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The treatment of sepsis caused by multidrug-resistant (MDR) Gram-negative bacterial infections remains challenging. With these pathogens exhibiting resistance to carbapenems and new generation cephalosporins, the traditional antibiotic polymyxin B (PMB) has reemerged as a critical treatment option. However, its severe neurotoxicity and nephrotoxicity greatly limit the clinical application. Therefore, we designed negatively charged high-density lipoprotein (HDL) mimicking nanodiscs as a PMB delivery system, which can simultaneously reduce toxicity and enhance drug efficacy. The negative charge prevented the PMB release in physiological conditions and binding to cell membranes, significantly reducing toxicity in mammalian cells and mice. Notably, nanodisc-PMB exhibits superior efficacy than free PMB in sepsis induced by carbapenem-resistant Acinetobacter baumannii (CRAB) strains. Nanodisc-PMB shows promise as a treatment for carbapenem-resistant Gram-negative bacterial sepsis, especially caused by Acinetobacter baumannii, and the nanodiscs could be repurposed for other toxic antibiotics as an innovative delivery system. STATEMENT OF SIGNIFICANCE: Multidrug-resistant Gram-negative bacteria, notably carbapenem-resistant Acinetobacter baumannii, currently pose a substantial challenge due to the scarcity of effective treatments, rendering Polymyxins a last-resort antibiotic option. However, their therapeutic application is significantly limited by severe neurotoxic and nephrotoxic side effects. Prevailing polymyxin delivery systems focus on either reducing toxicity or enhancing bioavailability yet fail to simultaneously achieve both. In this scenario, we have developed a distinctive HDL-mimicking nanodisc for polymyxin B, which not only significantly reduces toxicity but also improves efficacy against Gram-negative bacteria, especially in sepsis caused by CRAB. This research offers an innovative drug delivery system for polymyxin B. Such advancement could notably improve the therapeutic landscape and make a significant contribution to the arsenal against these notorious pathogens.
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Infecciones por Acinetobacter , Acinetobacter baumannii , Polimixina B , Sepsis , Polimixina B/farmacología , Polimixina B/química , Acinetobacter baumannii/efectos de los fármacos , Animales , Infecciones por Acinetobacter/tratamiento farmacológico , Sepsis/tratamiento farmacológico , Ratones , Nanoestructuras/química , Antibacterianos/farmacología , Antibacterianos/química , Humanos , Lipoproteínas HDL/químicaRESUMEN
Polymyxin B and polymyxin E (colistin) are presently considered the last line of defense against human infections caused by multidrug-resistant Gram-negative organisms such as carbapenemase-producer Enterobacterales, Acinetobacter baumannii, and Klebsiella pneumoniae. Yet resistance to this last-line drugs is a major public health threat and is rapidly increasing. Polymyxin S2 (S2) is a polymyxin B analogue previously synthesized in our institute with obviously high antibacterial activity and lower toxicity than polymyxin B and colistin. To predict the possible resistant mechanism of S2 for wide clinical application, we experimentally induced bacterial resistant mutants and studied the preliminary resistance mechanisms. Mut-S, a resistant mutant of K. pneumoniae ATCC BAA-2146 (Kpn2146) induced by S2, was analyzed by whole genome sequencing, transcriptomics, mass spectrometry and complementation experiment. Surprisingly, large-scale genomic inversion (LSGI) of approximately 1.1 Mbp in the chromosome caused by IS26 mediated intramolecular transposition was found in Mut-S, which led to mgrB truncation, lipid A modification and hence S2 resistance. The resistance can be complemented by plasmid carrying intact mgrB. The same mechanism was also found in polymyxin B and colistin induced drug-resistant mutants of Kpn2146 (Mut-B and Mut-E, respectively). This is the first report of polymyxin resistance caused by IS26 intramolecular transposition mediated mgrB truncation in chromosome in K. pneumoniae. The findings broaden our scope of knowledge for polymyxin resistance and enriched our understanding of how bacteria can manage to survive in the presence of antibiotics.
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BACKGROUND: Sepsis has become a global health concern owing to its increasing incidence and high mortality rate. In the present study, we investigated a novel drug candidate ASK0912 on its protective effects in mice with Acinetobacter baumannii 20-1-induced sepsis, and studied the related mechanisms. MATERIAL AND METHODS: To analyze the protective effect of ASK0912 on septic mice, survival rates, body temperature, organ and blood bacterial loads, white blood cell and platelet counts, organ damage, and cytokine levels were determined. RESULTS: ASK0912 remarkably increased the survival rate of mice with sepsis induced by A. baumannii 20-1 at a low dose of 0.6 mg/kg. Rectal temperature measurements showed that ASK0912 treatment prevented the body temperature decrease of septic mice to some extent. Treatment with ASK0912 can notably reduce the organ and blood bacterial loads and alleviate platelet count reduction due to sepsis. ASK0912 attenuated organ damage, including reduced levels of total bile acids, urea, and creatinine, aggregation of inflammatory cells, and mitigation of structural changes in septic mice, as demonstrated by biochemical analysis and hematoxylin & eosin staining. Additionally, multiplex assay showed that abnormally increased cytokine levels (IL-1ß, IL-3, IL-5, IL-6, IL-10, IL-13, MCP-1, RANTES, KC, MIP-1α, MIP-1ß, and G-CSF) in septic mice decreased after ASK0912 treatment. CONCLUSIONS: ASK0912 can not only improve the survival rate, hypothermia, lower the bacterial loads in the organs and blood, but also alleviate the pathophysiological manifestations such as intravascular coagulation abnormalities, organ damages, and immune system disorder of sepsis mice induced by A. baumannii 20-1.
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Acinetobacter baumannii , Sepsis , Ratones , Animales , Sepsis/complicaciones , Sepsis/tratamiento farmacológico , Citocinas , Quimiocina CCL4RESUMEN
OBJECTIVES: Contezolid acefosamil is a novel O-acyl phosphoramidate prodrug of contezolid. In the current study, we aimed to systemically evaluate the efficacy of contezolid acefosamil against infections caused by multiple Gram-positive pathogens, and compare the efficacy of the prodrug by oral and intravenous administrations. METHODS: The in vivo pharmacodynamic efficacy of contezolid acefosamil was evaluated in mouse models of systemic (with five S. aureus, three S. pneumoniae and two S. pyogenes bacterial isolates) and thigh (with two S. aureus isolates) infections using linezolid as the reference agent. RESULTS: In both models, contezolid acefosamil administrated either orally or intravenously, demonstrated high antibacterial efficacy similar to linezolid, and the antibacterial efficacy of oral and intravenous contezolid acefosamil were comparable. CONCLUSIONS: The high aqueous solubility and great efficacy of contezolid acefosamil support its clinical development as an injectable and oral antibiotic suitable for serious Gram-positive infections.
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Profármacos , Animales , Ratones , Linezolid , Profármacos/farmacología , Staphylococcus aureus , Antibacterianos/uso terapéutico , Administración Intravenosa , Pruebas de Sensibilidad Microbiana , Administración OralRESUMEN
Enterococci can cause various infectious diseases, including urinary tract infection, wound infection, and life-threatening endocarditis and meningitis. The emergence and transmission of vancomycin-resistant enterococci (VRE) have presented a challenge to clinical treatment. There is an urgent need to develop new strategies to fight against this pathogen. This study investigated the antibacterial and anti-biofilm activity of celastrol (CEL), a natural product originating from Tripterygium wilfordii Hook F, against enterococci, and its adjuvant capacity of restoring the susceptibility of VRE to vancomycin in vitro and in vivo. CEL inhibited all enterococcus strains tested, with MICs ranging from 0.5 to 4 µg/mL. More than 50% of biofilm was eliminated by CEL at 16 µg/mL after 24 h of exposure. The combination of CEL and vancomycin showed a synergistic effect against all 23 strains tested in checkerboard assays. The combination of sub-MIC levels of CEL and vancomycin showed a synergistic effect in a time-kill assay and exhibited significant protective efficacy in Galleria mellonella larval infection model compared with either drug used alone. The underlying mechanisms of CEL were explored by conducting biomolecular binding interactions and an enzyme inhibition assay of CEL on bacterial cell-division protein FtsZ. CEL presented strong binding and suppression ability to FtsZ, with Kd and IC50 values of 2.454 µM and 1.04 ± 0.17 µg/mL, respectively. CEL exhibits a significant antibacterial and synergic activity against VRE in vitro and in vivo and has the potential to be a new antibacterial agent or adjuvant to vancomycin as a therapeutic option in combating VRE. IMPORTANCE The emergence and transmission of VRE pose a significant medical and public health challenge. CEL, well-known for a wide range of biological activities, has not previously been investigated for its synergistic effect with vancomycin against VRE. In the present study, CEL exhibited antibacterial activity against enterococci, including VRE strains, and restored the activity of vancomycin against VRE in vitro and in vivo. Hence, CEL has the potential to be a new antibacterial adjuvant to vancomycin and could provide a promising therapeutic option in combating VRE.
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Enterococos Resistentes a la Vancomicina , Vancomicina , Vancomicina/farmacología , Antibacterianos/farmacología , Triterpenos Pentacíclicos/farmacología , Pruebas de Sensibilidad MicrobianaRESUMEN
An accurate and reliable susceptibility testing method for polymyxins is urgently needed not only for the clinical laboratory but also for new polymyxin-like lipopeptide development. Reference broth microdilution (rBMD), which was the recommended method by CLSI-EUCAST in clinics, has been proven not to be ideal, while the agar dilution (AD) method that was widely used in new antibiotics discovery has been neglected. In the present study, the AD method was compared with rBMD and broth macrodilution (BMAD) in susceptibility testing of polymyxin B and colistin against >200 Gram-negative isolates. AD showed strong agreement with BMAD for colistin (except for Klebsiella aerogenes and Pseudomonas aeruginosa); however, its performance was poor for polymyxin B or compared to rBMD. MICs of AD method were not affected when different types of Petri dishes were used, while glass-bottom microtiter plates could lower the MIC of polymyxins 2−8 times compared to tissue-culture-treated polystyrene plates when using rBMD, which demonstrated that tissue-culture-treated plates were not suitable. It was then validated with non-tissue-culture-treated plates. The culture volume was another influencing factor of accuracy for rBMD, and 200 µL seemed to be the most suitable volume for MIC detection of polymyxins. Additionally, no lack of growth phenomenon (skipped well) was observed for AD when it frequently occurred for both BMAD and rBMD. As for strains carrying mcr-1 gene, 100% of AD results were in essential agreement (EA) and categorical agreement (CA) with both rBMD and BMAD. Overall, rBMD is convenient and widely accepted for susceptibility testing of polymyxins. Although it may be too early to say that AD is superior compared to rBMD and BMAD, it did show some advantages in repeatability and anti-interference ability.
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A series of new N, N'-diarylurea derivatives were designed and synthesized, some of which exhibited potent antibacterial activity against the drug-susceptible and drug-resistant Gram-positive strains. Especially, compounds 2c, 2g-2l showed broader antibacterial spectrum and more potent antibacterial activity (MIC = 0.30-2.72 µM) against MRSA and MRSE than the control levofloxacin (MIC = 0.69-22.14 µM). In addition, compounds 2c, 2g, 2h and 2l exhibited much better antibacterial activity (MIC = 1.29-2.86 µM) against VRE (E. faecium) than sorafenib (MIC = 275.37 µM), PK150 (MIC = 5.07-10.13 µM) and SC78 (MIC = 2.40-4.79 µM). More importantly, the low cytotoxicity of compounds on cell lines HeLa and HepG2 implied a relatively wide therapeutic window, which was of high importance for further study.
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Antibacterianos , Staphylococcus aureus Resistente a Meticilina , Antibacterianos/farmacología , Levofloxacino/farmacología , Pruebas de Sensibilidad Microbiana , Sorafenib , Relación Estructura-ActividadRESUMEN
To study the effect of different tolerable levels of constitutive mcr-1 expression on Escherichia coli, and to provide direct evidence for moderate resistance mediated by mcr-1, construction of E. coli strains carrying mcr-1 on the chromosome with promoters of different strengths was conducted using λ-red recombination. Our results demonstrated that over-high expression of mcr-1 cannot be tolerated, and seven constructs with more than 200-fold mcr-1 transcriptional expression differences were obtained. The colistin MICs of the seven strains increased with the increase of MCR-1 levels, and the highest MIC was 8 µg/mL. Lower expression of mcr-1 didn't demonstrate many effects on bacteria, while higher tolerable expression of mcr-1 tended to show fitness costs in growth rate, competitive ability, and cell structures, but no obvious change of virulence was observed in mice. Bacteria demonstrated colistin MICs of 4-8 µg/mL at mcr-1 expression levels similar to clinical isolates, which were the mcr-1 expression levels with relatively lower fitness costs. IMPORTANCE The effects of relatively lower tolerable levels of mcr-1 were not evaluated thoroughly, and direct evidence for moderate resistance mediated by mcr-1 was lacking. In the present study, we made constructs carrying mcr-1 on the E. coli K12 chromosome under the control of serial constitutive promoters of different strengths and studied the effects of different tolerable levels of mcr-1 expression in vitro and in vivo. The results demonstrated that generally, except QH0007 (the construct with the highest mcr-1 expression that showed some extent of cell death), the fitness costs of tolerable mcr-1 expression on bacteria were not apparent or low. Bacteria demonstrated colistin MICs of 4-8 µg/mL at mcr-1 expression levels similar to clinical isolates, which corresponded to the lower levels of mcr-1 expression that can lead to colistin resistance, indicating the cleverness of bacteria to balance the benefit and cost of MCR-1-mediated colistin resistance.
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Infecciones por Escherichia coli , Proteínas de Escherichia coli , Ratones , Animales , Escherichia coli , Colistina/farmacología , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Farmacorresistencia Bacteriana/genética , Antibacterianos/farmacología , Infecciones por Escherichia coli/microbiología , Pruebas de Sensibilidad Microbiana , PlásmidosRESUMEN
The shortage of new antibiotics makes infections caused by gram-negative (G-) bacteria a significant clinical problem. The key enzymes involved in folate biosynthesis represent important targets for drug discovery, and new antifolates with novel mechanisms are urgently needed. By targeting to dihydrofolate reductase (DHFR), a series of 1,3-diamino-7H-pyrrol[3,2-f]quinazoline (PQZ) compounds were designed, and exhibited potent antibacterial activities in vitro, especially against multi-drug resistant G- strains. Multiple experiments indicated that PQZ compounds contain a different molecular mechanism against the typical DHFR inhibitor, trimethoprim (TMP), and the thymidylate synthase (TS) was identified as another potential but a relatively weak target. A significant synergism between the representative compound, OYYF-175, and sulfamethoxazole (SMZ) was observed with a strong cumulative and significantly bactericidal effect at extremely low concentrations (2 µg/mL for SMZ and 0.03 pg/mL for OYYF-175), which could be resulted from the simultaneous inhibition of dihydropteroate synthase (DHPS), DHFR and TS. PQZ compounds exhibited therapeutic effects in a mouse model of intraperitoneal infections caused by Escherichia coli (E. coli). The co-crystal structure of OYYF-175-DHFR was solved and the detailed interactions were provided. The inhibitors reported represent innovative chemical structures with novel molecular mechanism of action, which will benefit the generation of new, efficacious bactericidal compounds.
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Antibacterianos/farmacología , Descubrimiento de Drogas , Antagonistas del Ácido Fólico/farmacología , Ácido Fólico/metabolismo , Acinetobacter baumannii/efectos de los fármacos , Antibacterianos/síntesis química , Antibacterianos/química , Relación Dosis-Respuesta a Droga , Enterobacteriaceae/efectos de los fármacos , Antagonistas del Ácido Fólico/síntesis química , Antagonistas del Ácido Fólico/química , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Relación Estructura-Actividad , Enterococos Resistentes a la Vancomicina/efectos de los fármacosRESUMEN
Antivirulence strategy has been developed as a nontraditional therapy which would engender a lower evolutionary pressure toward the development of antimicrobial resistance. However, the majority of the antivirulence agents currently in development could not meet clinical needs due to their narrow antibacterial spectrum and limited indications. Therefore, our main purpose is to develop broad-spectrum antivirulence agents that could target on both Gram-positive and Gram-negative pathogens. We discovered ML364, a novel scaffold compound, could inhibit the productions of both pyocyanin of Pseudomonas aeruginosa and staphyloxanthin of Staphylococcus aureus. Further transcriptome sequencing and enrichment analysis showed that the quorum sensing (QS) system of pathogens was mainly disrupted by ML364 treatment. To date, autoinducer-2 (AI-2) of the QS system is the only non-species-specific signaling molecule that responsible for the cross-talk between Gram-negative and Gram-positive species. And further investigation showed that ML364 treatment could significantly inhibit the sensing of AI-2 or its nonborated form DPD signaling in Vibrio campbellii MM32 and attenuate the biofilm formation across multi-species pathogens including Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae and Staphylococcus aureus. The results of molecular docking and MM/GBSA free energy prediction showed that ML364 might have higher affinity with the receptors of DPD/AI-2, when compared with DPD molecule. Finally, the in vivo study showed that ML364 could significantly improve the survival rates of systemically infected mice and attenuate bacterial loads in the organs of mice. Overall, ML364 might interfere with AI-2 quorum sensing system to exert broad-spectrum antivirulence effect both in vitro and in vivo.
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Acinetobacter baumannii, especially multidrug resistant Acinetobacter baumannii, is a notable source of pressure in the areas of public health and antibiotic development. To overcome this problem, attention has been focused on membrane proteins. Different digestion methods and extraction detergents were examined for membrane proteome sample preparation, and label-free quantitative and targeted proteome analyses of the polymyxin B-induced Acinetobacter baumannii ATCC 19606 membrane proteome were performed based on nano LC-MS/MS. Ultracentrifugation of proteins at a speed of 150,000×g, digestion by trypsin, filter-aided sample preparation, and detergents such as lauryldimethylamine-N-oxide were proved as a fast and effective way for identification of membrane proteome by nano LC-MS/MS. Upon treatment with polymyxin B, expression levels of 15 proteins related to membrane structure, transporters, cell surface, and periplasmic space were found to be significantly changed. Furthermore, targeted proteome was also used to confirm these changes. A relatively rapid membrane proteome preparation method was developed, and a more comprehensive view of changes in the Acinetobacter baumannii membrane proteome under polymyxin B pressure was obtained.
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Acinetobacter baumannii , Antibacterianos/farmacología , Proteínas de la Membrana , Pruebas de Sensibilidad Microbiana , Polimixina B/farmacología , Polimixinas , Espectrometría de Masas en TándemRESUMEN
Antimicrobial resistance has been an increasingly serious threat to global public health. Anti-virulence strategies are being developed to manage antibiotic resistance because they apply a lower selective pressure for antimicrobial-resistant pathogens than that created using traditional bactericides. We aimed to discover novel small molecules that can reduce the production of virulence factors in Pseudomonas aeruginosa and determine the mechanism of action underlying these effects. A clinical compound library was screened, and ostarine was identified as a potential anti-virulence agent. The effects of ostarine were studied via antimicrobial susceptibility testing, bacterial growth assays, pyocyanin quantitation assays, transcriptomic analysis, quorum sensing signal molecule quantification, and real-time PCR assays. Ostarine treatment significantly decreased the synthesis of pyocyanin without any bactericidal action. Besides, ostarine treatment did not affect the relative growth rate and cell morphology of bacteria. Treatment with ostarine interfered with quorum sensing by decreasing the transcription of genes associated with quorum sensing systems and the production of signalling molecules. The inhibition of ostarine on pyocyanin production and gene expression can be alleviated when signalling molecules were supplemented externally. Overall, ostarine may act as a novel anti-virulence agent that can attenuate P. aeruginosa pyocyanin by interfering with quorum sensing systems.
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Anilidas/farmacología , Antibacterianos/farmacología , Pseudomonas aeruginosa/efectos de los fármacos , Piocianina/metabolismo , Farmacorresistencia Bacteriana , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidad , Piocianina/genética , Percepción de Quorum/efectos de los fármacos , Virulencia/efectos de los fármacos , Factores de VirulenciaRESUMEN
Resistance to colistin, especially mobilized colistin resistance (mcr), is a serious threat to public health since it may catalyze a return of the "pre-antibiotic era". Outer membrane vesicles (OMVs) play a role in antibiotic resistance in various ways. Currently, how OMVs participate in mcr-1-mediated colistin resistance has not been established. In this study, we showed that both OMVs from the mcr-1 negative and positive Escherichia coli (E. coli) strains conferred dose-dependent protection from colistin. However, OMVs from the mcr-1 positive strain conferred attenuated protection when compared to the OMVs of a mcr-1 negative strain at the same concentration. The attenuated protective effect of OMVs was related to the reduced ability to absorb colistin from the environment, thus promoting the killing of colistin sensitive E. coli strains. Lipid A modified with phosphoethanolamine was presented in the OMVs of the mcr-1 positive E. coli strain and resulted in decreased affinity to colistin and less protection. Meanwhile, E. coli strain carrying the mcr-1 gene packed more unmodified lipid A in OMVs and kept more phosphoethanolamine modified lipid A in the bacterial cells. Our study provides a first glimpse of the role of OMVs in mcr-1 -mediated colistin resistance.
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Colistina , Proteínas de Escherichia coli , Antibacterianos/farmacología , Colistina/farmacología , Farmacorresistencia Bacteriana , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Pruebas de Sensibilidad Microbiana , PlásmidosRESUMEN
The increasing incidence of tigecycline resistance undoubtedly constitutes a serious threat to global public health. The combination therapies had become the indispensable strategy against this threat. Herein, 11 clinical tigecycline-resistant Klebsiella pneumoniae which mainly has mutations in ramR, acrR, or macB were collected for tigecycline adjuvant screening. Interestingly, ML-7 hydrochloride (ML-7) dramatically potentiated tigecycline activity. We further picked up five analogs of ML-7 and evaluated their synergistic activities with tigecycline by using checkerboard assay. The results revealed that ML-7 showed certain synergy with tigecycline, while other analogs exerted attenuated synergistic effects among tigecycline-resistant isolates. Thus, ML-7 was selected for further investigation. The results from growth curves showed that ML-7 combined with tigecycline could completely inhibit the growth of bacteria, and the time-kill analysis revealed that the combination exhibited synergistic bactericidal activities for tigecycline-resistant isolates during 24 h. The ethidium bromide (EtBr) efflux assay demonstrated that ML-7 could inhibit the functions of efflux pump. Besides, ML-7 disrupted the proton motive force (PMF) via increasing ΔpH, which in turn lead to the inhibition of the functions of efflux pump, reduction of intracellular ATP levels, as well as accumulation of ROS. All of which promoted the death of bacteria. And further transcriptomic analysis revealed that genes related to the mechanism of ML-7 mainly enriched in ABC transporters. Taken together, these results revealed the potential of ML-7 as a novel tigecycline adjuvant to circumvent tigecycline-resistant Klebsiella pneumoniae.
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Infecciones por Klebsiella , Klebsiella pneumoniae , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Azepinas , Farmacorresistencia Bacteriana , Humanos , Infecciones por Klebsiella/tratamiento farmacológico , Pruebas de Sensibilidad Microbiana , Minociclina/farmacología , Naftalenos , Tigeciclina/farmacologíaRESUMEN
Aims: mcr-1 and blaNDM-1 co-harboring isolates have been reported, usually reside on different plasmids, suggesting co-transfer possibility of the two genes from separate donors to the same recipient strain. This study aims at screening and characterization of mcr-1 carrying Enterobacteriaceae in Northern China, and studying the transfer ability of mcr-1 alone and in company with blaNDM-1 from a second donor. Results: Three Escherichia coli strains and one Klebsiella pneumoniae strain carrying mcr-1 gene were screened out from 1992 isolates in our study. Co-existence of multiple resistance genes was found in the mcr-1-carrying strains, but none of them carried blaNDM-1. One E. coli demonstrated an single nucleotide polymorphism (SNP) (A-G) at -10 region of mcr-1, and one E. coli showed 2 SNPs (G-T and G-A) in the Shine-Dalgarno sequence-like region of mcr-1. The mcr-1 gene was located on plasmids of about 33-276 kb, and capable of transferring alone in three out of four mcr-1-positive isolates by conjugation. Co-transfer ability analysis demonstrated that mcr-1 from E. coli 13-68, which could not be transferred alone to E. coli C600, was successfully transferred in company with blaNDM-1 from K. pneumoniae ATCC BAA-2146. Conclusions: mcr-1 showed low incidence in our Enterobacteriaceae isolates. Co-transfer ability of mcr-1 and blaNDM-1 from separate donors provides direct evidence for the emergence of the mcr-1 and blaNDM-1 co-harboring isolates.
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Farmacorresistencia Bacteriana Múltiple/genética , Enterobacteriaceae/genética , Escherichia coli/genética , Genes Bacterianos/genética , Klebsiella pneumoniae/genética , Humanos , Pruebas de Sensibilidad Microbiana , Polimorfismo de Nucleótido Simple , Reacción en Cadena en Tiempo Real de la Polimerasa , beta-Lactamasas/genéticaRESUMEN
BACKGROUND: Infections caused by drug-resistant Staphylococcus aureus, especially vancomycin-intermediate Staphylococcus aureus (VISA), leave clinicians with limited therapeutic options for treatment. Persister cells is a leading cause of recalcitrant infection and antibiotic treatment failure, and there is no drug in clinical use that specifically targets persister cells currently. Here, we report a promising combination therapy of sodium new houttuyfonate (SNH) and berberine chloride (BBR) which is able to eradicate both growing and persistent drug-resistant Staphylococcus aureus. RESULTS: The susceptibility test showed SNH exhibited anti-MRSA activity with MIC90 at 64 µg/mL, while BBR showed weak anti-MRSA activity with MIC90 at 512 µg/mL. MICs of BBR in combination with 1/2 MIC SNH decreased by 4 to 64 folds compared with MICs of BBR alone. The results of time-killing assays revealed that the combined use of sub-MIC SNH and BBR offered an in vitro synergistic action against growing MRSA (including pathogenic MRSA) and VISA strains. More importantly, the combination of SNH and BBR was able to eradicate VISA Mu50 and pathogenic MRSA persister cells. The synergistic effect is likely related to the interruption of the cell membrane caused by SNH, which is confirmed by scanning electron microscope and membrane potential and permeability analysis. CONCLUSIONS: Our study provide a promising clinical curative strategy for combating drug-resistant S. aureus infections, especially for recalcitrant infections caused by persister cells.
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Antibacterianos/farmacología , Berberina/farmacología , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Ácidos Sulfónicos/farmacología , Staphylococcus aureus Resistente a Vancomicina/efectos de los fármacos , Combinación de Medicamentos , Sinergismo Farmacológico , Humanos , Resistencia a la Meticilina/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Infecciones Estafilocócicas/microbiología , Resistencia a la Vancomicina/efectos de los fármacosRESUMEN
INTRODUCTION: The clinical and molecular characteristics of hypervirulent Klebsiella pneumoniae (hvKp) in various provinces of China have been reported, however, there have been few reports in Hebei Province, North China. METHODOLOGY: The hvKp was identified by PCR amplification of hypervirulence-related genes, the hypermucoviscous phenotype was determined by the "string test", the drug susceptibility analysis was performed using the VITEK® 2 Compact Bacterial Identification and Monitoring System. Logistic regression was used to identify risk factors for hvKp infection. The molecular epidemiological characteristics of the strains were analyzed by pulsed-field gel electrophoresis (PFGE), and the capsular serotype of hvKp strain was detected by PCR. RESULTS: Overall, 52.21% (59/113) of K. pneumoniae isolates were hvKp, and the ratios of patients with older ages or a higher PMN cell count among hvKp infection were higher than those among classical Klebsiella pneumoniae (cKp) infection. hvKp are more susceptible to antibacterial drugs than cKp, and one ESBLs-producing hvKp strain was detected. The main capsular serotype of hvKp were K2, K57 and K1. PFGE indicated that the 59 strains of hvKp could be classified into 51 PFGE band types, forming 6 PFGE clusters. CONCLUSIONS: In this study, the detection rate of hvKp was 52.21% (59/113) identified by virulence genes. People with older ages or a higher PMN cell count are more likely to gain hvKp infection. ESBLs-producing hvKp is emerging, indicating the importance of epidemiologic surveillance and clinical awareness of this pathogen in this region.
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Infecciones por Klebsiella/epidemiología , Infecciones por Klebsiella/microbiología , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/patogenicidad , Factores de Virulencia/genética , Anciano , Antibacterianos/farmacología , Cápsulas Bacterianas/clasificación , China/epidemiología , Farmacorresistencia Bacteriana , Monitoreo Epidemiológico , Femenino , Hospitales/estadística & datos numéricos , Humanos , Infecciones por Klebsiella/inmunología , Klebsiella pneumoniae/clasificación , Klebsiella pneumoniae/efectos de los fármacos , Masculino , Persona de Mediana Edad , Factores de Riesgo , Serogrupo , VirulenciaRESUMEN
OBJECTIVE: To characterise a multidrug-resistant hypervirulent Klebsiella pneumoniae (MDR-hvKp) strain (Kpn1693) isolated from the sputum of a 67-year-old male patient diagnosed as having bronchiectasis with infection in Northern China. METHODS: Drug susceptibility testing was performed by broth microdilution method, and the hypervirulent phenotype of the strain was analysed by string test and a mice systemic infection model. The whole genome of Kpn1693 was sequenced using PacBio Sequel platform, de novo assembly was performed using SMRT Link v5.0.1 software and the GeneMarkS program was used to retrieve the related coding gene. Incompatibility types, virulence-associated genes and antimicrobial resistance genes were searched for using the PlasmidFinder database, virulence factors of pathogenic bacteria database (VFDB) and Comprehensive Antibiotic Research Database (CARD). The phylogenetic relationship of pKpn1693-CTXM with other IncFII plasmids carrying variants of MEGA X software. RESULTS: The Kpn1693 strain showed resistance to multiple antimicrobials and a hypervirulent phenotype. Bioinformatic analysis revealed that Kpn1693 had a 5 331 308-bp-long chromosome (cKpn1693), and harboured two plasmids of 192 347 bp (pK2044-like virulent plasmid, IncHI1B/IncFIB, pKpn1693-Vir) and 70 307 bp (blaCTX-M-24-bearing IncFII plasmid IncFII, pKpn1693-CTXM). CONCLUSION: We reported an MDR-hvKp strain with a blaCTX-M-24-carrying IncFII-type plasmid and a pK2044-like virulent plasmid, and this is the first report of a blaCTX-M-24-bearing IncFII plasmid carried by an MDR-hvKp.
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Resistencia a Múltiples Medicamentos , Infecciones por Klebsiella , Mycobacterium tuberculosis , Animales , China , Humanos , Klebsiella pneumoniae/genética , Masculino , Ratones , Pruebas de Sensibilidad Microbiana , Filogenia , Plásmidos/genética , beta-Lactamasas/genéticaRESUMEN
(E)-N,N-dimethyl-4-oxo-4-(4-(pyridin-4-yl)phenyl)but-2-enamide hydrochloride (IMB-YH-4py5-2H) is a novel Protein Kinase B (PknB) inhibitor with potent activity against Mycobacterium tuberculosis strains. In the present study, a sensitive and specific liquid chromatography/tandem mass spectrometry (LC-MS/MS) method was developed and validated to determine IMB-YH-4py5-2H in rat plasma. Sample pretreatment was achieved by liquid-liquid extraction with ethyl acetate, and separation was performed on an XTerra MS C18 column (2.1×50 mm, 3.5 µm) with gradient elution (methanol and 0.1% formic acid) at a flow rate of 0.3 mL/min. Detection was performed in multiple reaction monitoring (MRM) mode. Linear calibration curves were obtained over a concentration range of 1-100 ng/mL. The intra-day and inter-day precisions were lower than 8.46%, and the accuracies ranged from -8.71% to 12.36% at all quality control levels. The extraction recoveries were approximately 70%, and the matrix effects were negligible. All quality control samples were stable under different storage conditions. The validated method was successfully applied to a preclinical pharmacokinetic study in Sprague-Dawley rats. IMB-YH-4py5-2H demonstrated improved pharmacokinetic properties (higher exposure level) compared with its leading compound. IMB-YH-4py5-2H was also distributed throughout the lung pronouncedly, especially inside alveolar macrophages, indicating its effectiveness against lower respiratory infections.
Asunto(s)
Análisis Químico de la Sangre/métodos , Cromatografía Liquida/métodos , Límite de Detección , Piridinas/sangre , Piridinas/farmacocinética , Espectrometría de Masas en Tándem/métodos , Animales , Antituberculosos/sangre , Antituberculosos/aislamiento & purificación , Antituberculosos/farmacocinética , Piridinas/aislamiento & purificación , Ratas , Ratas Sprague-DawleyRESUMEN
Pharmacological efficacy is based on the drug concentration in target tissues, which usually cannot be represented by the plasma concentration. The purpose of this study was to compare the pharmacokinetic characteristics of gemifloxacin in plasma and skeletal muscle and evaluate its tissue penetration in both healthy and MRSA (methicillin-resistant Staphylococcus aureus)-infected rats. A microdialysis (MD) combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated to determine free gemifloxacin concentrations in rat plasma and skeletal muscle simultaneously. The in vivo recoveries of MD were 23.21% ± 3.42% for skeletal muscle and 20.62% ± 3.19% for plasma, and were concentration independent. We provided evidence that the method developed here meets FDA requirements. Additionally, this method was successfully applied to the determination of free gemifloxacin in rats. Muscle and blood dialysates were collected after an 18 mg/kg intravenous bolus dose. The mean areas under the concentration-time curves (AUCs) from 0 to 9 h for skeletal muscle and plasma were 3641.50 ± 915.65 h*ng/mL and 7068.32 ± 1964.19 h*ng/mL in MRSA-infected rats and 3774.72 ± 700.36 h*ng/mL and 6927.49 ± 1714.86 h*ng/mL in healthy rats, respectively. There was no significant difference (P>0.05) in gemifloxacin exposure between healthy rats and MRSA-infected rats for plasma or muscle. The low ratio of AUC0-9 muscle to AUC0-9 plasma suggested lower drug exposure in skeletal muscle than in plasma for both healthy and MRSA-infected rats. Our study suggested that the administration of gemifloxacin according to drug levels in plasma to treat local infection is unreasonable and might result in an inadequate dose regimen.