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1.
Cells ; 11(17)2022 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-36078157

RESUMO

Serine beta-lactamase-like protein (LACTB) is the only mammalian mitochondrial homolog evolved from penicillin-binding proteins and ß-lactamases (PBP-ßLs) in bacteria. LACTB, an active-site serine protease, polymerizes into stable filaments, which are localized to the intermembrane space (IMS) of mitochondrion and involved in the submitochondrial organization, modulating mitochondrial lipid metabolism. Cancer pathogenesis and progression are relevant to the alterations in mitochondrial metabolism. Metabolic reprogramming contributes to cancer cell behavior. This article (1) evidences the clinical implications of LACTB on neoplastic cell proliferation and migration and tumor growth and metastasis as well as LACTB's involvement in chemotherapeutic and immunotherapeutic responses; (2) sketches the structural basis for LACTB activity and function; and (3) highlights the relevant regulatory mechanisms to LACTB. The abnormal expression of LACTB has been associated with clinicopathological features of cancer tissues and outcomes of anticancer therapies. With the current pioneer researches on the tumor-suppressed function, structural basis, and regulatory mechanism of LACTB, the perspective hints at a great appeal of enzymic property, polymerization, mutation, and epigenetic and post-translational modifications in investigating LACTB's role in cancer pathogenesis. This perspective provides novel insights for LACTB as a metabolic regulator with potential to develop targeted cancer therapies or neoadjuvant therapeutic interventions.


Assuntos
Proteínas Mitocondriais , Neoplasias , Animais , Mamíferos/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Neoplasias/metabolismo , beta-Lactamases/genética , beta-Lactamases/metabolismo
2.
Front Cell Infect Microbiol ; 12: 981792, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36118031

RESUMO

Ceftazidime/Avibactam (CAZ/AVI) is frequently used to treat KPC-producing Pseudomonas aeruginosa (KPC-PA) and Enterobacterales. CAZ/AVI resistance is driven by several mechanisms. In P. aeruginosa this mainly occurs through alteration of AmpC, porins, and/or efflux pump overexpression, whereas in Enterobacterales it frequently occurs through D179Y substitution in the active site of KPC enzyme. This aminoacid change abolishes AVI binding to the KPC active site, hence inhibition is impaired. However, this substitution also decreases KPC-mediated resistance to carbapenems ("see-saw" effect). The goal of this work was to characterize the in vivo acquisition of CAZ/AVI resistance through D179Y substitution in a KPC-PA isolated from a hospitalized patient after CAZ/AVI treatment. Two KPC-PA isolates were obtained. The first isolate, PA-1, was obtained before CAZ/AVI treatment and was susceptible to CAZ/AVI. The second isolate, PA-2, was obtained after CAZ/AVI treatment and exhibited high-level CAZ/AVI resistance. Characterization of isolates PA-1 and PA-2 was performed through short and long-read whole genome sequencing analysis. The hybrid assembly showed that PA-1 and PA-2A had a single plasmid of 54,030 bp, named pPA-1 and pPA-2 respectively. Each plasmid harbored two copies of the bla KPC-containing Tn4401b transposon. However, while pPA-1 carried two copies of bla KPC-2, pPA-2 had one copy of bla KPC-2 and one copy of bla KPC-33, the allele with the D179Y substitution. Interestingly, isolate PA-2 did not exhibit the "see-saw" effect. The bla KPC-33 allele was detected only through hybrid assembly using a long-read-first approach. The present work describes a KPC-PA isolate harboring a plasmid-borne CAZ/AVI resistance mechanism based on two copies of bla KPC-2-Tn4401b and D179Y mutation in one of them, that is not associated with loss of resistance to carbapenems. These findings highlight the usefulness of a fine-tuned combined analysis of short and long-read data to detect similar emerging resistance mechanisms.


Assuntos
Ceftazidima , Pseudomonas aeruginosa , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Compostos Azabicíclicos , Carbapenêmicos/farmacologia , Ceftazidima/farmacologia , Combinação de Medicamentos , Humanos , Testes de Sensibilidade Microbiana , Mutação , Porinas/genética , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , beta-Lactamases/genética , beta-Lactamases/metabolismo
3.
J Proteomics ; 268: 104715, 2022 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-36058541

RESUMO

Carbapenem-resistant Klebsiella pneumoniae (CRKP) is an urgent threat to human health. Major outer membrane proteins (OMPs) porin mutation is one important resistance mechanism of CRKP, and may also affect the inhibition activity of ß-lactam and ß-lactamase inhibitor combinations. The ertapenem-resistant K. pneumoniae strain 2018B120 with major porin mutations was isolated from a clinical patient. Genomic and time-series proteomic analyses were conducted to retrieve the ertapenem-challenged response of 2018B120. The abundance changing of proteins from PTS systems,  ABC transporters, the autoinducer 2 (AI-2) quorum sensing system, and antioxidant systems can be observed. Overexpression of alternative porins was also noticed to balance major porins' defection. These findings added a detailed regulation network in bacterial resistance mechanisms and gave new insights into bypass adaptation mechanisms the porin deficient bacteria adopted under carbapenem antibiotics pressure. SIGNIFICANCE: Outer membrane porins deficiency is an important mechanism of carbapenem resistance in K. pneumoniae. Comprehensive genomic and proteomic profiling of an ertapenem-resistant K. pneumoniae strain 2018B120 gives a detailed systematic regulation network in bacterial resistance mechanisms. Overexpression of alternative porins to balance major porins' defection was noticed, giving new insights into bypass adaptation mechanisms of porin deficient bacteria.


Assuntos
Klebsiella pneumoniae , Porinas , Transportadores de Cassetes de Ligação de ATP/metabolismo , Antibacterianos/farmacologia , Antioxidantes/metabolismo , Proteínas de Bactérias/metabolismo , Carbapenêmicos/metabolismo , Carbapenêmicos/farmacologia , Ertapenem/metabolismo , Humanos , Testes de Sensibilidade Microbiana , Porinas/metabolismo , Proteômica , Inibidores de beta-Lactamases/metabolismo , beta-Lactamases/genética , beta-Lactamases/metabolismo , beta-Lactamas/metabolismo , beta-Lactamas/farmacologia
4.
Antimicrob Agents Chemother ; 66(9): e0049622, 2022 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-36066241

RESUMO

The CDC's Emerging Infections Program (EIP) conducted population- and laboratory-based surveillance of US carbapenem-resistant Pseudomonas aeruginosa (CRPA) from 2016 through 2018. To characterize the pathotype, 1,019 isolates collected through this project underwent antimicrobial susceptibility testing and whole-genome sequencing. Sequenced genomes were classified using the seven-gene multilocus sequence typing (MLST) scheme and a core genome (cg)MLST scheme was used to determine phylogeny. Both chromosomal and horizontally transmitted mechanisms of carbapenem resistance were assessed. There were 336 sequence types (STs) among the 1,019 sequenced genomes, and the genomes varied by an average of 84.7% of the cgMLST alleles used. Mutations associated with dysfunction of the porin OprD were found in 888 (87.1%) of the genomes and were correlated with carbapenem resistance, and a machine learning model incorporating hundreds of genetic variations among the chromosomal mechanisms of resistance was able to classify resistant genomes. While only 7 (0.1%) isolates harbored carbapenemase genes, 66 (6.5%) had acquired non-carbapenemase ß-lactamase genes, and these were more likely to have OprD dysfunction and be resistant to all carbapenems tested. The genetic diversity demonstrates that the pathotype includes a variety of strains, and clones previously identified as high-risk make up only a minority of CRPA strains in the United States. The increased carbapenem resistance in isolates with acquired non-carbapenemase ß-lactamase genes suggests that horizontally transmitted mechanisms aside from carbapenemases themselves may be important drivers of the spread of carbapenem resistance in P. aeruginosa.


Assuntos
Infecções por Pseudomonas , Pseudomonas aeruginosa , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Centers for Disease Control and Prevention, U.S. , Humanos , Testes de Sensibilidade Microbiana , Tipagem de Sequências Multilocus , Porinas/genética , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/epidemiologia , Estados Unidos/epidemiologia , beta-Lactamases/genética , beta-Lactamases/metabolismo
5.
Sci Rep ; 12(1): 14822, 2022 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-36050365

RESUMO

Infections caused by multi-drug resistant Escherichia coli cause significant morbidity and mortality especially in developing countries. In this study, we describe the molecular characteristics of E. coli isolated from clinical specimens and the patients' outcomes. Phenotypic methods were used in the identification and antimicrobial susceptibility testing of E. coli from clinical specimens from a tertiary hospital in Abuja, Nigeria. Whole genome sequencing was used to describe the antimicrobial resistance genes, serotypes, sequence types/clonal complexes, and mobile genetic elements. The mean age of the patients was 20.3 years with 70.1% females and majority of isolates 75% from urine, 21% from blood cultures, and 3% each from cerebrospinal fluid and endo-cervical swabs. Of the 107 non-duplicate E. coli isolates, 101 (94.3%) were resistant to ampicillin, 95 (88.8%) to trimethoprim/sulfamethoxazole, 86 (80.4%) to ceftriaxone, 60 (56.1%) to gentamicin, and eight (7.5%) to meropenem. There were 102 (95.3%) isolates that were multi-drug resistant (MDR). Expression of Extended Spectrum Beta Lactamase (ESBL) phenotype was detected in 54 (50%) and blaCTX-M-15 genes detected in 75 (70.1%) isolates. The carbapenemase genes blaNDM-1 and blaNDM-5 were detected in six (5.6%), while the AmpC gene- blaCMY-2, was detected in seven (6.5%) isolates. Two (1.9%) isolates simultaneously harboured the blaOXA-1, blaCMY-2, blaCTX-M-15, and blaNDM-5 genes. In total, 35 sequence types (STs) were found with the majority being ST131 (n = 23; 21.5%). The most common serotype was O25:H4 associated with all 23 strains of ST131, followed by O1:H6/ST648 (n = 6). The ST410, ST671, and ST101 strains displayed phenotypic resistance to wide array of antibiotic classes and harbored high numbers of antibiotic resistance genes via in-silico analysis. The ST410 strain in particular harbored a higher number of antibiotic resistance genes and was phenotypically resistant to a wider array of antibiotics. Four pairs of isolates were closely related with three isolates (ST131, ST38, ST652) having a pairwise SNP difference of zero. 71/72 75/76 52/14. The MDR E. coli lineages circulating in this setting pose a clinical and public health threat as they can hinder effective prevention and management of infections. The genetic diversity and MDR E. coli with the emergence of ST410 and ST101 clones is concerning because of the potential for rapid dissemination in hospitals and communities- further increasing the problems of antibiotic resistance. Continuous routine surveillance of E. coli infections for AMR in hospitals becomes imperative, aimed at development of effective antimicrobial stewardship programs, facilitating prudent use of antimicrobial agents, and limiting dissemination of resistant strains.


Assuntos
Infecções por Escherichia coli , Escherichia coli , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Infecções por Escherichia coli/tratamento farmacológico , Infecções por Escherichia coli/epidemiologia , Feminino , Humanos , Masculino , Testes de Sensibilidade Microbiana , Nigéria/epidemiologia , Centros de Atenção Terciária , beta-Lactamases/metabolismo
6.
Microbiol Spectr ; 10(4): e0214422, 2022 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-35946816

RESUMO

Carbapenem-resistant Acinetobacter baumannii (CRAB), which belonged to global clones 1 (GC1) or 2 (GC2), has been widely reported and become a global threat. However, non-GC1 and non-GC2 CRAB strains are not well-studied, especially for those with rare phenotype. Here, one pyomelanin-producing CRAB strain (A. baumannii DETAB-R21) was isolated from oral swab in the ICU. Antimicrobial susceptibility testing showed it was resistant to carbapenems, ceftazidime, levofloxacin, and ciprofloxacin. DETAB-R21 was ST164Pas and ST1418Oxf with KL47 and OCL5, respectively. Whole-genome sequencing (WGS) analysis revealed chromosome contained three copies of blaOXA-23 on three 4,805-bp Tn2006 composite transposons with various novel 9-bp target site duplications (TSD). A Tn125-like structure, including blaNDM-1, a novel 4,343 bp composite transposon encoding blaCARB-16, and three prophage regions were also identified. Importantly, hmgA was interrupted by a Tn2006 and contributed to pyomelanin production and further confirmed by hmgA overexpression. Furthermore, A. baumannii irradiated with UV light, DETAB-R21 showed a higher relatively survival rate compared to a control strain that did not produce pyomelanin. No effects of pyomelanin were observed on disinfectants susceptibility, growth, or virulence. In conclusion, pyomelanin-producing CRAB carrying the blaNDM-1 and blaOXA-23 genes embedded in the bacterial chromosome is of grave concern for health care settings, highlighting the need for effective measures to prevent further dissemination. IMPORTANCE Pyomelanin production is a quite rare phenotype in A. baumannii. Moreover, the mechanisms leading to the pyomelanin production was still unclear. Here, we for the first time, confirmed the mechanism of pyomelanin production, and further investigated the impact of pyomelanin on disinfectants susceptibility, growth, virulence, and UV irradiation. More importantly, many mobile genetic elements (MGEs), including three copies of Tn2006 composite transposons, one copy of blaNDM-1 on the Tn125-like structure and three prophage regions, were identified in the chromosome, demonstrated strong plasticity of A. baumannii genome. Our study provides important insights into the new rare ST164Pas A. baumannii strain with high level carbapenem resistance, which is of great threat for patients. These findings will provide important insights into the resistance gene transfer via transposition events and further spread in the clinic.


Assuntos
Infecções por Acinetobacter , Acinetobacter baumannii , Desinfetantes , beta-Lactamases/metabolismo , Infecções por Acinetobacter/microbiologia , Acinetobacter baumannii/genética , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Carbapenêmicos/farmacologia , Cromossomos , Desinfetantes/farmacologia , Humanos , Melaninas , Testes de Sensibilidade Microbiana , beta-Lactamases/genética
7.
Microbiol Spectr ; 10(4): e0220622, 2022 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-35916524

RESUMO

Although piperacillin-tazobactam (TZP) was shown to be less effective than carbapenems in treating bacteremia due to extended-spectrum ß-lactamase-producing (ESBL)-producing organisms in a randomized controlled trial, the fact that many of the causative organisms co-produced inhibitor-resistant OXA-1 along with ESBLs may have influenced the results. In this study, we compared the therapeutic effectiveness of TZP and carbapenem in treating ESBL-producing Escherichia coli bacteremia in areas with low frequency of OXA-1 co-production. Forty patients, 14 in the TZP treatment group and 26 in the carbapenem treatment group, were included in the analysis. There were no significant differences in patient background between the two groups. Urinary tract infection or cholangitis was the source of bacteremia in 26 patients (65%), and the Pitt bacteremia score was zero or one in 35 patients (87.5%). Only four (11.4%) of the 35 causative isolates available for microbiological analysis harbored blaOXA-1, and only three (8.6%) were non-susceptible to TZP. Seventeen (48.6%) isolates carried blaCTX-M-27, none of which carried other ß-lactamase genes. No significant difference in the frequency of treatment failure on day 14 of bacteremia was documented between the TZP and carbapenem treatment groups in both the crude analysis and the inverse probability of treatment weighting-adjusted analysis. This study demonstrates that TZP may be a treatment option for non-severe cases of ESBL-producing E. coli bacteremia in areas with low frequency of OXA-1 co-production. IMPORTANCE Although carbapenems are considered the drug of choice for severe infections caused by extended-spectrum ß-lactamase-producing (ESBL)-producing organisms, other therapeutic options are being explored to avoid increasing the selective pressure for carbapenem-resistant organisms. In this study, it was suggested that piperacillin-tazobactam may be as effective as carbapenems for the treatment of mild bacteremia caused by ESBL-producing Escherichia coli in areas where OXA-1 co-production by ESBL-producing E. coli is rare. The genetic background of each regional epidemic clone differs even among multidrug-resistant bacteria classified under the same name (e.g., ESBL-producing organisms), resulting in possible differences in the efficacy of therapeutic agents. Exploration of treatment options for multidrug-resistant organisms according to local epidemiology is worthwhile from the perspective of antimicrobial stewardship.


Assuntos
Bacteriemia , Infecções por Escherichia coli , beta-Lactamases/metabolismo , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Bacteriemia/tratamento farmacológico , Bacteriemia/microbiologia , Carbapenêmicos/farmacologia , Carbapenêmicos/uso terapêutico , Escherichia coli , Infecções por Escherichia coli/tratamento farmacológico , Infecções por Escherichia coli/microbiologia , Humanos , Combinação Piperacilina e Tazobactam/farmacologia , Combinação Piperacilina e Tazobactam/uso terapêutico , Resultado do Tratamento , beta-Lactamases/genética
8.
Bioorg Chem ; 128: 106048, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-35952448

RESUMO

Metallo-ß-lactamases (MßLs) hydrolyze almost all ß-lactam antibiotics, including last-resort carbapenems, and is emerging as a global antibiotic resistance threat. Discovering novel fluorescent molecules for visualizing MßLs has proved challenging. Herein, based on covalent and Zn(II)-binding scaffold of MßLs inhibitor, we designed and synthesized a novel series of environment-sensitive fluorescent probes ESA, DHA and DHS, to detect and inhibit the enzymatic activity of MßLs. Of these probes, ESA is a highly active NDM-1 inhibitor (IC50 = 81 nM), which exhibited excellent turn-on fluorescent properties to effectively distinguish NDM-1 (B1), ImiS (B2) and L1 (B3) in vitro. Cell imaging indicated that ESA can label and track the distribution process of the intracellular protein NDM-1 in living cells. Molecular docking further elucidated the environment-sensitive fluorescent response nature of ESA to the NDM-1. Significantly, ESA showed excellent synergistic antibacterial effect, combined with meropenem, to overcome NDM-1-mediated drug-resistant pathogens.


Assuntos
Corantes Fluorescentes , beta-Lactamases , Antibacterianos/química , Antibacterianos/farmacologia , Carbapenêmicos , Corantes Fluorescentes/farmacologia , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , beta-Lactamases/metabolismo
9.
Bioorg Med Chem ; 72: 116964, 2022 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-36030663

RESUMO

Metallo-ß-lactamases (MBLs) represent an increasingly serious threat to public health because of their increased prevalence worldwide in relevant opportunistic Gram-negative pathogens. MBLs efficiently inactivate widely used and most valuable ß-lactam antibiotics, such as oxyiminocephalosporins (ceftriaxone, ceftazidime) and the last-resort carbapenems. To date, no MBL inhibitor has been approved for therapeutic applications. We are developing inhibitors characterized by a 1,2,4-triazole-3-thione scaffold as an original zinc ligand and few promising series were already reported. Here, we present the synthesis and evaluation of a new series of compounds characterized by the presence of an arylalkyl substituent at position 4 of the triazole ring. The alkyl link was mainly an ethylene, but a few compounds without alkyl or with an alkyl group of various lengths up to a butyl chain were also synthesized. Some compounds in both sub-series were micromolar to submicromolar inhibitors of tested VIM-type MBLs. A few of them were broad-spectrum inhibitors, as they showed significant inhibitory activity on NDM-1 and, to a lesser extent, IMP-1. Among these, several inhibitors were able to significantly reduce the meropenem MIC on VIM-1- and VIM-4- producing clinical isolates by up to 16-fold. In addition, ACE inhibition was absent or moderate and one promising compound did not show toxicity toward HeLa cells at concentrations up to 250 µM. This series represents a promising basis for further exploration. Finally, molecular modelling of representative compounds in complex with VIM-2 was performed to study their binding mode.


Assuntos
Tionas , Inibidores de beta-Lactamases , Antibacterianos/farmacologia , Carbapenêmicos/farmacologia , Ceftazidima , Ceftriaxona , Etilenos , Células HeLa , Humanos , Inosina Monofosfato , Ligantes , Meropeném , Testes de Sensibilidade Microbiana , Triazóis/química , Triazóis/farmacologia , Zinco , Inibidores de beta-Lactamases/química , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases/metabolismo
10.
Antimicrob Agents Chemother ; 66(9): e0052722, 2022 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-35924913

RESUMO

Metallo-ß-lactamase (MBL)-producing Gram-negative bacteria cause infections associated with high rates of morbidity and mortality. Currently, a leading regimen to treat infections caused by MBL-producing bacteria is aztreonam combined with ceftazidime-avibactam. The purpose of the present study was to evaluate and rationally optimize the combination of aztreonam and ceftazidime-avibactam with and without polymyxin B against a clinical Klebsiella pneumoniae isolate producing NDM-1 and CTX-M by use of the hollow fiber infection model (HFIM). A novel de-escalation approach to polymyxin B dosing was also explored, whereby a standard 0-h loading dose was followed by maintenance doses that were 50% of the typical clinical regimen. In the HFIM, the addition of polymyxin B to aztreonam plus ceftazidime-avibactam significantly improved bacterial killing, leading to eradication, including for the novel de-escalation dosing strategy. Serial samples from the growth control and monotherapies were explored in a Galleria mellonella virulence model to assess virulence changes. Weibull regression showed that low-level ceftazidime resistance and treatment with monotherapy resulted in increased G. mellonella mortality (P < 0.05). A neutropenic rabbit pneumonia model demonstrated that aztreonam plus ceftazidime-avibactam with or without polymyxin B resulted in similar bacterial killing, and these combination therapies were statistically significantly better than monotherapies (P < 0.05). However, only the polymyxin B-containing combination therapy produced a statistically significant decrease in lung weights (P < 0.05), indicating a decreased inflammatory process. Altogether, adding polymyxin B to the combination of aztreonam plus ceftazidime-avibactam for NDM- and CTX-M-producing K. pneumoniae improved bacterial killing effects, reduced lung inflammation, suppressed resistance amplification, and limited virulence changes.


Assuntos
Ceftazidima , Klebsiella pneumoniae , Animais , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Compostos Azabicíclicos/farmacologia , Compostos Azabicíclicos/uso terapêutico , Aztreonam/farmacologia , Ceftazidima/farmacologia , Ceftazidima/uso terapêutico , Parede Celular/metabolismo , Combinação de Medicamentos , Klebsiella/metabolismo , Testes de Sensibilidade Microbiana , Polimixina B/farmacologia , Coelhos , beta-Lactamases/metabolismo
11.
Antimicrob Agents Chemother ; 66(9): e0069122, 2022 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-35943258

RESUMO

Biochemical properties of the novel subclass B3 metallo-ß-lactamase (MBL) PJM-1 expressed in Pseudoxanthomonas japonensis, which is often isolated from the environment, were determined. The 906-bp blaPJM-1 gene in P. japonensis is a species-specific MBL gene, and PJM, with 301 predicted amino acids, has 81.8% amino acid identity with AIM-1. In this study, PJM-1 was recombinantly expressed and purified. PJM-1 showed a low catalytic activity against ceftazidime and cefepime, and it was strongly inhibited by EDTA.


Assuntos
Ceftazidima , beta-Lactamases , Aminoácidos , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Cefepima , Ceftazidima/farmacologia , Ácido Edético , Xanthomonadaceae , beta-Lactamases/metabolismo
12.
PLoS One ; 17(8): e0273088, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35960734

RESUMO

The rise in antibiotic resistance has stimulated research into adjuvants that can improve the efficacy of broad-spectrum antibiotics. Lactoferrin is a candidate adjuvant; it is a multifunctional iron-binding protein with antimicrobial properties. It is known to show dose-dependent antimicrobial activity against Staphylococcus aureus through iron sequestration and repression of ß-lactamase expression. However, S. aureus can extract iron from lactoferrin through siderophores for their growth, which confounds the resolution of lactoferrin's method of action. We measured the minimum inhibitory concentration (MIC) for a range of lactoferrin/ ß-lactam antibiotic dose combinations and observed that at low doses (< 0.39 µM), lactoferrin contributes to increased S. aureus growth, but at higher doses (> 6.25 µM), iron-depleted native lactoferrin reduced bacterial growth and reduced the MIC of the ß-lactam-antibiotic cefazolin. This differential behaviour points to a bacterial population response to the lactoferrin/ ß-lactam dose combination. Here, with the aid of a mathematical model, we show that lactoferrin stratifies the bacterial population, and the resulting population heterogeneity is at the basis of the dose dependent response seen. Further, lactoferrin disables a sub-population from ß-lactam-induced production of ß-lactamase, which when sufficiently large reduces the population's ability to recover after being treated by an antibiotic. Our analysis shows that an optimal dose of lactoferrin acts as a suitable adjuvant to eliminate S. aureus colonies using ß-lactams, but sub-inhibitory doses of lactoferrin reduces the efficacy of ß-lactams.


Assuntos
Infecções Estafilocócicas , Staphylococcus aureus , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Humanos , Ferro/metabolismo , Lactoferrina/metabolismo , Lactoferrina/farmacologia , Testes de Sensibilidade Microbiana , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/metabolismo , beta-Lactamases/metabolismo , beta-Lactamas/farmacologia
13.
PLoS One ; 17(8): e0273504, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36006947

RESUMO

Antimicrobial peptides may be alternatives to traditional antibiotics with reduced bacterial resistance. The antimicrobial peptide GL13K was derived from the salivary protein BPIFA2. This study determined the relative activity of the L-and D-enantiomers of GL13K to wild-type and drug-resistant strains of three gram-negative species and against Pseudomonas aeruginosa biofilms. DGL13K displayed in vitro activity against extended-spectrum beta-lactamase (ESBL)-producing and Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae (MICs 16-32 µg/ml), MDR and XDR P. aeruginosa, and XDR Acinetobacter baumannii carrying metallo-beta-lactamases (MICs 8-32 µg/ml). P. aeruginosa showed low inherent resistance to DGL13K and the increased metabolic activity and growth caused by sub-MIC concentrations of GL13K peptides did not result in acquired bacterial resistance. Daily treatment for approximately two weeks did not increase the MIC of DGL13K or cause cross-resistance between LGL13K and DGL13K. These data suggest that DGL13K is a promising antimicrobial peptide candidate for further development.


Assuntos
Peptídeos Antimicrobianos , Farmacorresistência Bacteriana Múltipla , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Bactérias Gram-Negativas/metabolismo , Klebsiella pneumoniae/metabolismo , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa/metabolismo , beta-Lactamases/metabolismo
14.
Microb Pathog ; 170: 105686, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35917986

RESUMO

Carbapenem-resistant Klebsiella pneumoniae (CRKP) has proven to be an urgent threat to human health. Proteomics (TMT/LC-MS/MS) and bioinformatics approaches were employed to explore the potential mechanisms underlying carbapenem resistance. Proteomic profiling of CRKP and susceptible KP (sKP) isolates revealed the involvement of outer membrane, beta-lactam resistance pathway, and two-component systems (TCSs) in carbapenem resistance. 27 CRKP strains and 27 susceptible Klebsiella pneumoniae strains were isolated from inpatients at the Second Xiangya Hospital, China to verify the mechanisms. Modified carbapenem inactivation method (mCIM) and PCR of common carbapenem resistance genes confirmed that 77.8% (21/27) of CRKP isolates were carbapenemase-producing. Porin decrease in CRKP isolates was found by SDS-PAGE and mRNA levels of major porins (OmpK35 and OmpK36). RT-qPCR detection of two-component systems (envZ, ompR, phoP, phoQ, baeS and baeR) revealed down-regulation of EnvZ-OmpR, PhoPQ, BaeSR TCSs. Expression of the TCSs, except ompR, were closely correlated with OMPs with the R-value >0.7. Together, this study reaffirmed the significance of the ß-lactam resistance pathway in CRKP based on proteomic analysis. OmpK35/36 porin reduction and the controversial downregulation of EnvZ-OmpR, PhoPQ, and BaeSR TCSs were confirmed in carbapenem resistance of Klebsiella pneumoniae.


Assuntos
Enterobacteriáceas Resistentes a Carbapenêmicos , Infecções por Klebsiella , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Carbapenêmicos/metabolismo , Carbapenêmicos/farmacologia , Cromatografia Líquida , Regulação para Baixo , Humanos , Klebsiella pneumoniae , Testes de Sensibilidade Microbiana , Porinas/genética , Porinas/metabolismo , Proteômica , Espectrometria de Massas em Tandem , beta-Lactamases/metabolismo
15.
Microb Pathog ; 170: 105700, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35934203

RESUMO

The generation of antimicrobial-resistant bacteria largely depends on the use of antimicrobials not only in humans but also in pet animals and livestock. The present study was conducted to detect the occurrence of beta-lactamase and biofilm-producing- E.coli in healthy pet and backyard livestock. The study also intended on molecular docking experiments to confirm the nature of the catalytic mechanism in ß-lactamase enzymes, encoded by the various blaCTX-M genotypes and phylogenetic analysis to reveal clonal relationship of the animal origin E. coli isolates with human clinical strains. The rectal swabs were collected from healthy dogs (n = 254), cats (n = 108), sheep (n = 119) and goats (n = 143) in India. In total 247 (76.47%) E. coli strains were identified as ESBL producers. The possession of ESBL-producers was significantly more (p < 0.05) in pets than in the backyard livestock. Most of the strains possessed blaCTX-M-15 like clones. E. coli strains possessing blaCTX-M-15.2, blaCTX-M-157, blaCTX-M-181 and blaCTX-M-218 like clones, isolated from pets were not reported earlier. The study detected 56.65% of E. coli strains as moderate or strong biofilm producers possessing biofilm-associated genes (csgA, rcsA, rpoS, sdiA). ESBL-producing E. coli showed phenotypical resistance to tetracycline (93.1%), azithromycin (89.8%), ampicillin (84.2%), cefotaxime (80.9%), doxycycline (82.5%), co-trimoxazole (80.9%), ampicillin/cloxacillin (76.9%). The CTX-M variants obtained in this study were modelled by the SWISS-MODEL and verified. Ligand having minimum binding energy, show the highest affinity of ß-lactamases for cefotaxime and cefpodoxime. The Gibbs free energy release for all 14 different complex ranges between -6.9 (CTX-M-15.2+cefpodoxime) to -5.3 (CTX-M-218+cefpodoxime) Kcal/mol. Phylogenetic analysis of the animal origin ESBL-E. coli strains revealed a partial clonal relationship with the clinical isolates of local human patients. The present study described the significant presence of biofilm and ß-lactamase producing, multi-drug resistant E. coli in pet animals having public health importance.


Assuntos
Infecções por Escherichia coli , Escherichia coli , Ampicilina , Animais , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Biofilmes , Cefotaxima , Cães , Escherichia coli/metabolismo , Infecções por Escherichia coli/microbiologia , Humanos , Gado , Simulação de Acoplamento Molecular , Filogenia , Ovinos , Resistência beta-Lactâmica , beta-Lactamases/genética , beta-Lactamases/metabolismo
16.
J Antibiot (Tokyo) ; 75(10): 559-566, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-35986092

RESUMO

The blaNDM-1 gene encodes a carbapenemase, New Delhi metallo-ß-lactamase (NDM-1), and the ability to produce NDM-1 is spread among Enterobacteriaceae via horizontal gene transfer of plasmids. It has been widely accepted that blaNDM-1 is regulated by a hybrid promoter (PISAba125) consisting of a -10 box from the original blaNDM-1 and a -35 box from ISAba125. However, the conservation of this promoter and the vertical transmission of blaNDM genes by chromosomal integration have not been comprehensively analyzed. We retrieved the region containing the ORF of blaNDM-1 (>95% translated protein identity) and a region 120 bp upstream of the blaNDM-1 start codon from the complete sequence data of Enterobacteriaceae plasmids (n = 10,914) and chromosomes (n = 4908) deposited in GenBank, and the 310 extracted blaNDM genes were analyzed by an in-silico approach. The results showed that most blaNDM genes (99.0%) utilized the promoter, PISAba125. Interestingly, two blaNDM-1 genes from the genus Citrobacter utilized the ISCR1-derived outward-oriented promoters POUT (PISCR1). Furthermore, the insertion of ISAba125 and ISCR1 occurred upstream of the CCATATTT sequence, which is located upstream of the -10 box. We also confirmed that most of the blaNDM genes were disseminated by horizontal gene transfer of the plasmid, but 10 cases of the blaNDM genes were integrated into the chromosome via mobile genetic elements such as IS26, IS150, ISCR1, ICE, and Tn7-like elements. Thus, plasmid-mediated transmission of the PISAba125-blaNDM genes is predominant in Enterobacteriaceae. However, the spread of blaNDM genes with new promoters and vertical dissemination via chromosomal integrations may pose additional serious clinical problems.


Assuntos
Enterobacteriaceae , beta-Lactamases , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Enterobacteriaceae/genética , Transferência Genética Horizontal , Testes de Sensibilidade Microbiana , Plasmídeos/genética , beta-Lactamases/genética , beta-Lactamases/metabolismo
17.
Microbiology (Reading) ; 168(8)2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35943884

RESUMO

The discovery of penicillin by Alexander Fleming marked a new era for modern medicine, allowing not only the treatment of infectious diseases, but also the safe performance of life-saving interventions, like surgery and chemotherapy. Unfortunately, resistance against penicillin, as well as more complex ß-lactam antibiotics, has rapidly emerged since the introduction of these drugs in the clinic, and is largely driven by a single type of extra-cytoplasmic proteins, hydrolytic enzymes called ß-lactamases. While the structures, biochemistry and epidemiology of these resistance determinants have been extensively characterized, their biogenesis, a complex process including multiple steps and involving several fundamental biochemical pathways, is rarely discussed. In this review, we provide a comprehensive overview of the journey of ß-lactamases, from the moment they exit the ribosomal channel until they reach their final cellular destination as folded and active enzymes.


Assuntos
Penicilinas , beta-Lactamases , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Inibidores de beta-Lactamases , beta-Lactamases/genética , beta-Lactamases/metabolismo
18.
mBio ; 13(4): e0174922, 2022 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-35968954

RESUMO

ß-Lactamase production facilitates bacterial survival in nature and affects many infection therapies. However, much of its regulation remains unexplored. We used a genetics-based approach to identify a two-component system (TCS) present in a strain of Burkholderia thailandensis essential for the regulated expression of a class A ß-lactamase gene, penL, by sensing subtle envelope disturbance caused by ß-lactams, polymyxin B, or other chemical agents. The genes encoding stress responses and resistance to various antibiotics were coregulated, as were the catabolic genes that enabled the B. thailandensis strain to grow on penicillin G or phenylacetate, a degradation product of penicillin G. This regulon has likely evolved to facilitate bacterial survival in the soil microbiome that contains a multitude of antibiotic producers. Practically, this regulatory system makes this TCS, which we named BesRS, an excellent drug target for the purpose of increasing antibiotic efficacy in combination therapies for Burkholderia infections. IMPORTANCE ß-lactam antibiotics are the most frequently used drugs to treat infectious diseases. Although the production of ß-lactamases by bacteria is the main cause of treatments being compromised, much of the gene regulation mechanism governing the levels of these enzymes has not been fully explored. In this study, we report a novel ß-lactamase gene regulation mechanism that is governed by a two-component system responding to disturbances in the cell envelope. We showed gene regulation is a part of a regulon that includes genes involved in stress responses, resistance to various antibiotics, and a catabolic pathway for ß-lactams. This regulon may have been evolved to facilitate bacterial survival in the soil niches, which are highly competitive environments because of the presence of various antibiotic-producing microbes. The discovery of the ß-lactamase gene regulation mechanism opens new avenues for developing therapeutic strategies in the fight against antibiotic resistance.


Assuntos
Regulon , beta-Lactamases , Antibacterianos/farmacologia , Solo , beta-Lactamases/genética , beta-Lactamases/metabolismo , beta-Lactamas/farmacologia
19.
Int J Mol Sci ; 23(16)2022 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-36012433

RESUMO

The emergence of resistant bacteria takes place, endangering the effectiveness of antibiotics. A reason for antibiotic resistance is the presence of lactamases that catalyze the hydrolysis of ß-lactam antibiotics. An inhibitor of serine-ß-lactamases such as clavulanic acid binds to the active site of the enzymes, thus solving the resistance problem. A pressing issue, however, is that the reaction mechanism of metallo-ß-lactamases (MBLs) hydrolyzing ß-lactam antibiotics differs from that of serine-ß-lactamases due to the existence of zinc ions in the active site of MBLs. Thus, the development of potential inhibitors for MBLs remains urgent. Here, the ability to inhibit MBL from Bacillus anthracis (Bla2) was investigated in silico and in vitro using compounds possessing two hydroxamate functional groups such as 3-chloro-N-hydroxy-4-(7-(hydroxyamino)-7-oxoheptyl)benzamide (Compound 4) and N-hydroxy-4-(7-(hydroxyamino)-7-oxoheptyl)-3-methoxybenzamide (Compound 6). In silico docking and molecular dynamics simulations revealed that both Compounds 4 and 6 were coordinated with zinc ions in the active site, suggesting that the hydroxamate group attached to the aromatic ring of the compound plays a crucial role in the coordination to the zinc ions. In vitro kinetic analysis demonstrated that the mode of inhibitions for Compounds 4 and 6 were a competitive inhibition with Ki values of 6.4 ± 1.7 and 4.7 ± 1.4 kcal/mol, respectively. The agreement between in silico and in vitro investigations indicates that compounds containing dihyroxamate moieties may offer a new avenue to overcome antibiotic resistance to bacteria.


Assuntos
Bacillus anthracis , beta-Lactamases , Antibacterianos/química , Antibacterianos/farmacologia , Bacillus anthracis/metabolismo , Ácido Clavulânico , Ácidos Hidroxâmicos/farmacologia , Cinética , Serina , Zinco , Inibidores de beta-Lactamases/química , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases/metabolismo
20.
FEMS Microbiol Lett ; 369(1)2022 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-35883222

RESUMO

Pseudomonas aeruginosa is known to exhibit considerable resistance to the antimicrobial activity of the metal-sequestering protein calprotectin (CP). In this study, we demonstrate that although CP induces zinc deficiency in P. aeruginosa, a strain unable to import zinc through the two most important metal acquisition systems, namely ZnuABC and ZrmABCD, maintains significant growth capacity in the presence of high concentrations of CP. Furthermore, we have shown that nicotianamine, a molecule structurally similar to the metallophore pseudopaline, can favor the acquisition of the metal even in the presence of CP. To gain insights into the mechanisms through which metallophores can promote zinc acquisition, we analyzed the effect of nicotianamine on the activity of the metallo-ß-lactamase VIM-1. Our data suggest that metallophores released by bacteria in response to zinc deficiency can extract the protein-bound metal. The ability to interfere with the binding of metals to proteins, as well as favoring the acquisition of zinc, may contribute to increasing the resistance of P. aeruginosa to the antimicrobial action of CP.


Assuntos
Anti-Infecciosos , Infecções por Pseudomonas , Anti-Infecciosos/farmacologia , Humanos , Complexo Antígeno L1 Leucocitário/metabolismo , Complexo Antígeno L1 Leucocitário/farmacologia , Metais/metabolismo , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa , Zinco/metabolismo , Zinco/farmacologia , beta-Lactamases/metabolismo
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