RESUMEN
Background: Aminoglycoside-modifying enzymes (AMEs) play an essential role in bacterial resistance to aminoglycoside antimicrobials. With the development of sequencing techniques, more bacterial genomes have been sequenced, which has aided in the discovery of an increasing number of novel resistance mechanisms. Methods: The bacterial species was identified by 16S rRNA gene homology and average nucleotide identity (ANI) analyses. The minimum inhibitory concentration (MIC) of each antimicrobial was determined by the agar dilution method. The protein was expressed with the pCold I vector in E. coli BL21, and enzyme kinetic parameters were examined. The whole-genome sequence of the bacterium was obtained via the Illumina and PacBio sequencing platforms. Reconstruction of the phylogenetic tree, identification of conserved functional residues, and gene context analysis were performed using the corresponding bioinformatic techniques. Results: A novel aminoglycoside resistance gene, designated aph(3')-Ie, which confers resistance to ribostamycin, kanamycin, sisomicin and paromomycin, was identified in the chromosome of the animal bacterium Citrobacter gillenii DW61, which exhibited a multidrug resistance phenotype. APH(3')-Ie showed the highest amino acid identity of 74.90% with the functionally characterized enzyme APH(3')-Ia. Enzyme kinetics analysis demonstrated that it had phosphorylation activity toward four aminoglycoside substrates, exhibiting the highest affinity (K m, 4.22 ± 0.88 µM) and the highest catalytic efficiency [k cat/K m, (32.27 ± 8.14) × 104] for ribomycin. Similar to the other APH(3') proteins, APH(3')-Ie contained all the conserved functional sites of the APH family. The aph(3')-Ie homologous genes were present in C. gillenii isolates from different sources, including some of clinical significance. Conclusion: In this work, a novel chromosomal aminoglycoside resistance gene, designated aph(3')-Ie, conferring resistance to aminoglycoside antimicrobials, was identified in a rabbit isolate C. gillenii DW61. The elucidation of the novel resistance mechanism will aid in the effective treatment of infections caused by pathogens carrying such resistance genes.
Asunto(s)
Aminoglicósidos , Antibacterianos , Citrobacter , Pruebas de Sensibilidad Microbiana , Filogenia , ARN Ribosómico 16S , Animales , Antibacterianos/farmacología , Antibacterianos/metabolismo , Citrobacter/enzimología , Citrobacter/genética , Citrobacter/metabolismo , Citrobacter/clasificación , Aminoglicósidos/farmacología , Aminoglicósidos/metabolismo , ARN Ribosómico 16S/genética , Conejos , Farmacorresistencia Bacteriana Múltiple/genética , Genoma Bacteriano , Secuenciación Completa del Genoma , Sisomicina/farmacología , Sisomicina/análogos & derivados , Sisomicina/metabolismo , Kanamicina Quinasa/genética , Kanamicina Quinasa/metabolismo , Ribostamicina/metabolismo , Farmacorresistencia Bacteriana/genética , Kanamicina/farmacología , Escherichia coli/genética , Escherichia coli/metabolismo , Infecciones por Enterobacteriaceae/microbiologíaRESUMEN
The rapid development of antibiotic resistance, especially among difficult-to-treat Gram-negative bacteria, is recognized as a serious and urgent threat to public health. The detection and characterization of novel resistance mechanisms are essential to better predict the spread and evolution of antibiotic resistance. Corramycin is a novel and modified peptidic antibiotic with activity against several Gram-negative pathogens. We demonstrate that the kinase ComG, part of the corramycin biosynthetic gene cluster, phosphorylates and thereby inactivates corramycin, leading to the resistance of the host. Remarkably, we found that the closest structural homologues of ComG are aminoglycoside phosphotransferases; however, ComG shows no activity toward this class of antibiotics. The crystal structure of ComG in complex with corramycin reveals that corramycin adopts a ß-hairpin-like structure and allowed us to define the changes leading to a switch in substrate from sugar to peptide. Bioinformatic analyses suggest a limited occurrence of ComG-like proteins, which along with the absence of cross-resistance to clinically used drugs positions corramycin as an attractive antibiotic for further development.
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Antibacterianos , Bacterias Gramnegativas , Antibacterianos/química , Bacterias Gramnegativas/metabolismo , Kanamicina Quinasa/química , Kanamicina Quinasa/genética , Kanamicina Quinasa/metabolismo , PéptidosRESUMEN
Dichomitus squalens is an efficient white-rot fungus that generates a wide range of extracellular enzymes to degrade lignocellulose in nature. Although a protoplast-mediated transformation method for D. squalens has been developed, the transformation efficiency remains low. Here, we established a highly efficient Agrobacterium tumefaciens-mediated transformation (ATMT) procedure for D. squalens by transferring a binary vector harboring the neomycin phosphotransferase II (nptII) resistance gene fused with DsRed-Express2, under the control of the native glyceraldehyde-3-phosphate dehydrogenase (GPD) gene promoter. Key factors affecting the efficiency of transformation were tested. A. tumefaciens EHA105 strain with a cell density of 0.4 OD600nm and 96 h co-cultivation resulted in the highest transformation efficiency, with an average of 98 ± 11 transformants per co-cultivation plate. Besides, the strong expression of DsRed-Express2 indicates the effectiveness of the DsGPD promoter in driving gene expression in D. squalens. This ATMT system of D. squalens would be beneficial for its molecular genetic studies.
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Basidiomycota , Polyporaceae , Agrobacterium tumefaciens/genética , Kanamicina Quinasa/genética , Transformación GenéticaRESUMEN
Glyphosate herbicide treatment is essential to sustainable Eucalyptus plantation management in Brazil. Eucalyptus is highly sensitive to glyphosate, and Suzano/FuturaGene has genetically modified eucalyptus to tolerate glyphosate, with the aim of both protecting eucalyptus trees from glyphosate application damage and improving weed management. This study presents the biosafety results of the glyphosate-tolerant eucalyptus event 751K032, which expresses the selection marker neomycin phosphotransferase II (NPTII) enzyme and CP4-EPSPS, a glyphosate-tolerant variant of plant 5-enolpyruvyl-shikimate-3-phosphate synthase enzyme. The transgenic genetically modified (GM) event 751K032 behaved in the plantations like conventional non-transgenic eucalyptus clone, FGN-K, and had no effects on arthropods and soil microorganisms. The engineered NPTII and CP4 EPSPS proteins were heat-labile, readily digestible, and according to the bioinformatics analyses, unlikely to cause an allergenic or toxic reaction in humans or animals. This assessment of the biosafety of the glyphosate-tolerant eucalyptus event 751K032 concludes that it is safe to be used for wood production.
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Eucalyptus , Herbicidas , Animales , Humanos , Kanamicina Quinasa , Plantas Modificadas Genéticamente , Eucalyptus/genética , Herbicidas/toxicidadRESUMEN
The novel bacterial strain Marseille-P4005T was isolated from the stool sample of a healthy donor. It is a Gram-stain negative, non-motile, non-spore-forming rod. It grew optimally at 37 °C and at pH 7.0 on 5% sheep blood-enriched Columbia agar after preincubation in a blood-culture bottle supplemented with rumen and blood. This strain does not ferment monosaccharides (except D-tagatose), disaccharides, or polymeric carbohydrates. The major cellular fatty acids were hexadecenoic (24.6%), octadecanoic (22.8%), and tetradecanoic (20.1%) acids. Next-generation sequencing revealed a genome size of 3.2 Mbp with a 56.4 mol% G + C. Phylogenetic analysis based on the 16S rRNA gene highlighted Agathobaculum desmolans strain ATCC 43058T as the closest related strain. The OrthoANI, AAI, and digital DNA-DNA hybridization values were below the critical thresholds of 95%, 95-96%, and 70%, respectively, to define a novel bacterial species. Antibiotic resistance genes APH(3')-IIIa, erm(B), and tet(W) were detected with high identity percentages of 100%, 98.78%, and 97.18% for each gene, respectively. The APH(3')-IIIa gene confers resistance to amikacin, erm(B) gene confers resistance to erythromycin, lincomycin, and clindamycin, while tet(W) gene confers resistance to doxycycline and tetracycline. Based on KEGG BlastKOALA analyses, the annotation results showed that our strain could use glucose to produce L-lactate and pyruvate but not acetate or ethanol. Also, strain Marseille-P4005T was predicted to use phenylalanine to produce indole, a major intercellular signal molecule within the gut microbial ecosystem. Through having a gene coding for tryptophan synthase beta chain (trpB), strain Marseille-P4005T could produce L-tryptophan (an essential amino acid) from indole. Strain Marseille-P4005T showed its highest prevalence in the human gut (34.19%), followed by the reproductive system (17.98%), according to a query carried out on the Integrated Microbial NGS (IMNGS) platform. Based on phylogenetic, phenotypic, and genomic analyses, we classify strain Marseille-P4005T (= CSUR P4005 = CECT 9669), a novel species within the genus Agathobaculum, for which the name of Agathobaculum massiliense sp. nov. is proposed.
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Lactobacillales , Triptófano , Humanos , Triptófano/genética , Filogenia , ARN Ribosómico 16S/genética , Ecosistema , Kanamicina Quinasa/genética , Composición de Base , Genómica , Bacterias/genética , Lactobacillales/genética , Ácidos Grasos/química , Indoles , ADN , ADN Bacteriano/genética , ADN Bacteriano/química , Análisis de Secuencia de ADN , Técnicas de Tipificación BacterianaRESUMEN
BACKGROUND: Enterococci are considered as important causative pathogens of a variety of community and hospital-acquired infections. Due to the development of multidrug resistant (MDR) enterococci and the emergence of strains possessing high-level resistance to antimicrobial agents, treatment of their infections has been more complicated. In addition to more prevalent species of the Enterococcus genus, non-faecalis/non-faecium species are also responsible for severe healthcare-associated infections. Therefore, this study was designed to investigate high-level gentamicin resistance among the clinical isolates of non-faecalis and non-faecium enterococci in Shiraz, in the southwest of Iran. METHODS: A total of 28 non-faecalis/non-faecium spp. were isolated from various infections. They were identified by the conventional methods. Antimicrobial resistance patterns, multidrug resistance, and high-level gentamicin resistance were determined, according to CLSI guidelines and related definitions. Detection of aminoglycoside resistance genes was also performed using standard procedures. RESULTS: All of the isolates were MDR (100%), and 75% of them were high-level gentamicin resistant (HLGR) (MIC ≥ 500 µg/mL). The distributions of aac(6')-Ie-aph(2'')-Ia and aph(3')-IIIa resistance genes were 82.1% and 75%, respectively. The aph(2")-Ib, aph(2")-Ic, aph(2")-Id, and ant(4')-Ia genes were not found in any isolate. Although vancomycin resistance was observed in 19 (67.8%) isolates, all of the isolates were susceptible to linezolid and fosfomycin. CONCLUSIONS: Our data indicate a high rate of MDR non-faecalis/non-faecium isolates. Furthermore, high-level gentamicin resistance was notable and all of the HLGR isolates harbored at least one of aac(6')-Ie-aph(2'')-Ia or aph(3')-IIIa resistance gene.
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Antiinfecciosos , Fosfomicina , Aminoglicósidos/farmacología , Antibacterianos/farmacología , Antiinfecciosos/farmacología , Farmacorresistencia Bacteriana/genética , Enterococcus/genética , Fosfomicina/farmacología , Gentamicinas/farmacología , Humanos , Kanamicina Quinasa , Linezolid/farmacología , Pruebas de Sensibilidad MicrobianaRESUMEN
In this study, we evaluated the antimicrobial susceptibility, the presence of gene-encoding virulence factors and CRISPR systems, as well as the ability to produce lytic enzymes among clinical E. faecalis and E. faecium isolates (n = 44). All enterococci isolates showed phenotypes of multidrug resistance. E. faecalis and E. faecium isolates exhibited high-level aminoglycoside resistance phenotype, several of them harboring the aac(6')Ie-aph(2â³)Ia and aph(3')-IIIa genes. The gene vanA was the most frequent among vancomycin-resistant E. faecium. High prevalence of the virulence genes esp and efaA were observed; hyl gene was more associated with E. faecium, while ace and efaA genes were more frequently detected in E. faecalis. Caseinase activity was frequently detected among the isolates. Gelatinase and DNAse activities predominated among E. faecalis, while hemolytic capability was frequent among E. faecium isolates. Twenty-nine isolates showed at least one CRISPR system investigated. Several enterococci isolates harbored the aac(6')-Ie-aph(2â³)-Ia or aph(3')-IIIa genes and a CRISPR loci. CRISPR loci were positively correlated to efaA and gelE genes, and gelatinase and DNAse activities, while CRISPR loci absence was related to hyl gene presence. These results show that clinical isolates of E. faecalis and E. faecium harboring virulence genes show the concomitant presence of CRISPR loci and antibiotic resistance determinants.
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Enterococcus faecium , Infecciones por Bacterias Grampositivas , Aminoglicósidos , Antibacterianos/farmacología , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Desoxirribonucleasas/genética , Farmacorresistencia Bacteriana/genética , Enterococcus/genética , Enterococcus faecalis , Gelatinasas , Infecciones por Bacterias Grampositivas/epidemiología , Humanos , Kanamicina Quinasa/genética , Pruebas de Sensibilidad Microbiana , Vancomicina , Virulencia/genética , Factores de Virulencia/genéticaRESUMEN
Lanthipeptides are ribosomally synthesized and post-translationally modified peptides characterized by lanthionine (Lan) and/or methyllanthionine (MeLan) residues. Four classes of enzymes have been identified to install these structures in a substrate peptide. Recently, a novel class of lanthipeptides was discovered that lack genes for known class I-IV lanthionine synthases in their biosynthetic gene cluster (BGC). In this study, the dehydration of Ser/Thr during the biosynthesis of the class V lanthipeptide cacaoidin was reconstituted in vitro. The aminoglycoside phosphotransferase-like enzyme CaoK iteratively phosphorylates Ser/Thr residues on the precursor peptide CaoA, followed by phosphate elimination catalyzed by the HopA1 effector-like protein CaoY to achieve eight successive dehydrations. CaoY shows sequence similarity to the OspF family proteins and the lyase domains of class III/IV lanthionine synthetases, and mutagenesis studies identified residues that are critical for catalysis. An AlphaFold prediction of the structure of the dehydration enzyme complex engaged with its substrate suggests the importance of hydrophobic interactions between the CaoA leader peptide and CaoK in enzyme-substrate recognition. This model is supported by site-directed mutagenesis studies.
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Bacteriocinas , Liasas , Alanina/análogos & derivados , Bacteriocinas/metabolismo , Deshidratación , Kanamicina Quinasa/metabolismo , Ligasas/metabolismo , Liasas/metabolismo , Péptidos/química , Fosfatos , Señales de Clasificación de Proteína , SulfurosRESUMEN
Background: The intrinsic resistance mechanism plays an essential role in the bacterial resistance to a variety of the antimicrobials. The aim of this study is to find the chromosome-encoded novel antimicrobial resistance gene in the clinical isolate. Methods: The function of the predicted resistance gene was verified by gene cloning and antibiotic susceptibility test. Recombinant protein expression and enzyme kinetic studies were performed to explore the in vivo activity of the enzyme. Expression of the resistance gene exposed to antimicrobial was determined by RT-qPCR. Whole genome sequencing and bioinformatic analysis were applied to analyze the genetic context of the resistance gene. Results: The novel aminoglycoside (AG) resistance genes designated aph(9)-Ic and aph(9)-Ic1 confer resistance to spectinomycin, and a recombinant strain harboring aph(9)-Ic (pMD19-T-aph(9)-Ic/DH5α) showed a significantly increased minimum inhibitory concentration (MIC) level against spectinomycin compared with the control strains (DH5α and pMD19-T/DH5α). The result of the kinetic analysis of APH(9)-Ic was consistent with the MIC result for the recombinant pMD19-T-aph(9)-Ic/DH5α, showing the efficient catalytic activity for spectinomycin [kcat/Km ratio = (5.58 ± 0.31) × 104 M-1·s-1]. Whole-genome sequencing demonstrated that the aph(9)-Ic gene was located on the chromosome with a relatively conserved genetic environment, and no mobile genetic element was found in its surrounding region. Among all the function-characterized resistance genes, APH(9)-Ic shares the highest amino acid sequence identity of 33.75% with APH(9)-Ia. Conclusion: We characterized a novel AG resistance gene aph(9)-Ic and its variant aph(9)-Ic1 that mediated spectinomycin resistance from S. maltophilia. The identification of the novel AG resistance genes will assist us in elucidating the complexity of resistance mechanisms in microbial populations.
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Espectinomicina , Stenotrophomonas maltophilia , Kanamicina Quinasa/genética , Cinética , Stenotrophomonas maltophilia/genética , Antibacterianos/farmacología , Aminoglicósidos/farmacología , Pruebas de Sensibilidad Microbiana , Farmacorresistencia Bacteriana/genéticaRESUMEN
Kinases play central roles in many cellular processes, transferring the terminal phosphate groups of nucleoside triphosphates (NTPs) onto substrates. In the absence of substrates, kinases can also hydrolyse NTPs producing NDPs and inorganic phosphate. Hydrolysis is usually much less efficient than the native phosphoryl transfer reaction. This may be related to the fact that NTP hydrolysis is metabolically unfavorable as it unproductively consumes the cell's energy stores. It has been suggested that substrate interactions could drive changes in NTP binding pocket, activating catalysis only when substrates are present. Structural data show substrate-induced conformational rearrangements, however there is a lack of corresponding functional information. To better understand this phenomenon, we developed a suite of isothermal titration calorimetry (ITC) kinetics methods to characterize ATP hydrolysis by the antibiotic resistance enzyme aminoglycoside-3'-phosphotransferase-IIIa (APH(3')-IIIa). We measured Km, kcat, and product inhibition constants and single-turnover kinetics in the presence and absence of non-substrate aminoglycosides (nsAmgs) that are structurally similar to the native substrates. We found that the presence of an nsAmg increased the chemical step of cleaving the ATP γ-phosphate by at least 10- to 20-fold under single-turnover conditions, supporting the existence of interactions that link substrate binding to substantially enhanced catalytic rates. Our detailed kinetic data on the association and dissociation rates of nsAmgs and ADP shed light on the biophysical processes underlying the enzyme's Theorell-Chance reaction mechanism. Furthermore, they provide clues on how to design small-molecule effectors that could trigger efficient ATP hydrolysis and generate selective pressure against bacteria harboring the APH(3')-IIIa.
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Adenosina Trifosfato/metabolismo , Dominio Catalítico , Kanamicina Quinasa/metabolismo , Regulación Alostérica , Aminoglicósidos/metabolismo , Hidrólisis , Kanamicina Quinasa/química , Cinética , Unión ProteicaRESUMEN
OBJECTIVES: To describe a novel chromosomal aminoglycoside phosphotransferase named APH(3')-IId identified in an MDR Brucella intermedia ZJ499 isolate from a cancer patient. METHODS: Species identity was determined by PCR and MALDI-TOF MS analysis. WGS was performed to determine the genetic elements conferring antimicrobial resistance. Gene cloning, transcriptional analysis and targeted gene deletion, as well as protein purification and kinetic analysis, were performed to investigate the mechanism of resistance. RESULTS: APH(3')-IId consists of 266 amino acids and shares the highest identity (48.25%) with the previously known APH(3')-IIb. Expression of aph(3')-IId in Escherichia coli decreased susceptibility to kanamycin, neomycin, paromomycin and ribostamycin. The aph(3')-IId gene in ZJ499 was transcriptionally active under laboratory conditions and the relative abundance of this transcript was unaffected by treatment with the above four antibiotics. However, deletion of aph(3')-IId in ZJ499 results in decreased MICs of these drugs. The purified APH(3')-IId showed phosphotransferase activity against kanamycin, neomycin, paromomycin and ribostamycin, with catalytic efficiencies (kcat/Km) ranging from â¼105 to 107 M-1 s-1. Genetic environment and comparative genomic analyses suggested that aph(3')-IId is probably a ubiquitous gene in Brucella, with no mobile genetic elements detected in its surrounding region. CONCLUSIONS: APH(3')-IId is a novel chromosomal aminoglycoside phosphotransferase and plays an important role in the resistance of B. intermedia ZJ499 to kanamycin, neomycin, paromomycin and ribostamycin. To the best of our knowledge, APH(3')-IId represents the fourth characterized example of an APH(3')-II enzyme.
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Aminoglicósidos , Brucella , Farmacorresistencia Bacteriana Múltiple , Kanamicina Quinasa , Aminoglicósidos/farmacología , Antibacterianos/farmacología , Brucella/efectos de los fármacos , Brucella/enzimología , Humanos , Kanamicina/farmacología , Kanamicina Quinasa/genética , CinéticaRESUMEN
BACKGROUND: The C-3',4'-dideoxygenation structure in gentamicin can prevent deactivation by aminoglycoside 3'-phosphotransferase (APH(3')) in drug-resistant pathogens. However, the enzyme catalyzing the dideoxygenation step in the gentamicin biosynthesis pathway remains unknown. RESULTS: Here, we report that GenP catalyzes 3' phosphorylation of the gentamicin biosynthesis intermediates JI-20A, JI-20Ba, and JI-20B. We further demonstrate that the pyridoxal-5'-phosphate (PLP)-dependent enzyme GenB3 uses these phosphorylated substrates to form 3',4'-dideoxy-4',5'-ene-6'-oxo products. The following C-6'-transamination and the GenB4-catalyzed reduction of 4',5'-olefin lead to the formation of gentamicin C. To the best of our knowledge, GenB3 is the first PLP-dependent enzyme catalyzing dideoxygenation in aminoglycoside biosynthesis. CONCLUSIONS: This discovery solves a long-standing puzzle in gentamicin biosynthesis and enriches our knowledge of the chemistry of PLP-dependent enzymes. Interestingly, these results demonstrate that to evade APH(3') deactivation by pathogens, the gentamicin producers evolved a smart strategy, which utilized their own APH(3') to activate hydroxyls as leaving groups for the 3',4'-dideoxygenation in gentamicin biosynthesis.
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Antibacterianos/biosíntesis , Antibacterianos/metabolismo , Vías Biosintéticas/fisiología , Gentamicinas/biosíntesis , Gentamicinas/metabolismo , Antibacterianos/química , Biocatálisis , Vías Biosintéticas/genética , Gentamicinas/química , Kanamicina Quinasa/metabolismo , Micromonospora/enzimología , Micromonospora/genética , FosforilaciónRESUMEN
Among various species of marine bacteria, those belonging to the genus Halomonas have several promising applications and have been studied well. However, not much information has been available on their antibiotic resistance. In our efforts to learn about the antibiotic resistance of strain Halomonas socia CKY01, which showed production of various hydrolases and growth promotion by osmolytes in previous study, we found that it exhibited resistance to multiple antibiotics including kanamycin, ampicillin, oxacillin, carbenicillin, gentamicin, apramycin, tetracycline, and spectinomycin. However, the H. socia CKY01 resistance pattern to kanamycin, gentamicin, apramycin, tetracycline, and spectinomycin differed in the presence of 10% NaCl and 1% NaCl in the culture medium. To determine the mechanism underlying this NaCl concentration-dependent antibiotic resistance, we compared four aminoglycoside resistance genes under different salt conditions while also performing time-dependent reverse transcription PCR. We found that the aph2 gene encoding aminoglycoside phosphotransferase showed increased expression under the 10% rather than 1% NaCl conditions. When these genes were overexpressed in an Escherichia coli strain, pETDuet-1::aph2 showed a smaller inhibition zone in the presence of kanamycin, gentamicin, and apramycin than the respective control, suggesting aph2 was involved in aminoglycoside resistance. Our results demonstrated a more direct link between NaCl and aminoglycoside resistance exhibited by the H. socia CKY01 strain.
Asunto(s)
Aminoglicósidos/farmacología , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Halomonas/efectos de los fármacos , Cloruro de Sodio/metabolismo , Aminoglicósidos/análisis , Antibacterianos/análisis , Proteínas Bacterianas/metabolismo , Farmacorresistencia Bacteriana , Regulación Bacteriana de la Expresión Génica , Gentamicinas/farmacología , Halomonas/genética , Halomonas/metabolismo , Kanamicina/farmacología , Kanamicina Quinasa/genética , Kanamicina Quinasa/metabolismo , Nebramicina/análogos & derivados , Nebramicina/farmacología , Cloruro de Sodio/análisisRESUMEN
Aminoglycosides and ß-lactams are the most commonly used antimicrobial agents in clinical practice. This occurs because they are capable of acting in the treatment of acute bacterial infections. However, the effectiveness of antibiotics has been constantly threatened due to bacterial pathogens producing resistance enzymes. Among them, the aminoglycoside-modifying enzymes (AMEs) and ß-lactamase enzymes are the most frequently reported resistance mechanisms. AMEs can inactivate aminoglycosides by adding specific chemical molecules in the compound, whereas ß-lactamases hydrolyze the ß-lactams ring, preventing drug-target interaction. Thus, these enzymes provide a scenario of multidrug-resistance and a significant threat to public health at a global level. In response to this challenge, in recent decades, several studies have focused on the development of inhibitors that can restore aminoglycosides and ß-lactams activity. In this context, peptides appear as a promising approach in the field of inhibitors for future antibacterial therapies, as multiresistant bacteria may be susceptible to these molecules. Therefore, this review focused on the most recent findings related to peptide-based inhibitors that act on AMEs and ß-lactamases, and how these molecules could be used for future treatment strategies.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/uso terapéutico , Infecciones Bacterianas/tratamiento farmacológico , Ácido Clavulánico/uso terapéutico , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Sulbactam/uso terapéutico , Tazobactam/uso terapéutico , Aminoglicósidos/metabolismo , Aminoglicósidos/uso terapéutico , Antibacterianos/metabolismo , Antibacterianos/uso terapéutico , Infecciones Bacterianas/microbiología , Infecciones Bacterianas/patología , Diseño de Fármacos , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Gramnegativas/enzimología , Bacterias Gramnegativas/crecimiento & desarrollo , Bacterias Grampositivas/efectos de los fármacos , Bacterias Grampositivas/enzimología , Bacterias Grampositivas/crecimiento & desarrollo , Humanos , Kanamicina Quinasa/antagonistas & inhibidores , Kanamicina Quinasa/química , Kanamicina Quinasa/metabolismo , Nucleotidiltransferasas/antagonistas & inhibidores , Nucleotidiltransferasas/química , Nucleotidiltransferasas/metabolismo , beta-Lactamasas/química , beta-Lactamasas/metabolismo , beta-Lactamas/metabolismo , beta-Lactamas/uso terapéuticoRESUMEN
BACKGROUND: High antimicrobial use (AMU) and antimicrobial resistance (AMR) in veal calves remain a source of concern. As part of the EFFORT project, the association between AMU and the abundance of faecal antimicrobial resistance genes (ARGs) in veal calves in three European countries was determined. METHODS: In 2015, faecal samples of veal calves close to slaughter were collected from farms located in France, Germany and the Netherlands (20 farms in France, 20 farms in the Netherlands and 21 farms in Germany; 25 calves per farm). Standardized questionnaires were used to record AMU and farm characteristics. In total, 405 faecal samples were selected for DNA extraction and quantitative polymerase chain reaction to quantify the abundance (16S normalized concentration) of four ARGs [aph(3')-III, ermB, sul2 and tetW] encoding for resistance to frequently used antimicrobials in veal calves. Multiple linear mixed models with random effects for country and farm were used to relate ARGs to AMU and farm characteristics. RESULTS: A significant positive association was found between the use of trimethoprim/sulfonamides and the concentration of sul2 in faeces from veal calves. A higher weight of calves on arrival at the farm was negatively associated with aph(3')-III and ermB. Lower concentrations of aph(3')-III were found at farms with non-commercial animals present. Furthermore, farms using only water for the cleaning of stables had a significantly lower abundance of faecal ermB and tetW compared with other farms. CONCLUSION: A positive association was found between the use of trimethoprim/sulfonamides and the abundance of sul2 in faeces in veal calves. Additionally, other relevant risk factors associated with ARGs in veal calves were identified, such as weight on arrival at the farm and cleaning practices.
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Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Bacterias/genética , Sulfonamidas/farmacología , Trimetoprim/farmacología , Animales , Antibacterianos/administración & dosificación , Proteínas Bacterianas/genética , Proteínas Portadoras/genética , Bovinos , Enfermedades de los Bovinos/microbiología , Combinación de Medicamentos , Heces/microbiología , Francia , Alemania , Kanamicina Quinasa/genética , Metiltransferasas/genética , Países Bajos , Uso Excesivo de Medicamentos Recetados , Reacción en Cadena en Tiempo Real de la Polimerasa , Encuestas y CuestionariosRESUMEN
CRISPR-Cas9 is a powerful tool for generating targeted mutations and genomic deletions. However, precise gene insertion or sequence replacement remains a major hurdle before application of CRISPR-Cas9 technology is fully realized in plant breeding. Here, we report high-frequency, selectable marker-free intra-genomic gene targeting (GT) in maize. Heat shock-inducible Cas9 was used for generating targeted double-strand breaks and simultaneous mobilization of the donor template from pre-integrated T-DNA. The construct was designed such that release of the donor template and subsequent DNA repair activated expression of the selectable marker gene within the donor locus. This approach generated up to 4.7% targeted insertion of the donor sequence into the target locus in T0 plants, with up to 86% detected donor template release and 99% mutation rate being observed at the donor loci and the genomic target site, respectively. Unlike previous in planta or intra-genomic homologous recombination reports in which the original chimeric GT plants required extensive progeny screening in the next generation to identify non-chimeric GT individuals, our method provides non-chimeric heritable GT in one generation.
Asunto(s)
Sistemas CRISPR-Cas , Marcación de Gen/métodos , Zea mays/genética , Marcadores Genéticos , Kanamicina Quinasa/genética , Mutagénesis InsercionalRESUMEN
The aminoglycoside phosphotransferase (APH(3')-IIIa) kinases form a clinically central group of antibiotic-resistant enzymes. Computationally, we have studied the catalytic mechanism of the APH(3')-IIIa enzyme at the atomic-level. The proposed reaction mechanism involves protonation of Asp190 by the kanamycin 3'-hydroxyl group mediated through an explicit neighboring water molecule, which leads to a simultaneous nucleophilic attack on the γ-phosphate of the ATP by the deprotonated kanamycin 3'-hydroxyl group. The second step is a proton abstraction from the protonated Asp190 to the phosphate group of the phosphorylated kanamycin mediated by an explicit water molecule. The calculated Gibbs energy of activation (ΔG⧧) of the rate-determining step for the phosphorylation reaction is 77 kJ mol-1 at the M06-2X/6-311++G(2df,p)//ONIOM(M06-2X/6-31+G(d):HF/6-31G(d)) level of theory. This study has provided a new understanding of the APH(3')-IIIa catalytic mechanism that agrees with the available experimental data (ΔG⧧ = 75 ± 4 kJ mol-1) and could provide a starting point for the rational design of mechanism-based inhibitors of aminoglycoside modifying enzyme to circumvent antibiotic resistance.
Asunto(s)
Antibacterianos , Kanamicina , Catálisis , Kanamicina Quinasa/metabolismo , FosforilaciónRESUMEN
Neurospora crassa is an excellent model fungus for studies on molecular genetics, biochemistry, physiology, and molecular cell biology. Along with the rapid progress of Neurospora research, new tools facilitating more efficient and accurate genetic analysis are in high demand. Here, we tested whether the dominant selective makers widely used in yeasts are applicable in N. crassa. Among them, we found that the strains of N. crassa are sensitive to the aminoglycoside antibiotics, G418 and nourseothricin. 1000 µg/mL of G418 or 50 µg/mL of nourseothricin is sufficient to inhibit Neurospora growth completely. When the neomycin phosphotransferase gene (neo) used in mammalian cells is expressed, N. crassa shows potent resistance to G418. This establishes G418-resistant marker as a dominant selectable marker to use in N. crassa. Similarly, when the nourseothricin acetyltransferase gene (nat) from Streptomyces noursei is induced by qa-2 promoter in the presence of quinic acid (QA), N. crassa shows potent resistance to nourseothricin. When nat is constitutively expressed by full-length or truncated versions of the promoter from the N. crassa cfp gene (NCU02193), or by the trpC promoter of Aspergillus nidulans, the growth of N. crassa in the presence of nourseothricin is proportional to the expression levels of Nat. Finally, these two markers are used to knock-out wc-2 or al-1 gene from the N. crassa genome. The successful development of these two markers in this study expands the toolbox for N. crassa and very likely for other filamentous fungi as well.
Asunto(s)
Farmacorresistencia Fúngica/genética , Marcadores Genéticos , Neurospora crassa/efectos de los fármacos , Neurospora crassa/genética , Acetiltransferasas/genética , Antibacterianos/farmacología , Elementos Transponibles de ADN , Farmacorresistencia Fúngica/efectos de los fármacos , Regulación Fúngica de la Expresión Génica , Técnicas de Inactivación de Genes , Genes Dominantes , Gentamicinas/farmacología , Kanamicina Quinasa/genética , Microorganismos Modificados Genéticamente , Regiones Promotoras Genéticas , Ácido Quínico/farmacología , Estreptotricinas/farmacologíaRESUMEN
Eukaryotic filamentous yellow-green algae from the Tribonema genus are considered to be excellent candidates for biofuels and value-added products, owing to their ability to grow under autotrophic, mixotrophic, and heterotrophic conditions and synthesize large amounts of fatty acids, especially unsaturated fatty acids. To elucidate the molecular mechanism of fatty acids and/or establish the organism as a model strain, the development of genetic methods is important. Towards this goal, here, we constructed a genetic transformation method to introduce exogenous genes for the first time into the eukaryotic filamentous alga Tribonema minus via particle bombardment. In this study, we constructed pSimple-tub-eGFP and pEASY-tub-nptâ ¡ plasmids in which the green fluorescence protein (eGFP) gene and the neomycin phosphotransferase â ¡-encoding G418-resistant gene (nptâ ¡) were flanked by the T. minus-derived tubulin gene (tub) promoter and terminator, respectively. The two plasmids were introduced into T. minus cells through particle-gun bombardment under various test conditions. By combining agar and liquid selecting methods to exclude the pseudotransformants under long-term antibiotic treatment, plasmids pSimple-tub-eGFP and pEASY-tub- nptâ ¡ were successfully transformed into the genome of T. minus, which was verified using green fluorescence detection and the polymerase chain reaction, respectively. These results suggest new possibilities for efficient genetic engineering of T. minus for future genetic improvement.
Asunto(s)
Células Eucariotas/metabolismo , Microalgas/genética , Estramenopilos/genética , Transformación Genética , Biocombustibles , Ácidos Grasos/metabolismo , Ingeniería Genética/métodos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Microbiología Industrial/métodos , Kanamicina Quinasa/genética , Kanamicina Quinasa/metabolismo , Microalgas/metabolismo , Estramenopilos/metabolismoRESUMEN
Recent research has shown that plants can uptake long dsRNAs and dsRNA-derived siRNAs that target important genes of infecting fungi or viruses when applied on the surface of plant leaves. The external RNAs were capable of local and systemic movement inducing plant resistance against the pathogens. Few studies have been made for plant gene regulation by foliar application of RNAs. In this study, several types of ssRNA and siRNA duplexes targeting the neomycin phosphotransferase II (NPTII) transgene were in vitro-synthesized and externally applied to the leaf surface of 4-week-old transgenic Arabidopsis thaliana plants. External application of the synthetic NPTII-encoding siRNAs down-regulated NPTII transcript levels in transgenic A. thaliana 1 and 7 days post-treatment with a higher and more consistent effect being observed for siRNAs methylated at 3' ends. We also analyzed the effects of external NPTII-encoding dsRNA precursors and a dsRNA-derived heterogenous siRNA mix. Digestion of the NPTII-dsRNA to the heterogeneous siRNAs did not improve efficiency of the transgene suppression effect. Key Points⢠Foliar application of siRNAs down-regulated a commonly used transgene in Arabidopsis. ⢠A more consistent effect was observed for methylated siRNAs. ⢠The findings are important for development of plant gene regulation approaches.