RESUMO
The use of ß-lactam/ß-lactamase inhibitors constitutes an important strategy to counteract ß-lactamases in multidrug-resistant (MDR) Gram-negative bacteria. Recent reports have described ceftazidime-/avibactam-resistant isolates producing CTX-M variants with different amino acid substitutions (e.g., P167S, L169Q, and S130G). Relebactam (REL) combined with imipenem has proved very effective against Enterobacterales producing ESBLs, serine-carbapenemases, and AmpCs. Herein, we evaluated the inhibitory efficacy of REL against CTX-M-96, a CTX-M-15-type variant. The CTX-M-96 structure was obtained in complex with REL at 1.03 Å resolution (PDB 8EHH). REL was covalently bound to the S70-Oγ atom upon cleavage of the C7-N6 bond. Compared with apo CTX-M-96, binding of REL forces a slight displacement of the deacylating water inwards the active site (0.81 Å), making the E166 and N170 side chains shift to create a proper hydrogen bonding network. Binding of REL also disturbs the hydrophobic patch formed by Y105, P107, and Y129, likely due to the piperidine ring of REL that creates clashes with these residues. Also, a remarkable change in the positioning of the N104 sidechain is also affected by the piperidine ring. Therefore, differences in the kinetic behavior of REL against class A ß-lactamases seem to rely, at least in part, on differences in the residues being involved in the association and stabilization of the inhibitor before hydrolysis. Our data provide the biochemical and structural basis for REL effectiveness against CTX-M-producing Gram-negative pathogens and essential details for further DBO design. Imipenem/REL remains an important choice for dealing with isolates co-producing CTX-M with other ß-lactamases.
Assuntos
Compostos Azabicíclicos , Inibidores de beta-Lactamases , beta-Lactamases , Compostos Azabicíclicos/farmacologia , Compostos Azabicíclicos/química , beta-Lactamases/genética , beta-Lactamases/metabolismo , beta-Lactamases/química , Inibidores de beta-Lactamases/farmacologia , Inibidores de beta-Lactamases/química , Cristalografia por Raios X , Antibacterianos/farmacologia , Imipenem/farmacologia , Imipenem/química , Ceftazidima/farmacologia , Testes de Sensibilidade Microbiana , Domínio CatalíticoRESUMO
INTRODUCTION: Novel beta-lactam/beta-lactamase inhibitor (BIBLI) combinations are commercially available and have been used for treating carbapenem-resistant Klebsiella pneumoniae (CRKP) infections. Continuous surveillance of susceptibility profiles and resistance mechanism identification are necessary to monitor the evolution of resistance within these agents. OBJECTIVE: The purpose of this study was to evaluate the susceptibility rates of ceftazidime/avibactam, imipenem/relebactam and meropenem/vaborbactam in CRKP isolated from patients with bloodstream infections who underwent screening for a randomized clinical trial in Brazil. METHODS: Minimum inhibitory concentrations (MICs) were determined for meropenem, ceftazidime/avibactam, imipenem/relebactam and meropenem/vaborbactam using the gradient diffusion strip method. Carbapenemase genes were detected by multiplex real-time polymerase chain reaction. Klebsiella pneumoniae carbapenemase (KPC)-producing isolates showing resistance to any BLBLI and New Delhi Metallo-beta-lactamase (NDM)-producing isolates with susceptibility to any BLBLI isolates were further submitted for whole-genome sequencing. RESULTS: From a total of 69 CRKP isolates, 39 were positive for blaKPC, 19 for blaNDM and 11 for blaKPC and blaNDM. KPC-producing isolates demonstrated susceptibility rates above 94â¯% for all BLBLIs. Two isolates with resistance to meropenem/vaborbactam demonstrated a Gly and Asp duplication at the porin OmpK36 as well as a truncated OmpK35. All NDM-producing isolates, including KPC and NDM coproducers, demonstrated susceptibility rates to ceftazidime/avibactam, imipenem/relebactam and meropenem/vaborbactam of 0â¯%, 9.1-21.1â¯% and 9.1-26.3â¯%, respectively. Five NDM-producing isolates that presented susceptibility to BLBLIs also had porin alterations CONCLUSIONS: This study showed that, although high susceptibility rates to BLBLIs were found, KPC-2 isolates were able to demonstrate resistance probably as a result of porin mutations. Additionally, NDM-1 isolates showed susceptibility to BLBLIs in vitro.
Assuntos
Antibacterianos , Compostos Azabicíclicos , Enterobacteriáceas Resistentes a Carbapenêmicos , Ceftazidima , Combinação de Medicamentos , Infecções por Klebsiella , Klebsiella pneumoniae , Testes de Sensibilidade Microbiana , Inibidores de beta-Lactamases , beta-Lactamases , Humanos , Brasil , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/isolamento & purificação , Inibidores de beta-Lactamases/farmacologia , Infecções por Klebsiella/microbiologia , Compostos Azabicíclicos/farmacologia , Ceftazidima/farmacologia , beta-Lactamases/genética , Antibacterianos/farmacologia , Enterobacteriáceas Resistentes a Carbapenêmicos/efeitos dos fármacos , Enterobacteriáceas Resistentes a Carbapenêmicos/genética , Enterobacteriáceas Resistentes a Carbapenêmicos/isolamento & purificação , Proteínas de Bactérias/genética , Meropeném/farmacologia , Imipenem/farmacologia , Bacteriemia/microbiologia , Ácidos Borônicos/farmacologia , Compostos Heterocíclicos com 1 AnelRESUMO
BACKGROUND: Infections caused by Stenotrophomonas maltophilia and related species are increasing worldwide. Unfortunately, treatment options are limited, whereas the antimicrobial resistance is increasing. METHODS: We included clinical isolates identified as S. maltophilia by VITEK 2 Compact. Ceftazidime/avibactam, meropenem/vaborbactam, imipenem/relebactam, cefiderocol, quinolones, and tetracycline family members were evaluated by broth microdilution method and compared with first-line treatment drugs. Minimum inhibitory concentrations (MICs) were reported for all antibiotics. We sequenced the Whole Genome of cefiderocol resistant strains (CRSs) and annotated their genes associated with cefiderocol resistance (GACR). Presumptive phylogenetic identification employing the 16S marker was performed. RESULTS: One hundred and one clinical strains were evaluated, sulfamethoxazole and trimethoprim, levofloxacin and minocycline showed susceptibilities of 99.01%, 95.04% and 100% respectively. Ceftazidime was the antibiotic with the highest percentage of resistance in all samples (77.22%). Five strains were resistant to cefiderocol exhibiting MIC values ≥ 2 µg/mL (4.95%). The ß-lactamase inhibitors meropenem/vaborbactam and imipenem/relebactam, failed to inhibit S. maltophilia, preserving both MIC50 and MIC90 ≥64 µg/mL. Ceftazidime/avibactam restored the activity of ceftazidime decreasing the MIC range. Tigecycline had the lowest MIC range, MIC50 and MIC90. Phylogeny based on 16S rRNA allowed to identify to cefiderocol resistant strains as putative species clustered into Stenotrophomonas maltophilia complex (Smc). In these strains, we detected GARCs such as Mutiple Drug Resistance (MDR) efflux pumps, L1-type ß-lactamases, iron transporters and type-1 fimbriae. CONCLUSION: Antimicrobial resistance to first-line treatment is low. The in vitro activity of new ß-lactamase inhibitors against S. maltophilia is poor, but avibactam may be a potential option. Cefiderocol could be considered as a potential new option for multidrug resistant infections. Tetracyclines had the best in vitro activity of all antibiotics evaluated.
Assuntos
Ácidos Borônicos , Ceftazidima , Stenotrophomonas maltophilia , Ceftazidima/farmacologia , Cefiderocol , Meropeném , Inibidores de beta-Lactamases/farmacologia , Inibidores de beta-Lactamases/uso terapêutico , Stenotrophomonas , Filogenia , RNA Ribossômico 16S , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Compostos Azabicíclicos/farmacologia , Combinação de Medicamentos , Imipenem/farmacologia , Testes de Sensibilidade Microbiana , beta-Lactamases/genéticaRESUMO
Antimicrobial resistance is a global public health threat. Metallo-ß-lactamases (MBLs) inactivate ß-lactam antibiotics, including carbapenems, are disseminating among Gram-negative bacteria, and lack clinically useful inhibitors. The evolving bisthiazolidine (BTZ) scaffold inhibits all three MBL subclasses (B1-B3). We report design, synthesis, and evaluation of BTZ analogues. Structure-activity relationships identified the BTZ thiol as essential, while carboxylate is replaceable, with its removal enhancing potency by facilitating hydrophobic interactions within the MBL active site. While the introduction of a flexible aromatic ring is neutral or detrimental for inhibition, a rigid (fused) ring generated nM benzobisheterocycle (BBH) inhibitors that potentiated carbapenems against MBL-producing strains. Crystallography of BBH:MBL complexes identified hydrophobic interactions as the basis of potency toward B1 MBLs. These data underscore BTZs as versatile, potent broad-spectrum MBL inhibitors (with activity extending to enzymes refractory to other inhibitors) and provide a rational approach to further improve the tricyclic BBH scaffold.
Assuntos
Antibacterianos , Inibidores de beta-Lactamases , Inibidores de beta-Lactamases/farmacologia , Inibidores de beta-Lactamases/química , Antibacterianos/farmacologia , Antibacterianos/química , beta-Lactamases/química , Carbapenêmicos , Bactérias Gram-NegativasRESUMO
The design of inhibitors against metallo-ß-lactamases (MBLs), the largest family of carbapenemases, has been a strategic goal in designing novel antimicrobial therapies. In this regard, the development of bicyclic boronates, such as taniborbactam (TAN) and xeruborbactam, is a major achievement that may help in overcoming the threat of MBL-producing and carbapenem-resistant Gram-negative pathogens. Of concern, a recent report has shown that New Delhi MBL-9 (NDM-9) escapes the inhibitory action of TAN by a single amino acid substitution with respect to New Delhi MBL-1 (NDM-1), the most widely disseminated MBL. Here, we report a docking and computational analysis that identifies that "escape variants" against TAN can arise by disruption of the electrostatic interaction of negative charges in the active site loops of MBLs with the N-(2-aminoethyl)cyclohexylamine side chain of TAN. These changes result in non-productive binding modes of TAN that preclude reaction with the MBLs, a phenomenon that is not restricted to NDM-9. This analysis demonstrates that single amino acid substitutions in non-essential residues in MBL loops can unexpectedly elicit resistance to TAN.
Assuntos
Antibacterianos , Ácidos Borínicos , Ácidos Carboxílicos , Antibacterianos/farmacologia , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases/metabolismo , Ácidos Borínicos/farmacologia , Resistência beta-Lactâmica , Testes de Sensibilidade MicrobianaRESUMO
The bacterial species Staphylococcus aureus presents a variety of resistance mechanisms, among which the expression of ß-lactamases and efflux pumps stand out for providing a significant degree of resistance to clinically relevant antibiotics. The 1,8-naphthyridines are nitrogen heterocycles with a broad spectrum of biological activities and, as such, are promising research targets. However, the potential roles of these compounds on bacterial resistance management remain to be better investigated. Therefore, the present study evaluated the antibacterial activity of 1,8-naphthyridine sulfonamides, addressing their ability to act as inhibitors of ß-lactamases and efflux pump (QacA/B and QacC) against the strains SA-K4414 and SA-K4100 of S. aureus. All substances were prepared at an initial concentration of 1024 µg/mL, and their minimum inhibitory concentrations (MIC) were determined by the broth microdilution method. Subsequently, their effects on ß-lactamase- and efflux pump-mediated antibiotic resistance was evaluated from the reduction of the MIC of ethidium bromide (EtBr) and ß-lactam antibiotics, respectively. The 1,8-naphthyridines did not present direct antibacterial activity against the strains SA-K4414 and SA-K4100 of S. aureus. On the other hand, when associated with antibiotics against both strains, the compounds reduced the MIC of EtBr and ß-lactam antibiotics, suggesting that they may act by inhibiting ß-lactamases and efflux pumps such as QacC and QacA/B. However, further research is required to elucidate the molecular mechanisms underlying these observed effects.
Assuntos
Antibacterianos , Staphylococcus aureus , Inibidores de beta-Lactamases , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases/metabolismo , beta-Lactamas/farmacologia , Testes de Sensibilidade Microbiana , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Staphylococcus aureus/efeitos dos fármacosRESUMO
BACKGROUND: Gram-negative and Gram-positive bacteria produce beta-lactamase as factors to overcome beta-lactam antibiotics, causing their hydrolysis and impaired antimicrobial action. Class A beta-lactamase contains the chromosomal sulfhydryl reagent variable (SHV, point mutation variants of SHV-1), LEN (Klebsiella pneumoniae strain LEN-1), and other K. pneumoniae beta-lactamase (OKP) found mostly in Klebsiella's phylogroups. The SHV known as extended-spectrum ß-lactamase can inactivate most beta-lactam antibiotics. Class A also includes the worrisome plasmid-encoded Klebsiella pneumoniae carbapenemase (KPC-2), a carbapenemase that can inactivate most beta-lactam antibiotics, carbapenems, and some beta-lactamase inhibitors. OBJECTIVES: So far, there is no 3D crystal structure for OKP-B, so our goal was to perform structural characterisation and molecular docking studies of this new enzyme. METHODS: We applied a homology modelling method to build the OKP-B-6 structure, which was compared with SHV-1 and KPC-2 according to their electrostatic potentials at the active site. Using the DockThor-VS, we performed molecular docking of the SHV-1 inhibitors commercially available as sulbactam, tazobactam, and avibactam against the constructed model of OKP-B-6. FINDINGS: From the point of view of enzyme inhibition, our results indicate that OKP-B-6 should be an extended-spectrum beta-lactamase (ESBL) susceptible to the same drugs as SHV-1. MAIN CONCLUSIONS: This conclusion advantageously impacts the clinical control of the bacterial pathogens encoding OKP-B in their genome by using any effective, broad-spectrum, and multitarget inhibitor against SHV-containing bacteria.
Assuntos
Sulbactam , Inibidores de beta-Lactamases , Antibacterianos/farmacologia , Carbapenêmicos/farmacologia , Klebsiella , Klebsiella pneumoniae , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Sulbactam/farmacologia , Reagentes de Sulfidrila/farmacologia , Tazobactam/farmacologia , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases/genéticaRESUMO
Diazabicyclooctanone inhibitors such as ETX2514 and avibactam have shown enhanced inhibitory performance to fight the antibiotic resistance developed by pathogens. However, avibactam is ineffective against Acinetobacter baumannii infections, unlike ETX2514. The molecular basis for this difference has not been tackled from a molecular approach, precluding the knowledge of relevant information. In this article, the mechanisms involved in the inhibition of OXA-24 by ETX2514 and avibactam are studied theoretically by hybrid QM/MM calculations. The results show that both inhibitors share the same inhibition mechanisms, comprising acylation a deacylation stages. The involved mechanisms include the same number of steps, transition states and intermediates; although they differ in the involved activation barriers. This difference accounts for the dissimilar inhibitory ability of both inhibitors. The molecular reason for this is the endocyclic double bond in the piperidine ring of ETX2514 increasing the ring strain and chemical reactivity on the N6 and C7 atoms, besides the methyl substituent, which enhance the hydrophobic character of the ring. Furthermore, Lys218 and the carboxylated Lys84 of ETX2514, play a crucial role in the mechanism by coordinating their protonation states in an on/off (protonated/deprotonated) manner, favoring the proton transference between the residues and the inhibitor.
Assuntos
Antibacterianos , Inibidores de beta-Lactamases , Antibacterianos/química , Antibacterianos/farmacologia , Compostos Azabicíclicos , Testes de Sensibilidade Microbiana , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases/químicaRESUMO
Bacterial resistance is responsible for a wide variety of health problems, both in children and adults. The persistence of symptoms and infections are mainly treated with ß-lactam antibiotics. The increasing resistance to those antibiotics by bacterial pathogens generated the emergence of extended-spectrum ß-lactamases (ESBLs), an actual public health problem. This is due to rapid mutations of bacteria when exposed to antibiotics. In this case, ß-lactamases are enzymes used by bacteria to hydrolyze the beta-lactam rings present in the antibiotics. Therefore, it was necessary to explore novel molecules as potential ß-lactamases inhibitors to find antibacterial compounds against infection caused by ESBLs. A computational methodology based on molecular docking and molecular dynamic simulations was used to find new microalgae metabolites inhibitors of ß-lactamase. Six 3D ß-lactamase proteins were selected, and the molecular docking revealed that the metabolites belonging to the same structural families, such as phenylacridine (4-Ph), quercetin (Qn), and cryptophycin (Cryp), exhibit a better binding score and binding energy than commercial clinical medicine ß-lactamase inhibitors, such as clavulanic acid, sulbactam, and tazobactam. These results indicate that 4-Ph, Qn, and Cryp molecules, homologous from microalgae metabolites, could be used, likely as novel ß-lactamase inhibitors or as structural templates for new in-silico pharmaceutical designs, with the possibility of combatting ß-lactam resistance.
Assuntos
Bactérias/enzimologia , Fatores Biológicos/farmacologia , Microalgas/química , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases/metabolismo , Bactérias/efeitos dos fármacos , Fatores Biológicos/química , Depsipeptídeos/química , Depsipeptídeos/farmacologia , Modelos Moleculares , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Estrutura Molecular , Conformação Proteica , Quercetina/química , Quercetina/farmacologia , Resistência beta-Lactâmica , Inibidores de beta-Lactamases/química , beta-Lactamases/químicaRESUMO
The past two decades have been marked by a global spread of bacterial resistance to ß-lactam drugs and carbapenems derivatives are the ultimate treatment against multidrug-resistant bacteria. ß-lactamase expression is related to resistance which demands the development of bacterial resistance blockers. Drug inhibitor combinations of serine-ß-lactamase and ß-lactam were successful employed in therapy despite their inactivity against New Delhi metallo-beta-lactamase (NDM). Until now, few compounds are active against NDM-producing bacteria and no specific inhibitors are available yet. The rational strategy for NDM inhibitors development starts with in vitro assays aiming to seek compounds that could act synergistically with ß-lactam antibiotics. Thus, eight thiosemicarbazone derivatives were synthesized and investigated for their ability to reverse the resistant phenotype in NDM in Enterobacter cloacae. Phenotypic screening indicated that four isatin-beta-thiosemicarbazones showed Fractional Inhibitory Concentration (FIC) ≤ 250 µM in the presence of meropenem (4 µg/mL). The most promising compound (FIC= 31.25 µM) also presented synergistic effect (FICI = 0.34). Docking and molecular dynamics studies on NDM-thiosemicarbazone complex suggested that 2,3-dihydro-1H-indol-2-one subunit interacts with catalytic zinc and interacted through hydrogen bonds with Asp124 acting like a carboxylic acid bioisostere. Additionally, thiosemicarbazone tautomer with oxidized sulfur (thione) seems to act as a spacer rather than zinc chelator, and the aromatic moieties are stabilized by pi-pi and cation-pi interactions with His189 and Lys221 residues. Our results addressed some thiosemicarbazone structural changes to increase its biological activity against NDM and highlight its scaffold as promising alternatives to treat bacterial resistance.Communicated by Ramaswamy H. Sarma.