RESUMO
Trimethoprim (TMP) is a low-cost, widely prescribed antibiotic. Its effectiveness is increasingly challenged by the spread of genes coding for TMP-resistant dihydrofolate reductases: dfrA, and the lesser-known, evolutionarily unrelated dfrB. Despite recent reports of novel variants conferring high level TMP resistance (dfrB10 to dfrB21), the prevalence of dfrB is still unknown due to underreporting, heterogeneity of the analyzed genetic material in terms of isolation sources, and limited bioinformatic processing. In this study, we explored a coherent set of shotgun metagenomic sequences to quantitatively estimate the abundance of dfrB gene variants in aquatic environments. Specifically, we scanned sequences originating from influents and effluents of municipal sewage treatment plants as well as river-borne microbiomes. Our analyses reveal an increased prevalence of dfrB1, dfrB2, dfrB3, dfrB4, dfrB5, and dfrB7 in wastewater microbiomes as compared to freshwater. These gene variants were frequently found in genomic neighborship with other resistance genes, transposable elements, and integrons, indicating their mobility. By contrast, the relative abundances of the more recently discovered variants dfrB9, dfrB10, and dfrB13 were significantly higher in freshwater than in wastewater microbiomes. Moreover, their direct neighborship with other resistance genes or markers of mobile genetic elements was significantly less likely. Our findings suggest that natural freshwater communities form a major reservoir of the recently discovered dfrB gene variants. Their proliferation and mobilization in response to the exposure of freshwater communities to selective TMP concentrations may promote the prevalence of high-level TMP resistance and thus limit the future effectiveness of antimicrobial therapies.
Assuntos
Resistência a Trimetoprima , Águas Residuárias , Resistência a Trimetoprima/genética , Genes Bacterianos , Trimetoprima/farmacologia , Antibacterianos/farmacologiaRESUMO
BACKGROUND: Although urinary tract infections (UTIs) are extremely common, isolation of causative uropathogens is not always routinely performed, with antibiotics frequently prescribed empirically. This study determined the susceptibility of urinary isolates from two Health and Social Care Trusts (HSCTs) in Northern Ireland to a range of antibiotics commonly used in the treatment of UTIs. Furthermore, we determined if detection of trimethoprim resistance genes (dfrA) could be used as a potential biomarker for rapid detection of phenotypic trimethoprim resistance in urinary pathogens and from urine without culture. METHODS: Susceptibility of E. coli and Klebsiella spp. isolates (n = 124) to trimethoprim, amoxicillin, ceftazidime, ciprofloxacin, co-amoxiclav and nitrofurantoin in addition to susceptibility of Proteus mirabilis (n = 61) and Staphylococcus saprophyticus (n = 17) to trimethoprim was determined by ETEST® and interpreted according to EUCAST breakpoints. PCR was used to detect dfrA genes in bacterial isolates (n = 202) and urine samples(n = 94). RESULTS: Resistance to trimethoprim was observed in 37/124 (29.8%) E. coli and Klebsiella spp. isolates with an MIC90 > 32 mg/L. DfrA genes were detected in 29/37 (78.4%) trimethoprim-resistant isolates. Detection of dfrA was highly sensitive (93.6%) and specific (91.4%) in predicting phenotypic trimethoprim resistance among E. coli and Klebsiella spp. isolates. The dfrA genes analysed were detected using a culture-independent PCR method in 16/94 (17%) urine samples. Phenotypic trimethoprim resistance was apparent in isolates cultured from 15/16 (94%) dfrA-positive urine samples. There was a significant association (P < 0.0001) between the presence of dfrA and trimethoprim resistance in urine samples containing Gram-negative bacteria (Sensitivity = 75%; Specificity = 96.9%; PPV = 93.8%; NPV = 86.1%). CONCLUSIONS: This study demonstrates that molecular detection of dfrA genes is a good indicator of trimethoprim resistance without the need for culture and susceptibility testing.
Assuntos
Antibacterianos , Infecções Urinárias , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Farmacorresistência Bacteriana , Escherichia coli , Humanos , Testes de Sensibilidade Microbiana , Trimetoprima/farmacologia , Resistência a Trimetoprima/genética , Infecções Urinárias/microbiologiaRESUMO
Two plasmid-encoded dihydrofolate reductase (DHFR) isoforms, DfrA1 and DfrA5, that give rise to high levels of resistance in Gram-negative bacteria were structurally and biochemically characterized to reveal the mechanism of TMP resistance and to support phylogenic groupings for drug development against antibiotic resistant pathogens. Preliminary screening of novel antifolates revealed related chemotypes that showed high levels of inhibitory potency against Escherichia coli chromosomal DHFR (EcDHFR), DfrA1, and DfrA5. Kinetics and biophysical analysis, coupled with crystal structures of trimethoprim bound to EcDHFR, DfrA1 and DfrA5, and two propargyl-linked antifolates (PLA) complexed with EcDHFR, DfrA1 and DfrA5, were determined to define structural features of the substrate binding pocket and guide synthesis of pan-DHFR inhibitors.
Assuntos
Antagonistas do Ácido Fólico , Resistência a Trimetoprima , Escherichia coli/genética , Escherichia coli/metabolismo , Ácido Fólico/análogos & derivados , Antagonistas do Ácido Fólico/química , Antagonistas do Ácido Fólico/metabolismo , Antagonistas do Ácido Fólico/farmacologia , Plasmídeos/genética , Tetra-Hidrofolato Desidrogenase/química , Tetra-Hidrofolato Desidrogenase/genética , Tetra-Hidrofolato Desidrogenase/metabolismo , Resistência a Trimetoprima/genéticaRESUMO
OBJECTIVE: Shewanella algae is a zoonotic marine bacterium that causes a variety of infections in immunocompromised patients or those who have been exposed to seawater. The development of trimethoprim/sulfamethoxazole (TMP/SMX) resistance in S. algae are found in human and environment isolates during the past ten years, and thus the treatment options are decreasing. METHODOLOGY: In the study, we conduct a comparative genomic study to identify the resistant mechanism of TMP/SMX-resistance in S. algae. RESULTS: We found the resistance of TMP/SMX in S. algae is associated with the existence of sul1 and dfrA12 within the class 1 integron. The gene cassette dfra12-aadA2-qacEΔ1/sul1 within the class 1 integron is highly conserved. In addition, the class 1 integron and encapsulated sul1 are significantly enriched in Enterobacteriaceae in NCBI and UniProt databases. CONCLUSION: Our study suggests that the horizontal transfer of TMP/SMX resistance via class 1 integron is most frequently occurred within Enterobacteriaceae and has spread to a wide range of sources including soil, poultry, and marine water.
Assuntos
Shewanella , Combinação Trimetoprima e Sulfametoxazol , Humanos , Shewanella/genética , Resistência a Trimetoprima/genética , GenômicaRESUMO
To track the spread of antibiotic resistance genes, accurate identification of individual genes is essential. Acquired trimethoprim resistance genes encoding trimethoprim-insensitive homologues of the sensitive dihydrofolate reductases encoded by the folA genes of bacteria are increasingly found in genome sequences. However, naming and numbering in publicly available records (journal publications or entries in the GenBank non-redundant DNA database) has not always been unambiguous. In addition, the nomenclature has evolved over time. Here, the changes in nomenclature and the most commonly encountered problems and pitfalls affecting dfrA gene identification arising from historically incorrect or inaccurate numbering are explained. The complete set of dfrA genes/DfrA proteins found in Gram-negative bacteria for which readily searchable sequence information is currently available has been compiled using less than 98% identity for both the gene and the derived protein sequence as the criteria for assignment of a new number. In most cases, trimethoprim resistance has been demonstrated. The gene context, predominantly in a gene cassette or near the ori end of CR1 or CR2, is also covered. The RefSeq database that underpins the programs used to automatically identify resistance genes in genome data sets has been curated to assign all sequences listed to the correct number. This led to the assignment of corrected or new gene numbers to several mis-assigned sequences. The unique numbers assigned for the dfrA/DfrA set are now listed in the RefSeq database, which we propose provides a way forward that should end future duplication of numbers and the confusion that causes.
Assuntos
Resistência a Trimetoprima , Trimetoprima , Antibacterianos/farmacologia , Bactérias Gram-Negativas/genética , Tetra-Hidrofolato Desidrogenase/genética , Trimetoprima/farmacologia , Resistência a Trimetoprima/genéticaRESUMO
The antibiotic trimethoprim (TMP) is used to treat a variety of Escherichia coli infections, but its efficacy is limited by the rapid emergence of TMP-resistant bacteria. Previous laboratory evolution experiments have identified resistance-conferring mutations in the gene encoding the TMP target, bacterial dihydrofolate reductase (DHFR), in particular mutation L28R. Here, we show that 4'-desmethyltrimethoprim (4'-DTMP) inhibits both DHFR and its L28R variant, and selects against the emergence of TMP-resistant bacteria that carry the L28R mutation in laboratory experiments. Furthermore, antibiotic-sensitive E. coli populations acquire antibiotic resistance at a substantially slower rate when grown in the presence of 4'-DTMP than in the presence of TMP. We find that 4'-DTMP impedes evolution of resistance by selecting against resistant genotypes with the L28R mutation and diverting genetic trajectories to other resistance-conferring DHFR mutations with catalytic deficiencies. Our results demonstrate how a detailed characterization of resistance-conferring mutations in a target enzyme can help identify potential drugs against antibiotic-resistant bacteria, which may ultimately increase long-term efficacy of antimicrobial therapies by modulating evolutionary trajectories that lead to resistance.
Assuntos
Infecções por Escherichia coli/tratamento farmacológico , Infecções por Escherichia coli/microbiologia , Resistência a Trimetoprima/genética , Trimetoprima/análogos & derivados , Substituição de Aminoácidos , Antibacterianos/química , Antibacterianos/farmacologia , Cristalografia por Raios X , Evolução Molecular Direcionada , Desenho de Fármacos , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Antagonistas do Ácido Fólico/química , Antagonistas do Ácido Fólico/farmacologia , Genes Bacterianos , Genótipo , Humanos , Modelos Moleculares , Mutação , Tetra-Hidrofolato Desidrogenase/química , Tetra-Hidrofolato Desidrogenase/genética , Trimetoprima/química , Trimetoprima/farmacologiaRESUMO
ABSTRACT: The aim of this study was to discuss the correlation between the sulfamethoxazole-trimethoprim resistance of Shigella flexneri (S. flexneri) and the antibiotic resistance genes sul1, sul2, and sul3 and SXT element.From May 2013 to October 2018, 102 isolates of S. flexneri were collected from the clinical samples in Jinan. The Kirby-Bauer (K-B) test was employed to determine the antibiotic susceptibility of the S. flexneri isolates. The antibiotic resistance rate was analyzed with the WHONET5.4 software. The isolates were subject to the PCR amplification of the sul genes (sul1, sul2, and sul3) and the SXT element. On the basis of the sequencing results, the correlation between the sulfamethoxazole-trimethoprim resistance of the S. flexneri isolates and the sul genes was analyzed.The antibiotic resistance rates of the 102 S. flexneri isolates to ampicillin, streptomycin, chloramphenicol, tetracycline, and sulfamethoxazole-trimethoprim were 90.2%, 90.2%, 88.2%, 88.2%, and 62.7%, respectively. The antibiotic resistance rates of these isolates to cefotaxime, ceftazidime, and ciprofloxacin varied between 20% and 35%. However, these isolates were 100% susceptible to cefoxitin. Positive fragments were amplified from 59.8% (61/102) of the 102 S. flexneri isolates, the sizes of the sul1 and sul2 genes being 338âbp and 286âbp, respectively. The sequence alignment revealed the presence of the sul1 and sul2 genes encoding for dihydrofolate synthase. The carrying rate of the sul1 gene was 13.7% (14/102), and that of the sul2 gene was 48.0% (49/102). No target gene fragments were amplified from the 3 isolates resistant to sulfamethoxazole-trimethoprim. The sul3 gene and SXT element were not amplified from any of the isolates. The testing and statistical analysis showed that the resistance of the S. flexneri isolates to sulfamethoxazole-trimethoprim correlated to the sul1 and sul2 genes.The acquired antibiotic resistance genes sul1 and sul2 were closely associated with the resistance of the 102 S. flexneri isolates to sulfamethoxazole-trimethoprim.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Disenteria Bacilar/tratamento farmacológico , Shigella flexneri/genética , Resistência a Trimetoprima/genética , Combinação Trimetoprima e Sulfametoxazol/farmacologia , Elementos de DNA Transponíveis/genética , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Disenteria Bacilar/microbiologia , Fezes/microbiologia , Humanos , Testes de Sensibilidade Microbiana , Reação em Cadeia da Polimerase , Shigella flexneri/efeitos dos fármacos , Shigella flexneri/isolamento & purificação , Combinação Trimetoprima e Sulfametoxazol/uso terapêuticoRESUMO
AIMS: Antimicrobial resistance genes (ARGs) are often associated with mobile genetic elements (MGEs), which facilitate their movement within and between bacterial populations. Detection of mobility is therefore important to understand the dynamics of MGE dissemination and their associated genes, especially in resistant clinical isolates that often have multiple ARGs associated with MGEs. Therefore, this study aimed to develop an entrapment vector to capture active MGEs and ARGs in clinical isolates of Escherichia coli. METHODS AND RESULTS: We engineered an entrapment vector, called pBACpAK, to capture MGEs in clinical E. coli isolates. It contains a cI-tetA positive selection cartridge in which the cI gene encodes a repressor that inhibits the expression of tetA. Therefore, any disruption of cI, for example, by insertion of a MGE, will allow tetA to be expressed and result in a selectable tetracycline-resistant phenotype. The pBACpAK was introduced into clinical E. coli isolates and grown on tetracycline-containing agar to select for clones with the insertion of MGEs into the entrapment vector. Several insertion sequences were detected within pBACpAK, including IS26, IS903B and ISSbo1. A novel translocatable unit (TU), containing IS26 and dfrA8 was also captured, and dfrA8 was shown to confer trimethoprim resistance when it was cloned into E. coli DH5α. CONCLUSIONS: The entrapment vector, pBACpAK was developed and shown to be able to capture MGEs and their associated ARGs from clinical E. coli isolates. We have captured, for the first time, a TU encoding antibiotic resistance. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first time that a TU and associated resistance gene has been captured from clinical E. coli isolates using an entrapment vector. The pBACpAK has the potential to be used not only as a tool to capture MGEs in clinical E. coli isolates, but also to study dynamics, frequency and potentiators of mobility for MGEs.
Assuntos
Farmacorresistência Bacteriana/genética , Escherichia coli/genética , Sequências Repetitivas Dispersas/genética , Antibacterianos/farmacologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Escherichia coli/isolamento & purificação , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Genes Bacterianos , Vetores Genéticos , Humanos , Resistência a Trimetoprima/efeitos dos fármacos , Resistência a Trimetoprima/genéticaRESUMO
Stenotrophomonas maltophilia has emerged as an important opportunistic pathogen in the last decade. Increased resistance to sulfamethoxazole/trimethoprim (SMX/TMP) has been reported in S. maltophilia strains in the past few years, leading to few therapeutic options. We conducted a prospective multicenter study at two Brazilian teaching hospitals that identified S. maltophilia isolates and evaluated their antimicrobial susceptibility profile, SMX/TMP resistance genes and their clonality profile. A total of 106 non-repeated clinical samples of S. maltophilia were evaluated. Resistance to SMX/TMP was identified in 21.6% of the samples, and previous use of SMX/TMP occurred in 19 (82.6%). PCR detected the sul1 gene in 14 of 106 strains (13.2%). Of these isolates, nine displayed resistance to SMX/TMP. The resistant strains presented a polyclonal profile. This opportunistic pathogen has emerged in immunocompromised hosts, with few therapeutic options, which is aggravated by the description of emerging resistance mechanisms, although with a polyclonal distribution profile.
Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana/genética , Infecções por Bactérias Gram-Negativas/tratamento farmacológico , Stenotrophomonas maltophilia/efeitos dos fármacos , Stenotrophomonas maltophilia/genética , Combinação Trimetoprima e Sulfametoxazol/farmacologia , Antibacterianos/uso terapêutico , Proteínas de Bactérias/genética , Brasil , DNA Bacteriano/genética , Farmacorresistência Bacteriana/efeitos dos fármacos , Infecções por Bactérias Gram-Negativas/epidemiologia , Infecções por Bactérias Gram-Negativas/microbiologia , Humanos , Hospedeiro Imunocomprometido , Testes de Sensibilidade Microbiana , Reação em Cadeia da Polimerase , Estudos Prospectivos , Stenotrophomonas maltophilia/isolamento & purificação , Resistência a Trimetoprima/genética , Combinação Trimetoprima e Sulfametoxazol/uso terapêuticoRESUMO
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 Acinetobacter baumannii, 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.
Assuntos
Acinetobacter baumannii/genética , Antibacterianos/farmacologia , Tetra-Hidrofolato Desidrogenase/genética , Resistência a Trimetoprima/genética , Trimetoprima/farmacologia , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/isolamento & purificação , Evolução Biológica , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Ácido Fólico/biossíntese , Humanos , Testes de Sensibilidade Microbiana , Sulfonamidas/farmacologiaRESUMO
Antibiotic resistance presents a serious and still growing threat to human health. Environmental exposure levels required to select for resistance are unknown for most antibiotics. Here, we evaluated different experimental approaches and ways to interpret effect measures, in order to identify what concentration of trimethoprim that are likely to select for resistance in aquatic environments. When grown in complex biofilms, selection for resistant E. coli increased at 100 µg/L, whereas there was only a non-significant trend with regards to changes in taxonomic composition within the tested range (0-100 µg/L). Planktonic co-culturing of 149 different E. coli strains isolated from sewage again confirmed selection at 100 µg/L. Finally, pairwise competition experiments were performed with engineered E. coli strains carrying different trimethoprim resistance genes (dfr) and their sensitive counterparts. While strains with introduced resistance genes grew slower than the sensitive ones at 0 and 10 µg/L, a significant reduction in cost was found already at 10 µg/L. Defining lowest effect concentrations by comparing proportion of resistant strains to sensitive ones at the same time point, rather than to their initial ratios, will reflect the advantage a resistance factor can bring, while ignoring exposure-independent fitness costs. As costs are likely to be highly dependent on the specific environmental and genetic contexts, the former approach might be more suitable as a basis for defining exposure limits with the intention to prevent selection for resistance. Based on the present and other studies, we propose that 1 µg/L would be a reasonably protective exposure limit for trimethoprim in aquatic environments.
Assuntos
Escherichia coli , Resistência a Trimetoprima , Antibacterianos/farmacologia , Resistência Microbiana a Medicamentos , Escherichia coli/genética , Humanos , Trimetoprima/toxicidade , Resistência a Trimetoprima/genéticaRESUMO
Bdellovibrio bacteriovorus is a small Gram-negative bacterium and an obligate predator of other Gram-negative bacteria. Prey resistance to B. bacteriovorus attack is rare and transient. This consideration together with its safety and low immunogenicity makes B. bacteriovorus a valid alternative to antibiotics, especially in the treatment of multidrug resistant pathogens. In this study we developed a novel technique to estimate B. bacteriovorus sensitivity against antibiotics in order to make feasible the development and testing of co-therapies with antibiotics that would increase its antimicrobial efficacy and at the same time reduce the development of drug resistance. Results from tests performed with this technique show that among all tested antibiotics, trimethoprim has the lowest antimicrobial effect on B. bacteriovorus. Additional experiments revealed that the mechanism of trimethoprim resistance in B. bacteriovorus depends on the low affinity of this compound for the B. bacteriovorus dihydrofolate reductase (Bd DHFR).
Assuntos
Antibacterianos/metabolismo , Bdellovibrio bacteriovorus/crescimento & desenvolvimento , Bdellovibrio bacteriovorus/metabolismo , Antibiose/genética , Bdellovibrio/genética , Bdellovibrio/crescimento & desenvolvimento , Bdellovibrio bacteriovorus/genética , Farmacorresistência Bacteriana/genética , Bactérias Gram-Negativas/efeitos dos fármacos , Testes de Sensibilidade Microbiana/métodos , Trimetoprima/farmacologia , Resistência a Trimetoprima/genéticaAssuntos
Antibacterianos/farmacologia , Infecções Comunitárias Adquiridas/microbiologia , Farmacorresistência Bacteriana/genética , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Resistência a Trimetoprima/genética , Combinação Trimetoprima e Sulfametoxazol/farmacologia , Austrália , Técnicas de Laboratório Clínico , Humanos , Staphylococcus aureus Resistente à Meticilina/genética , Testes de Sensibilidade Microbiana , Grupos Populacionais , Kit de Reagentes para Diagnóstico , Reprodutibilidade dos Testes , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/efeitos dos fármacosRESUMO
Whole-genome sequencing of trimethoprim-resistant Escherichia coli strains MF2165 and PF9285 from healthy Swiss fattening calves revealed a so far uncharacterized dihydrofolate reductase gene, dfrA35 Functionality and association with trimethoprim resistance were demonstrated by cloning and expressing dfrA35 in E. coli The DfrA35 protein showed the closest amino acid identity (49.4%) to DfrA20 from Pasteurella multocida and to the Dfr determinants DfrG (41.2%), DfrD (40.8%), and DfrK (40.0%) found in Gram-positive bacteria. The dfrA35 gene was integrated within a florfenicol/chloramphenicol-sulfonamide resistance ISCR2 element (floR-ISCR2-dfrA35-sul2) next to a Tn21-like transposon that contained genes with resistance to sulfonamides (sul1), streptomycin (aadA1), gentamicin/tobramycin/kanamycin (aadB), and quaternary ammonium compounds (qacEΔ1). A search of GenBank databases revealed that dfrA35 was present in 26 other E. coli strains from different origins as well as in AcinetobacterIMPORTANCE The presence of dfrA35 associated with ISCR2 in Escherichia coli from animals, as well as its presence in other E. coli strains from different sources and countries and in Acinetobacter, highlights the global spread of this gene and its potential for further dissemination. The genetic link of ISCR2-dfrA35 with other antibiotic and disinfectant resistance genes showed that multidrug-resistant E. coli may be selected and maintained by the use of either one of several antimicrobials.
Assuntos
Antibacterianos/farmacologia , Proteínas de Escherichia coli/genética , Escherichia coli/efeitos dos fármacos , Tetra-Hidrofolato Desidrogenase/genética , Resistência a Trimetoprima/genética , Trimetoprima/farmacologia , Fatores Etários , Animais , Bovinos , Escherichia coli/genética , Infecções por Escherichia coli/microbiologiaRESUMO
Evolutionary fitness landscapes of several antibiotic target proteins have been comprehensively mapped showing strong high-order epistasis between mutations, but understanding these effects at the biochemical and structural levels remained open. Here, we carried out an extensive experimental and computational study to quantitatively understand the evolutionary dynamics of Escherichia coli dihydrofolate reductase (DHFR) enzyme in the presence of trimethoprim-induced selection. To facilitate this, we developed a new in vitro assay for rapidly characterizing DHFR steady-state kinetics. Biochemical and structural characterization of resistance-conferring mutations targeting a total of ten residues spanning the substrate binding pocket of DHFR revealed distinct changes in the catalytic efficiencies of mutated DHFR enzymes. Next, we measured biochemical parameters (Km, Ki, and kcat) for a mutant library carrying all possible combinations of six resistance-conferring DHFR mutations and quantified epistatic interactions between them. We found that the high-order epistasis in catalytic power of DHFR (kcat and Km) creates a rugged fitness landscape under trimethoprim selection. Taken together, our data provide a concrete illustration of how epistatic coupling at the level of biochemical parameters can give rise to complex fitness landscapes, and suggest new strategies for developing mutant specific inhibitors.
Assuntos
Epistasia Genética , Aptidão Genética , Seleção Genética , Tetra-Hidrofolato Desidrogenase/genética , Resistência a Trimetoprima/genética , Escherichia coli , Simulação de Dinâmica Molecular , Mutação , Tetra-Hidrofolato Desidrogenase/metabolismoRESUMO
Nocardia spp. are Gram-positive opportunistic pathogens that affect largely immunocompromised patients, leading to serious pulmonary or systemic infections. Combination therapy using the folate biosynthesis pathway inhibitors trimethoprim (TMP) and sulfamethoxazole (SMX) is commonly used as an antimicrobial therapy. Not surprisingly, as antibiotic therapies for nocardiosis can extend for many months, resistance to TMP-SMX has emerged. Using experimental evolution, we surveyed the genetic basis of adaptation to TMP-SMX across 8 strains of Nocardia nova and 2 strains of Nocardia cyriacigeorgica By employing both continuous experimental evolution to provide longitudinal information on the order of changes and characterization of resistant endpoint isolates, we observe changes that are consistent with modifications of two enzymes of the folate biosynthesis pathway: dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) (FolP), with the mutations often being clustered near the active site of the enzymes. While changes to DHFR and DHPS might be expected, we also noted that mutations in a previously undescribed homolog of DHPS (DHPS2 or FolP2) that was annotated as being "nonfunctional" were also sufficient to generate TMP-SMX resistance, which serves as a cautionary tale for the use of automated annotation by investigators and for the future discovery of drugs against this genus. Additionally, folP2 overlapped glucosyl-3-phosphoglycerate synthase. Remarkably, an adaptive frameshift mutation within the overlapping region resulted in a new in-frame fusion to the downstream gene to produce a potentially new bifunctional enzyme. How a single potentially bifunctional DHPS2 enzyme might confer resistance is unclear. However, it highlights the unexpected ways in which adaptive evolution finds novel solutions for selection.
Assuntos
Antibacterianos/farmacologia , Di-Hidropteroato Sintase/genética , Farmacorresistência Bacteriana Múltipla/genética , Nocardia/efeitos dos fármacos , Nocardia/genética , Tetra-Hidrofolato Desidrogenase/genética , Resistência a Trimetoprima/genética , Combinação Trimetoprima e Sulfametoxazol/farmacologia , Sequência de Aminoácidos , Sequência de Bases , Mutação da Fase de Leitura/genética , Glucosiltransferases/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Estudos Longitudinais , Testes de Sensibilidade Microbiana , Nocardia/isolamento & purificação , Nocardiose/tratamento farmacológicoRESUMO
Trimethoprim with sulfamethoxazole is a therapeutic agent combination used to treat infections caused by the facultative intracellular foodborne pathogen Listeria monocytogenes. The aim of this study was to assess the frequency of resistance of L. monocytogenes arising due to exposure to trimethoprim and subsequently investigate the molecular mechanisms of resistance. After exposure of a culture of L. monocytogenes ATCC 13932 to trimethoprim at 10-fold the minimal inhibitory concentration spontaneous resistant mutants were recovered, giving a frequency of resistance development of 6.85 ± 0.92 × 10-8. The isolates exhibited a 32-64-fold decrease in susceptibility compared with the parental strain. These results indicate the capacity of L. monocytogenes to develop low-level resistance toward trimethoprim after exposure to the drug. The trimethoprim resistance genes (dhfr) and their promoter regions from all trimethoprim-resistant isolates were amplified and sequenced, leading to the identification of four single amino acid substitutions (Met20-Val, Pro21-Leu, Thr46-Asn, Val95-Leu) and two double substitutions (Met20-Ile+Thr46-Asn and Thr46-Asn+Leu85-Phe) in DHFR. Of the identified mutations, the Thr46-Asn substitution has not been previously reported as the mechanism of resistance to trimethoprim in other bacteria; thus this substitution seems to be unique to L. monocytogenes. The expression of the mutated L. monocytogenes dhfr genes in Escherichia coli led to decreased susceptibility of the heterological host, therefore proving that the identified point mutations in dhfr serve as the molecular mechanism of acquired resistance of L. monocytogenes to trimethoprim.
Assuntos
Genes Bacterianos , Listeria monocytogenes/efeitos dos fármacos , Listeria monocytogenes/genética , Resistência a Trimetoprima/genética , Trimetoprima/farmacologia , Substituição de Aminoácidos , DNA Bacteriano/genética , Escherichia coli , Doenças Transmitidas por Alimentos/tratamento farmacológico , Técnicas de Genotipagem , Testes de Sensibilidade Microbiana , Regiões Promotoras Genéticas , Sulfametoxazol/farmacologia , Tetra-Hidrofolato Desidrogenase/genética , Tetra-Hidrofolato Desidrogenase/metabolismoRESUMO
The trimethoprim and sulfamethoxazole combination, co-trimoxazole, plays a vital role in the treatment of Burkholderia pseudomallei infections. Previous studies demonstrated that the B. pseudomallei BpeEF-OprC efflux pump confers widespread trimethoprim resistance in clinical and environmental isolates, but this is not accompanied by significant resistance to co-trimoxazole. Using the excluded select-agent strain B. pseudomallei Bp82, we now show that in vitro acquired trimethoprim versus co-trimoxazole resistance is mainly mediated by constitutive BpeEF-OprC expression due to bpeT mutations or by BpeEF-OprC overexpression due to bpeS mutations. Mutations in bpeT affect the carboxy-terminal effector-binding domain of the BpeT LysR-type activator protein. Trimethoprim resistance can also be mediated by dihydrofolate reductase (FolA) target mutations, but this occurs rarely unless BpeEF-OprC is absent. BpeS is a transcriptional regulator that is 62% identical to BpeT. Mutations affecting the BpeS DNA-binding or carboxy-terminal effector-binding domains result in constitutive BpeEF-OprC overexpression, leading to trimethoprim and sulfamethoxazole efflux and thus to co-trimoxazole resistance. The majority of laboratory-selected co-trimoxazole-resistant mutants often also contain mutations in folM, encoding a pterin reductase. Genetic analyses of these mutants established that both bpeS mutations and folM mutations contribute to co-trimoxazole resistance, although the exact role of folM remains to be determined. Mutations affecting bpeT, bpeS, and folM are common in co-trimoxazole-resistant clinical isolates, indicating that mutations affecting these genes are clinically significant. Co-trimoxazole resistance in B. pseudomallei is a complex phenomenon, which may explain why resistance to this drug is rare in this bacterium.IMPORTANCEBurkholderia pseudomallei causes melioidosis, a tropical disease that is difficult to treat. The bacterium's resistance to antibiotics limits therapeutic options. The paucity of orally available drugs further complicates therapy. The oral drug of choice is co-trimoxazole, a combination of trimethoprim and sulfamethoxazole. These antibiotics target two distinct enzymes, FolA (dihydrofolate reductase) and FolP (dihydropteroate synthase), in the bacterial tetrahydrofolate biosynthetic pathway. Although co-trimoxazole resistance is minimized due to two-target inhibition, bacterial resistance due to folA and folP mutations does occur. Co-trimoxazole resistance in B. pseudomallei is rare and has not yet been studied. Co-trimoxazole resistance in this bacterium employs a novel strategy involving differential regulation of BpeEF-OprC efflux pump expression that determines the drug resistance profile. Contributing are mutations affecting folA, but not folP, and folM, a folate pathway-associated gene whose function is not yet well understood and which has not been previously implicated in folate inhibitor resistance in clinical isolates.
Assuntos
Antibacterianos/farmacologia , Burkholderia pseudomallei/efeitos dos fármacos , Farmacorresistência Bacteriana Múltipla/genética , Antagonistas do Ácido Fólico/farmacologia , Ácido Fólico/metabolismo , Combinação Trimetoprima e Sulfametoxazol/farmacologia , Trimetoprima/farmacologia , Burkholderia pseudomallei/genética , Burkholderia pseudomallei/metabolismo , Humanos , Melioidose/tratamento farmacológico , Melioidose/microbiologia , Redes e Vias Metabólicas/efeitos dos fármacos , Redes e Vias Metabólicas/genética , Testes de Sensibilidade Microbiana , Mutação , Tetra-Hidrofolato Desidrogenase/genética , Resistência a Trimetoprima/genética , Combinação Trimetoprima e Sulfametoxazol/metabolismoRESUMO
Whole-genome sequencing of trimethoprim-resistant Escherichia coli clinical isolates identified a member of the trimethoprim-resistant type II dihydrofolate reductase gene family (dfrB). The dfrB4 gene was located within a class I integron flanked by multiple resistance genes. This arrangement was previously reported in a 130.6-kb multiresistance plasmid. The DfrB4 protein conferred a >2,000-fold increased trimethoprim resistance on overexpression in E. coli Our results are consistent with the finding that dfrB4 contributes to clinical trimethoprim resistance.
Assuntos
Anti-Infecciosos Urinários/farmacologia , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Integrons/genética , Tetra-Hidrofolato Desidrogenase/genética , Resistência a Trimetoprima/genética , Trimetoprima/farmacologia , Farmacorresistência Bacteriana/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/isolamento & purificação , Humanos , Testes de Sensibilidade Microbiana , Infecções Urinárias/tratamento farmacológico , Infecções Urinárias/microbiologiaRESUMO
The global emergence of Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae poses a major public health threat requiring immediate and aggressive action. Some older generation antibiotics, such as trimethoprim, serve as alternatives for treatment of infections. Here, we determined the complete nucleotide sequence of plasmid pHS091147, which co-harbored the carbapenemase (blaKPC-2) and trimethoprim resistance genes (dfrA25) from a Klebsiella pneumoniae sequence type (ST) 11 clone recovered in Shanghai, China. pHS091147 had three replication genes, several plasmid-stability genes and an intact type IV secretion system gene cluster. Besides blaKPC-2 and dfrA25, pHS091147 carried several other resistance genes, including ß-lactamase genes blaTEM-1 and blaCTX-M-14, sulphonamide resistance gene sul1, a quinolone resistance gene remnant (ΔqnrB2), and virulence associated gene iroN. Notably, the multidrug-resistance region was a chimeric structure composed of three subregions, which shared strong sequence homology with several plasmids previously assigned in Genbank. To our knowledge, this is the first report of the co-localization of blaKPC-2 and dfrA25 on a novel putative multi-replicon plasmid in a Klebsiella pneumoniae ST11 clone.