Draft genome sequence of Stenotrophomonas maltophilia strain UV74 reveals extensive variability within its genomic group

We report the draft genome sequence of Stenotrophomonas maltophilia UV74, isolated from a vascular ulcer. This draft genome sequence shall contribute to the understanding of the evolution and pathogenicity of this species, particularly regarding isolates of clinical origin

Quorum sensing signaling and quenching in the multidrug-resistant pathogen stenotrophomonas maltophilia

Stenotrophomonas maltophilia is an opportunistic Gram-negative pathogen with increasing incidence in clinical settings. The most critical aspect of S. maltophilia is its frequent resistance to a majority of the antibiotics of clinical use. Quorum Sensing (QS) systems coordinate bacterial populations and act as major regulatory mechanisms of pathogenesis in both pure cultures and poly-microbial communities. Disruption of QS systems, a phenomenon known as Quorum Quenching (QQ), represents a new promising paradigm for the design of novel antimicrobial strategies. In this context, we review the main advances in the field of QS in S. maltophilia by paying special attention to Diffusible Signal Factor (DSF) signaling, Acyl Homoserine Lactone (AHL) responses and the controversial Ax21 system. Advances in the DSF system include regulatory aspects of DSF synthesis and perception by both rpf -1 and rpf -2 variant systems, as well as their reciprocal communication. Interaction via DSF of S. maltophilia with unrelated organisms including bacteria, yeast and plants is also considered. Finally, an overview of the different QQ mechanisms involving S. maltophilia as quencher and as object of quenching is presented, revealing the potential of this species for use in QQ applications. This review provides a comprehensive snapshot of the interconnected QS network that S. maltophilia uses to sense and respond to its surrounding biotic or abiotic environment. Understanding such cooperative and competitive communication mechanisms is essential for the design of effective anti QS strategies

Genetic variants of the DSF quórum sensing system in Stenotrophomonas maltophilia influence virulence and resistance phenotypes among genotypically diverse clinical isoaltes

The pathogenicity of Stenotrophomonas maltophilia is regulated in part by its quorum sensing (QS) system. The main QS signaling molecule in S. maltophilia is known as diffusible signal factor (DSF), and the rpf gene cluster is responsible for its synthesis and perception. Two cluster variants have been previously described, rpf-1 and rpf-2, which differ basically in the conditions under which DSF is produced. Here, correlations between the rpf variant and antibiotic susceptibility, LPS electrophoretic profiles and virulence-related phenotypes were evaluated for a collection of 78 geographically and genetically diverse clinical strains of S. maltophilia. In general there were associations between previously established genogroups and the genetic variant of the rpf cluster. However, only few genotype-phenotype correlations could be observed. Resistance to the β-lactam antibiotics ceftazidime and ticarcillin was associated with strains carrying the rpf-1 variant, whereas strains of variant rpf-2, particularly those of genogroup C, showed higher resistance levels to colistin. Strains of variant rpf-2 were also significantly more virulent to Galleria mellonella larvae than those of rpf-1, most likely due to an increased ability of rpf-2 strains to form biofilms. A comparative genomic analysis revealed the presence of proteins unique to individual genogroups. In particular, the strains of genogroup C share an operon that encodes for a new virulence determinant in S. maltophilia related to the synthesis of an alternative Flp/Tad pilus. Overall, this study establishes a link between the DSF-based QS system and the virulence and resistance phenotypes in this species, and identifies potential high-risk clones circulating in European hospitals

Genetic Variants of the DSF Quorum Sensing System in Stenotrophomonas maltophilia Influence Virulence and Resistance Phenotypes Among Genotypically Diverse Clinical Isolates

Altres ajuts: Departament de Salut, Generalitat de Catalunya: SLT002/16/00349The pathogenicity of Stenotrophomonas maltophilia is regulated in part by its quorum sensing (QS) system. The main QS signaling molecule in S. maltophilia is known as diffusible signal factor (DSF), and the rpf gene cluster is responsible for its synthesis and perception. Two cluster variants have been previously described, rpf -1 and rpf -2, which differ basically in the conditions under which DSF is produced. Here, correlations between the rpf variant and antibiotic susceptibility, LPS electrophoretic profiles and virulence-related phenotypes were evaluated for a collection of 78 geographically and genetically diverse clinical strains of S. maltophilia. In general there were associations between previously established genogroups and the genetic variant of the rpf cluster. However, only few genotype-phenotype correlations could be observed. Resistance to the β-lactam antibiotics ceftazidime and ticarcillin was associated with strains carrying the rpf -1 variant, whereas strains of variant rpf -2, particularly those of genogroup C, showed higher resistance levels to colistin. Strains of variant rpf -2 were also significantly more virulent to Galleria mellonella larvae than those of rpf -1, most likely due to an increased ability of rpf -2 strains to form biofilms. A comparative genomic analysis revealed the presence of proteins unique to individual genogroups. In particular, the strains of genogroup C share an operon that encodes for a new virulence determinant in S. maltophilia related to the synthesis of an alternative Flp/Tad pilus. Overall, this study establishes a link between the DSF-based QS system and the virulence and resistance phenotypes in this species, and identifies potential high-risk clones circulating in European hospitals

Improved mini-Tn 7 Delivery Plasmids for Fluorescent Labeling of

Fluorescently labeled bacterial cells have become indispensable for many aspects of microbiological research, including studies on biofilm formation as an important virulence factor of various opportunistic bacteria of environmental origin such as . Using a Tn 7 -based genomic integration system, we report the construction of improved mini-Tn 7 delivery plasmids for labeling of with sfGFP, mCherry, tdTomato and mKate2 by expressing their codon-optimized genes from a strong, constitutive promoter and an optimized ribosomal binding site. Transposition of the mini-Tn 7 transposons into single neutral sites located on average 25 nucleotides downstream of the 3'-end of the conserved glmS gene of different wild-type strains did not have any adverse effects on the fitness of their fluorescently labeled derivatives. This was demonstrated by comparative analyses of growth, resistance profiles against 18 antibiotics of different classes, the ability to form biofilms on abiotic and biotic surfaces, also independent of the fluorescent protein expressed, and virulence in . It is also shown that the mini-Tn 7 elements remained stably integrated in the genome of over a prolonged period of time in the absence of antibiotic selection pressure. Overall, we provide evidence that the new improved mini-Tn 7 delivery plasmids are valuable tools for generating fluorescently labeled strains that are indistinguishable in their properties from their parental wild-type strains. IMPORTANCE The bacterium is an important opportunistic nosocomial pathogen that can cause bacteremia and pneumonia in immunocompromised patients with a high rate of mortality. It is now considered as a clinically relevant and notorious pathogen in cystic fibrosis patients but has also been isolated from lung specimen of healthy donors. The high intrinsic resistance to a wide range of antibiotics complicates treatment and most likely contributes to the increasing incidence of infections worldwide. One important virulence-related trait of is the ability to form biofilms on any surface, which may result in the development of increased transient phenotypic resistance to antimicrobials. The significance of our work is to provide a mini-Tn 7 -based labeling system for to study the mechanisms of biofilm formation or host-pathogen interactions with live bacteria under non-destructive conditions

Factors de Resistència i Virulència Relacionats amb el Quorum Sensing i la Regulació del Metabolisme i Homeòstasi Lipídica en Stenotrophomonas maltophilia

Stenotrophomonas maltophilia és un patogen oportunista que està adquirint importància donat l'augment en la prevalença d'infeccions nosocomials. Tot i ser un microorganisme ambiental amb una virulència limitada, S. maltophilia presenta una resistència intrínseca a múltiples antibiòtics podent causar infeccions persistents especialment en pacients immunocompromesos i pacients amb fibrosi quística. En els darrers anys s'ha profunditzat en l'estudi dels mecanismes de virulència implicats en les infeccions per S. maltophilia i dels seus mecanismes de regulació. Se sap que el Quòrum Sensing (QS) és l'eina principal de comunicació intercel·lular emprada per a microorganismes patògens per a regular funcions implicades en la seva virulència. S. maltophilia presenta un sistema de QS mediat per la molècula autoinductora (AI) de naturalesa lipídica DSF (Diffusible Signal Factor) i aquest està implicat en l'expressió de factors de virulència. Més enllà d'aquest QS mediat per DSF, també té un regulador que detecta molècules AI de la família AHL (N-acil-L-homoserina lactona) anomenat SmoR. Aquest treball s'ha centrat en primer lloc en caracteritzar la resposta a nivell d'expressió gènica global als dos tipus de senyals AIs per a trobar mecanismes implicats en patogènesi i virulència. També s'ha investigat el grup de gens que poden estar regulats per SmoR per tal de determinar les funcions biològiques d'aquest regulador. Aquesta primera part ha permés determinar la resposta a nivell transcriptòmic a situacions d'elevada densitat cel·lular, com la fase estacionària de creixement i la presència exògena d'AHLs i DSF. Cal destacar la interconnexió observada entre tots dos sistemes, indicant un punt de regulació comuna, i la presència de diferents reguladors transcripcionals. Un dels gens implicats, amb una gran sobreexpressió en totes les condicions estudiades, ha permés caracteritzar un regulador global del metabolisme d'àcids grassos en S. maltophilia, la proteïna Smlt2053. S'ha identificat com a un ortòleg del regulador PsrA en Pseudomonas aeruginosa, que està implicat en la regulació global de la fisiologia bacteriana. En S. maltophilia, però, tot sembla indicar que la seva principal funció és la regulació de la β-oxidació d'àcids grassos. La fita més important ha estat identificar aquest regulador com a un receptor citoplasmàtic de DSF, la senyal principal de QS en Xanthomonadaceae i produïda endògenament per S. maltophilia. A més, també és capaç de sensar l'àcid gras més abundant en aquesta espècie, l'àcid iso-pentadecanoic (iso-C15:0). Amb tot, aquest regulador presenta un vincle entre el QS i el metabolisme energètic, suggerint que S. maltophilia utilitza les senyals de QS com a font de carboni en els ambients complexes on sol habitar. Finalment, s'ha caracteritzat el sistema Mla (maintenance of lípid asymmetry) en S. maltophilia. Aquest sistema participa en el transport de fosfolípids deslocalitzats de la membrana externa cap al citoplasma, on sembla que poden ser reciclats pel metabolisme lipídic. A S. maltophilia aquest sistema s'ha vist implicat en fenotips de morfologia cel·lular, resistència a antibiòtics, formació de biofilm i permeabilitat de la membrana. La caracterització de la proteïna periplasmàtica d'unió a substrat MlaC d'aquest sistema ha permés verificar la seva afinitat, com a mínim, per fosfolípids de 14 carbonis, incloent l'àcid gras iso-C15:0. Amb tot, es posa de manifest la rellevància dels àcids grassos i altres molècules de naturalesa lipídica com a senyalitzadors cel·lulars, implicat en una gran diversitat de fenotips en S. maltophilia. La regulació del metabolisme i homeòstasi lipídica en S. maltophilia podrien constituir dianes per a una intervenció antimicrobiana o una atenuació de la seva virulència.Stenotrophomonas maltophilia is an opportunistic pathogen that is becoming relevant given the increase in the prevalence of nosocomial infections. Despite being an environmental microorganism with limited virulence, S. maltophilia presents an intrinsic resistance to multiple antibiotics and can cause persistent infections, especially in immunocompromised patients and patients with cystic fibrosis. In recent years, the study of the virulence mechanisms involved in S. maltophilia infections and their regulatory mechanisms have been deeply investigated. Quorum Sensing (QS) is known to be the main intercellular communication system used for pathogenic microorganisms to regulate functions involved in their virulence. S. maltophilia presents a QS system mediated by the autoinducer (AI) molecule of lipid nature DSF (Diffusible Signal Factor) which is involved in the expression of virulence factors. Beyond this QS system mediated by DSF, S. maltophilia also has a regulator that sense AI molecules from the AHL (N-Acyl-L-homoserine lactone) family, known as SmoR. This work has first focused in the characterization of the global gene expression under the presence of these two AI signals, to find mechanisms involved in pathogenesis and virulence. The group of genes that might be regulated by SmoR have also been investigated to determine the biological role of this regulator. This first part has allowed to determine the response at transcriptomic level to situations of high cell density, such as the stationary growth phase and the exogenous presence of AHLs and DSF. It is worth highlighting the observed interconnection between both systems, indicating a common regulatory pathway and the presence of different transcriptional regulators. One of the genes involved, with considerable overexpression in all the conditions studied, has allowed to characterize a global regulator of fatty acid metabolism in S. maltophilia, the protein Smlt2053. In S. maltophilia, however, results seem to indicate that its main function is the regulation of the fatty acid β-oxidation. The main milestone has been to identify this regulator as a cytoplasmic DSF receptor, the main signal of QS in Xanthomonadaceae which is endogenously produced by S. maltophilia. In addition, it is also able to sense the most abundant fatty acid in this species, iso-pentadecanoic acid (iso-C15:0). Altogether, this regulator presents a link between QS and energy metabolism, suggesting that S. maltophilia uses QS signals as a carbon source in the complex environments where it usually inhabits. Finally, the Mla system (maintenance of lipid asymmetry) has been characterized in S. maltophilia. This system participates in the transport of delocalized phospholipids from the outer membrane to the cytoplasm, where it seems that they can be recycled by lipid metabolism. In S. maltophilia this system has shown to be involved in diverse phenotypes, such as cell morphology, antibiotic resistance, biofilm formation and membrane permeability. The characterization of the periplasmic substrate-binding protein MlaC of this system has allowed to verify its affinity, at least, for phospholipids of 14 carbons, including iso-C15:0. Overall, the relevance of fatty acids and other molecules of lipidic nature as cell communication signals is revealed, which are also involved in a great diversity of phenotypes in S. maltophilia. The regulation of lipid metabolism and homeostasis in S. maltophilia could constitute a target for antimicrobials or virulence attenuation

Factors de Resistència i Virulència Relacionats amb el Quorum Sensing i la Regulació del Metabolisme i Homeòstasi Lipídica en Stenotrophomonas maltophilia

Stenotrophomonas maltophilia és un patogen oportunista que està adquirint importància donat l’augment en la prevalença d’infeccions nosocomials. Tot i ser un microorganisme ambiental amb una virulència limitada, S. maltophilia presenta una resistència intrínseca a múltiples antibiòtics podent causar infeccions persistents especialment en pacients immunocompromesos i pacients amb fibrosi quística. En els darrers anys s’ha profunditzat en l’estudi dels mecanismes de virulència implicats en les infeccions per S. maltophilia i dels seus mecanismes de regulació. Se sap que el Quòrum Sensing (QS) és l’eina principal de comunicació intercel·lular emprada per a microorganismes patògens per a regular funcions implicades en la seva virulència. S. maltophilia presenta un sistema de QS mediat per la molècula autoinductora (AI) de naturalesa lipídica DSF (Diffusible Signal Factor) i aquest està implicat en l’expressió de factors de virulència. Més enllà d’aquest QS mediat per DSF, també té un regulador que detecta molècules AI de la família AHL (N-acil-L-homoserina lactona) anomenat SmoR. Aquest treball s’ha centrat en primer lloc en caracteritzar la resposta a nivell d’expressió gènica global als dos tipus de senyals AIs per a trobar mecanismes implicats en patogènesi i virulència. També s’ha investigat el grup de gens que poden estar regulats per SmoR per tal de determinar les funcions biològiques d’aquest regulador. Aquesta primera part ha permés determinar la resposta a nivell transcriptòmic a situacions d’elevada densitat cel·lular, com la fase estacionària de creixement i la presència exògena d’AHLs i DSF. Cal destacar la interconnexió observada entre tots dos sistemes, indicant un punt de regulació comuna, i la presència de diferents reguladors transcripcionals. Un dels gens implicats, amb una gran sobreexpressió en totes les condicions estudiades, ha permés caracteritzar un regulador global del metabolisme d’àcids grassos en S. maltophilia, la proteïna Smlt2053. S’ha identificat com a un ortòleg del regulador PsrA en Pseudomonas aeruginosa, que està implicat en la regulació global de la fisiologia bacteriana. En S. maltophilia, però, tot sembla indicar que la seva principal funció és la regulació de la β-oxidació d’àcids grassos. La fita més important ha estat identificar aquest regulador com a un receptor citoplasmàtic de DSF, la senyal principal de QS en Xanthomonadaceae i produïda endògenament per S. maltophilia. A més, també és capaç de sensar l’àcid gras més abundant en aquesta espècie, l’àcid iso-pentadecanoic (iso-C15:0). Amb tot, aquest regulador presenta un vincle entre el QS i el metabolisme energètic, suggerint que S. maltophilia utilitza les senyals de QS com a font de carboni en els ambients complexes on sol habitar. Finalment, s’ha caracteritzat el sistema Mla (maintenance of lípid asymmetry) en S. maltophilia. Aquest sistema participa en el transport de fosfolípids deslocalitzats de la membrana externa cap al citoplasma, on sembla que poden ser reciclats pel metabolisme lipídic. A S. maltophilia aquest sistema s’ha vist implicat en fenotips de morfologia cel·lular, resistència a antibiòtics, formació de biofilm i permeabilitat de la membrana. La caracterització de la proteïna periplasmàtica d’unió a substrat MlaC d’aquest sistema ha permés verificar la seva afinitat, com a mínim, per fosfolípids de 14 carbonis, incloent l’àcid gras iso-C15:0. Amb tot, es posa de manifest la rellevància dels àcids grassos i altres molècules de naturalesa lipídica com a senyalitzadors cel·lulars, implicat en una gran diversitat de fenotips en S. maltophilia. La regulació del metabolisme i homeòstasi lipídica en S. maltophilia podrien constituir dianes per a una intervenció antimicrobiana o una atenuació de la seva virulència.Stenotrophomonas maltophilia is an opportunistic pathogen that is becoming relevant given the increase in the prevalence of nosocomial infections. Despite being an environmental microorganism with limited virulence, S. maltophilia presents an intrinsic resistance to multiple antibiotics and can cause persistent infections, especially in immunocompromised patients and patients with cystic fibrosis. In recent years, the study of the virulence mechanisms involved in S. maltophilia infections and their regulatory mechanisms have been deeply investigated. Quorum Sensing (QS) is known to be the main intercellular communication system used for pathogenic microorganisms to regulate functions involved in their virulence. S. maltophilia presents a QS system mediated by the autoinducer (AI) molecule of lipid nature DSF (Diffusible Signal Factor) which is involved in the expression of virulence factors. Beyond this QS system mediated by DSF, S. maltophilia also has a regulator that sense AI molecules from the AHL (N-Acyl-L-homoserine lactone) family, known as SmoR. This work has first focused in the characterization of the global gene expression under the presence of these two AI signals, to find mechanisms involved in pathogenesis and virulence. The group of genes that might be regulated by SmoR have also been investigated to determine the biological role of this regulator. This first part has allowed to determine the response at transcriptomic level to situations of high cell density, such as the stationary growth phase and the exogenous presence of AHLs and DSF. It is worth highlighting the observed interconnection between both systems, indicating a common regulatory pathway and the presence of different transcriptional regulators. One of the genes involved, with considerable overexpression in all the conditions studied, has allowed to characterize a global regulator of fatty acid metabolism in S. maltophilia, the protein Smlt2053. In S. maltophilia, however, results seem to indicate that its main function is the regulation of the fatty acid β-oxidation. The main milestone has been to identify this regulator as a cytoplasmic DSF receptor, the main signal of QS in Xanthomonadaceae which is endogenously produced by S. maltophilia. In addition, it is also able to sense the most abundant fatty acid in this species, iso-pentadecanoic acid (iso-C15:0). Altogether, this regulator presents a link between QS and energy metabolism, suggesting that S. maltophilia uses QS signals as a carbon source in the complex environments where it usually inhabits. Finally, the Mla system (maintenance of lipid asymmetry) has been characterized in S. maltophilia. This system participates in the transport of delocalized phospholipids from the outer membrane to the cytoplasm, where it seems that they can be recycled by lipid metabolism. In S. maltophilia this system has shown to be involved in diverse phenotypes, such as cell morphology, antibiotic resistance, biofilm formation and membrane permeability. The characterization of the periplasmic substrate-binding protein MlaC of this system has allowed to verify its affinity, at least, for phospholipids of 14 carbons, including iso-C15:0. Overall, the relevance of fatty acids and other molecules of lipidic nature as cell communication signals is revealed, which are also involved in a great diversity of phenotypes in S. maltophilia. The regulation of lipid metabolism and homeostasis in S. maltophilia could constitute a target for antimicrobials or virulence attenuation.Universitat Autònoma de Barcelona. Programa de Doctorat en Microbiologi

Quorum Sensing Signaling and Quenching in the Multidrug-Resistant Pathogen Stenotrophomonas maltophilia

Stenotrophomonas maltophilia is an opportunistic Gram-negative pathogen with increasing incidence in clinical settings. The most critical aspect of S. maltophilia is its frequent resistance to a majority of the antibiotics of clinical use. Quorum Sensing (QS) systems coordinate bacterial populations and act as major regulatory mechanisms of pathogenesis in both pure cultures and poly-microbial communities. Disruption of QS systems, a phenomenon known as Quorum Quenching (QQ), represents a new promising paradigm for the design of novel antimicrobial strategies. In this context, we review the main advances in the field of QS in S. maltophilia by paying special attention to Diffusible Signal Factor (DSF) signaling, Acyl Homoserine Lactone (AHL) responses and the controversial Ax21 system. Advances in the DSF system include regulatory aspects of DSF synthesis and perception by both rpf-1 and rpf-2 variant systems, as well as their reciprocal communication. Interaction via DSF of S. maltophilia with unrelated organisms including bacteria, yeast and plants is also considered. Finally, an overview of the different QQ mechanisms involving S. maltophilia as quencher and as object of quenching is presented, revealing the potential of this species for use in QQ applications. This review provides a comprehensive snapshot of the interconnected QS network that S. maltophilia uses to sense and respond to its surrounding biotic or abiotic environment. Understanding such cooperative and competitive communication mechanisms is essential for the design of effective anti QS strategies

Quorum sensing signaling and quenching in the multidrug-resistant pathogen stenotrophomonas maltophilia

Stenotrophomonas maltophilia is an opportunistic Gram-negative pathogen with increasing incidence in clinical settings. The most critical aspect of S. maltophilia is its frequent resistance to a majority of the antibiotics of clinical use. Quorum Sensing (QS) systems coordinate bacterial populations and act as major regulatory mechanisms of pathogenesis in both pure cultures and poly-microbial communities. Disruption of QS systems, a phenomenon known as Quorum Quenching (QQ), represents a new promising paradigm for the design of novel antimicrobial strategies. In this context, we review the main advances in the field of QS in S. maltophilia by paying special attention to Diffusible Signal Factor (DSF) signaling, Acyl Homoserine Lactone (AHL) responses and the controversial Ax21 system. Advances in the DSF system include regulatory aspects of DSF synthesis and perception by both rpf -1 and rpf -2 variant systems, as well as their reciprocal communication. Interaction via DSF of S. maltophilia with unrelated organisms including bacteria, yeast and plants is also considered. Finally, an overview of the different QQ mechanisms involving S. maltophilia as quencher and as object of quenching is presented, revealing the potential of this species for use in QQ applications. This review provides a comprehensive snapshot of the interconnected QS network that S. maltophilia uses to sense and respond to its surrounding biotic or abiotic environment. Understanding such cooperative and competitive communication mechanisms is essential for the design of effective anti QS strategies