RESUMO
Chromosomal and transferable AmpC ß-lactamases represent top resistance mechanisms in different gram-negatives, but knowledge regarding the latter, mostly concerning regulation and virulence-related implications, is far from being complete. To fill this gap, we used Klebsiella pneumoniae (KP) and two different plasmid-encoded AmpCs [DHA-1 (AmpR regulator linked, inducible) and CMY-2 (constitutive)] as models to perform a study in which we show that blockade of peptidoglycan recycling through AmpG permease inactivation abolished DHA-1 inducibility but did not affect CMY-2 production and neither did it alter KP pathogenic behavior. Moreover, whereas regular production of both AmpC-type enzymes did not attenuate KP virulence, when blaDHA-1 was expressed in an ampG-defective mutant, Galleria mellonella killing was significantly (but not drastically) attenuated. Spontaneous DHA-1 hyperproducer mutants were readily obtained in vitro, showing slight or insignificant virulence attenuations together with high-level resistance to ß-lactams only mildly affected by basal production (e.g., ceftazidime, ceftolozane/tazobactam). By analyzing diverse DHA-1-harboring clinical KP strains, we demonstrate that the natural selection of these hyperproducers is not exceptional (>10% of the collection), whereas mutational inactivation of the typical AmpC hyperproduction-related gene mpl was the most frequent underlying mechanism. The potential silent dissemination of this kind of strains, for which an important fitness cost-related contention barrier does not seem to exist, is envisaged as a neglected threat for most ß-lactams effectiveness, including recently introduced combinations. Analyzing whether this phenomenon is applicable to other transferable ß-lactamases and species as well as determining the levels of conferred resistance poses an essential topic to be addressed.IMPORTANCEAlthough there is solid knowledge about the regulation of transferable and especially chromosomal AmpC ß-lactamases in Enterobacterales, there are still gaps to fill, mainly related to regulatory mechanisms and virulence interplays of the former. This work addresses them using Klebsiella pneumoniae as model, delving into a barely explored conception: the acquisition of a plasmid-encoded inducible AmpC-type enzyme whose production can be increased through selection of chromosomal mutations, entailing dramatically increased resistance compared to basal expression but minor associated virulence costs. Accordingly, we demonstrate that clinical K. pneumoniae DHA-1 hyperproducer strains are not exceptional. Through this study, we warn for the first time that this phenomenon may be a neglected new threat for ß-lactams effectiveness (including some recently introduced ones) silently spreading in the clinical context, not only in K. pneumoniae but potentially also in other pathogens. These facts must be carefully considered in order to design future resistance-preventive strategies.
Assuntos
Antibacterianos , Proteínas de Bactérias , Klebsiella pneumoniae , Proteínas de Membrana Transportadoras , Testes de Sensibilidade Microbiana , Peptidoglicano , Plasmídeos , beta-Lactamases , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/patogenicidade , Klebsiella pneumoniae/metabolismo , Peptidoglicano/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Virulência , beta-Lactamases/genética , beta-Lactamases/metabolismo , Antibacterianos/farmacologia , Plasmídeos/genética , Animais , Infecções por Klebsiella/microbiologia , Mariposas/microbiologiaRESUMO
Control of infections caused by carbapenem-resistant Klebsiella pneumoniae continues to be challenging. The success of this pathogen is favored by its ability to acquire antimicrobial resistance and to spread and persist in both the environment and in humans. The emergence of clinically important clones, such as sequence types 11, 15, 101, and 258, has been reported worldwide. However, the mechanisms promoting the dissemination of such high-risk clones are unknown. Unraveling the factors that play a role in the pathobiology and epidemicity of K. pneumoniae is therefore important for managing infections. To address this issue, we studied a carbapenem-resistant ST-15 K. pneumoniae isolate (Kp3380) that displayed a remarkable adherent phenotype with abundant pilus-like structures. Genome sequencing enabled us to identify a chaperone-usher pili system (Kpi) in Kp3380. Analysis of a large K. pneumoniae population from 32 European countries showed that the Kpi system is associated with the ST-15 clone. Phylogenetic analysis of the operon revealed that Kpi belongs to the little-characterized γ2-fimbrial clade. We demonstrate that Kpi contributes positively to the ability of K. pneumoniae to form biofilms and adhere to different host tissues. Moreover, the in vivo intestinal colonizing capacity of the Kpi-defective mutant was significantly reduced, as was its ability to infect Galleria mellonella The findings provide information about the pathobiology and epidemicity of Kpi+K. pneumoniae and indicate that the presence of Kpi may explain the success of the ST-15 clone. Disrupting bacterial adherence to the intestinal surface could potentially target gastrointestinal colonization.
Assuntos
Fímbrias Bacterianas/genética , Klebsiella pneumoniae/genética , Chaperonas Moleculares/genética , Células A549 , Animais , Antibacterianos , Aderência Bacteriana/efeitos dos fármacos , Aderência Bacteriana/genética , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Carbapenêmicos/farmacologia , Linhagem Celular , Modelos Animais de Doenças , Farmacorresistência Bacteriana Múltipla/genética , Células Epiteliais/microbiologia , Europa (Continente) , Feminino , Deleção de Genes , Genes Bacterianos/genética , Humanos , Infecções por Klebsiella , Klebsiella pneumoniae/citologia , Klebsiella pneumoniae/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Tipagem de Sequências Multilocus , Óperon , FilogeniaRESUMO
Pseudomonas aeruginosa is one of the leading causes of nosocomial pneumonia and its associated mortality. Moreover, extensively drug-resistant high-risk clones are globally widespread, presenting a major challenge to the healthcare systems. Despite this, no vaccine is available against this high-concerning pathogen. Here we tested immunogenicity and protective efficacy of an experimental live vaccine against P. aeruginosa pneumonia, consisting of an auxotrophic strain which lacks the key enzyme involved in D-glutamate biosynthesis, a structural component of the bacterial cell wall. As the amounts of free D-glutamate in vivo are trace substances in most cases, blockage of the cell wall synthesis occurs, compromising the growth of this strain, but not its immunogenic properties. Indeed, when delivered intranasally, this vaccine stimulated production of systemic and mucosal antibodies, induced effector memory, central memory and IL-17A-producing CD4+ T cells, and recruited neutrophils and mononuclear phagocytes into the airway mucosa. A significant improvement in mice survival after lung infection caused by ExoU-producing PAO1 and PA14 strains was observed. Nearly one third of the mice infected with the XDR high-risk clone ST235 were also protected. These findings highlight the potential of this vaccine for the control of acute pneumonia caused by this bacterial pathogen.
Assuntos
Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa/imunologia , Vacinas Atenuadas/imunologia , Administração Intranasal , Animais , Anticorpos Antibacterianos/imunologia , Feminino , Imunidade nas Mucosas , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pneumonia Bacteriana/imunologia , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/patogenicidade , Sistema Respiratório/imunologia , Infecções Respiratórias/imunologia , Vacinas Atenuadas/farmacologiaRESUMO
OBJECTIVES: To characterize the antimicrobial susceptibility, molecular epidemiology and carbapenem resistance mechanisms in Pseudomonas aeruginosa isolates recovered from respiratory tract samples from patients with ventilator-associated pneumonia enrolled in the MagicBullet clinical trial. METHODS: Isolates were collected from 53 patients from 12 hospitals in Spain, Italy and Greece. Susceptibility was determined using broth microdilution and Etest. MALDI-TOF MS was used to detect carbapenemase activity and carbapenemases were identified by PCR and sequencing. Molecular epidemiology was investigated using PFGE and MLST. RESULTS: Of the 53 isolates, 2 (3.8%) were considered pandrug resistant (PDR), 19 (35.8%) were XDR and 16 (30.2%) were MDR. Most (88.9%) of the isolates from Greece were MDR, XDR or PDR, whereas fewer of the isolates from Spain (33.3%) and Italy (43.5%) showed antibiotic resistance. Three Greek isolates were resistant to colistin. Overall, the rates of resistance of P. aeruginosa isolates to imipenem, ciprofloxacin, ceftolozane/tazobactam and ceftazidime/avibactam were 64.1%, 54.7%, 22.6% and 24.5%, respectively. All isolates resistant to ceftolozane/tazobactam and ceftazidime/avibactam (Greece, n = 10; and Italy, n = 2) carried blaVIM-2. Spanish isolates were susceptible to the new drug combinations. Forty-eight restriction patterns and 27 STs were documented. Sixty percent of isolates belonged to six STs, including the high-risk clones ST-111, ST-175 and ST-235. CONCLUSIONS: MDR/XDR isolates were highly prevalent, particularly in Greece. The most effective antibiotic against P. aeruginosa was colistin, followed by ceftolozane/tazobactam and ceftazidime/avibactam. blaVIM-2 is associated with resistance to ceftolozane/tazobactam and ceftazidime/avibactam, and related to highly resistant phenotypes. ST-111 was the most frequent and disseminated clone and the clonal diversity was lower in XDR and PDR strains.
Assuntos
Farmacorresistência Bacteriana Múltipla , Pneumonia Associada à Ventilação Mecânica/epidemiologia , Pneumonia Associada à Ventilação Mecânica/etiologia , Infecções por Pseudomonas/epidemiologia , Infecções por Pseudomonas/etiologia , Pseudomonas aeruginosa/efeitos dos fármacos , Proteínas de Bactérias/genética , Grécia/epidemiologia , Humanos , Incidência , Concentração Inibidora 50 , Itália/epidemiologia , Testes de Sensibilidade Microbiana , Epidemiologia Molecular , Tipagem de Sequências Multilocus , Filogenia , Pneumonia Associada à Ventilação Mecânica/tratamento farmacológico , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/classificação , Pseudomonas aeruginosa/genética , Espanha/epidemiologia , Resistência beta-Lactâmica , beta-Lactamases/genéticaRESUMO
The FIKK family of kinases is unique to parasites of the Apicomplexan order, which includes all malaria parasites. Plasmodium falciparum, the most virulent form of human malaria, has a family of 19 FIKK kinases, most of which are exported into the host red blood cell during malaria infection. Here, we confirm that FIKK 8 is a non-exported member of the FIKK kinase family. Through expression and purification of the recombinant kinase domain, we establish that emodin is a relatively high-affinity (IC50=2µM) inhibitor of PfFk8. Closely related anthraquinones do not inhibit PfFk8, suggesting that the particular substitution pattern of emodin is critical to the inhibitory pharmacophore. This first report of a P. falciparum FIKK kinase inhibitor lays the groundwork for developing specific inhibitors of the various members of the FIKK kinase family in order to probe their physiological function.
Assuntos
Emodina/química , Emodina/farmacologia , Plasmodium falciparum/enzimologia , Proteínas Quinases/química , Proteínas Quinases/farmacologia , Proteínas de Protozoários/antagonistas & inibidores , Sequência de Aminoácidos , Antraquinonas/química , Emodina/metabolismo , Ativação Enzimática/efeitos dos fármacos , Eritrócitos/metabolismo , Eritrócitos/parasitologia , Humanos , Concentração Inibidora 50 , Microscopia de Fluorescência , Plasmodium falciparum/efeitos dos fármacos , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/genética , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismoRESUMO
OBJECTIVES: Two natural variants of ADC-type ß-lactamases of Acinetobacter spp., ADC-1 and ADC-5, differ by nine mutations in their protein sequence. ADC-5 hydrolyses cefoxitin better than ADC-1 and the opposite is true for ceftazidime. We produced single and combined mutations in ADC-5 and characterized the variants microbiologically and biochemically to determine which amino acid residues are involved in the hydrolysis of ß-lactam antibiotics in this family of ß-lactamases. METHODS: Site-directed mutagenesis, with blaADC-5 as a source of DNA, was used to generate nine single mutated and three combined mutated enzymes. The proteins (wild-type and derivatives) were then expressed in isogenic conditions in Escherichia coli. MICs of ß-lactams were determined using Etest strips. ADC-1, ADC-5, ADC-5-P167S and ADC-5-P167S/D242G/Q163K/G342R were also purified and the kinetic parameters determined for ceftazidime, cefoxitin, cefalotin and ampicillin. RESULTS: Single mutations did not significantly convert the hydrolysis spectrum of the ADC-5 enzyme into that of the ADC-1 enzyme, although among all studied mutants only the quadruple mutant (ADC-5-P167S/D242G/Q163K/G342R) displayed microbiological and biochemical properties consistent with those of ADC-1. CONCLUSIONS: Although some single mutations are known to affect cefepime hydrolysis in ADC-type ß-lactamases, little is known about ceftazidime and cefoxitin hydrolysis in this family of ß-lactamases. Hydrolysis of these antibiotics appears to be positively and negatively affected, respectively, by the Q163K, P167S, D242G and G342R amino acid replacements.
Assuntos
Acinetobacter/enzimologia , Antibacterianos/metabolismo , Cefoxitina/metabolismo , Ceftazidima/metabolismo , Mutação , beta-Lactamases/genética , beta-Lactamases/metabolismo , Acinetobacter/efeitos dos fármacos , Análise Mutacional de DNA , Escherichia coli/genética , Expressão Gênica , Hidrólise , Cinética , Testes de Sensibilidade Microbiana , Mutagênese Sítio-Dirigida , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
Background: Systemic lupus erythematosus (SLE) is a chronic autoimmune disease that can affect multiple organs and body systems. Objective: To describe the sociodemographic, clinical, and biochemical characteristics of the Lupus-IMMS-Mexico (LUPUS-IMMex) patient cohort from a tertiary-level center. Material and methods: Observational descriptive study of 160 patients with diagnosis of SLE belonging to the aforementioned cohort. Various variables were analyzed at the time of diagnosis. For quantitative variables, normality tests were applied, followed by measures of central tendency and dispersion according to their distribution. For categorical variables, frequencies and percentages were calculated. Results: 81.87% of the patients were female, with a median age at diagnosis of 28 years. 18.12% had a family history of SLE, and concurrently with SLE, 32.50% had hypertension, and 11.25% had antiphospholipid syndrome. The most common clinical manifestation was joint involvement (68.12%), renal (49.37%) and hematological (43.75%) manifestations. Conclusions: SLE affects millions globally. Lack of awareness leads to delayed diagnoses, suboptimal management, and diminished quality of life. After analyzing 160 patients with SLE, their clinical, socioeconomic, and therapeutic characteristics are largely like other cohorts, with differences attributable to ethnic and geographical influences. Informing patients about SLE and providing reliable resources are essential for self-care. Awareness promotes research, therapies, and enhances medical care and the lives of patients globally.
Introducción: el lupus eritematoso sistémico (LES) es una enfermedad autoinmunitaria crónica que puede afectar a múltiples órganos y sistemas del cuerpo. Objetivo: describir las características sociodemográficas, clínicas y bioquímicas de la cohorte de pacientes Lupus-IMMS-México (LUPUS-IMMex) de un hospital de tercer nivel. Material y métodos: estudio descriptivo observacional de 160 pacientes con diagnóstico de LES de la cohorte mencionada. Se analizaron diversas variables al momento del diagnóstico. Para variables cuantitativas se aplicaron pruebas de normalidad y posteriormente medidas de tendencia central y dispersión de acuerdo con su distribución. Para variables categóricas se calcularon frecuencias y porcentajes. Resultados: 81.87% de los pacientes fueron del sexo femenino, con mediana de edad al diagnóstico de 28 años. El 18.12% tenían antecedentes familiares de LES y concomitante al LES, hipertensión (32.50%) y síndromes antifosfolípidos (11.25%). Las afecciones clínicas más frecuentes fueron la articular (68.12%), la renal (49.37%) y la hematológica (43.75%). Conclusiones: el LES afecta a millones de personas globalmente. La falta de conciencia lleva a diagnósticos tardíos, manejo deficiente y baja calidad de vida. Tras analizar 160 pacientes con LES, sus características clínicas, socioeconómicas y terapéuticas son mayormente similares a otras cohortes, con diferencias atribuibles a influencias étnicas y geográficas. Informar a los pacientes sobre el LES y brindar recursos confiables es esencial para el autocuidado. La sensibilización fomenta la investigación, las terapias y mejora la atención médica y la vida de pacientes a nivel global.
Assuntos
Lúpus Eritematoso Sistêmico , Humanos , Lúpus Eritematoso Sistêmico/diagnóstico , Lúpus Eritematoso Sistêmico/epidemiologia , Lúpus Eritematoso Sistêmico/sangue , Feminino , Masculino , Adulto , México/epidemiologia , Pessoa de Meia-Idade , Adulto Jovem , Estudos de Coortes , AdolescenteRESUMO
The need for alternative drugs to treat methicillin-resistant Staphylococcus aureus (MRSA) bacteraemia has led to a focus on ceftaroline, for which clinical data remain scarce. Herein, the efficacy of ceftaroline fosamil for the treatment of experimental MRSA bacteraemia was compared with that of approved therapies. Five MRSA strains were tested in an immunocompetent BALB/c bacteraemia model. Serum pharmacokinetics of ceftaroline fosamil were determined using HPLC/MS Q-TOF. Two hours after infection with the MRSA strains, mice were administered 50 mg/kg of ceftaroline fosamil every 6 h, for 24 h. This regimen yielded a T>MIC of 61.5% for an MIC of 1 mg/L and proved efficacious against all strains, including an hVISA strain with non-susceptibility to daptomycin, as indicated by the reduction (mean ± s.d.) in log10 CFU/mL in blood of 2.34 ± 0.33 and log10 CFU/g in kidney of 2.08 ± 0.22. Similarly, treatment with daptomycin yielded a log reduction of 2.30 ± 0.60 in blood and 2.14 ± 0.31 in kidney. The decrease in bacterial density was less accentuated after treatment with vancomycin, which yielded 1.84 ± 0.73 and 1.95 ± 0.32 log reductions in blood and kidney, respectively. The results of the study showed that the efficacy of ceftaroline fosamil against MRSA bacteraemia in mice is not inferior to that of vancomycin and daptomycin, and indicated the potential use of ceftaroline fosamil against difficult-to-treat S. aureus bacteraemia. Considering these promising data, clinical trials should be conducted to ascertain the efficacy of the drug for treating bloodstream infections in humans.
Assuntos
Bacteriemia , Daptomicina , Staphylococcus aureus Resistente à Meticilina , Infecções Estafilocócicas , Humanos , Animais , Camundongos , Vancomicina/uso terapêutico , Vancomicina/farmacocinética , Daptomicina/uso terapêutico , Daptomicina/farmacocinética , Antibacterianos/uso terapêutico , Antibacterianos/farmacocinética , Bacteriemia/tratamento farmacológico , Bacteriemia/microbiologia , Modelos Animais de Doenças , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus , Cefalosporinas/uso terapêutico , Cefalosporinas/farmacocinética , Testes de Sensibilidade Microbiana , CeftarolinaRESUMO
Multidrug efflux pumps have emerged as important mechanisms of antimicrobial resistance in bacterial pathogens. In order to cause infection, pathogenic bacteria require mechanisms to avoid the effects of host-produced compounds, and express efflux pumps may accomplish this task. In this study, we evaluated the effect of the inactivation of AcrAB-TolC on antimicrobial resistance, fitness, and virulence in Enterobacter cloacae, an opportunistic pathogen usually involved in nosocomial infections. Two different clinical isolates of E. cloacae were used, EcDC64 (multidrug resistance overexpressing the AcrAB-TolC efflux pump) and Jc194 (basal AcrAB-TolC expression). The acrA and tolC genes were deleted in strains EcDC64 and Jc194 to produce, respectively, EcΔacrA and EcΔtolC and JcΔacrA and JcΔtolC knockout (KO) derivatives. Antibiotic susceptibility testing was performed with all isolates, and we discovered that these mechanisms are involved in the resistance of E. cloacae to several antibiotics. Competition experiments were also performed with wild-type and isogenic KO strains. The competition index (CI), defined as the mutant/wild-type ratio, revealed that the acrA and tolC genes both affect the fitness of E. cloacae, as fitness was clearly reduced in the acrA and tolC KO strains. The median CI values obtained in vitro and in vivo were, respectively, 0.42 and 0.3 for EcDC64/EcΔacrA, 0.24 and 0.38 for EcDC64/EcΔtolC, 0.15 and 0.11 for Jc194/JcΔacrA, and 0.38 and 0.39 for Jc194/JcΔtolC. Use of an intraperitoneal mouse model of systemic infection revealed reduced virulence in both E. cloacae clinical strains when either the acrA or tolC gene was inactivated. In conclusion, the structural components of the AcrAB-TolC efflux pump appear to play a role in antibiotic resistance as well as environmental adaptation and host virulence in clinical isolates of E. cloacae.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Farmacorresistência Bacteriana/fisiologia , Enterobacter cloacae/efeitos dos fármacos , Enterobacter cloacae/metabolismo , Animais , Clonagem Molecular , Enterobacter cloacae/patogenicidade , Infecções por Enterobacteriaceae/microbiologia , Aptidão Genética , Camundongos , Camundongos Endogâmicos C57BL , Testes de Sensibilidade Microbiana , Plasmídeos/genética , Reação em Cadeia da Polimerase , Virulência/genética , Virulência/fisiologiaRESUMO
ß-Lactamases and penicillin-binding proteins (PBPs) have evolved from a common ancestor. ß-Lactamases are enzymes that degrade ß-lactam antibiotics, whereas PBPs are involved in the synthesis and processing of peptidoglycan, which forms an elastic network in the bacterial cell wall. This study analyzed the interaction between ß-lactamases and peptidoglycan and the impact on fitness and biofilm production. A representative set of all classes of ß-lactamases was cloned in the expression vector pBGS18 under the control of the CTX-M promoter and expressed in Escherichia coli MG1655. The peptidoglycan composition of all clones was evaluated, and quantitative changes were found in E. coli strains expressing OXA-24, OXA-10-like, and SFO-1 (with its upstream regulator AmpR) ß-lactamases; the level of cross-linked muropeptides decreased, and their average length increased. These changes were associated with a statistically significant fitness cost, which was demonstrated in both in vitro and in vivo experiments. The observed changes in peptidoglycan may be explained by the presence of residual DD-endopeptidase activity in these ß-lactamases, which may result in hydrolysis of the peptide cross bridge. The biological cost associated with these changes provides important data regarding the interaction between ß-lactamases and the metabolism of peptidoglycan and may provide an explanation for the epidemiology of these ß-lactamases in Enterobacteriaceae.
Assuntos
Escherichia coli/efeitos dos fármacos , Escherichia coli/patogenicidade , Peptidoglicano/química , beta-Lactamases/biossíntese , beta-Lactamases/genética , Animais , Biofilmes , Clonagem Molecular , Farmacorresistência Bacteriana/genética , Escherichia coli/genética , Infecções por Escherichia coli/microbiologia , Feminino , Camundongos , Microscopia Eletrônica de Transmissão , Peptidoglicano/metabolismo , Fenótipo , Plasmídeos/genéticaRESUMO
Control of membrane permeability is a key step in regulating the intracellular concentration of antibiotics. Efflux pumps confer innate resistance to a wide range of toxic compounds such as antibiotics, dyes, detergents, and disinfectants in members of the Enterobacteriaceae. The AcrAB-TolC efflux pump is involved in multidrug resistance in Enterobacter cloacae. However, the underlying mechanism that regulates the system in this microorganism remains unknown. In Escherichia coli, the transcription of acrAB is upregulated under global stress conditions by proteins such as MarA, SoxS, and Rob. In the present study, two clinical isolates of E. cloacae, EcDC64 (a multidrug-resistant strain overexpressing the AcrAB-TolC efflux pump) and Jc194 (a strain with a basal AcrAB-TolC expression level), were used to determine whether similar global stress responses operate in E. cloacae and also to establish the molecular mechanisms underlying this response. A decrease in susceptibility to erythromycin, tetracycline, telithromycin, ciprofloxacin, and chloramphenicol was observed in clinical isolate Jc194 and, to a lesser extent in EcDC64, in the presence of salicylate, decanoate, tetracycline, and paraquat. Increased expression of the acrAB promoter in the presence of the above-described conditions was observed by flow cytometry and reverse transcription-PCR, by using a reporter fusion protein (green fluorescent protein). The expression level of the AcrAB promoter decreased in E. cloacae EcDC64 derivates deficient in SoxS, RobA, and RamA. Accordingly, the expression level of the AcrAB promoter was higher in E. cloacae Jc194 strains overproducing SoxS, RobA, and RamA. Overall, the data showed that SoxS, RobA, and RamA regulators were associated with the upregulation of acrAB, thus conferring antimicrobial resistance as well as a stress response in E. cloacae. In summary, the regulatory proteins SoxS, RobA, and RamA were cloned and sequenced for the first time in this species. The involvement of these proteins in conferring antimicrobial resistance through upregulation of acrAB was demonstrated in E. cloacae.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Enterobacter cloacae/genética , Enterobacter cloacae/metabolismo , Transativadores/genética , Transativadores/fisiologia , Antibacterianos/farmacologia , Proteínas de Bactérias/biossíntese , Clonagem Molecular , Meios de Cultura , DNA Bacteriano/química , DNA Bacteriano/genética , Resistência Microbiana a Medicamentos/efeitos dos fármacos , Ensaio de Desvio de Mobilidade Eletroforética , Enterobacter cloacae/efeitos dos fármacos , Citometria de Fluxo , Genes Reporter , Teste de Complementação Genética , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Plasmídeos , Reação em Cadeia da PolimeraseRESUMO
We report a facile, economic, and ecofriendly method for selective recovery of Er, Gd, Sn, and In from liquid crystal display (LCD) screen wastes by ultrasound-assisted leaching, followed by magnetic separation. Thermodynamic analysis showed that the pyrophosphate ion is an excellent leaching agent for Er, Gd, and In at pH values below 8. Dissolved screen waste powder was subjected to leaching at room temperature using aqueous solutions of 0.05 M of sodium pyrophosphate (Na4P2O7) as the leaching agent; hydrogen peroxide (H2O2) (3 v/v %) was added as an auxiliary reducing agent, and an ultrasonic source was used in the process. Once completed, magnetic separation was applied to the leached residue. The average contents of Er, In, Sn, and Gd in the LCD screen were found to be 477, 2422, 835, and 93 mg·kg-1, respectively, of which 93, 97, 72, and 99% were selectively recovered from the waste material by this method at pH 3 after 2 h of leaching. The proposed method emerges as an easy and selective process for leaching Er from LCD screen wastes and concentrating In, Sn, and Gd in a separable magnetic solid.
RESUMO
The antimicrobial and immunomodulatory capacities of the peptide Css54 and the chemokine MCP-1 were tested. The first, a peptide isolated from the venom of the scorpion Centruroides suffusus suffusus was synthesized chemically. In contrast, the second is a monocyte chemoattractant expressed as a recombinant protein in our lab. It was observed in vitro that Css54 inhibited the growth of Salmonella enterica serovar Typhimurium (6.2 µg/mL). At high concentrations, it was toxic to macrophages (25 µg/mL), activated macrophage phagocytosis (1.5 µg/mL), and bound Salmonella LPS (3 µg/mL). On the other hand, the recombinant MCP-1 neither inhibited the growth of Salmonella Typhimurium nor was it toxic to macrophages (up to 25 µg/mL), nor activated macrophage phagocytosis or bound Salmonella LPS (up to 3 µg/mL). Although it was observed in vivo in mice Balb/C that both Css54 and MCP-1 did not resolve the intraperitoneal infection by S. Typhimurium, Css54 decreased the expression of IL-6 and increased IL-10, IL-12p70, and TNF-α levels; meanwhile, MCP-1 decreased the expression of IFN-γ and increased IL-12p70 and TNF-α. It was also observed that the combination of both molecules Css54 and MCP-1 increased the expression of IL-10 and TNF-α.
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The emergence of 16S rRNA methyltransferases (RMTs) in Gram-negative pathogens bearing other clinically relevant resistance mechanisms, such as carbapenemase-producing Enterobacterales (CPE), is becoming an alarming concern. We investigated the prevalence, antimicrobial susceptibility, resistance mechanisms, molecular epidemiology and genetic support of RMTs in CPE isolates from Spain. This study included a collection of 468 CPE isolates recovered during 2018 from 32 participating Spanish hospitals. MICs were determined using the broth microdilution method, the agar dilution method (fosfomycin) or MIC gradient strips (plazomicin). All isolates were subjected to hybrid whole-genome sequencing (WGS). Sequence types (STs), core genome phylogenetic relatedness, horizontally acquired resistance mechanisms, plasmid analysis and the genetic environment of RMTs were determined in silico from WGS data in all RMT-positive isolates. Among the 468 CPE isolates evaluated, 24 isolates (5.1%) recovered from nine different hospitals spanning five Spanish regions showed resistance to all aminoglycosides and were positive for an RMT (21 RmtF, 2 ArmA and 1 RmtC). All RMT-producers showed high-level resistance to all aminoglycosides, including plazomicin, and in most cases exhibited an extensively drug-resistant susceptibility profile. The RMT-positive isolates showed low genetic diversity and were global clones of Klebsiella pneumoniae (ST147, ST101, ST395) and Enterobacter cloacae (ST93) bearing blaOXA-48, blaNDM-1 or blaVIM-1 carbapenemase genes. RMTs were harboured in five different multidrug resistance plasmids and linked to efficient mobile genetic elements. Our findings highlight that RMTs are emerging among clinical CPE isolates from Spain and their spread should be monitored to preserve the future clinical utility of aminoglycosides and plazomicin.
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Farmacorresistência Bacteriana/genética , Enterobacteriaceae/genética , Enterobacteriaceae/metabolismo , Metiltransferases/genética , Metiltransferases/metabolismo , RNA Ribossômico 16S/genética , beta-Lactamases/genética , beta-Lactamases/metabolismo , Variação Genética , Estudo de Associação Genômica Ampla , Genótipo , Humanos , EspanhaRESUMO
INTRODUCTION: The COVID-19 pandemic led to the cancellation of non-essential surgical procedures in March 2020. With the resumption of surgical activity, patients undergoing surgery were one of the first population groups to be systematically tested for PCR. The aim of this study was to determine the prevalence of asymptomatic SARS-CoV-2 carriers after the resumption of non-essential surgical activity. METHODS: Retrospective multicenter observational study of patients scheduled for surgery or undergoing emergency surgery in Catalonia between 20 April and 31 May 2020. The microbiological results of preoperative PCR tests and clinical records were reviewed, and an epidemiological survey was conducted on patients with positive PCR for SARS-CoV-2. RESULTS: A total of 10,838 patients scheduled for surgery or who underwent emergency surgery were screened for COVID-19. One hundred and eighteen patients (1.09%) were positive for SARS-CoV-2 in the 72 h prior to surgery. The prevalence of asymptomatic carriers was 0.7% (IC95%: 0.6%-0.9%). The first week of the study presented the highest prevalence of asymptomatic carriers [1.9% (CI95%:1.1%-3.2%)]. CONCLUSIONS: The low levels of asymptomatic carriers of COVID-19 infection obtained in the surgical population of hospitals in Catalonia after the resumption of surgical activity, shows that most patients were able to undergo surgical procedures without the risks of COVID-19 associated complications in the perioperative period.
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COVID-19 , COVID-19/epidemiologia , Hospitais , Humanos , Pandemias , Prevalência , SARS-CoV-2 , Espanha/epidemiologiaRESUMO
An extraction method was designed and scaled up to produce multicomponent polyphenolic extracts from blueberries (Vaccinium corymbosum) of three different varieties. The process was specifically drawn up to comply with green chemistry principles. Extracts were obtained for the direct assessment of their antimicrobial and antiadhesive activities, and their direct use in the control of infections caused by concerning multidrug-resistant nosocomial pathogens. Analytical characterization was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Similar qualitative profiles were obtained in the three studied varieties with some significant quantitative differences. Up to 22 different polyphenols were identified with a clear predominance of anthocyani(di)ns followed by flavanols, non-flavonoids, and far behind by flavan-3-ols and procyanidins. The individual content of the main polyphenols was also discussed. A pilot scale extract has been also produced as a proof-of-concept, showing that scaling-up triples the content of bioactive phytochemicals. The effect of the polyphenolic extracts was analyzed against seven multidrug-resistance bacterial species by performing biofilm formation and growth and killing curves assays. All the studied varieties showed antibacterial and antiadhesive activities, being the extract containing the highest concentration of bioactive polyphenols, the most active with a high bactericidal effect.
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BACKGROUND: Infections increase morbidity and mortality in patients with autoimmune disorders; however, this association has not been established in rheumatic diseases and SARS-CoV-2 infection. OBJECTIVE: To describe the clinical characteristics and mortality of patients with rheumatic diseases and severe COVID-19. MATERIAL AND METHODS: Observational and descriptive case series in patients with rheumatic diseases and severe SARS-CoV-2 infection, confirmed by PCR or pulmonary tomography, hospitalized in Mexico City from March to August 2020. RESULTS: 15 patients with a mean age of 57 years (SD ± 11) were included, 66.6% were women, and 80% had a positive PCR test. The time from onset of symptoms to hospitalization, on average, was 7.2 days (SD ± 2.1). 46.6% died. Patients who died had a lower mean platelet level compared to survivors. The inflammatory reactants were higher in the deceased. There were no statistically significant differences in mortality for the variables related to rheumatic disease. CONCLUSIONS: The differences in mortality of patients with severe COVID-19 in this series of cases seem to be related to the infection and not to the rheumatic disease.
INTRODUCCIÓN: las infecciones incrementan la morbimortalidad de los pacientes con trastornos autoinmunes; sin embargo, esta asociación no ha sido establecida en enfermedades reumáticas e infección por SARS-CoV-2. OBJETIVO: describir las características clínicas y la mortalidad de pacientes con enfermedades reumáticas y COVID-19 grave. MATERIAL Y MÉTODOS: serie de casos observacional y descriptiva, en pacientes con enfermedades reumáticas e infección grave por SARS-CoV-2, confirmada por PCR o tomografía pulmonar, hospitalizados en la Ciudad de México de marzo a agosto de 2020. RESULTADOS: se incluyeron 15 pacientes con edad media de 57 años (DE ± 11), el 66.6% eran mujeres, el 80% tuvieron prueba de PCR positiva. El tiempo de inicio de síntomas hasta la hospitalización, en promedio, fue de 7.2 días (DE ± 2.1). Falleció el 46.6%. Los pacientes que murieron tenían un nivel medio de plaquetas más bajo en comparación con los sobrevivientes. Los reactantes inflamatorios fueron más altos en los fallecidos. No hubo diferencias estadísticamente significativas en cuanto a la mortalidad para las variables relacionadas a la enfermedad reumática. CONCLUSIONES: las diferencias en mortalidad de pacientes con COVID-19 grave en esta serie de casos parecen estar relacionadas con la infección y no con la enfermedad reumática.
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COVID-19 , Doenças Reumáticas , Feminino , Hospitalização , Humanos , México/epidemiologia , Pessoa de Meia-Idade , Doenças Reumáticas/complicações , Doenças Reumáticas/diagnóstico , SARS-CoV-2RESUMO
Corals are vulnerable to increasing ocean temperatures. It is known that elevated temperatures lead to the breakdown of an essential mutualistic relationship with photosynthetic algae. The molecular mechanisms of this temperature-dependent loss of symbiosis are less well understood. Here, the thermal stability of a critical metabolic enzyme, glyceraldehyde-3-phosphate dehydrogenase, from the stony coral Acropora millepora was found to increase significantly in the presence of its cofactor NAD+. Determination of the structure of the cofactor-enzyme complex (PDB ID 6PX2) revealed variable NAD+ occupancy across the four monomers of the tetrameric enzyme. The structure of the fully occupied monomers was compared to those with partial cofactor occupancy, identifying regions of difference that may account for the increased thermal stability.
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Pseudomonas aeruginosa, a major cause of nosocomial infections, is considered a paradigm of antimicrobial resistance, largely due to hyperproduction of chromosomal cephalosporinase AmpC. Here, we explore the ability of 6-pyridylmethylidene penicillin-based sulfones 1-3 to inactivate the AmpC ß-lactamase and thus rescue the activity of the antipseudomonal ceftazidime. These compounds increased the susceptibility to ceftazidime in a collection of clinical isolates and PAO1 mutant strains with different ampC expression levels and also improved the inhibition kinetics relative to avibactam, displaying a slow deacylation rate and involving the formation of an indolizine adduct. Bromide 2 was the inhibitor with the lowest KI (15.6 nM) and the highest inhibitory efficiency (kinact/KI). Computational studies using diverse AmpC enzymes revealed that the aromatic moiety in 1-3 targets a tunnel-like site adjacent to the catalytic serine and induces the folding of the H10 helix, indicating the potential value of this not-always-evident pocket in drug design.