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1.
Zhonghua Zhong Liu Za Zhi ; 42(3): 216-221, 2020 Mar 23.
Artigo em Chinês | MEDLINE | ID: mdl-32252200

RESUMO

Objective: To investigate the effect of compound matrine injection on morphine tolerance in mice with lung cancer in situ and the expressions of multidrug resistance gene 1 (MDR1) and P-glycoprotein (P-gp). Methods: A mouse model of lung cancer in situ and morphine tolerance mode was established. The mice were injected with gradient concentration of compound matrine. The pain thresholds under different conditions were measured by thermal radiation tail-flick method. The mRNA level of MDR1 was tested by reverse transcription polymerase chain reaction (RT-PCR) and the protein level of P-gp was detected by western blot. The DNA binding activity of cyclophosphoadenosine response element binding protein (CREB) to the promoter of MDR1 gene was detected by electrophoretic mobility shift assay (EMSA). Results: The maximum analgesic percentage (MPE) of the mice in the morphine group was (85.21±6.53)% on the 8th day, and decreased to (38.45±5.52)% and (28.14±4.52)% on the 10th and 12th day, respectively, which indicated the morphine tolerance of mice with lung cancer in situ.The MPE of the mice in the group treated with morphine and compound matrine injection (300 mg/kg) was (79.34±6.50)% on the 8th day, and decreased to (62.16±5.53)% and (40.20±4.50)% on the 10th and 12th day, respectively.The results of RT-PCR assay showed that the relative expression levels of MDR1 mRNA in the brain tissues of mice in the morphine group, saline group, morphine combined with compound matrine injection (300 mg/kg) group and compound matrine injection (200 mg/kg) group were 2.33±0.79, 1.04±0.38, 1.37±0.38, and 1.43±0.53, respectively. There were statistically significant differences between the morphine group and the normal saline group, the morphine group and the morphine combined with compound matrine injection (300 mg/kg) group (P<0.05). There was no significant difference between the normal saline group and the compound matrine injection (200 mg/kg) group (P=0.05). The results of western blot showed that the relative expression levels of P-gp protein in the brain tissue of mice in the morphine group, saline group, and morphine combined with compound matrine injection (300 mg/kg) group were 1.86±0.40, 1.00±0.23, and 1.27±0.27, respectively. The expression of P-gp protein in the morphine group was significantly higher than those of the normal saline group and the morphine combined with compound matrine injection (300 mg/kg) group (P<0.05). The DNA-binding activity of CREB in the saline group was (0.23±0.07) Pu, significantly lower than (0.89±0.23) Pu of morphine combined with naloxone group and (0.80±0.23) Pu of morphine group (P<0.05). While the CREB DNA binding activity of morphine combined with compound matrine injection (300 mg/kg) group was (0.79±0.21) Pu, implicated that compound matrine had marginal effect on the DNA-binding activity of CREB (P>0.05). Conclusion: Compound matrine injection can significantly improve morphine tolerance and drug resistance of lung cancer through inhibiting the upregulations of MDR1 and P-gp induced by morphine.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Alcaloides/efeitos adversos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Genes MDR , Neoplasias Pulmonares/fisiopatologia , Morfina/farmacologia , Quinolizinas/efeitos adversos , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Alcaloides/administração & dosagem , Animais , Neoplasias Pulmonares/genética , Camundongos , Quinolizinas/administração & dosagem
2.
J Microbiol ; 58(2): 123-130, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31875929

RESUMO

Antibiotic resistance genes (ARGs) are emerging contaminants that pose a potential threat to human health worldwide. Urban wastewater treatment plants (WWTPs) are a main source of both antibiotic-resistant bacteria and ARGs released into the environment. Nevertheless, the propagation of ARGs and their underlying mechanisms and the dynamics of mobile genetic elements (MGEs) in WWTPs have rarely been investigated in South Korea. In this study, shotgun metagenomic analysis was used to identify comprehensive ARGs and their mechanisms, bacterial communities, and MGEs from 4 configurations with 2 activated sludge (AS) and 2 anaerobic digestion sludge (ADS) samples. A total of 181 ARG subtypes belonging to 22 ARG types were broadly detected, and the ARG abundances in the AS samples were 1.3-2.0 orders of magnitude higher than in the ADS samples. Multidrug and bacitracin resistance genes were the predominant ARG types in AS samples, followed by ARGs against sulfonamide, tetracycline, and ß-lactam. However, the composition of ARG types in ADS samples was significantly changed. The abundance of multidrug and ß-lactam resistance genes was drastically reduced in the ADS samples. The resistance genes of MLS were the predominant, followed by ARGs against sulfonamide and tetracycline in the ADS samples. In addition, plasmids were the dominant MGEs in the AS samples, while integrons (intI1) were the dominant MGEs in the ADS samples. These results provide valuable information regarding the prevalence of ARG types and MGEs and the difference patterns between the AS and ADS systems.


Assuntos
Resistência Microbiana a Medicamentos/genética , Genes Bacterianos , Metagenoma , Esgotos/microbiologia , Águas Residuárias/microbiologia , Bactérias/genética , Genes MDR , Humanos , Metagenômica , Plasmídeos , República da Coreia , Resistência a Tetraciclina/genética , Saúde da População Urbana , Resistência beta-Lactâmica/genética
3.
Korean J Parasitol ; 57(4): 369-377, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31533403

RESUMO

Artemisinin-based combination therapy (ACT) resistance is widespread throughout the Greater Mekong Subregion. This raises concern over the antimalarial treatment in Thailand since it shares borders with Cambodia, Laos, and Myanmar where high ACT failure rates were reported. It is crucial to have information about the spread of ACT resistance for efficient planning and treatment. This study was to identify the molecular markers for antimalarial drug resistance: Pfkelch13 and Pfmdr1 mutations from 5 provinces of southern Thailand, from 2012 to 2017, of which 2 provinces on the Thai- Myanmar border (Chumphon and Ranong), one on Thai-Malaysia border (Yala) and 2 from non-border provinces (Phang Nga and Surat Thani). The results showed that C580Y mutation of Pfkelch13 was found mainly in the province on the Thai-Myanmar border. No mutations in the PfKelch13 gene were found in Surat Thani and Yala. The Pfmdr1 gene isolated from the Thai-Malaysia border was a different pattern from those found in other areas (100% N86Y) whereas wild type strain was present in Phang Nga. Our study indicated that the molecular markers of artemisinin resistance were spread in the provinces bordering along the Thai-Myanmar, and the pattern of Pfmdr1 mutations from the areas along the international border of Thailand differed from those of the non-border provinces. The information of the molecular markers from this study highlighted the recent spread of artemisinin resistant parasites from the endemic area, and the data will be useful for optimizing antimalarial treatment based on regional differences.


Assuntos
Antimaláricos/farmacologia , Artemisininas/farmacologia , Marcadores Genéticos , Malária Falciparum/tratamento farmacológico , Plasmodium falciparum/genética , Antimaláricos/administração & dosagem , Antimaláricos/uso terapêutico , Artemisininas/administração & dosagem , Artemisininas/uso terapêutico , Sequência de Bases , DNA de Protozoário/química , Combinação de Medicamentos , Resistência a Medicamentos/genética , Genes MDR/genética , Humanos , Repetição Kelch/genética , Malária Falciparum/sangue , Malária Falciparum/parasitologia , Mutação , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/isolamento & purificação , Reação em Cadeia da Polimerase , Tailândia
4.
Microb Pathog ; 134: 103596, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31212036

RESUMO

To establish infection in the host, pathogens have evolved sophisticated systems to cope with environmental conditions and to protect cells against host immunity. TolC is the outer membrane channel component of type 1 secretion systems and multidrug efflux pumps that plays critical roles during the infection process in many pathogens. However, little is known about the exact roles of TolC1 in the pathogenicity of A. pleuropneumoniae, an etiological agent of the porcine contagious pleuropneumoniae that causes severe respiratory disease. In this study, deletion of tolC1 causes apparent ultrastructural defects in A. pleuropneumoniae cell examined by transmission electron microscopy. The tolC1 mutant is hypersensitivity to oxidative, osmotic and acid challenges by in vitro stress assays. Analysis on secreted proteins shows that the excretion of ApxIIA and an ApxIVA-like protein, ApxIVA-S, is abolished in the absence of TolC1. This result confirms the essential role of TolC1 in the secretion of Apx toxins and this is the first identification of an ApxIVA-like protein in in vitro culture of A. pleuropneumoniae. Besides, disruption of TolC1 leads to a significant attenuation of virulence in mice by an intraperitoneal route of A. pleuropneumoniae. The basis for the attenuation is further investigated using a mouse intranasal infection model, which reveals an impaired ability to colonize and induce lesions in the lungs for the loss of TolC1 of A. pleuropneumoniae. In conclusion, our findings demonstrate significant roles of TolC1 in facilitating bacterial survival in hostile conditions, maximum colonization as well as pathogenicity during the infection of A. pleuropneumoniae.


Assuntos
Infecções por Actinobacillus/microbiologia , Actinobacillus pleuropneumoniae/fisiologia , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Fatores de Virulência/metabolismo , Infecções por Actinobacillus/patologia , Actinobacillus pleuropneumoniae/citologia , Actinobacillus pleuropneumoniae/patogenicidade , Animais , Proteínas da Membrana Bacteriana Externa/classificação , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Modelos Animais de Doenças , Deleção de Genes , Genes MDR , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , Pulmão/microbiologia , Pulmão/patologia , Camundongos , Pressão Osmótica , Estresse Oxidativo , Proteoma/análise , Proteoma/isolamento & purificação , Proteínas Recombinantes , Estresse Fisiológico , Transcriptoma , Sistemas de Secreção Tipo I/química , Sistemas de Secreção Tipo I/genética , Sistemas de Secreção Tipo I/metabolismo , Virulência , Fatores de Virulência/genética
5.
mSphere ; 4(3)2019 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-31118304

RESUMO

The increased prevalence of antimicrobial resistance (AMR) among Enterobacteriaceae has had major clinical and economic impacts on human medicine. Many of the multidrug-resistant (multiresistant) Enterobacteriaceae found in humans are community acquired, and some of them are possibly linked to food animals (i.e., livestock raised for meat and dairy products). In this study, we examined whether numerically dominant commensal Escherichia coli strains from humans (n = 63 isolates) and domestic animals (n = 174 isolates) in the same community and with matching phenotypic AMR patterns were clonally related or shared the same plasmids. We identified 25 multiresistant isolates (i.e., isolates resistant to more than one antimicrobial) that shared identical phenotypic resistance patterns. We then investigated the diversity of E. coli clones, AMR genes, and plasmids carrying the AMR genes using conjugation, replicon typing, and whole-genome sequencing. All of the multiresistant E. coli isolates (from children and domestic animals) analyzed had at least 90 or more whole-genome SNP differences between one another, suggesting that none of the strains was recently transferred. While the majority of isolates shared the same antimicrobial resistance genes and replicons, DNA sequencing indicated that these genes and replicons were found on different plasmid structures. We did not find evidence of the clonal spread of AMR in this community: instead, AMR genes were carried on diverse clones and plasmids. This presents a significant challenge for understanding the movement of AMR in a community.IMPORTANCE Even though Escherichia coli strains may share nearly identical phenotypic AMR profiles and AMR genes and overlap in space and time, the diversity of clones and plasmids challenges research that aims to identify sources of AMR. Horizontal gene transfer appears to play a more significant role than clonal expansion in the spread of AMR in this community.


Assuntos
Animais Domésticos/microbiologia , Infecções por Escherichia coli/veterinária , Escherichia coli/genética , Transferência Genética Horizontal , Genes MDR , Simbiose , Animais , Antibacterianos/farmacologia , Pré-Escolar , Farmacorresistência Bacteriana , Equador , Escherichia coli/efeitos dos fármacos , Infecções por Escherichia coli/microbiologia , Fezes/microbiologia , Humanos , Lactente , Testes de Sensibilidade Microbiana , Plasmídeos/genética , População Rural , Análise de Sequência de DNA
6.
Infect Control Hosp Epidemiol ; 40(3): 333-340, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30887943

RESUMO

OBJECTIVE: The smr and qacA/B genes in Staphylococcus aureus confer tolerance to antiseptics and are associated with nosocomial acquisition of infection and underlying medical conditions. Such antiseptic tolerance (AT) genes have also been reported in coagulase-negative staphylococci (CoNS) and enterococci, however, few data are available regarding their prevalence. We sought to describe the frequency of AT genes among bloodstream isolates of S. aureus, CoNS and enterococci at Texas Children's Hospital (TCH). METHODS: Banked CoNS, S. aureus and enterococci isolated from blood cultures collected bewteen October 1, 2016, and October 1, 2017, were obtained from the TCH clinical microbiology laboratory. All isolates underwent polymerase chain reaction (PCR) assay for the qacA/B and smr genes. Medical records were reviewed for all cases. RESULTS: In total, 103 CoNS, 19 Enterococcus spp, and 119 S. aureus isolates were included in the study, and 80.6% of the CoNS possessed at least 1 AT gene compared to 37% of S. aureus and 43.8% of E. faecalis isolates (P < .001). Among CoNS bloodstream isolates, the presence of either AT gene was strongly associated with nosocomial infection (P < .001). The AT genes in S. aureus were associated with nosocomial infection (P = .025) as well as the diagnosis of central-line-associated bloodstream infection (CLABSI; P = .04) and recent hospitalizations (P < .001). We found no correlation with genotypic AT in E. faecalis and any clinical variable we examined. CONCLUSIONS: Antiseptic tolerance is common among bloodstream staphylococci and E. faecalis isolates at TCH. Among CoNS, the presence of AT genes is strongly correlated with nosocomial acquisition of infection, consistent with previous studies in S. aureus. These data suggest that the healthcare environment contributes to AT among staphylococci.


Assuntos
Anti-Infecciosos Locais/administração & dosagem , Infecção Hospitalar , Genes MDR/genética , Infecções por Bactérias Gram-Positivas/sangue , Infecções Estafilocócicas/sangue , Antiporters/sangue , Antiporters/genética , Proteínas de Bactérias/sangue , Proteínas de Bactérias/genética , Criança , Infecção Hospitalar/epidemiologia , Enterococcus , Feminino , Infecções por Bactérias Gram-Positivas/epidemiologia , Hospitais Pediátricos , Humanos , Masculino , Proteínas de Membrana Transportadoras/sangue , Proteínas de Membrana Transportadoras/genética , Infecções Estafilocócicas/epidemiologia , Staphylococcus , Staphylococcus aureus/genética
7.
Genome Biol Evol ; 11(3): 776-785, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30715343

RESUMO

Shigella flexneri is a major etiological agent of shigellosis in developing countries, primarily occurring in children under 5 years of age. We have sequenced, for the first time, the complete genome of S. flexneri serotype 3b (strain SFL1520). We used a hybrid sequencing method--both long-read MinION Flow (Oxford Nanopore Technologies) and short-read MiSeq (Illumina) sequencing to generate a high-quality reference genome. The SFL1520 chromosome was found to be ∼4.58 Mb long, with 4,729 coding sequences. Despite sharing a substantial number of genes with other publicly available S. flexneri genomes (2,803), the SFL1520 strain contains 1,926 accessory genes. The phage-related genes accounted for 8% of the SFL1520 genome, including remnants of the Sf6 bacteriophage with an intact O-acetyltransferase gene specific to serotype 3b. The SFL1520 chromosome was also found to contain a multiple-antibiotic resistance cassette conferring resistance to ampicillin, chloramphenicol, streptomycin, and tetracycline, which was potentially acquired from a plasmid via transposases. The phylogenetic analysis based on core genes showed a high level of similarity of SFL1520 with other S. flexneri serotypes; however, there were marked differences in the accessory genes of SFL1520. In particular, a large number of unique genes were identified in SFL1520 suggesting significant horizontal gene acquisition in a relatively short time period. The major virulence traits of SFL1520 (such as serotype conversion and antimicrobial resistance) were associated with horizontal gene acquisitions highlighting the role of horizontal gene transfer in S. flexneri diversity and evolution.


Assuntos
Transferência Genética Horizontal , Genes MDR , Genoma Bacteriano , Shigella flexneri/genética , Ilhas Genômicas , Filogenia , Shigella flexneri/patogenicidade
8.
Pathog Glob Health ; 113(1): 20-26, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30722761

RESUMO

Non-typhoidal Salmonella (NTS) are foodborne pathogens that are responsible for self-limiting gastroenteritis in humans. The present study aims at the molecular characterisation and comparative genomics of Salmonella enterica serovar Senftenberg strain P5558 isolated from the pus samples of a patient suffering from stump infection. The isolate was subjected to serotyping and antimicrobial susceptibility test to understand the phenotypical characteristics. Whole genome sequencing (WGS) was carried out and comparative genomics using computational tools showed the antimicrobial resistance and virulence gene profile of the isolates from the genome sequence data. Typing experiments confirmed that the isolate belong to S. Senftenberg with sequence type ST14. Resistance against ß-lactams is associated with the presence of blaTEM-1, blaOXA-9, blaCMY-2 and blaNDM-1 genes. Similarly resistance to aminoglycoside was associated with five aminoglycoside modifying enzymes aac(6')-Ia, aac(6')-Ib, aph(3')-Ib, aph(6')-Ib and ant(3'')-Ia, sulfonamide with sul-1 and sul-2 and chloramphenicol with florR gene. Substitutions in gyrA (S83Y, D87G) and parC (S80I) genes found to be the reason for fluoroquinolone resistance. The plasmid profiling showed the isolate has four resistance plasmids in which plasmid p5558-NDM (IncA/C) harbours major resistance genes including blaNDM-1 and blaCMY-2. Determination of virulence gene profile revealed that the genome carries all major Salmonella pathogenicity islands and virulence factors. From our findings it is clear that the isolate possess characteristic pathogenicity islands (SPI 1-6, 13, 14), major virulence factors and acquired resistance genes. Comparative analysis suggests the evolution and distribution of the MDR gene encoding plasmids in NTS.


Assuntos
Infecções por Salmonella/microbiologia , Salmonella enterica/genética , beta-Lactamases/genética , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana Múltipla/genética , Genes MDR/genética , Humanos , Masculino , Testes de Sensibilidade Microbiana/métodos , Fosfoproteínas/genética , Salmonella enterica/classificação , Salmonella enterica/efeitos dos fármacos , Salmonella enterica/patogenicidade , Sorotipagem/métodos , Virulência/genética , Fatores de Virulência/genética , Sequenciamento Completo do Genoma/métodos , Adulto Jovem
9.
J Microbiol ; 57(3): 185-194, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30806976

RESUMO

Tripartite efflux pumps and the type I secretion system of Gram-negative bacteria are large protein complexes that span the entire cell envelope. These complexes expel antibiotics and other toxic substances or transport protein toxins from bacterial cells. Elucidating the binary and ternary complex structures at an atomic resolution are crucial to understanding the assembly and working mechanism. Recent advances in cryoelectron microscopy along with the construction of chimeric proteins drastically shifted the assembly models. In this review, we describe the current assembly models from a historical perspective and emphasize the common assembly mechanism for the assembly of diverse tripartite pumps and type I secretion systems.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana , Bactérias Gram-Negativas/fisiologia , Sistemas de Secreção Tipo I/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Microscopia Crioeletrônica , Genes MDR/genética , Bactérias Gram-Negativas/genética , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Proteínas Periplásmicas/genética , Proteínas Periplásmicas/metabolismo , Multimerização Proteica , Sistemas de Secreção Tipo I/genética
10.
PLoS One ; 14(2): e0212077, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30763358

RESUMO

Photorhabdus luminescens is an enterobacterium establishing a mutualistic symbiosis with nematodes, that also kills insects after septicaemia and connective tissue colonization. The role of the bacterial mdtABC genes encoding a putative multidrug efflux system from the resistance/nodulation/cell division family was investigated. We showed that a mdtA mutant and the wild type had similar levels of resistance to antibiotics, antimicrobial peptides, metals, detergents and bile salts. The mdtA mutant was also as pathogenic as the wild-type following intrahaemocoel injection in Locusta migratoria, but had a slightly attenuated phenotype in Spodoptera littoralis. A transcriptional fusion of the mdtA promoter (PmdtA) and the green fluorescent protein (gfp) encoding gene was induced by copper in bacteria cultured in vitro. The PmdtA-gfp fusion was strongly induced within bacterial aggregates in the haematopoietic organ during late stages of infection in L. migratoria, whereas it was only weakly expressed in insect plasma throughout infection. A medium supplemented with haematopoietic organ extracts induced the PmdtA-gfp fusion ex vivo, suggesting that site-specific mdtABC expression resulted from insect signals from the haematopoietic organ. Finally, we showed that protease inhibitors abolished ex vivo activity of the PmdtA-gfp fusion in the presence of haematopoietic organ extracts, suggesting that proteolysis by-products play a key role in upregulating the putative MdtABC efflux pump during insect infection with P. luminescens.


Assuntos
Proteínas de Bactérias/genética , Perfilação da Expressão Gênica , Locusta migratoria/microbiologia , Peptídeo Hidrolases/metabolismo , Photorhabdus/genética , Photorhabdus/fisiologia , Animais , Antibacterianos/farmacologia , Cobre/farmacologia , Genes MDR/genética , Testes de Sensibilidade Microbiana , Mutação , Óperon/genética , Fenótipo , Photorhabdus/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Transcrição Genética/efeitos dos fármacos
11.
Microb Drug Resist ; 25(5): 631-638, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30614757

RESUMO

Benzalkonium chloride (BAC) is widely used as a disinfectant and preservative. This study investigated the effect on antimicrobial susceptibility and the cellular changes that occurred after exposure of Klebsiella pneumoniae clinical isolates to sublethal concentrations of BAC. Minimum inhibitory concentration and minimum bactericidal concentration of BAC were determined for the collected 50 K. pneumoniae clinical isolates by broth microdilution method, and the tested isolates were adapted to increasing sublethal concentrations of BAC. The effect of adaptation on MICs of the tested 16 antimicrobial agents, the cell ultrastructure, efflux, and membrane depolarization of the tested isolates were examined. Interestingly, most K. pneumoniae isolates that adapted to BAC showed increased antimicrobial resistance, various morphological and structural changes, increased membrane depolarization, and enhanced efflux activity. The findings of this study suggest that the extensive use of BAC at sublethal concentrations could contribute to the emergence of antibiotic resistance in K. pneumoniae clinical isolates that might complicate the therapy of infections caused by this pathogen. In conclusion, the hazard associated with the prolonged exposure to sublethal concentrations of BAC represents a public health risk and therefore it should be a focus in both hospital and community sanitation practices.


Assuntos
Antibacterianos/farmacologia , Anti-Infecciosos Locais/farmacologia , Compostos de Benzalcônio/farmacologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Genes MDR/efeitos dos fármacos , Klebsiella pneumoniae/efeitos dos fármacos , Aminoglicosídeos/farmacologia , Carbapenêmicos/farmacologia , Cefalosporinas/farmacologia , Farmacorresistência Bacteriana/genética , Fluoroquinolonas/farmacologia , Expressão Gênica , Humanos , Infecções por Klebsiella/tratamento farmacológico , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/isolamento & purificação , Klebsiella pneumoniae/ultraestrutura , Macrolídeos/farmacologia , Proteínas de Membrana Transportadoras/agonistas , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Testes de Sensibilidade Microbiana , Proteínas Associadas à Resistência a Múltiplos Medicamentos/agonistas , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Nitrobenzenos/farmacologia , Penicilinas/farmacologia , Tetraciclinas/farmacologia
12.
Leuk Res ; 76: 15-23, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30476680

RESUMO

Acute myeloid leukemia is an aggressive disease that arises from clonal expansion of malignant hematopoietic precursor cells of the bone marrow. Deletions on the long arm of chromosome 9 (del(9q)) are observed in 2% of acute myeloid leukemia patients. Our deletion analysis in a cohort of 31 del(9q) acute myeloid leukemia patients further supports the importance of a minimally deleted region composed of seven genes potentially involved in leukemogenesis: GKAP1, KIF27, C9ORF64, HNRNPK, RMI1, SLC28A3 and NTRK2. Importantly, among them HNRNPK, encoding heterogeneous nuclear ribonucleoprotein K is proposed to function in leukemogenesis. We show that expression of HNRNPK and the other genes of the minimally deleted region is significantly reduced in patients with del(9q) compared with normal karyotype acute myeloid leukemia. Also, two mRNAs interacting with heterogeneous nuclear ribonucleoprotein K, namely CDKN1A and CEBPA are significantly downregulated. While the deletion size is not correlated with outcome, associated genetic aberrations are important. Patients with an additional t(8;21) show a good prognosis. RUNX1-RUNX1T1, which emerges from the t(8;21) leads to transcriptional down-regulation of CEBPA. Acute myeloid leukemia patients with mutations in CEBPA have a good prognosis as well. Interestingly, in del(9q) patients with CEBPA mutation mRNA levels of HNRNPK and the other genes located in the minimally deleted region is restored to normal karyotype level. Our data indicate that a link between CEBPA and the genes of the minimally deleted region, among them HNRNPK contributes to leukemogenesis in acute myeloid leukemia with del(9q).


Assuntos
Deleção Cromossômica , Cromossomos Humanos Par 9 , Leucemia Mieloide Aguda/genética , Cariótipo Anormal , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais , Proteínas Estimuladoras de Ligação a CCAAT/genética , Feminino , Expressão Gênica , Genes MDR , Humanos , Leucemia Mieloide Aguda/diagnóstico , Masculino , Pessoa de Meia-Idade , Mutação
13.
Colloids Surf B Biointerfaces ; 175: 523-529, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30579053

RESUMO

Fungal resistance is the major problem related to fluconazole treatments. This study aims to develop innovative lipid core nanocapsules and nanostructured lipid carriers containing fluconazole, to study in vitro antifungal activity and to assess the possibility of resistance reversion in Candida albicans, C. glabrata, C. krusei, and C. tropicalis isolates. The action mechanism of nanoparticles was investigated through efflux pumps and scanning electron microscopy studies. The lipid core nanocapsules and nanostructured lipid carriers were prepared by interfacial deposition of preformed polymer and high-pressure homogenization methods, respectively. Both nanostructures presented sizes below 250 nm, SPAN < 1.6, negative zeta potential, pH slightly acid, high drug content and controlled drug release. The nanostructured lipid carriers were unable to reverse the fungal resistance. Lipid core nanoparticles displayed advantages such as a reduction in the effective dose of fluconazole and resistance reversion in all isolates tested - with multiple mechanisms of resistance. The main role of the supramolecular structure and the composition of the nanoparticles on antifungal mechanisms of action were discussed. The results achieved through this study have an impact on clinical therapy, with a potential application in the treatment of fungal infections caused by resistant isolates of Candida spp.


Assuntos
Antifúngicos/farmacologia , Candida/efeitos dos fármacos , Preparações de Ação Retardada/química , Farmacorresistência Fúngica/efeitos dos fármacos , Fluconazol/farmacologia , Proteínas Fúngicas/antagonistas & inibidores , Nanopartículas/química , Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Candida/genética , Candida/crescimento & desenvolvimento , Candida/metabolismo , Candida albicans/efeitos dos fármacos , Candida albicans/genética , Candida albicans/crescimento & desenvolvimento , Candida albicans/metabolismo , Candida glabrata/efeitos dos fármacos , Candida glabrata/genética , Candida glabrata/crescimento & desenvolvimento , Candida glabrata/metabolismo , Candida tropicalis/efeitos dos fármacos , Candida tropicalis/genética , Candida tropicalis/crescimento & desenvolvimento , Candida tropicalis/metabolismo , Caprilatos/química , Composição de Medicamentos/métodos , Farmacorresistência Fúngica/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Expressão Gênica , Genes MDR/efeitos dos fármacos , Hexoses/química , Concentração de Íons de Hidrogênio , Testes de Sensibilidade Microbiana , Nanopartículas/ultraestrutura , Palmitatos/química , Tamanho da Partícula , Triglicerídeos/química , Verapamil/farmacologia
14.
Appl Environ Microbiol ; 85(4)2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30530714

RESUMO

Bacterial pathogens that carry antibiotic resistance alleles sometimes pay a cost in the form of impaired growth in antibiotic-free conditions. This cost of resistance is expected to be a key parameter for understanding how resistance spreads and persists in pathogen populations. Analysis of individual resistance alleles from laboratory evolution and natural isolates has shown they are typically costly, but these costs are highly variable and influenced by genetic variation at other loci. It therefore remains unclear how strongly resistance is linked to impaired antibiotic-free growth in bacteria from natural and clinical scenarios, where resistance alleles are likely to coincide with other types of genetic variation. To investigate this, we measured the growth of 92 natural and clinical Escherichia coli isolates across three antibiotic-free environments. We then tested whether variation of antibiotic-free growth among isolates was predicted by their resistance to 10 antibiotics, while accounting for the phylogenetic structure of the data. We found that isolates with similar resistance profiles had similar antibiotic-free growth profiles, but it was not simply that higher average resistance was associated with impaired growth. Next, we used whole-genome sequences to identify antibiotic resistance genes and found that isolates carrying a greater number of resistance gene types grew relatively poorly in antibiotic-free conditions, even when the resistance genes they carried were different. This suggests that the resistance of bacterial pathogens is linked to growth costs in nature, but it is the total genetic burden and multivariate resistance phenotype that predict these costs, rather than individual alleles or mean resistance across antibiotics.IMPORTANCE Managing the spread of antibiotic resistance in bacterial pathogens is a major challenge for global public health. Central to this challenge is understanding whether resistance is linked to impaired bacterial growth in the absence of antibiotics, because this determines whether resistance declines when bacteria are no longer exposed to antibiotics. We studied 92 isolates of the key bacterial pathogen Escherichia coli; these isolates varied in both their antibiotic resistance genes and other parts of the genome. Taking this approach, rather than focusing on individual genetic changes associated with resistance as in much previous work, revealed that growth without antibiotics was linked to the number of specialized resistance genes carried and the combination of antibiotics to which isolates were resistant but was not linked to average antibiotic resistance. This approach provides new insights into the genetic factors driving the long-term persistence of antibiotic-resistant bacteria, which is important for future efforts to predict and manage resistance.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/genética , Genes MDR/genética , Alelos , Desinfetantes/farmacologia , Escherichia coli/isolamento & purificação , Infecções por Escherichia coli/microbiologia , Variação Genética , Metais/farmacologia , Testes de Sensibilidade Microbiana , Fenótipo , Filogenia , Sequenciamento Completo do Genoma
15.
Gene ; 689: 172-182, 2019 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-30562604

RESUMO

The non-steroidal anti-inflammatory drug (NSAID) diclofenac, known to cause hyperuricemia and concomitant visceral gout in Gyps vultures is suggested to be a result of interference with renal uric acid excretion. Three species of Gyps vultures are on the verge of extinction due to nephrotoxic veterinary diclofenac having entered the food chain, notwithstanding the fact that the toxicity of different avian species to the NSAIDs like diclofenac varies. The multidrug resistance protein 4 (MRP4), an organic anion transporter in birds has unique role in unidirectional efflux of urate into proximal renal tubular lumen for excretion and maintenance of homeostasis. We characterized MRP4 channel at molecular level to predict its structural based ligand binding activity in Gallus domesticus (Indian domestic chicken) and Gyps himalayensis (Himalayan griffon vulture). MRP4 gene was amplified using reverse transcribed cDNA from renal tissue sample in overlapping fragments. The obtained amplicons were cloned, sequenced, assembled and analyzed. Multiple alignment and blast analysis revealed point variations and presence of additional stretch of 57 bp towards the 3' end which was confirmed in Real time PCR. Predicted MRP4 polypeptides revealed presence of characteristic 12 transmembrane helices (TMH) with two nucleotide binding domains (NBD). Additional 19 amino acids in transcript variant was found to be localized in NBD2 that might influence the transporter function. The homology modeling and pocket identification throws ample light on varying transport efficacy and paves the way for depicting its role of these amino acids in effect of diclofenac on urate transport in further studies.


Assuntos
Galinhas/genética , Falconiformes/genética , Variação Genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Sequência de Aminoácidos , Animais , Expressão Gênica , Genes MDR , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Ácido Úrico/metabolismo
16.
Mem. Inst. Oswaldo Cruz ; 114: e190105, 2019. tab, graf
Artigo em Inglês | LILACS | ID: biblio-1012671

RESUMO

BACKGROUND Healthcare-associated infections caused by bacteria such as Pseudomonas aeruginosa are a major public health problem worldwide. Gene regulatory networks (GRN) computationally represent interactions among regulatory genes and their targets. They are an important approach to help understand bacterial behaviour and to provide novel ways of overcoming scientific challenges, including the identification of potential therapeutic targets and the development of new drugs. OBJECTIVES The goal of this study was to reconstruct the multidrug-resistant (MDR) P. aeruginosa GRN and to analyse its topological properties. METHODS The methodology used in this study was based on gene orthology inference using the reciprocal best hit method. We used the genome of P. aeruginosa CCBH4851 as the basis of the reconstruction process. This MDR strain is representative of the sequence type 277, which was involved in an endemic outbreak in Brazil. FINDINGS We obtained a network with a larger number of regulatory genes, target genes and interactions as compared to the previously reported network. Topological analysis results are in accordance with the complex network representation of biological processes. MAIN CONCLUSIONS The properties of the network were consistent with the biological features of P. aeruginosa. To the best of our knowledge, the P. aeruginosa GRN presented here is the most complete version available to date.


Assuntos
Humanos , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/genética , Infecções por Pseudomonas/imunologia , Genes Reguladores/imunologia , Brasil/epidemiologia , Genes MDR/genética
17.
Science ; 362(6415): 686-690, 2018 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-30409883

RESUMO

Antibiotic resistance is often the result of mutations that block drug activity; however, bacteria also evade antibiotics by transiently expressing genes such as multidrug efflux pumps. A crucial question is whether transient resistance can promote permanent genetic changes. Previous studies have established that antibiotic treatment can select tolerant cells that then mutate to achieve permanent resistance. Whether these mutations result from antibiotic stress or preexist within the population is unclear. To address this question, we focused on the multidrug pump AcrAB-TolC. Using time-lapse microscopy, we found that cells with higher acrAB expression have lower expression of the DNA mismatch repair gene mutS, lower growth rates, and higher mutation frequencies. Thus, transient antibiotic resistance from elevated acrAB expression can promote spontaneous mutations within single cells.


Assuntos
Antibacterianos/farmacologia , Proteínas de Transporte/genética , Farmacorresistência Bacteriana/genética , Proteínas de Escherichia coli/genética , Escherichia coli/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica , Genes MDR , Proteína MutS de Ligação de DNA com Erro de Pareamento/genética , Proteínas de Bactérias/genética , Escherichia coli/genética , Mutação , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/genética , Seleção Genética
18.
Environ Int ; 121(Pt 2): 1217-1226, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30389380

RESUMO

BACKGROUND: Antibiotic resistance poses an increasing threat to public health. Horizontal gene transfer (HGT) promoted by antibiotics is recognized as a significant pathway to disseminate antibiotic resistance genes (ARGs). However, it is unclear whether non-antibiotic, anti-microbial (NAAM) chemicals can directly promote HGT of ARGs in the environment. OBJECTIVES: We aimed to investigate whether triclosan (TCS), a widely-used NAAM chemical in personal care products, is able to stimulate the conjugative transfer of antibiotic multi-resistance genes carried by plasmid within and across bacterial genera. METHODS: We established two model mating systems, to investigate intra-genera transfer and inter-genera transfer. Escherichia coli K-12 LE392 carrying IncP-α plasmid RP4 was used as the donor, and E. coli K-12 MG1655 or Pseudomonas putida KT2440 were the intra- and inter-genera recipients, respectively. The mechanisms of the HGT promoted by TCS were unveiled by detecting oxidative stress and cell membrane permeability, in combination with Nanopore sequencing, genome-wide RNA sequencing and proteomic analyses. RESULTS: Exposure of the bacteria to environmentally relevant concentrations of TCS (from 0.02 µg/L to 20 µg/L) significantly stimulated the conjugative transfer of plasmid-encoded multi-resistance genes within and across genera. The TCS exposure promoted ROS generation and damaged bacterial membrane, and caused increased expression of the SOS response regulatory genes umuC, dinB and dinD in the donor. In addition, higher expression levels of ATP synthesis encoding genes in E. coli and P. putida were found with increased TCS dosage. CONCLUSIONS: TCS could enhance the conjugative ARGs transfer between bacteria by triggering ROS overproduction at environmentally relevant concentrations. These findings improve our awareness of the hidden risks of NAAM chemicals on the spread of antibiotic resistance.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana/genética , Poluentes Ambientais/farmacologia , Escherichia coli K12/efeitos dos fármacos , Transferência Genética Horizontal , Genes MDR , Triclosan/farmacologia , Conjugação Genética , Escherichia coli K12/genética , Genes Bacterianos , Humanos , Plasmídeos , Pseudomonas putida/efeitos dos fármacos , Pseudomonas putida/genética
19.
Cell Mol Biol (Noisy-le-grand) ; 64(13): 79-83, 2018 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-30403600

RESUMO

RND (Resistance-Nodulation-Division) family transporters have a vital role in both intrinsic and acquired multi-drug resistance in Gram-negative bacteria. It is important to find a conserved domain in the RND family between different pathogenic bacteria for diagnostic and therapeutic purpose. Total sequences of three-component system RND efflux pumps were retrieved from NCBI nucleotide and protein database and were subjected to conservation and variation analysis using the multiple sequence alignment feature of the CLC workbench. The phylogenetic tree for main transporters was drawn and the three-dimensional structure was also evaluated. From the sequence conservation analysis, highly conserved residues with 282 base pair (94 amino acid) long were identified. The location of the highly conserved domain is positioned in the domain 1 crystallographic structure of AcrB Escherichia coli and MexB Pseudomonas aeruginosa. The main transporter component phylogenetic tree shows the clusters of different genotypes and their evolutionary association.  Each of three components of RND proteins is crucial for drug efflux, and the absence of even one component makes the entire complex totally nonfunctional. Therefore, this highly conserved region can be used to disable the RND multidrug efflux pumps. In addition, this highly conserved can also be used for diagnostic aspects.


Assuntos
Sequência Conservada , Genes MDR , Bactérias Gram-Negativas/metabolismo , Proteínas de Membrana Transportadoras/química , Sequência de Aminoácidos , Sequência Consenso , Bactérias Gram-Negativas/genética , Filogenia , Domínios Proteicos , Alinhamento de Sequência
20.
Sci Rep ; 8(1): 14741, 2018 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-30282985

RESUMO

In the past few decades Acinetobacter baumannii has emerged as a notorious nosocomial pathogen because of its ability to acquire genetic material and persist in extreme environments. Recently, human serum albumin (HSA) was shown to significantly increase natural transformation frequency in A. baumannii. This observation led us to perform transcriptomic analysis of strain A118 under HSA induction to identify genes that are altered by HSA. Our results revealed the statistically significant differential expression of 296 protein-coding genes, including those associated with motility, biofilm formation, metabolism, efflux pumps, capsule synthesis, and transcriptional regulation. Phenotypic analysis of these traits showed an increase in surface-associated motility, a decrease in biofilm formation, reduced activity of a citric acid cycle associated enzyme, and increased survival associated with zinc availability. Furthermore, the expression of genes known to play a role in pathogenicity and antibiotic resistance were altered. These genes included those associated with RND-type efflux pumps, the type VI secretion system, iron acquisition/metabolism, and ß-lactam resistance. Together, these results illustrate how human products, in particular HSA, may play a significant role in both survival and persistence of A. baumannii.


Assuntos
Acinetobacter baumannii/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Genoma Bacteriano , Albumina Sérica Humana/farmacologia , Resistência beta-Lactâmica/efeitos dos fármacos , Acinetobacter baumannii/genética , Acinetobacter baumannii/metabolismo , Acinetobacter baumannii/patogenicidade , Cápsulas Bacterianas/efeitos dos fármacos , Cápsulas Bacterianas/genética , Cápsulas Bacterianas/metabolismo , Biofilmes , Ciclo do Ácido Cítrico/efeitos dos fármacos , Ciclo do Ácido Cítrico/genética , Perfilação da Expressão Gênica , Genes MDR/efeitos dos fármacos , Humanos , Transporte de Íons/efeitos dos fármacos , Ferro/metabolismo , Viabilidade Microbiana/efeitos dos fármacos , Transformação Bacteriana/efeitos dos fármacos , Sistemas de Secreção Tipo VI/genética , Sistemas de Secreção Tipo VI/metabolismo , Zinco/metabolismo , Resistência beta-Lactâmica/genética , beta-Lactamas/farmacologia
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