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1.
Commun Biol ; 5(1): 459, 2022 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-35562546

RESUMO

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/genética
2.
BMC Microbiol ; 22(1): 144, 2022 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-35610571

RESUMO

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/microbiologia
3.
EBioMedicine ; 74: 103742, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34902789

RESUMO

BACKGROUND: Understanding drivers of antibiotic resistance evolution is fundamental for designing optimal treatment strategies and interventions to reduce the spread of antibiotic resistance. Various cytotoxic drugs used in cancer chemotherapy have antibacterial properties, but how bacterial populations are affected by these selective pressures is unknown. Here we test the hypothesis that the widely used cytotoxic drug methotrexate affects the evolution and selection of antibiotic resistance. METHODS: First, we determined methotrexate susceptibility (IC90) and selective abilities in a collection of Escherichia coli and Klebsiella pneumoniae strains with and without pre-existing trimethoprim resistance determinants. We constructed fluorescently labelled pairs of E. coli MG1655 differing only in trimethoprim resistance determinants and determined the minimum selective concentrations of methotrexate using flow-cytometry. We further used an experimental evolution approach to investigate the effects of methotrexate on de novo trimethoprim resistance evolution. FINDINGS: We show that methotrexate can select for acquired trimethoprim resistance determinants located on the chromosome or a plasmid. Additionally, methotrexate co-selects for genetically linked resistance determinants when present together with trimethoprim resistance on a multi-drug resistance plasmid. These selective effects occur at concentrations 40- to >320-fold below the methotrexate minimal inhibitory concentration. INTERPRETATION: Our results strongly suggest a selective role of methotrexate for virtually any antibiotic resistance determinant when present together with trimethoprim resistance on a multi-drug resistance plasmid. The presented results may have significant implications for patient groups strongly depending on effective antibiotic treatment. FUNDING: PJJ was supported by UiT The Arctic University of Norway and the Northern Norway Regional Health Authority (SFP1292-16/HNF1586-21) and JPI-EC-AMR (Project 271,176/H10). DIA was supported by the Swedish Research Council (grant 2017-01,527). The publication charges for this article have been funded by a grant from the publication fund of UiT The Arctic University of Norway.


Assuntos
Proteínas de Bactérias/genética , Escherichia coli/efeitos dos fármacos , Klebsiella pneumoniae/efeitos dos fármacos , Metotrexato/farmacologia , Cromossomos Bacterianos/genética , Escherichia coli/genética , Evolução Molecular , Citometria de Fluxo , Klebsiella pneumoniae/genética , Testes de Sensibilidade Microbiana , Noruega , Plasmídeos/genética , Resistência a Trimetoprima , Sequenciamento Completo do Genoma
5.
J Antimicrob Chemother ; 76(11): 2748-2756, 2021 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-34180526

RESUMO

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/genética
6.
Mol Plant Pathol ; 22(7): 843-857, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33942460

RESUMO

Genetic transformation is a valuable and essential method that provides powerful insights into the gene function of microorganisms and contributes to the construction of engineered bacteria. Here, we developed a novel genetic transformation system to easily knock out a highly GC-rich gene (74.71% GC) from Burkholderia pyrrocinia JK-SH007, a biocontrol strain of poplar canker disease. This system revealed a reliable selectable marker (trimethoprim resistance gene, Tmp) and a simplified, efficient transformation method (6,363.64 CFU/µg, pHKT2) that was developed via freeze-thawing. The knockout recombineering of B. pyrrocinia JK-SH007 was achieved through a suicide plasmid with a three-fragment mutagenesis construct. The three-fragment cassette for mutagenesis was generated by overlap extension and touchdown PCRs and composed of Tmp flanked by GC-rich upstream and downstream fragments from B. pyrrocinia JK-SH007. The mutant strain (ΔBpEG), which was verified by PCR, lost 93.3% of its ability to degrade carboxymethyl cellulose over 40 days. Overall, this system may contribute to future research on B. pyrrocinia traits.


Assuntos
Burkholderia/genética , Doenças das Plantas/prevenção & controle , Populus/microbiologia , Transformação Genética , Agentes de Controle Biológico , Burkholderia/metabolismo , Carboximetilcelulose Sódica/metabolismo , Congelamento , Sequência Rica em GC , Técnicas de Inativação de Genes , Marcadores Genéticos/genética , Recombinação Homóloga , Mutação , Doenças das Plantas/microbiologia , Plasmídeos/genética , Resistência a Trimetoprima
7.
Nat Commun ; 12(1): 2949, 2021 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-34011959

RESUMO

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/farmacologia
9.
Medicine (Baltimore) ; 100(10): e24970, 2021 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-33725864

RESUMO

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 338 bp and 286 bp, 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 terapêutico
10.
J Appl Microbiol ; 130(3): 832-842, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32881179

RESUMO

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/genética
11.
Artigo em Inglês | MEDLINE | ID: mdl-33295480

RESUMO

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 terapêutico
12.
Microb Genom ; 6(12)2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33206038

RESUMO

Urinary tract infections (UTIs) are the most common bacterial infections requiring medical attention and a leading justification for antibiotic prescription. Trimethoprim is prescribed empirically for uncomplicated cases. UTIs are primarily caused by extraintestinal pathogenic Escherichia coli (ExPEC) and ExPEC strains play a central role in disseminating antimicrobial-resistance genes worldwide. Here, we describe the whole-genome sequences of trimethoprim-resistant ExPEC and/or ExPEC from recurrent UTIs (67 in total) from patients attending a regional Australian hospital from 2006 to 2008. Twenty-three sequence types (STs) were observed, with ST131 predominating (28 %), then ST69 and ST73 (both 7 %). Co-occurrence of trimethoprim-resistance genes with genes conferring resistance to extended-spectrum ß-lactams, heavy metals and quaternary ammonium ions was a feature of the ExPEC described here. Seven trimethoprim-resistance genes were identified, most commonly dfrA17 (38 %) and dfrA12 (18 %). An uncommon dfrB4 variant was also observed. Two blaCTX-M variants were identified - blaCTX-M-15 (16 %) and blaCTX-M-14 (10 %). The former was always associated with dfrA12, the latter with dfrA17, and all blaCTX-M genes co-occurred with chromate-resistance gene chrA. Eighteen class 1 integron structures were characterized, and chrA featured in eight structures; dfrA genes featured in seventeen. ST131 H30Rx isolates possessed distinct antimicrobial gene profiles comprising aac(3)-IIa, aac(6)-Ib-cr, aph(3')-Ia, aadA2, blaCTX-M-15, blaOXA-1 and dfrA12. The most common virulence-associated genes (VAGs) were fimH, fyuA, irp2 and sitA (all 91 %). Virulence profile clustering showed ST131 H30 isolates carried similar VAGs to ST73, ST405, ST550 and ST1193 isolates. The sole ST131 H27 isolate carried molecular predictors of enteroaggregative E. coli/ExPEC hybrid strains (aatA, aggR, fyuA). Seven isolates (10 %) carried VAGs suggesting ColV plasmid carriage. Finally, SNP analysis of serial UTI patients experiencing worsening sequelae demonstrated a high proportion of point mutations in virulence factors.


Assuntos
Proteínas de Bactérias/genética , Farmacorresistência Bacteriana Múltipla , Infecções por Escherichia coli/microbiologia , Escherichia coli Extraintestinal Patogênica/genética , Resistência a Trimetoprima , Infecções Urinárias/microbiologia , Austrália , Infecções por Escherichia coli/tratamento farmacológico , Escherichia coli Extraintestinal Patogênica/efeitos dos fármacos , Humanos , Masculino , Metais Pesados/farmacologia , Polimorfismo de Nucleotídeo Único , Compostos de Amônio Quaternário/farmacologia , Recidiva , Infecções Urinárias/tratamento farmacológico , Fatores de Virulência/genética , Sequenciamento Completo do Genoma , beta-Lactamas/farmacologia
14.
Microb Genom ; 6(11)2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32969787

RESUMO

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/farmacologia
15.
Environ Int ; 144: 106083, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32890888

RESUMO

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/genética
16.
Artigo em Inglês | MEDLINE | ID: mdl-32547961

RESUMO

Small protein B(SmpB) cooperates with transfer-messenger RNA (tmRNA) for trans-translation to ensure the quality control of protein synthesis in prokaryotes. Furthermore, they regulate cell metabolism separately. According to research, SmpB functions as a transcription factor, and tmRNA acts as a small RNA. Purine pathway has been reported to be related to trimethoprim resistance, including hypoxanthine synthesis, adenosine metabolism and guanosine metabolism. Another reason of drug tolerance is the efflux pump of the bacterium. In transcriptomic data, it was shown that the expression of some related enzymes in adenosine metabolism were raised significantly in smpB deletion strain than that of wild type, which led to the differential trimethoprim resistance of Aeromonas veronii (A. veronii). Furthermore, the metabolic products of adenosine AMP, cAMP, and deoxyadenosine were accumulated significantly. However, the expressions of the enzymes related to hypoxanthine synthesis and guanosine metabolism were elevated significantly in ssrA (small stable RNA, tmRNA) deletion strain, which eventually caused an augmented metabolic product xanthine. In addition, the deletion of ssrA also affected the significant downregulations of efflux pump acrA/acrB. The minimal inhibitory concentrations (MIC) were overall decreased after the trimethoprim treatment to the wild type, ΔsmpB and ΔssrA. And the difference in sensitivity between ΔsmpB and ΔssrA was evident. The MIC of ΔsmpB was descended significantly than those of wild type and ΔssrA in M9 medium supplemented with 1 mM adenosine, illustrating that the adenosine metabolism pathway was principally influenced by SmpB. Likewise, the strain ΔssrA conferred more sensitivity than wild type and ΔsmpB in M9 medium supplemented with 1mM guanosine. By overexpressing acrA/acrB, the tolerance to trimethoprim was partially recovered in ΔssrA. These results revealed that SmpB and tmRNA acted on different branches in purine metabolism, conferring the diverse trimethoprim resistance to A. veronii. This study suggests that the trans-translation system might be an effective target in clinical treatment of A. veronii and other multi-antibiotic resistance bacteria with trimethoprim.


Assuntos
Aeromonas veronii , Resistência a Trimetoprima , Aeromonas veronii/genética , Biossíntese de Proteínas , Purinas , RNA Bacteriano/metabolismo
17.
Sci Rep ; 10(1): 5315, 2020 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-32210253

RESUMO

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/genética
18.
Prog Urol ; 29(16): 943-946, 2019 Dec.
Artigo em Francês | MEDLINE | ID: mdl-31757712

RESUMO

OBJECTIVE: The objective of this study was to assess the level of resistance of trimethoprim alone (TMP) with respect to E. coli strains isolated from the urines of women with simple acute cystitis in community. PATIENTS AND METHODS: Prospective study realized for 9 months in 2017-18. A total of 351 urine samples were analyzed. Culture has been made according to the usual techniques and antibiogram was carried out according to the recommendations of the CA-SFM. RESULTS: The rate of resistance to TMP was 16.5% (58/351). Only 11 strains of E. coli (3%) producing ESBL were found, 5 of which were sensitive to TMP. CONCLUSION: The resistance rate of E. coli to TMP remains below 20%, the threshold for choosing a probabilistic treatment of a non-serious infection. Considering the good tolerance of TMP and its weak effect on the microbiota during a short treatment, one can propose TMP alone in the probabilistic treatment of simple acute cystitis.


Assuntos
Cistite/tratamento farmacológico , Cistite/microbiologia , Infecções por Escherichia coli/tratamento farmacológico , Escherichia coli/efeitos dos fármacos , Resistência a Trimetoprima , Trimetoprima/farmacologia , Trimetoprima/uso terapêutico , Infecções Urinárias/tratamento farmacológico , Infecções Urinárias/microbiologia , Doença Aguda , Adolescente , Adulto , Feminino , França , Humanos , Estudos Prospectivos
19.
Eur J Clin Microbiol Infect Dis ; 38(12): 2283-2290, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31494829

RESUMO

In recent years, high frequencies of trimethoprim resistance in urinary tract infections (UTIs) caused by E. coli are have been reported. Co-resistance to other antimicrobial drugs may play a role in this increase. Therefore, we investigated whether previous use of other antimicrobial drugs was associated with trimethoprim resistance. We conducted a nested case-control study with urinary cultures with E. coli from participants of the Rotterdam Study sent in by general practitioners to the regional laboratory between 1 January 2000 and 1 April 2016. Multivariable logistic regression analysis was performed to study the association between prior prescriptions of several antimicrobial drug groups and trimethoprim resistance using individual participant data. Urinary cultures of 1264 individuals with a UTI caused by E. coli were included. When adjusted for previous other antimicrobial drug use, a history of > 3 prescriptions of extended-spectrum penicillins (OR 1.68; 95% CI 1.10-2.55) was significantly associated with trimethoprim resistance of E. coli as was the use of > 3 prescriptions of sulfonamides and trimethoprim (OR 2.22; 95% CI 1.51-3.26). The use of > 3 prescriptions of nitrofuran derivatives was associated with a lower frequency of trimethoprim resistance (OR 0.60; 95% CI 0.39-0.92), after adjustment for other antimicrobial drug prescriptions. We found that previous use of extended-spectrum penicillins is associated with trimethoprim resistance. On the contrary, previous nitrofurantoin use was associated with a lower frequency of trimethoprim resistance. Especially in individuals with recurrent UTI, co-resistance should be taken into account and susceptibility testing before starting trimethoprim should be considered.


Assuntos
Antibacterianos/uso terapêutico , Infecções por Escherichia coli/tratamento farmacológico , Escherichia coli/efeitos dos fármacos , Resistência a Trimetoprima , Infecções Urinárias/tratamento farmacológico , Idoso , Idoso de 80 Anos ou mais , Antibacterianos/farmacologia , Estudos de Casos e Controles , Farmacorresistência Bacteriana Múltipla , Escherichia coli/isolamento & purificação , Infecções por Escherichia coli/epidemiologia , Infecções por Escherichia coli/microbiologia , Feminino , Medicina Geral , Humanos , Masculino , Testes de Sensibilidade Microbiana , Países Baixos/epidemiologia , Nitrofurantoína/farmacologia , Nitrofurantoína/uso terapêutico , Penicilinas/farmacologia , Penicilinas/uso terapêutico , Padrões de Prática Médica/estatística & dados numéricos , Fatores de Tempo , Trimetoprima/farmacologia , Trimetoprima/uso terapêutico , Infecções Urinárias/epidemiologia , Infecções Urinárias/microbiologia
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