos , GMP Cíclico/análogos & derivados , Doxorrubicina/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , GMP Cíclico/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/fisiologiaRESUMO
Stress response plays an important role on microbial adaptation under hostile environmental conditions. It is generally unclear how the signaling transduction pathway mediates a stress response in planktonic and biofilm modes of microbial communities simultaneously. Here, we showed that metalloid tellurite (TeO3(2-)) exposure induced the intracellular content of the secondary messenger cyclic di-GMP (c-di-GMP) of Pseudomonas aeruginosa. Two diguanylate cyclases (DGCs), SadC and SiaD, were responsible for the increased intracellular content of c-di-GMP. Enhanced c-di-GMP levels by TeO3(2-) further increased P. aeruginosa biofilm formation and resistance to TeO3(2-). P. aeruginosa ΔsadCΔsiaD and PAO1/p(lac)-yhjH mutants with low intracellular c-di-GMP content were more sensitive to TeO3(2-) exposure and had low relative fitness compared to the wild-type PAO1 planktonic and biofilm cultures exposed to TeO3(2-). Our study provided evidence that c-di-GMP level can play an important role in mediating stress response in microbial communities during both planktonic and biofilm modes of growth.
Assuntos
Biofilmes/efeitos dos fármacos , GMP Cíclico/análogos & derivados , Pseudomonas aeruginosa/efeitos dos fármacos , Telúrio/toxicidade , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , GMP Cíclico/metabolismo , Farmacorresistência Bacteriana , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Microscopia Eletrônica de Varredura , Fósforo-Oxigênio Liases/genética , Fósforo-Oxigênio Liases/metabolismo , Proteoma/análise , Pseudomonas aeruginosa/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Espectrometria por Raios XRESUMO
We previously reported that the non-digestible carbohydrates inulin and apple pectin promoted Listeria monocytogenes infection in guinea pigs, whereas xylo- and galacto-oligosaccharides (XOS and GOS), prevented infection by this pathogen. In the present study, mechanisms that could explain the previous in vivo observations were explored. Mixing bacterial cultures with XOS significantly (P < 0.05) decreased the ability of two out of three strains of L. monocytogenes to adhere to Caco-2 cells. Additionally, 2 h incubation with XOS followed by washing of the bacteria significantly (P < 0.05) decreased the ability of all three strains to adhere to Caco-2 cells. Consistently, expression of the adhesion-relevant genes inlA and lap was reduced by the presence of XOS. The observation that XOS inhibit the adhesion of Listeria to the intestinal epithelium in vitro may explain the reported preventive effect of XOS on Listeria infection in guinea pigs in vivo, while the preventive effect of GOS was not explicable by the assays chosen here.
Assuntos
Aderência Bacteriana/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Enterócitos/microbiologia , Glucuronatos/farmacologia , Listeria monocytogenes/fisiologia , Listeriose/microbiologia , Oligossacarídeos/farmacologia , Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Células CACO-2 , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Humanos , Listeria monocytogenes/efeitos dos fármacos , Listeria monocytogenes/genéticaRESUMO
We here present a method based on qRT-PCR to quantify E. coli LF82 in intestinal human samples. Two different primer-probe sets were designed to detect LF82, and a third to target total E. coli. The assay showed high robustness and specificity for detection of LF82 in the presence of intestinal tissue.
Assuntos
Doença de Crohn/microbiologia , Escherichia coli/isolamento & purificação , Mucosa Intestinal/microbiologia , Reação em Cadeia da Polimerase/métodos , Primers do DNA/genética , Escherichia coli/genética , HumanosRESUMO
Three different Listeria monocytogenes strains, LO28 (a laboratory strain with truncated InlA), 4446 (a clinical isolate) and 7291 (a food isolate), were compared in a guinea-pig model designed to mimic food-borne exposure. The objectives were (1) to verify the applicability of the animal model for distinguishing between Listeria with different virulence properties and (2) to explore whether it was possible to reduce the required number of animals by dosing with mixed cultures instead of monocultures. Consistent with in vitro observations of infectivity in Caco-2 cells, faecal densities and presence in selected organs were considerably lower for LO28 than for the other two strains. Additionally, the animal study revealed a difference in prevalence in faeces as well as in internal organs between the clinical isolate and the food isolate, which was not reproduced in vitro. Dosage with monocultures of Listeria strains gave similar results as dosage with a mixture of the three strains; thus, the mixed infection approach was a feasible way to reduce the number of animals needed for determination of listerial virulence.