Origin of the mobile di-hydro-pteroate synthase gene determining sulfonamide resistance in clinical isolates

Sulfonamides are synthetic chemotherapeutic agents that work as competitive inhibitors of the di-hydro-pteroate synthase (DHPS) enzyme, encoded by the folP gene. Resistance to sulfonamides is widespread in the clinical setting and predominantly mediated by plasmid- and integron-borne sul1-3 genes encoding mutant DHPS enzymes that do not bind sulfonamides. In spite of their clinical importance, the genetic origin of sul1-3 genes remains unknown. Here we analyze sul genes and their genetic neighborhoods to uncover sul signature elements that enable the elucidation of their genetic origin. We identify a protein sequence Sul motif associated with sul-encoded proteins, as well as consistent association of a phosphoglucosamine mutase gene (glmM) with the sul2 gene. We identify chromosomal folP genes bearing these genetic markers in two bacterial families: the Rhodobiaceae and the Leptospiraceae. Bayesian phylogenetic inference of FolP/Sul and GlmM protein sequences clearly establishes that sul1-2 and sul3 genes originated as a mobilization of folP genes present in, respectively, the Rhodobiaceae and the Leptospiraceae, and indicate that the Rhodobiaceae folP gene was transferred from the Leptospiraceae. Analysis of %GC content in folP/sul gene sequences supports the phylogenetic inference results and indicates that the emergence of the Sul motif in chromosomally encoded FolP proteins is ancient and considerably predates the clinical introduction of sulfonamides. In vitro assays reveal that both the Rhodobiaceae and the Leptospiraceae, but not other related chromosomally encoded FolP proteins confer resistance in a sulfonamide-sensitive Escherichia coli background, indicating that the Sul motif is associated with sulfonamide resistance. Given the absence of any known natural sulfonamides targeting DHPS, these results provide a novel perspective on the emergence of resistance to synthetic chemotherapeutic agents, whereby preexisting resistant variants in the vast bacterial pangenome may be rapidly selected for and disseminated upon the clinical introduction of novel chemotherapeuticals

Flexible comparative genomics of prokaryotic transcriptional regulatory networks

Comparative genomics methods enable the reconstruction of bacterial regulatory networks using available experimental data. In spite of their potential for accelerating research into the composition and evolution of bacterial regulons, few comparative genomics suites have been developed for the automated analysis of these regulatory systems. Available solutions typically rely on precomputed databases for operon and ortholog predictions, limiting the scope of analyses to processed complete genomes, and several key issues such as the transfer of experimental information or the integration of regulatory information in a probabilistic setting remain largely unaddressed. Here we introduce CGB, a flexible platform for comparative genomics of prokaryotic regulons. CGB has few external dependencies and enables fully customized analyses of newly available genome data. The platform automates the merging of experimental information and uses a gene-centered, Bayesian framework to generate and integrate easily interpretable results. We demonstrate its flexibility and power by analyzing the evolution of type III secretion system regulation in pathogenic Proteobacteria and by characterizing the SOS regulon of a new bacterial phylum, the Balneolaeota. Our results demonstrate the applicability of the CGB pipeline in multiple settings. CGB's ability to automatically integrate experimental information from multiple sources and use complete and draft genomic data, coupled with its non-reliance on precomputed databases and its easily interpretable display of gene-centered posterior probabilities of regulation provide users with an unprecedented level of flexibility in launching comparative genomics analyses of prokaryotic transcriptional regulatory networks. The analyses of type III secretion and SOS response regulatory networks illustrate instances of convergent and divergent evolution of these regulatory systems, showcasing the power of formal ancestral state reconstruction at inferring the evolutionary history of regulatory networks

Origin of the Mobile Di-Hydro-Pteroate Synthase Gene Determining Sulfonamide Resistance in Clinical Isolates

Sulfonamides are synthetic chemotherapeutic agents that work as competitive inhibitors of the di-hydro-pteroate synthase (DHPS) enzyme, encoded by the folP gene. Resistance to sulfonamides is widespread in the clinical setting and predominantly mediated by plasmid- and integron-borne sul1-3 genes encoding mutant DHPS enzymes that do not bind sulfonamides. In spite of their clinical importance, the genetic origin of sul1-3 genes remains unknown. Here we analyze sul genes and their genetic neighborhoods to uncover sul signature elements that enable the elucidation of their genetic origin. We identify a protein sequence Sul motif associated with sul-encoded proteins, as well as consistent association of a phosphoglucosamine mutase gene (glmM) with the sul2 gene. We identify chromosomal folP genes bearing these genetic markers in two bacterial families: the Rhodobiaceae and the Leptospiraceae. Bayesian phylogenetic inference of FolP/Sul and GlmM protein sequences clearly establishes that sul1-2 and sul3 genes originated as a mobilization of folP genes present in, respectively, the Rhodobiaceae and the Leptospiraceae, and indicate that the Rhodobiaceae folP gene was transferred from the Leptospiraceae. Analysis of %GC content in folP/sul gene sequences supports the phylogenetic inference results and indicates that the emergence of the Sul motif in chromosomally encoded FolP proteins is ancient and considerably predates the clinical introduction of sulfonamides. In vitro assays reveal that both the Rhodobiaceae and the Leptospiraceae, but not other related chromosomally encoded FolP proteins confer resistance in a sulfonamide-sensitive Escherichia coli background, indicating that the Sul motif is associated with sulfonamide resistance. Given the absence of any known natural sulfonamides targeting DHPS, these results provide a novel perspective on the emergence of resistance to synthetic chemotherapeutic agents, whereby preexisting resistant variants in the vast bacterial pangenome may be rapidly selected for and disseminated upon the clinical introduction of novel chemotherapeuticals

Exploration into the origins and mobilization of di-hydrofolate reductase genes and the emergence of clinical resistance to trimethoprim

Trimethoprim is a synthetic antibacterial agent that targets folate biosynthesis by competitively binding to the di-hydrofolate reductase enzyme (DHFR). Trimethoprim is often administered synergistically with sulfonamide, another chemotherapeutic agent targeting the di-hydropteroate synthase (DHPS) enzyme in the same pathway. Clinical resistance to both drugs is widespread and mediated by enzyme variants capable of performing their biological function without binding to these drugs. These mutant enzymes were assumed to have arisen after the discovery of these synthetic drugs, but recent work has shown that genes conferring resistance to sulfonamide were present in the bacterial pangenome millions of years ago. Here, we apply phylogenetics and comparative genomics methods to study the largest family of mobile trimethoprim-resistance genes (dfrA). We show that most of the dfrA genes identified to date map to two large clades that likely arose from independent mobilization events. In contrast to sulfonamide resistance (sul) genes, we find evidence of recurrent mobilization in dfrA genes. Phylogenetic evidence allows us to identify novel dfrA genes in the emerging pathogen , and we confirm their resistance phenotype in vitro. We also identify a cluster of dfrA homologues in cryptic plasmid and phage genomes, but we show that these enzymes do not confer resistance to trimethoprim. Our methods also allow us to pinpoint the chromosomal origin of previously reported dfrA genes, and we show that many of these ancient chromosomal genes also confer resistance to trimethoprim. Our work reveals that trimethoprim resistance predated the clinical use of this chemotherapeutic agent, but that novel mutations have likely also arisen and become mobilized following its widespread use within and outside the clinic. Hence, this work confirms that resistance to novel drugs may already be present in the bacterial pangenome, and stresses the importance of rapid mobilization as a fundamental element in the emergence and global spread of resistance determinants

Importance of twitching and surface-associated motility in the virulence of Acinetobacter baumannii

Acinetobacter baumannii is a pathogen of increasing clinical importance worldwide, especially given its ability to readily acquire resistance determinants. Motile strains of this bacterium can move by either or both of two types of motility: (i) twitching, driven by type IV pili, and (ii) surface-associated motility, an appendage-independent form of movement. A. baumannii strain MAR002 possesses both twitching and surface-associated motility. In this study, we isolated spontaneous rifampin-resistant mutants of strain MAR002 in which point mutations in the rpoB gene were identified that resulted in an altered motility pattern. Transcriptomic analysis of mutants lacking twitching, surface-associated motility, or both led to the identification of deregulated genes within each motility phenotype, based on their level of expression and their biological function. Investigations of the corresponding knockout mutants revealed several genes involved in the motility of A. baumannii strain MAR002, including two involved in twitching (encoding a minor pilin subunit and an RND [resistance nodulation division] component), one in surface-associated motility (encoding an amino acid permease), and eight in both (encoding RND and ABC components, the energy transducer TonB, the porin OprD, the T6SS component TagF, an IclR transcriptional regulator, a PQQ-dependent sugar dehydrogenase, and a putative pectate lyase). Virulence assays showed the reduced pathogenicity of mutants with impairments in both types of motility or in surface-associated motility alone. By contrast, the virulence of twitching-affected mutants was not affected. These results shed light on the key role of surface-associated motility and the limited role of twitching in the pathogenicity of A. baumannii

Impact of mutagenesis and lateral gene transfer processes in bacterial susceptibility to phage in food biocontrol and phage therapy

IntroductionThe emergence of resistance and interference mechanisms to phage infection can hinder the success of bacteriophage-based applications, but the significance of these mechanisms in phage therapy has not been determined. This work studies the emergence of Salmonella isolates with reduced susceptibility to a cocktail of three phages under three scenarios: i) Salmonella cultures (LAB), ii) biocontrol of cooked ham slices as a model of food safety (FOOD), and iii) oral phage therapy in broilers (PT).MethodsS. Typhimurium ATCC 14028 RifR variants with reduced phage susceptibility were isolated from the three scenarios and conventional and molecular microbiology techniques were applied to study them.Results and discussionIn LAB, 92% of Salmonella isolates lost susceptibility to all three phages 24 h after phage infection. This percentage was lower in FOOD, with 4.3% of isolates not susceptible to at least two of the three phages after seven days at 4°C following phage treatment. In PT, 9.7% and 3.3 % of isolates from untreated and treated broilers, respectively, displayed some mechanism of interference with the life cycle of some of the phages. In LAB and FOOD scenarios, resistant variants carrying mutations in rfc and rfaJ genes involved in lipopolysaccharide synthesis (phage receptor) were identified. However, in PT, the significant decrease of EOP, ECOI, and burst size observed in isolates was prompted by lateral gene transfer of large IncI1 plasmids, which may encode phage defense mechanisms. These data indicate that the acquisition of specific conjugative plasmids has a stronger impact than mutagenesis on the emergence of reduced phage-susceptibility bacteria in certain environments. In spite of this, neither mechanism seems to significantly impair the success of Salmonella biocontrol and oral phage therapy

A 3-biomarker 2-point-based risk stratification strategy in acute heart failure

[Abstract] Introduction and Objectives: Most multi-biomarker strategies in acute heart failure (HF) have only measured biomarkers in a single-point time. This study aimed to evaluate the prognostic yielding of NT-proBNP, hsTnT, Cys-C, hs-CRP, GDF15, and GAL-3 in HF patients both at admission and discharge. Methods: We included 830 patients enrolled consecutively in a prospective multicenter registry. Primary outcome was 12-month mortality. The gain in the C-index, calibration, net reclassification improvement (NRI), and integrated discrimination improvement (IDI) was calculated after adding each individual biomarker value or their combination on top of the best clinical model developed in this study (C-index 0.752, 0.715–0.789) and also on top of 4 currently used scores (MAGGIC, GWTG-HF, Redin-SCORE, BCN-bioHF). Results: After 12-month, death occurred in 154 (18.5%) cases. On top of the best clinical model, the addition of NT-proBNP, hs-CRP, and GDF-15 above the respective cutoff point at admission and discharge and their delta during compensation improved the C-index to 0.782 (0.747–0.817), IDI by 5% (p < 0.001), and NRI by 57% (p < 0.001) for 12-month mortality. A 4-risk grading categories for 12-month mortality (11.7, 19.2, 26.7, and 39.4%, respectively; p < 0.001) were obtained using combination of these biomarkers. Conclusion: A model including NT-proBNP, hs-CRP, and GDF-15 measured at admission and discharge afforded a mortality risk prediction greater than our clinical model and also better than the most currently used scores. In addition, this 3-biomarker panel defined 4-risk categories for 12-month mortality.Instituto de Salud Carlos III; RD06-0003-0000Instituto de Salud Carlos III; RD12/0042/000

A 3-Biomarker 2-Point-Based Risk Stratification Strategy in Acute Heart Failure

Altres ajuts: ISCIII/RD06-0003-0000Altres ajuts: ISCIII/RD12/0042/0002Introduction and Objectives: Most multi-biomarker strategies in acute heart failure (HF) have only measured biomarkers in a single-point time. This study aimed to evaluate the prognostic yielding of NT-proBNP, hsTnT, Cys-C, hs-CRP, GDF15, and GAL-3 in HF patients both at admission and discharge. Methods: We included 830 patients enrolled consecutively in a prospective multicenter registry. Primary outcome was 12-month mortality. The gain in the C-index, calibration, net reclassification improvement (NRI), and integrated discrimination improvement (IDI) was calculated after adding each individual biomarker value or their combination on top of the best clinical model developed in this study (C-index 0.752, 0.715-0.789) and also on top of 4 currently used scores (MAGGIC, GWTG-HF, Redin-SCORE, BCN-bioHF). Results: After 12-month, death occurred in 154 (18.5%) cases. On top of the best clinical model, the addition of NT-proBNP, hs-CRP, and GDF-15 above the respective cutoff point at admission and discharge and their delta during compensation improved the C-index to 0.782 (0.747-0.817), IDI by 5% (p < 0.001), and NRI by 57% (p < 0.001) for 12-month mortality. A 4-risk grading categories for 12-month mortality (11.7, 19.2, 26.7, and 39.4%, respectively; p < 0.001) were obtained using combination of these biomarkers. Conclusion: A model including NT-proBNP, hs-CRP, and GDF-15 measured at admission and discharge afforded a mortality risk prediction greater than our clinical model and also better than the most currently used scores. In addition, this 3-biomarker panel defined 4-risk categories for 12-month mortality

Origen y evolución de los genes que confieren resistencia clínica a sulfamidas y a trimetoprim

La resistència als compostos antibacterians és un greu problema a la sanitat moderna, tant humana com animal. Està ampliament demostrat que la resistència als antibiòtics va sorgir fa milions d’anys a bacteris productors d’aquests compostos o als seus competidors. No obstant això, la resistència als agents quimioterapèutics no es pot explicar pel mateix mecanisme, ja que no són produïts de forma natural. Per tant, clàssicament s’ha assumit que la resistència a aquest tipus de compostos va emergir i va evolucionar ràpidament a partir de la seva aplicación a la teràpia antibacteriana. La resistència clínica a les sulfamides i al trimetoprim es deu principalment a la presència dels gens mòbils sul i dfr, respectivament. Aquests gens codifiquen versions alternatives i funcionals dels enzims dihidropteroat sintasa i dihidrofolat reductasa que no són inhibits per l’acció d’aquests antibacterians, a diferència dels gens bacterians folP i folA que codifiquen les variants cromosòmiques d’aquests enzims i que són típicament sensibles als citats agents antibacterians. L’objectiu central de la present Tesis Doctoral ha estat la identificació de l’origen dels gens que confereixen resistència clínica a les sulfamides i al trimetoprim, així com l’anàlisis del procés evolutiu que ha donat lloc a les variants enzimàtiques actuals. Així, s’han analitzat les seqüències dels gens sul i el seu entorn amb l’objectiu de detectar elements que permetessin l’elucidació del seu origen. Com a conseqüència d’aquesta anàlisi, s’ha identificat un motiu Sul present a les seqüències de les proteïnes codificades pels gens mòbils sul1, sul2 i sul3, que confereixen resistència a les sulfamides, així com la associació física a múltiples plasmidis del gen sul2 amb un fragment del gen glmM que codifica una fosfoglucomutasa. L’estudi comparatiu des les seqüències dels gens folP cromosòmics de tot el domini Bacteria ha permet identificar la presencia del motiu Sul a les proteïnes codificades pels gens folP de dues famílies bacterianes: Rhodobiaceae i Leptospiraceae. L’estudi filogenètic de les proteïnes FolP/Sul i GlmM ha establert clarament que els gens sul1-2 i sul3 es van originar mitjançant sengles processos de mobilització independents dels gens folP cromosòmics presents, respectivament, a Rhodobiaceae i Leptospiraceae. Tanmateix, els experiments in vitro han revelat que els gens folP codificats a Rhodobiaceae i Leptospiraceae confereixen resistència a les sulfamides. Aquests resultats indiquen que la resistència a les sulfamides és ancestral i anterior a la introducció clínica d’aquest agent antibcterià. D’altra banda, es va analitzar l’origen i la evolució dels gens mòbils dfrA que confereixen resistència al trimetoprim. Al contrari del que s’ha descrit pels gens sul, els resultats obtinguts de l’anàlisis de les seqüències dels gens dfrA mòbils indiquen que aquests s’han originat mitjançant events múltiples de transferència lateral de gens folA cromosòmics. Així mateix, s’ha pogut establir que la majoria dels gens cromosòmics folA predecessors dels dfrA també confereixen resistència al trimetoprim. Tot això revela que la resistència al trimetoprim determinada per elements mòbils es pot explicar per dos processos independents i complementaris: i) la captació de gens folA cromosòmics, portadors d’una mutación ancestral responsable d’aquesta resistència i que es va originar en absència del quimitoeràpic, i ii) la transferència de mutants espontanis cromosòmics del gen folA, presents a una població bacteriana coetània amb l’exposició al citat antibacterià, i havent estat seleccionats per aquesta exposició.La resistencia a los compuestos antibacterianos es un grave problema en la sanidad moderna, tanto humana como animal. Está ampliamente demostrado que la resistencia a los antibióticos surgió hace millones de años en bacterias productoras de estos compuestos o en sus competidoras. No obstante, la resistencia a los agentes antibacterianos sintéticos no se puede explicar por el mismo mecanismo, ya que no se producen de forma natural. Por consiguiente, se ha asumido que la resistencia a este tipo de compuestos emergió y ha evolucionado rápidamente a partir de su aplicación en la terapia antibacteriana. La resistencia clínica a las sulfamidas y al trimetoprim se debe principalmente a la presencia de los genes móviles sul y dfr, respectivamente. Estos genes codifican versiones alternativas y funcionales de las enzimas dihidropteroato sintasa y dihidrofolato reductasa que no son inhibidas por la acción de estos antibacterianos, a diferencia de los genes bacterianos folP y folA que codifican las variantes cromosómicas de estos enzimas, típicamente sensibles a los citados agentes quimioterapéuticos. El objetivo central de la presente Tesis Doctoral ha sido la identificación del origen de los genes que confieren resistencia clínica a las sulfamidas y al trimetoprim, así como el análisis del proceso evolutivo que ha dado lugar a las actuales variantes enzimáticas. Para ello, se han analizado las secuencias de los genes sul y su entorno para detectar elementos que permitiesen la elucidación de su origen. De este modo, se ha identificado un motivo Sul presente en las secuencias de las proteínas codificadas por los genes móviles sul1, sul2 y sul3, que confieren resistencia a las sulfamidas, así como la asociación física en múltiples plásmidos del gen sul2 con un fragmento del gen glmM que codifica una fosfoglucomutasa. El estudio comparativo de las secuencias de los genes folP cromosómicos de todo el dominio Bacteria ha permitido identificar la presencia del motivo Sul en las proteínas codificadas en los genes folP de dos familias bacterianas: Rhodobiaceae y Leptospiraceae. El análisis filogenético de las proteínas FolP/Sul y GlmM ha establecido claramente que los genes sul1-2 y sul3 se originaron mediante sendos procesos de movilización independientes de los genes folP cromosómicos presentes, respectivamente, en Rhodobiaceae y Leptospiraceae. Asimismo, los ensayos in vitro han revelado que los genes folP codificados en Rhodobiaceae y en Leptospiraceae confieren resistencia a sulfamidas. Estos resultados indican que la resistencia a las sulfamidas es ancestral y anterior a la introducción clínica de este antibacteriano. Por otro lado, se analizó el origen y evolución de los genes móviles dfrA que confieren resistencia al trimetoprim. Al contrario de lo descrito para los genes sul, los resultados obtenidos en el análisis de las secuencias existentes de genes dfrA móviles indican que éstos se han originado mediante múltiples eventos de transferencia lateral de genes folA cromosómicos. Asimismo, se ha podido establecer que muchos de los genes cromosómicos folA predecesores de los dfrA también confieren resistencia a trimetoprim. Todo ello revela que la resistencia a trimetroprim determinada por elementos móviles se puede explicar por dos procesos independientes y complementarios: i) la captación de genes folA cromosómicos movilizados, portadores de una mutación ancestral responsable de dicha resistencia originada en ausencia del quimioterápico, y ii) la transferencia de genes mutantes espontáneos folA cromosómicos, presentes en una población bacteriana coetánea con la exposición al citado antibacteriano, y habiendo sido seleccionados por dicha exposición.Antibacterial resistance is a pressing problem in human and animal modern healthcare. It is widely accepted that antibiotic resistance originated million years ago in either the microbes that naturally produce the antibiotics or their competitors. However, resistance to synthetic chemotherapeutic agents cannot be explained by this mechanism, since these drugs are not naturally produced. Resistance to chemotherapeutic compounds is hence assumed to have emerged and evolved rapidly following the clinical introduction of these compounds. Resistance to sulfonamides and trimethoprim is mediated by mobile sul and dfr genes, respectively. These genes encode resistant variants of the di-hydro-pteroate synthase and di-hydrofolate reductase enzymes, in contrast to chromosomal folP and folA genes encoding variants sensitive to these agents. The overall objective of this Doctoral Thesis is the identification of the chromosomal origin of genes conferring clinical resistance to sulfonamides and trimethoprim, and the study of the evolutionary processes governing their emergence and dissemination. In this work, sul genes and their genetic neighborhoods have been analyzed to uncover elements that enable the elucidation of their genetic origin. This analysis has led to the identification of a Sul motif associated with sul-encoded proteins, as well as the observation of a consistent genomic association of the sul2 gene with a phosphoglucosamine mutase gene (glmM) . The study of chromosomal folP genes across the Bacteria domain revealed the presence of the Sul motif in the chromosomal folP genes of two families: the Rhodobiaceae and the Leptospiraceae. Phylogenetic analysis of FolP/Sul and GlmM proteins established that the sul1-2 and sul3 genes originated through the independent mobilization of chromosomal folP genes encoded by the Rhodobiaceae and the Leptospiraceae, respectively. In vitro experiments revealed that both the Rhodobiaceae and the Leptospiraceae chromosomal folP genes confer resistance to sulfonamides. These results suggest that resistance to sulfonamides is ancient and predates the clinical introduction of these drugs. Likewise, the origin and evolution of mobile dfrA genes conferring clinical resistance to trimethoprim have been analysed. The results have shown that, in contrast to sul genes, the dfrA gene arose from multiple mobilization events from chromosomal folA. Furthermore, it has been established that most chromosomal folA genes that are predecessors of mobile dfrA genes also confer trimethoprim resistance. Taken together, these results indicate that the resistance to trimethoprim determined by mobile elements can be explained by two independent and complementary processes: i) the mobilization of chromosomal folA genes carrying an ancestral mutation originated in the absence of trimethoprim and responsible for trimethoprim resistance, and ii) the mobilization of spontaneous mutants in chromosomal folA genes, present in a bacterial population coetaneous with the exposure to trimethoprim, and having been selected by that exposure. Given the absence of sulfonamides and trimethoprim in nature, these results provide a new perspective on the emergence and dissemination of resistance to new-generation chemotherapeutic agents. Thus, this work concludes that resistant genes may exist in the vast bacterial pangenome and can be selected for and mobilized upon exposure to these new drugs.Universitat Autònoma de Barcelona. Programa de Doctorat en Microbiologi