Heterogeneous Colistin-Resistance Phenotypes Coexisting in Stenotrophomonas maltophilia Isolates Influence Colistin Susceptibility Testing

The polymyxin antibiotic colistin shows in vitro activity against Stenotrophomonas maltophilia. However, an increased incidence of colistin-resistant isolates has been recently observed. In addition, in vitro evaluation of colistin susceptibility for this organism has been problematic. The aims of this study were to investigate the colistin-resistance phenotypes displayed by S. maltophilia and their potential association with the challenging determination of colistin susceptibilities for this organism by even the recommended method. Colistin-resistance phenotypes were inferred by use of the recommended broth microdilution method in different clinical isolates of S. maltophilia. Most of the strains showed non-interpretable minimum inhibitory concentrations (MICs) for colistin due to an incomplete growth inhibition in wells of the microdilution plate. In addition, the subpopulation of bacteria resistant to colistin showed an increased ability to form biofilms on the plastic surface of MIC plates. The observed incomplete growth inhibition in the microdilution plates is compatible with a progressive adaptation to colistin or a heterogeneous susceptibility to this antibiotic. Therefore, to determine the existence of heteroresistance or adaptive resistance, four colistin-resistant clinical isolates were subjected to serial Etest assays, growth rate analyses, and the population analysis profile test. The experiments indicated that these S. maltophilia isolates display a colistin-resistant sub-population that survives and multiplies in the presence of the antibiotic. Interestingly, this phenomenon might not be explainable by the natural background mutation rate alone since the development of a resistant sub-population occurred upon the contact with the antibiotic and it was reversible. This complex colistin-resistance phenotype is exhibited differently by the different isolates and significantly affected colistin susceptibility testing. Furthermore, it can coexist with adaptive resistance to colistin as response to pre-incubation with sub-inhibitory concentrations of the antibiotic. Overall, the combined action of heterogeneous colistin-resistance mechanisms in S. maltophilia isolates, including colistin-induced biofilm formation, may hamper the correct interpretation of colistin susceptibility tests, thus having potentially serious implications on antimicrobial-therapy decision making

Draft genome sequence of Stenotrophomonas maltophilia strain M30, isolated from a chronic pressure ulcer in an elderly patient

Stenotrophomonas maltophilia is an emerging opportunistic pathogen with an increasing prevalence of multidrug-resistant strains. Here, we report the draft genome sequence of S. maltophilia strain M30, isolated from a pressure ulcer in an elderly patient

Stenotrophomonas maltophilia responds to exogenous AHL signals through the LuxR solo SmoR (Smlt1839)

Quorum Sensing (QS) mediated by Acyl Homoserine Lactone (AHL) molecules are probably the most widespread and studied among Gram-negative bacteria. Canonical AHL systems are composed by a synthase (LuxI family) and a regulator element (LuxR family), whose genes are usually adjacent in the genome. However, incomplete AHL-QS machinery lacking the synthase LuxI is frequently observed in Proteobacteria, and the regulator element is then referred as LuxR solo. It has been shown that certain LuxR solos participate in interspecific communication by detecting signals produced by different organisms. In the case of Stenotrophomonas maltophilia, a preliminary genome sequence analysis revealed numerous putative luxR genes, none of them associated to a luxI gene. From these, the hypothetical LuxR solo Smlt1839, here designated SmoR, presents a conserved AHL binding domain and a helix-turn-helix DNA binding motif. Its genomic organization-adjacent to hchA gene-indicate that SmoR belongs to the new family "LuxR regulator chaperone HchA-associated." AHL-binding assays revealed that SmoR binds to AHLs in-vitro, at least to oxo-C8-homoserine lactone, and it regulates operon transcription, likely by recognizing a conserved palindromic regulatory box in the hchA upstream region. Supplementation with concentrated supernatants from Pseudomonas aeruginosa, which contain significant amounts of AHLs, promoted swarming motility in S. maltophilia. Contrarily, no swarming stimulation was observed when the P. aeruginosa supernatant was treated with the lactonase AiiA from Bacillus subtilis, confirming that AHL contributes to enhance the swarming ability of S. maltophilia. Finally, mutation of smoR resulted in a swarming alteration and an apparent insensitivity to the exogenous AHLs provided by P. aeruginosa. In conclusion, our results demonstrate that S. maltophilia senses AHLs produced by neighboring bacteria through the LuxR solo SmoR, regulating population behaviors such as swarming motility

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

Decoding the genetic and functional diversity of the DSF Quorum-Sensing system in Stenotrophomonas maltophilia

Stenotrophomonas maltophilia uses the Diffusible Signal Factor (DSF) quorum sensing (QS) system to mediate intra- and inter-specific signaling and regulate virulence-related processes. The components of this system are encoded by the rpf cluster, with genes rpfF and rpfC encoding for the DSF synthase RpfF and sensor RpfC, respectively. Recently, we have shown that there exist two variants of the rpf cluster (rpf-1 and rpf-2), distinguishing two groups of S. maltophilia strains. Surprisingly, only rpf-1 strains produce detectable DSF, correlating with their ability to control biofilm formation, swarming motility and virulence. The evolutive advantage of acquiring two different rpf clusters, the phylogenetic time point and mechanism of this acquisition and the conditions that activate DSF production in rpf-2 strains, are however not known. Examination of this cluster in various species suggests that its variability originated most probably by genetic exchange between rhizosphere bacteria. We propose that rpf-2 variant strains make use of a strategy recently termed as "social cheating." Analysis of cellular and extracellular fatty acids (FAs) of strains E77 (rpf-1) and M30 (rpf-2) suggests that their RpfFs have also a thioesterase activity that facilitates the release of unspecific FAs to the medium in addition to DSF. Production of DSF in rpf-1 strains appears in fact to be modulated by some of these extracellular FAs in addition to other factors such as temperature and nutrients, while in rpf-2 strains DSF biosynthesis is derepressed only upon detection of DSF itself, suggesting that they require cohabitation with DSF-producer bacteria to activate their DSF regulatory machinery. Finally, we show that the mixed rpf-1/rpf-2 population presents synergism in DSF production and virulence capacity in an in vivo infection model. Recovery and quantification of DSF from co-infected animals correlates with the observed mortality rate

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

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

Fenotips de resistència a antibiòtics en Stenotrophomonas maltophilia: Impacte en la susceptibilitat a la colistina

Stenotrophomonas maltophilia és un patogen oportunista de rellevància creixent a causa de la seva ascendent prevalença com a nosocomial. Tot i que es tracta d’un microorganisme ambiental amb una virulència limitada, S. maltophilia presenta resistència intrínseca a múltiples antibiòtics i pot produir múltiples infeccions, especialment en pacients immunocompromesos o amb fibrosi quística. Aquest treball de tesi s’engloba dins d’un projecte enfocat a la recerca i estudi de determinants de virulència i resistència a S. maltophilia, i el seu objectiu general és l’estudi de fenotips de resistència, en particular front colistina. La colistina és un antibiòtic polipeptídic que s'està utilitzant cada vegada més com a opció alternativa o "d'últim recurs" per tractar infeccions causades per organismes gramnegatius multiresistents. La primera part del treball confirma l'heterogeneïtat fenotípica i genotípica de S. maltophilia en una col·lecció de 78 aïllats clínics europeus. Els genotips s'han determinat mitjançant l’estudi del perfil MLST i el gen rpfF. També s’ha avaluat la susceptibilitat antimicrobiana, la formació de biofilms i la virulència. De manera general, no existeix associació entre les variables genotípiques i els fenotips de resistència i virulència avaluats, tot i que hi ha un grup genòmic, àmpliament distribuït geogràficament, que destaca per la seva capacitat de formar biofilm i la seva virulència. També, s’ha demostrat que les soques amb variant rpfF-2 són més formadores de biofilm que les rpfF-1. S’ha confirmat que els antibiòtics més eficaços contra S. maltophilia són el trimetroprim-sulfamethoxazol i la minociclina, malgrat hi hagi un 68,8% de soques multiresistents (MDR) i detectar-se una gran heterogeneïtat en la resistència a la colistina dins de cada soca. A la segona part, s’han estudiat les possibles causes de la resistència heterogènia a la colistina. Per avaluar la resistència adaptativa i l’heteroresistència, els aïllats van ser sotmesos a assajos Etest consecutius, a anàlisis de taxes de creixement i a assajos PAP (Perfil d’anàlisi poblacional). Els resultats han revelat que la majoria (78,7%) dels aïllats presenten una varietat de mecanismes de resistència poblacional diferents, incloent resistència adaptativa i heteroresistència. Curiosament, el desenvolupament de la subpoblació resistent ha estat reversible. Aquest fenotip complex de resistència a colistina, que hem anomenat “heteroresistència adaptativa”, es presenta de manera diferent entre els aïllats i afecta significativament les proves recomanades de susceptibilitat a la colistina. Finalment, s’han descrit els possibles determinants genètics que participen en els fenotips de resistència a la colistina. Específicament, s'han validat experimentalment els homòlegs cromosòmics dels gens mcr d’E. coli (smlt2804 i smlt3571), i els que codifiquen per a la bomba d’expulsió SmeGH (smlt3170-3171) i un component del sistema regulador ParRS. Mitjançant mutants de la soca K279a, s’ha demostrat que smlt3571 està directament implicat en la resistència a la colistina ja que la soca deficient per aquest gen és més susceptible. D'altra banda, smlt2804 probablement té un paper important en la resistència adaptativa a colistina també mitjançant modificació del lipopolisacàrid (LPS). Així mateix, s’ha demostrat que ParRS contribueix a la resistència adaptativa a colistina però depenent de la concentració de Mg2+. La bomba d’expulsió SmeGH no s’ha associat a la resistència a la colistina, però s’ha determinat que l'eliminació de SmeGH redueix la formació de biofilms induïda per antibiòtics. Tenint present tot el que s’ha exposat, S. maltophilia té la capacitat d’adaptar-se i esdevenir més resistent a la colistina mitjançant una combinació de mecanismes reguladors que podrien provocar la modificació del LPS. Aquests mecanismes, a més a més, varien tant a nivell poblacional dins d’una mateixa soca com entre les diferents soques circulants. Per aquests motius, doncs, la colistina no hauria de ser considerada com una opció terapèutica rutinària contra aquest bacteri. Malgrat això, no s’ha de descartar com fàrmac d’últim recurs i, consegüentment, són necessaris més estudis sobre els mecanismes de resistència a aquest fàrmac, així com també, estudis per al disseny de noves estratègies antimicrobianes.Stenotrophomonas maltophilia is an opportunistic pathogen of growing relevance due to its increasing prevalence in the nosocomial settings. Albeit an enviromental microorganism with limited virulence, S. maltophilia shows intrinsic resistance to multiple antibiotics and can produce a broad spectrum of clinical infections, especially in immunocompromised patients or people with cystic fibrosis. This thesis is a part of a project dealing with the search and study of virulence and resistance determinants in S. maltophilia, and its main objective is to study resistance phenotypes, particularly to colistin. Colistin is a polypeptide antibiotic that is being used increasingly as alternative or “last-resort” option to treat infections by multiresistant Gram-negative bacteria. In the first part of this work, the phenotypic and genotypic heterogeneity of S. maltophilia is confirmed in a collection of 78 european clinical isolates. Genotypes were determined by analyzing MLST profiles and sequences of the rpfF gene. Antimicrobial susceptibility, biofilm formation and virulence were also determined in all isolates. In general, no associations between genotypic and phenotypic variables were found, although there was a genomic group, widely distributed geographically, which stood out due to the ability to form biofilm and the virulence of its members. In addition, rpfF-2 strains were more biofilm producer than rpfF-1.It was confirmed that the most effective antibiotics against S. maltophilia are trimethroprim-sulfamethoxazole and minocycline, despite most of isolates (68.8%) were considered MDR and to identify heterogeneous resistance phenotypes to colistin. In the second part, we investigated that heterogeneous resistance to colistin. To evaluate adaptive resistance and heteroresistance, the isolates were subjected to serial Etest assays, growth rate analyses, and the population analysis profile test. Our studies revealed that most of our S. maltophilia isolates (78.7%) display different populational resistance mechanisms including adaptative resistance and heteroresistance. Interestingly, the development of the resistant subpopulation was reversible. This complex colistin-resistance phenotype, we have termed adaptive hetero-resistance, was displayed differently by the isolates and significantly affected to recommended colistin susceptibility testing. Finally, in the last part of this work, possible genetic determinants for colistin resistance were described. Among them we validated two chromosomal homologs of the E. coli mcr genes (smlt2804 and smlt3571), and those encoding for the SmeGH efflux pump (smlt3170-3171) and a component of the ParRS regulatory system. By site-specific mutagenesis in the K279a strain, we have shown that smlt3571 is directly involved in colistin resistance since a strain defective in this enzyme is more susceptible to this antibiotic. On the other hand, smlt2804 probably plays an important role in adaptive resistance to colistin also by modification of lipopolysaccharide (LPS). Moreover, we demonstrated that ParRS contributed to adaptive resistance to colistin, but in this case in response to the extracellular concentration of Mg2+. The SmeGH efflux pump was not associated to colistin resistance, but we demonstrated that deletion of the smeGH operon reduce antibiotic-induced biofilm formation. Taking all together, we have shown that S. maltophilia has the ability to adapt and to become more resistant to colistin by a combination of regulatory mechanisms that could cause, ultimately, modifications in the LPS. Moreover, these mechanisms vary both at a population level within the same strain and between different circulating strains. For these reasons, colistin should not be considered as a routine therapeutic option against this bacterium. However, it should not be ruled out as a last-resort drug and, consequently, further studies about the mechanisms of colistin resistance are needed, as well as for the design of novel antimicrobial strategies

Heterogeneous Colistin-Resistance Phenotypes Coexisting in Stenotrophomonas maltophilia Isolates Influence Colistin Susceptibility Testing

The polymyxin antibiotic colistin shows in vitro activity against Stenotrophomonas maltophilia. However, an increased incidence of colistin-resistant isolates has been recently observed. In addition, in vitro evaluation of colistin susceptibility for this organism has been problematic. The aims of this study were to investigate the colistin-resistance phenotypes displayed by S. maltophilia and their potential association with the challenging determination of colistin susceptibilities for this organism by even the recommended method. Colistin-resistance phenotypes were inferred by use of the recommended broth microdilution method in different clinical isolates of S. maltophilia. Most of the strains showed non-interpretable minimum inhibitory concentrations (MICs) for colistin due to an incomplete growth inhibition in wells of the microdilution plate. In addition, the subpopulation of bacteria resistant to colistin showed an increased ability to form biofilms on the plastic surface of MIC plates. The observed incomplete growth inhibition in the microdilution plates is compatible with a progressive adaptation to colistin or a heterogeneous susceptibility to this antibiotic. Therefore, to determine the existence of heteroresistance or adaptive resistance, four colistin-resistant clinical isolates were subjected to serial Etest assays, growth rate analyses, and the population analysis profile test. The experiments indicated that these S. maltophilia isolates display a colistin-resistant sub-population that survives and multiplies in the presence of the antibiotic. Interestingly, this phenomenon might not be explainable by the natural background mutation rate alone since the development of a resistant sub-population occurred upon the contact with the antibiotic and it was reversible. This complex colistin-resistance phenotype is exhibited differently by the different isolates and significantly affected colistin susceptibility testing. Furthermore, it can coexist with adaptive resistance to colistin as response to pre-incubation with sub-inhibitory concentrations of the antibiotic. Overall, the combined action of heterogeneous colistin-resistance mechanisms in S. maltophilia isolates, including colistin-induced biofilm formation, may hamper the correct interpretation of colistin susceptibility tests, thus having potentially serious implications on antimicrobial-therapy decision makin

Heterogeneous colistin-resistance phenotypes coexisting in stenotrophomonas maltophiliaisolates influence colistin susceptibility testing

The polymyxin antibiotic colistin shows in vitro activity against Stenotrophomonas maltophilia. However, an increased incidence of colistin-resistant isolates has been recently observed. In addition, in vitro evaluation of colistin susceptibility for this organism has been problematic. The aims of this study were to investigate the colistin-resistance phenotypes displayed by S. maltophilia and their potential association with the challenging determination of colistin susceptibilities for this organism by even the recommended method. Colistin-resistance phenotypes were inferred by use of the recommended broth microdilution method in different clinical isolates of S. maltophilia. Most of the strains showed non-interpretable minimum inhibitory concentrations (MICs) for colistin due to an incomplete growth inhibition in wells of the microdilution plate. In addition, the subpopulation of bacteria resistant to colistin showed an increased ability to form biofilms on the plastic surface of MIC plates. The observed incomplete growth inhibition in the microdilution plates is compatible with a progressive adaptation to colistin or a heterogeneous susceptibility to this antibiotic. Therefore, to determine the existence of heteroresistance or adaptive resistance, four colistin-resistant clinical isolates were subjected to serial Etest assays, growth rate analyses, and the population analysis profile test. The experiments indicated that these S. maltophilia isolates display a colistin-resistant sub-population that survives and multiplies in the presence of the antibiotic. Interestingly, this phenomenon might not be explainable by the natural background mutation rate alone since the development of a resistant sub-population occurred upon the contact with the antibiotic and it was reversible. This complex colistin-resistance phenotype is exhibited differently by the different isolates and significantly affected colistin susceptibility testing. Furthermore, it can coexist with adaptive resistance to colistin as response to pre-incubation with sub-inhibitory concentrations of the antibiotic. Overall, the combined action of heterogeneous colistin-resistance mechanisms in S. maltophilia isolates, including colistin-induced biofilm formation, may hamper the correct interpretation of colistin susceptibility tests, thus having potentially serious implications on antimicrobial-therapy decision makin