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1.
Biochemistry ; 63(10): 1278-1286, 2024 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-38690676

RESUMO

Metallo-ß-lactamases (MBL) deactivate ß-lactam antibiotics through a catalytic reaction caused by two zinc ions at the active center. Since MBLs deteriorate a wide range of antibiotics, they are dangerous factors for bacterial multidrug resistance. In this work, organic synthesis, computational design, and crystal structure analysis were performed to obtain potent MBL inhibitors based on a previously identified hit compound. The hit compound comprised 3,4-dihydro-2(1H)-quinolinone linked with a phenyl-ether-methyl group via a thiazole ring. In the first step, the thiazole ring was replaced with a tertiary amine to avoid the planar structure. In the second step, we virtually modified the compound by keeping the quinolinone backbone. Every modified compound was bound to a kind of MBL, imipenemase-1 (IMP-1), and the binding pose was optimized by a molecular mechanics calculation. The binding scores were evaluated for the respective optimized binding poses. Given the predicted binding poses and calculated binding scores, candidate compounds were determined for organic syntheses. The inhibitory activities of the synthesized compounds were measured by an in vitro assay for two kinds of MBLs, IMP-1 and New Delhi metallo-ß-lactamase (NDM-1). A quinolinone connected with an amine bound with methyl-phenyl-ether-propyl and cyclohexyl-ethyl showed a 50% inhibitory concentration of 4.8 µM. An X-ray crystal analysis clarified the binding structure of a synthesized compound to IMP-1. The δ-lactam ring of quinolinone was hydrolyzed, and the generated carboxyl group was coordinated with zinc ions. The findings on the chemical structure and binding pose are expected to be a base for developing MBL inhibitors.


Assuntos
Inibidores de beta-Lactamases , beta-Lactamases , beta-Lactamases/química , beta-Lactamases/metabolismo , Inibidores de beta-Lactamases/farmacologia , Inibidores de beta-Lactamases/química , Cristalografia por Raios X , Desenho de Fármacos , Simulação de Acoplamento Molecular , Antibacterianos/farmacologia , Antibacterianos/química , Quinolonas/química , Quinolonas/farmacologia , Quinolonas/metabolismo
2.
Sci Total Environ ; 931: 172873, 2024 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-38692330

RESUMO

Carbapenem resistance's global proliferation poses a significant public health challenge. The primary resistance mechanism is carbapenemase production. In this study, we discovered a novel carbapenemase, RATA, located on the chromosome of Riemerella anatipestifer isolates. This enzyme shares ≤52 % amino acid sequence identity with other known ß-lactamases. Antimicrobial susceptibility tests and kinetic assays demonstrated that RATA could hydrolyze not only penicillins and extended-spectrum cephalosporins but also monobactams, cephamycins, and carbapenems. Furthermore, its activity was readily inhibited by ß-lactamase inhibitors. Bioinformatic analysis revealed 46 blaRATA-like genes encoding 27 variants in the NCBI database, involving 21 different species, including pathogens, host-associated bacteria, and environmental isolates. Notably, blaRATA-positive strains were globally distributed and primarily collected from marine environments. Concurrently, taxonomic analysis and GC content analysis indicated that blaRATA orthologue genes were predominantly located on the chromosomes of Flavobacteriaceae and shared a similar GC content as Flavobacteriaceae. Although no explicit mobile genetic elements were identified by genetic environment analysis, blaRATA-2 possessed the ability of horizontal transfer in R. anatipestifer via natural transformation. This work's data suggest that RATA is a new chromosome-encoded class A carbapenemase, and Flavobacteriaceae from marine environments could be the primary reservoir of the blaRATA gene.


Assuntos
Proteínas de Bactérias , beta-Lactamases , beta-Lactamases/genética , beta-Lactamases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Antibacterianos/farmacologia , Testes de Sensibilidade Microbiana , Carbapenêmicos/farmacologia
3.
Biotechnol J ; 19(5): e2400023, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38719589

RESUMO

The discovery of antibiotics has noticeably promoted the development of human civilization; however, antibiotic resistance in bacteria caused by abusing and overusing greatly challenges human health and food safety. Considering the worsening situation, it is an urgent demand to develop emerging nontraditional technologies or methods to address this issue. With the expanding of synthetic biology, optogenetics exhibits a tempting prospect for precisely regulating gene expression in many fields. Consequently, it is attractive to employ optogenetics to reduce the risk of antibiotic resistance. Here, a blue light-controllable gene expression system was established in Escherichia coli based on a photosensitive DNA-binding protein (EL222). Further, this strategy was successfully applied to repress the expression of ß-lactamase gene (bla) using blue light illumination, resulting a dramatic reduction of ampicillin resistance in engineered E. coli. Moreover, blue light was utilized to induce the expression of the mechanosensitive channel of large conductance (MscL), triumphantly leading to the increase of streptomycin susceptibility in engineered E. coli. Finally, the increased susceptibility of ampicillin and streptomycin was simultaneously induced by blue light in the same E. coli cell, revealing the excellent potential of this strategy in controlling multidrug-resistant (MDR) bacteria. As a proof of concept, our work demonstrates that light can be used as an alternative tool to prolong the use period of common antibiotics without developing new antibiotics. And this novel strategy based on optogenetics shows a promising foreground to combat antibiotic resistance in the future.


Assuntos
Antibacterianos , Escherichia coli , Luz , Escherichia coli/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Antibacterianos/farmacologia , Optogenética/métodos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Ampicilina/farmacologia , beta-Lactamases/genética , beta-Lactamases/metabolismo , Farmacorresistência Bacteriana/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Estreptomicina/farmacologia , Luz Azul
4.
Sci Rep ; 14(1): 10066, 2024 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-38698009

RESUMO

The global threat of antibiotic resistance has increased the importance of the detection of antibiotics. Conventional methods to detect antibiotics are time-consuming and require expensive specialized equipment. Here, we present a simple and rapid biosensor for detecting ampicillin, a commonly used antibiotic. Our method is based on the fluorescent properties of chitosan-coated Mn-doped ZnS micromaterials combined with the ß-lactamase enzyme. The biosensors exhibited the highest sensitivity in a linear working range of 13.1-72.2 pM with a limit of detection of 8.24 pM in deionized water. In addition, due to the biological specificity of ß-lactamase, the proposed sensors have demonstrated high selectivity over penicillin, tetracycline, and glucose through the enhancing and quenching effects at wavelengths of 510 nm and 614 nm, respectively. These proposed sensors also showed promising results when tested in various matrices, including tap water, bottled water, and milk. Our work reports for the first time the cost-effective (Mn:ZnS)Chitosan micromaterial was used for ampicillin detection. The results will facilitate the monitoring of antibiotics in clinical and environmental contexts.


Assuntos
Ampicilina , Técnicas Biossensoriais , Quitosana , Manganês , Sulfetos , Compostos de Zinco , Ampicilina/análise , Ampicilina/química , Quitosana/química , Técnicas Biossensoriais/métodos , Compostos de Zinco/química , Manganês/química , Sulfetos/química , Antibacterianos/análise , Antibacterianos/química , beta-Lactamases/análise , beta-Lactamases/metabolismo , beta-Lactamases/química , Leite/química , Limite de Detecção , Espectrometria de Fluorescência/métodos , Corantes Fluorescentes/química , Animais
5.
New Microbiol ; 47(1): 1-14, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38700878

RESUMO

Antibiotics are designed to effectively treat bacterial infections while minimizing harm to the human body. They work by targeting specific components of bacteria or by disrupting essential processes such as cell wall synthesis, membrane function, protein production, and metabolic pathways. However, the misuse and overuse of antibiotics have led to the emergence of drug resistance in humans, animals, and agriculture, contributing to the global spread of this problem. Drug resistance can be either innate or acquired, with acquired resistance involving changes in the bacterial chromosomes or transferable elements. Bacterial species employ various mechanisms of drug resistance, including modifying the antibiotic targets, inactivating the drug, reducing uptake or increasing efflux, overexpressing the target, utilizing alternative pathways, and forming biofilms. One significant concern in the realm of drug resistance revolves around the emergence and proliferation of extended-spectrum beta-lactamases (ESBLs), a gene that is found in most gram-negative bacteria, primarily carried by Escherichia coli and Klebsiella pneumoniae in healthcare settings. ESBL-mediated resistance poses challenges for diagnosis, treatment, infection control, and antibiotic stewardship. Accurate detection of ESBL genes is crucial, and phenotypic methods are commonly used for initial screening. However, these methods have limitations, and confirmatory molecular techniques such as PCR and DNA sequencing are employed to accurately identify ESBL genes. Despite the significant global concerns surrounding ESBLs, they have spread worldwide, mainly facilitated by healthcare settings, inappropriate antimicrobial use, and host susceptibility. Addressing this issue requires implementing comprehensive measures, including enhanced surveillance, strict infection control practices, antibiotic stewardship programs, rapid diagnostic methods, alternative therapies, public education initiatives, and research focused on developing new drugs. Furthermore, collaboration among the healthcare, public health, and research sectors is pivotal in effectively combating the escalating threat posed by ESBL-mediated resistance. Antibiotics have revolutionized medical care by effectively treating bacterial infections. However, the emergence of ESBL gene resistance poses a global challenge that requires an integrated approach to prevent a threatening future.


Assuntos
Antibacterianos , beta-Lactamases , beta-Lactamases/genética , beta-Lactamases/metabolismo , Antibacterianos/farmacologia , Humanos , Animais , Farmacorresistência Bacteriana/genética , Fenótipo , Bactérias/efeitos dos fármacos , Bactérias/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo
6.
PLoS Pathog ; 20(5): e1012187, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38718038

RESUMO

The emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) has significant challenges to human health and clinical treatment, with KPC-2-producing CRKP being the predominant epidemic strain. Therefore, there is an urgent need to identify new therapeutic targets and strategies. Non-coding small RNA (sRNA) is a post-transcriptional regulator of genes involved in important biological processes in bacteria and represents an emerging therapeutic strategy for antibiotic-resistant bacteria. In this study, we analyzed the transcription profile of KPC-2-producing CRKP using RNA-seq. Of the 4693 known genes detected, the expression of 307 genes was significantly different from that of carbapenem-sensitive Klebsiella pneumoniae (CSKP), including 133 up-regulated and 174 down-regulated genes. Both the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment and Gene Ontology (GO) analysis showed that these differentially expressed genes (DEGs) were mainly related to metabolism. In addition, we identified the sRNA expression profile of KPC-2-producing CRKP for the first time and detected 115 sRNAs, including 112 newly discovered sRNAs. Compared to CSKP, 43 sRNAs were differentially expressed in KPC-2-producing CRKP, including 39 up-regulated and 4 down-regulated sRNAs. We chose sRNA51, the most significantly differentially expressed sRNA in KPC-2-producing CRKP, as our research subject. By constructing sRNA51-overexpressing KPC-2-producing CRKP strains, we found that sRNA51 overexpression down-regulated the expression of acrA and alleviated resistance to meropenem and ertapenem in KPC-2-producing CRKP, while overexpression of acrA in sRNA51-overexpressing strains restored the reduction of resistance. Therefore, we speculated that sRNA51 could affect the resistance of KPC-2-producing CRKP by inhibiting acrA expression and affecting the formation of efflux pumps. This provides a new approach for developing antibiotic adjuvants to restore the sensitivity of CRKP.


Assuntos
Carbapenêmicos , Klebsiella pneumoniae , beta-Lactamases , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/metabolismo , Klebsiella pneumoniae/efeitos dos fármacos , beta-Lactamases/genética , beta-Lactamases/metabolismo , Carbapenêmicos/farmacologia , Humanos , Regulação Bacteriana da Expressão Gênica , Antibacterianos/farmacologia , Infecções por Klebsiella/microbiologia , Infecções por Klebsiella/tratamento farmacológico , Infecções por Klebsiella/genética , Enterobacteriáceas Resistentes a Carbapenêmicos/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Pequeno RNA não Traduzido/genética , RNA Bacteriano/genética , Testes de Sensibilidade Microbiana
8.
BMC Genomics ; 25(1): 508, 2024 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-38778284

RESUMO

BACKGROUND: Enzymatic degradation mediated by beta-lactamases constitutes one of the primary mechanisms of resistance to beta-lactam antibiotics in gram-negative bacteria. This enzyme family comprises four molecular classes, categorized into serine beta-lactamases (Classes A, C, and D) and zinc-dependent metallo-beta-lactamases (Class B). Gram-negative bacteria producing beta-lactamase are of significant concern, particularly due to their prevalence in nosocomial infections. A comprehensive understanding of the evolution and dissemination of this enzyme family is essential for effective control of these pathogens. In this study, we conducted the prospecting, phylogenetic analysis, and in silico analysis of beta-lactamases and homologous proteins identified in 1827 bacterial genomes with phenotypic data on beta-lactam resistance. These genomes were distributed among Klebsiella pneumoniae (45%), Acinetobacter baumannii (31%), Pseudomonas aeruginosa (14%), Escherichia coli (6%), and Enterobacter spp. (4%). Using an HMM profile and searching for conserved domains, we mined 2514, 8733, 5424, and 2957 proteins for molecular classes A, B, C, and D, respectively. This set of proteins encompasses canonical subfamilies of beta-lactamases as well as hypothetical proteins and other functional groups. Canonical beta-lactamases were found to be phylogenetically distant from hypothetical proteins, which, in turn, are closer to other representatives of the penicillin-binding-protein (PBP-like) and metallo-beta-lactamase (MBL) families. The catalytic amino acid residues characteristic of beta-lactamases were identified from the sequence alignment and revealed that motifs are less conserved in homologous groups than in beta-lactamases. After comparing the frequency of protein groups in genomes of resistant strains with those of sensitive ones applying Fisher's exact test and relative risk, it was observed that some groups of homologous proteins to classes B and C are more common in the genomes of resistant strains, particularly to carbapenems. We identified the beta-lactamase-like domain widely distributed in gram-negative species of the ESKAPEE group, which highlights its importance in the context of beta-lactam resistance. Some hypothetical homologous proteins have been shown to potentially possess promiscuous activity against beta-lactam antibiotics, however, they do not appear to expressly determine the resistance phenotype. The selective pressure due to the widespread use of antibiotics may favor the optimization of these functions for specialized resistance enzymes.


Assuntos
Bactérias Gram-Negativas , Filogenia , beta-Lactamases , beta-Lactamases/metabolismo , beta-Lactamases/genética , beta-Lactamases/química , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/enzimologia , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , beta-Lactamas/farmacologia , beta-Lactamas/metabolismo , Antibacterianos/farmacologia , Genoma Bacteriano , Resistência beta-Lactâmica/genética , Antibióticos beta Lactam
9.
PLoS One ; 19(5): e0303872, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38771780

RESUMO

BACKGROUND: Antimicrobial resistance (AMR) is among the top public health concerns in the globe. Estimating the prevalence of multidrug resistance (MDR), MDR index (MDR-I) and extended-spectrum beta-lactamase (ESBL)-producing lactose fermenting Enterobacteriaceae (LFE) is important in designing strategies to combat AMR. Thus, this study was designed to determine the status of MDR, MDR-I and ESBL-producing LFE isolated from the human-dairy interface in the northwestern part of Ethiopia, where such information is lacking. METHODOLOGY: A cross-sectional study was conducted from June 2022 to August 2023 by analyzing 362 samples consisting of raw pooled milk (58), milk container swabs (58), milker's hand swabs (58), farm sewage (57), milker's stool (47), and cow's feces (84). The samples were analyzed using standard bacteriological methods. The antimicrobial susceptibility patterns and ESBL production ability of the LFE isolates were screened using the Kirby-Bauer disk diffusion method, and candidate isolates passing the screening criteria were phenotypically confirmed by using cefotaxime (30 µg) and cefotaxime /clavulanic acid (30 µg/10 µg) combined-disk diffusion test. The isolates were further characterized genotypically using multiplex polymerase chain reaction targeting the three ESBL-encoding- genes namely blaTEM, blaSHV, and blaCTX-M. RESULTS: A total of 375 bacterial isolates were identified and the proportion of MDR and ESBL-producing bacterial isolates were 70.7 and 21.3%, respectively. The MDR-I varied from 0.0 to 0.81 with an average of 0.30. The ESBL production was detected in all sample types. Genotypically, the majority of the isolates (97.5%), which were positive on the phenotypic test, were carrying one or more of the three genes. CONCLUSION: A high proportion of the bacterial isolates were MDR; had high MDR-I and were positive for ESBL production. The findings provide evidence that the human-dairy interface is one of the important reservoirs of AMR traits. Therefore, the implementation of AMR mitigation strategies is highly needed in the area.


Assuntos
Antibacterianos , Farmacorresistência Bacteriana Múltipla , Enterobacteriaceae , Lactose , beta-Lactamases , Humanos , Etiópia , beta-Lactamases/genética , beta-Lactamases/metabolismo , Enterobacteriaceae/genética , Enterobacteriaceae/efeitos dos fármacos , Enterobacteriaceae/isolamento & purificação , Enterobacteriaceae/enzimologia , Lactose/metabolismo , Farmacorresistência Bacteriana Múltipla/genética , Estudos Transversais , Antibacterianos/farmacologia , Animais , Testes de Sensibilidade Microbiana , Bovinos , Infecções por Enterobacteriaceae/microbiologia , Cefotaxima/farmacologia , Leite/microbiologia , Fermentação , Fezes/microbiologia
10.
Sci Rep ; 14(1): 10897, 2024 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-38740876

RESUMO

Urinary tract infection (UTI) is the most prevalent urological condition worldwide. Choosing appropriate antibiotics for patients who have fever before receiving a culture result is challenging. This retrospective study enrolled patients 394 patients hospitalized at Gangneung Asan Hospital for UTI from May 2017 to April 2021. Fever at 48 h of hospitalization was the analysis point, as this is when the response to antibiotic therapy manifest, although the results of antibiogram are not available. Multivariate analysis was performed to assess the correlation between ESBL producing bacteria (EPB) and fever at 48 h. Overall, 36.3% of patients had EPB and 27.9% had fever at 48 h. In multivariate analysis, a significant positive association was found between EPB and fever (odds ratio 1.17, 95% CI 1.05-1.30, P = 0.004) Female had negative association with multivariate model (OR 0.83, 95% CI 0.73-0.94, P = 0.004). Diabetes did not demonstrate a significant association with EPB. (OR 1.10, 95% CI 0.99-1.22, P = 0.072). Fever at 48 h is associated with EPB and could be considered a predictive factor for EPB infection in patients with UTI. Antibiotic escalation may be considered in patients with fever at 48 h.


Assuntos
Antibacterianos , Febre , Infecções Urinárias , beta-Lactamases , Humanos , Infecções Urinárias/microbiologia , Infecções Urinárias/tratamento farmacológico , Feminino , Masculino , beta-Lactamases/metabolismo , Estudos Retrospectivos , Idoso , Pessoa de Meia-Idade , Antibacterianos/uso terapêutico , Antibacterianos/farmacologia , Febre/microbiologia , Febre/tratamento farmacológico , Idoso de 80 Anos ou mais , Adulto
11.
PLoS One ; 19(5): e0303753, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38758757

RESUMO

NDM-producing carbapenem-resistant bacterial infections became a challenge for clinicians. Combination therapy of aztreonam and ceftazidime-avibactam is a prudent choice for these infections. However, there is still no recommendation of a practically feasible method for testing aztreonam and ceftazidime-avibactam synergy. We proposed a simple method for testing aztreonam and ceftazidime-avibactam synergy and compared it with reference broth micro-dilution and other methods. Carbapenem-resistant Enterobacterales clinical isolates were screened for the presence of the NDM gene by the Carba R test. NDM harbouring isolates were tested for aztreonam and ceftazidime-avibactam synergy by broth microdilution (reference method), E strip-disc diffusion, double disc diffusion, and disc replacement methods. In the newly proposed method, the MHA medium was supplemented with ceftazidime-avibactam (corresponding to an aztreonam concentration of 4µg/ml). The MHA medium was then inoculated with the standard inoculum (0.5 McFarland) of the test organism. An AZT disc (30 µg) was placed on the supplemented MHA medium, and the medium was incubated overnight at 37°C. Aztreonam zone diameter on the supplemented MHA medium (in the presence of ceftazidime-avibactam) was compared with that from a standard disc diffusion plate (without ceftazidime-avibactam), performed in parallel. Interpretation of synergy was based on the restoration of aztreonam zone diameter (in the presence of ceftazidime-avibactam) crossing the CLSI susceptibility breakpoint, i.e., ≥ 21 mm. Of 37 carbapenem-resistant NDM-producing isolates, 35 (94.6%) were resistant to aztreonam and tested synergy positive by the proposed method. Its sensitivity and specificity were 97.14% and 100%, respectively. Cohen's kappa value showed substantial agreement of the reference method with the proposed method (κ = 0.78) but no other methods. The proposed method is simple, easily interpretable, and showed excellent sensitivity, specificity, and agreement with the reference method. Therefore, the new method is feasible and reliable for testing aztreonam synergy with avibactam in NDM-producing Enterobacterales.


Assuntos
Antibacterianos , Compostos Azabicíclicos , Aztreonam , Ceftazidima , Combinação de Medicamentos , Enterobacteriaceae , Testes de Sensibilidade Microbiana , beta-Lactamases , Ceftazidima/farmacologia , Aztreonam/farmacologia , Compostos Azabicíclicos/farmacologia , beta-Lactamases/metabolismo , beta-Lactamases/genética , Testes de Sensibilidade Microbiana/métodos , Antibacterianos/farmacologia , Enterobacteriaceae/efeitos dos fármacos , Enterobacteriaceae/enzimologia , Enterobacteriaceae/genética , Humanos , Sinergismo Farmacológico , Infecções por Enterobacteriaceae/microbiologia , Infecções por Enterobacteriaceae/tratamento farmacológico
12.
Virulence ; 15(1): 2348251, 2024 12.
Artigo em Inglês | MEDLINE | ID: mdl-38697754

RESUMO

OBJECTIVES: This study aimed at revealing the underlying mechanisms of the loss and gain of ceftazidime-avibactam susceptibility in a non-carbapenemase-producing hypervirulent Klebsiella pneumoniae (hvKp). METHODS: Here we longitudinally recovered 3 non-carbapenemase-producing K1-ST23 hvKp strains at a one-month interval (KP29105, KP29499 and KP30086) from an elderly male. Antimicrobial susceptibility testing, whole genome sequencing, transcriptomic sequencing, gene cloning, plasmid conjugation, quantitative real-time PCR (qRT-PCR), and SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) were conducted. RESULTS: Among the 3 hvKp strains, KP29105 was resistant to the third- and fourth-generation cephalosporins, KP29499 acquired resistance to both ceftazidime-avibactam and carbapenems, while KP30086 restored its susceptibility to ceftazidime-avibactam, imipenem and meropenem but retained low-level resistance to ertapenem. KP29105 and KP29499 carried plasmid-encoded genes blaCTX-M-15 and blaCTX-M-71, respectively, but KP30086 lost both. Cloning of gene blaCTX-M-71 and conjugation experiment of blaCTX-M-71-carrying plasmid showed that the transformant and transconjugant were susceptible to ceftazidime-avibactam but had a more than 8-fold increase in MICs. Supplementation with an outer membrane permeabilizer could reduce the MIC of ceftazidime-avibactam by 32 folds, indicating that porins play a key role in ceftazidime-avibactam resistance. The OmpK35 of the 3 isolates was not expressed, and the OmpK36 of KP29499 and KP30086 had a novel amino acid substitution (L359R). SDS-PAGE and qRT-PCR showed that the expression of porin OmpK36 of KP29499 and KP30086 was significantly down-regulated compared with KP29105. CONCLUSIONS: In summary, we reported the rare ceftazidime-avibactam resistance in a non-carbapenemase-producing hvKp strain. Resistance plasmid carrying blaCTX-M-71 and mutated OmpK36 had a synergetic effect on the resistance.


Assuntos
Antibacterianos , Compostos Azabicíclicos , Proteínas de Bactérias , Ceftazidima , Combinação de Medicamentos , Infecções por Klebsiella , Klebsiella pneumoniae , Testes de Sensibilidade Microbiana , Ceftazidima/farmacologia , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/patogenicidade , Klebsiella pneumoniae/enzimologia , Compostos Azabicíclicos/farmacologia , Antibacterianos/farmacologia , Masculino , Infecções por Klebsiella/microbiologia , Infecções por Klebsiella/tratamento farmacológico , Humanos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , beta-Lactamases/genética , beta-Lactamases/metabolismo , Idoso , Farmacorresistência Bacteriana Múltipla/genética , Virulência , Plasmídeos/genética , Sequenciamento Completo do Genoma
13.
Nat Commun ; 15(1): 4093, 2024 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-38750030

RESUMO

Plasmids carrying antibiotic resistance genes (ARG) are the main mechanism of resistance dissemination in Enterobacterales. However, the fitness-resistance trade-off may result in their elimination. Chromosomal integration of ARGs preserves resistance advantage while relieving the selective pressure for keeping costly plasmids. In some bacterial lineages, such as carbapenemase producing sequence type ST38 Escherichia coli, most ARGs are chromosomally integrated. Here we reproduce by experimental evolution the mobilisation of the carbapenemase blaOXA-48 gene from the pOXA-48 plasmid into the chromosome. We demonstrate that this integration depends on a plasmid-induced fitness cost, a mobile genetic structure embedding the ARG and a novel antiplasmid system ApsAB actively involved in pOXA-48 destabilization. We show that ApsAB targets high and low-copy number plasmids. ApsAB combines a nuclease/helicase protein and a novel type of Argonaute-like protein. It belongs to a family of defense systems broadly distributed among bacteria, which might have a strong ecological impact on plasmid diffusion.


Assuntos
Escherichia coli , Plasmídeos , beta-Lactamases , Plasmídeos/genética , beta-Lactamases/genética , beta-Lactamases/metabolismo , Escherichia coli/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Antibacterianos/farmacologia , Farmacorresistência Bacteriana/genética , Cromossomos Bacterianos/genética
14.
PLoS One ; 19(5): e0303353, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38743684

RESUMO

INTRODUCTION: The study of Klebsiella quasipneumoniae, Klebsiella variicola, and AmpC production in extended-spectrum ß-lactamase (ESBL)-producing Klebsiella in Japan is limited, and existing data are insufficient. This study aims to characterize Klebsiella species, determine AmpC production rates, and analyze antimicrobial resistance patterns in ESBL-producing Klebsiella isolates in Japan. METHODS: A total of 139 clinical isolates of ESBL-producing Klebsiella were collected in Japan, along with their corresponding antimicrobial susceptibility profiles. The isolates were identified using a web-based tool. ESBL genes within the isolates were identified using multiplex PCR. Screening for AmpC-producing isolates was performed using cefoxitin disks, followed by multiplex PCR to detect the presence of AmpC genes. Antimicrobial resistance patterns were analyzed across the predominant ESBL genotypes. RESULTS: The web-based tool identified 135 isolates (97.1%) as Klebsiella pneumoniae and 4 (2.9%) as K. quasipneumoniae subsp. similipneumoniae, with no instances of K. variicola detected. Among K. pneumoniae, the CTX-M-1 group emerged as the predominant genotype (83/135, 61.5%), followed by K. quasipneumoniae subsp. similipneumoniae (3/4, 75.0%). The CTX-M-9 group was the second most prevalent genotype in K. pneumoniae (45/135, 33.3%). The high resistance rates were observed for quinolones (ranging from 46.7% to 63.0%) and trimethoprim/sulfamethoxazole (78.5%). The CTX-M-1 group exhibited higher resistance to ciprofloxacin (66/83, 79.5%) compared to the CTX-M-9 group (18/45, 40.0%), a trend also observed for levofloxacin and trimethoprim/sulfamethoxazole. Among the 16 isolates that tested positive during AmpC screening, only one K. pneumoniae isolates (0.7%) were confirmed to carry the AmpC gene. CONCLUSION: Klebsiella pneumoniae with the CTX-M-1 group is the most common ESBL-producing Klebsiella in Japan and showed a low proportion of AmpC production. These isolates are resistant to quinolones and trimethoprim/sulfamethoxazole, highlighting the challenge of managing this pathogen. The findings underscore the importance of broader research and continuous monitoring to address the resistance patterns of ESBL-producing Klebsiella.


Assuntos
Antibacterianos , Proteínas de Bactérias , Infecções por Klebsiella , Klebsiella pneumoniae , Klebsiella , Testes de Sensibilidade Microbiana , beta-Lactamases , beta-Lactamases/genética , beta-Lactamases/metabolismo , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/isolamento & purificação , Klebsiella pneumoniae/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Klebsiella/genética , Klebsiella/efeitos dos fármacos , Klebsiella/isolamento & purificação , Klebsiella/enzimologia , Japão , Estudos Retrospectivos , Humanos , Infecções por Klebsiella/microbiologia , Infecções por Klebsiella/tratamento farmacológico , Infecções por Klebsiella/epidemiologia , Antibacterianos/farmacologia , Farmacorresistência Bacteriana/genética , Masculino , Feminino , População do Leste Asiático
15.
Nat Commun ; 15(1): 3947, 2024 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-38729951

RESUMO

Gram-negative bacteria (GNB) are a major cause of neonatal sepsis in low- and middle-income countries (LMICs). Although the World Health Organization (WHO) reports that over 80% of these sepsis deaths could be prevented through improved treatment, the efficacy of the currently recommended first- and second-line treatment regimens for this condition is increasingly affected by high rates of drug resistance. Here we assess three well known antibiotics, fosfomycin, flomoxef and amikacin, in combination as potential antibiotic treatment regimens by investigating the drug resistance and genetic profiles of commonly isolated GNB causing neonatal sepsis in LMICs. The five most prevalent bacterial isolates in the NeoOBS study (NCT03721302) are Klebsiella pneumoniae, Acinetobacter baumannii, E. coli, Serratia marcescens and Enterobacter cloacae complex. Among these isolates, high levels of ESBL and carbapenemase encoding genes are detected along with resistance to ampicillin, gentamicin and cefotaxime, the current WHO recommended empiric regimens. The three new combinations show excellent in vitro activity against ESBL-producing K. pneumoniae and E. coli isolates. Our data should further inform and support the clinical evaluation of these three antibiotic combinations for the treatment of neonatal sepsis in areas with high rates of multidrug-resistant Gram-negative bacteria.


Assuntos
Acinetobacter baumannii , Antibacterianos , Bactérias Gram-Negativas , Infecções por Bactérias Gram-Negativas , Klebsiella pneumoniae , Testes de Sensibilidade Microbiana , Sepse Neonatal , Humanos , Antibacterianos/uso terapêutico , Antibacterianos/farmacologia , Sepse Neonatal/microbiologia , Sepse Neonatal/tratamento farmacológico , Recém-Nascido , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/isolamento & purificação , Infecções por Bactérias Gram-Negativas/tratamento farmacológico , Infecções por Bactérias Gram-Negativas/microbiologia , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/isolamento & purificação , Acinetobacter baumannii/genética , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/isolamento & purificação , Klebsiella pneumoniae/genética , Amicacina/farmacologia , Amicacina/uso terapêutico , Fosfomicina/farmacologia , Fosfomicina/uso terapêutico , beta-Lactamases/genética , beta-Lactamases/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Países em Desenvolvimento , Farmacorresistência Bacteriana Múltipla/genética , Quimioterapia Combinada , Serratia marcescens/efeitos dos fármacos , Serratia marcescens/genética , Serratia marcescens/isolamento & purificação , Enterobacter cloacae/efeitos dos fármacos , Enterobacter cloacae/genética , Enterobacter cloacae/isolamento & purificação , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo
16.
Euro Surveill ; 29(21)2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38785090

RESUMO

Fosfomycin-resistant FosA8-producing Enterobacterales are uncommon strains with extremely low incidence in Europe, based on only three reports in the literature. We detected FosA8-producing Escherichia coli ST131 in clinical isolates from two patients admitted in February 2023 to a rehabilitation unit in Italy. The occurrence of rare fosA-like genes in the high-risk clone ST131 is of clinical relevance. The dissemination of FosA-producing E. coli, although still at low levels, should be continuously monitored.


Assuntos
Antibacterianos , Infecções por Escherichia coli , Escherichia coli , Humanos , Itália/epidemiologia , Escherichia coli/isolamento & purificação , Escherichia coli/genética , Escherichia coli/efeitos dos fármacos , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/epidemiologia , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Testes de Sensibilidade Microbiana , Fosfomicina/farmacologia , Fosfomicina/uso terapêutico , Masculino , beta-Lactamases/genética , beta-Lactamases/metabolismo , Feminino , Farmacorresistência Bacteriana , Tipagem de Sequências Multilocus
17.
Emerg Microbes Infect ; 13(1): 2353310, 2024 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-38712879

RESUMO

OXA-48-like enzymes represent the most frequently detected carbapenemases in Enterobacterales in Western Europe, North Africa and the Middle East. In contrast to other species, the presence of OXA-48-like in Proteus mirabilis leads to an unusually susceptible phenotype with low MICs for carbapenems and piperacillin-tazobactam, which is easily missed in the diagnostic laboratory. So far, there is little data available on the genetic environments of the corresponding genes, blaOXA-48-like, in P. mirabilis. In this study susceptibility phenotypes and genomic data of 13 OXA-48-like-producing P. mirabilis were investigated (OXA-48, n = 9; OXA-181, n = 3; OXA-162, n = 1). Ten isolates were susceptible to meropenem and ertapenem and three isolates were susceptible to piperacillin-tazobactam. The gene blaOXA-48 was chromosomally located in 7/9 isolates. Thereof, in three isolates blaOXA-48 was inserted into a P. mirabilis genomic island. Of the three isolates harbouring blaOXA-181 one was located on an IncX3 plasmid and two were located on a novel MOBF plasmid, pOXA-P12, within the new transposon Tn7713. In 5/6 isolates with plasmidic location of blaOXA-48-like, the plasmids could conjugate to E. coli recipients in vitro. Vice versa, blaOXA-48-carrying plasmids could conjugate from other Enterobacterales into a P. mirabilis recipient. These data show a high diversity of blaOXA-48-like genetic environments compared to other Enterobacterales, where genetic environments are quite homogenous. Given the difficult-to-detect phenotype of OXA-48-like-producing P. mirabilis and the location of blaOXA-48-like on mobile genetic elements, it is likely that OXA-48-like-producing P. mirabilis can disseminate, escape most surveillance systems, and contribute to a hidden spread of OXA-48-like.


Assuntos
Antibacterianos , Proteínas de Bactérias , Testes de Sensibilidade Microbiana , Infecções por Proteus , Proteus mirabilis , beta-Lactamases , Proteus mirabilis/genética , Proteus mirabilis/enzimologia , Proteus mirabilis/isolamento & purificação , Proteus mirabilis/efeitos dos fármacos , beta-Lactamases/genética , beta-Lactamases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Antibacterianos/farmacologia , Humanos , Infecções por Proteus/microbiologia , Plasmídeos/genética , Ilhas Genômicas , Carbapenêmicos/farmacologia
18.
Front Cell Infect Microbiol ; 14: 1297312, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38690325

RESUMO

Background: During the coronavirus disease 2019 (COVID-19) pandemic, in patients treated for SARS-CoV-2 infection, infections with the Klebsiella pneumoniae bacteria producing New Delhi metallo-B-lactamase (NDM) carbapenemase in the USA, Brazil, Mexico, and Italy were observed, especially in intensive care units (ICUs). This study aimed to assess the impact of Klebsiella pneumoniae NDM infection and other bacterial infections on mortality in patients treated in ICUs due to COVID-19. Methods: The 160 patients who qualified for the study were hospitalized in ICUs due to COVID-19. Three groups were distinguished: patients with COVID-19 infection only (N = 72), patients with COVID-19 infection and infection caused by Klebsiella pneumoniae NDM (N = 30), and patients with COVID-19 infection and infection of bacterial etiology other than Klebsiella pneumoniae NDM (N = 58). Mortality in the groups and chosen demographic data; biochemical parameters analyzed on days 1, 3, 5, and 7; comorbidities; and ICU scores were analyzed. Results: Bacterial infection, including with Klebsiella pneumoniae NDM type, did not elevate mortality rates. In the group of patients who survived the acute phase of COVID-19 the prolonged survival time was demonstrated: the median overall survival time was 13 days in the NDM bacterial infection group, 14 days in the other bacterial infection group, and 7 days in the COVID-19 only group. Comparing the COVID-19 with NDM infection and COVID-19 only groups, the adjusted model estimated a statistically significant hazard ratio of 0.28 (p = 0.002). Multivariate analysis revealed that age, APACHE II score, and CRP were predictors of mortality in all the patient groups. Conclusion: In patients treated for SARS-CoV-2 infection acquiring a bacterial infection due to prolonged hospitalization associated with the treatment of COVID-19 did not elevate mortality rates. The data suggests that in severe COVID-19 patients who survived beyond the first week of hospitalization, bacterial infections, particularly Klebsiella pneumoniae NDM, do not significantly impact mortality. Multivariate analysis revealed that age, APACHE II score, and CRP were predictors of mortality in all the patient groups.


Assuntos
COVID-19 , Farmacorresistência Bacteriana Múltipla , Unidades de Terapia Intensiva , Infecções por Klebsiella , Klebsiella pneumoniae , SARS-CoV-2 , beta-Lactamases , Humanos , COVID-19/mortalidade , COVID-19/microbiologia , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/enzimologia , Masculino , Feminino , Infecções por Klebsiella/mortalidade , Infecções por Klebsiella/tratamento farmacológico , Infecções por Klebsiella/microbiologia , Infecções por Klebsiella/epidemiologia , beta-Lactamases/metabolismo , beta-Lactamases/genética , Pessoa de Meia-Idade , Idoso , Adulto , Antibacterianos/uso terapêutico , Antibacterianos/farmacologia , Idoso de 80 Anos ou mais
19.
Bioorg Chem ; 147: 107328, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38583248

RESUMO

Discovering novel NDM-1 inhibitors is an urgent task for treatment of 'superbug' infectious diseases. In this study, we found that naturally occurring houttuynin and its sulfonate derivatives might be effective NDM-1 inhibitors with novel mechanism, i.e. the attribute of partially covalent inhibition of sulfonate derivatives of houttuynin against NDM-1. Primary structure-activity relationship study showed that both the long aliphatic side chain and the warhead of aldehyde group are vital for the efficiency against NDM-1. The homologs with longer chains (SNH-2 to SNH-5) displayed stronger inhibitory activities with IC50 range of 1.1-1.5 µM, while the shorter chain the weaker inhibition. Further synergistic experiments in cell level confirmed that all these 4 compounds (at 32 µg/mL) recovered the antibacterial activity of meropenem (MER) against E. coli BL21/pET15b-blaNDM-1.


Assuntos
Antibacterianos , Relação Dose-Resposta a Droga , Escherichia coli , Testes de Sensibilidade Microbiana , Relação Estrutura-Atividade , Antibacterianos/farmacologia , Antibacterianos/química , Antibacterianos/síntese química , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Estrutura Molecular , beta-Lactamases/metabolismo , Inibidores de beta-Lactamases/farmacologia , Inibidores de beta-Lactamases/química , Inibidores de beta-Lactamases/síntese química , Produtos Biológicos/farmacologia , Produtos Biológicos/química , Produtos Biológicos/síntese química , Humanos , Proteínas de Escherichia coli
20.
J Chem Theory Comput ; 20(8): 3335-3348, 2024 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-38563746

RESUMO

Protein-protein interactions mediate most molecular processes in the cell, offering a significant opportunity to expand the set of known druggable targets. Unfortunately, targeting these interactions can be challenging due to their typically flat and featureless interaction surfaces, which often change as the complex forms. Such surface changes may reveal hidden (cryptic) druggable pockets. Here, we analyze a set of well-characterized protein-protein interactions harboring cryptic pockets and investigate the predictive power of current computational methods. Based on our observations, we developed a new computational strategy, SWISH-X (SWISH Expanded), which combines the established cryptic pocket identification capabilities of SWISH with the rapid temperature range exploration of OPES MultiThermal. SWISH-X is able to reliably identify cryptic pockets at protein-protein interfaces while retaining its predictive power for revealing cryptic pockets in isolated proteins, such as TEM-1 ß-lactamase.


Assuntos
Proteínas , beta-Lactamases , beta-Lactamases/química , beta-Lactamases/metabolismo , Proteínas/química , Proteínas/metabolismo , Ligação Proteica , Sítios de Ligação , Simulação de Dinâmica Molecular
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