Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 10.100
Filtrar
1.
Ann Clin Microbiol Antimicrob ; 19(1): 8, 2020 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-32169075

RESUMO

Treatment of infections by Pseudomonas aeruginosa forming biofilms after antimicrobial testing on planktonic bacteria can result in substantial failure. Therefore, we offer a robust and simple experimental platform to test the impact of antimicrobials on biofilms. Antibiotic response patterns varied uniquely within biofilm formation capacity and minimal biofilm eradication concentrations (MBECs) has a significantly better discriminatory power than minimum inhibitory concentrations (MICs) to differentiate the overall efficiency of antibiotics to eradicate biofilm. Our resazurin-based 96-well-plate platform is able to emulate bacterial responses to antibiotics under biofilm conditions in a fast, simple, and cost-effective screening method adaptable to automation, and warrants trials in the clinic.


Assuntos
Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Testes de Sensibilidade Microbiana/métodos , Infecções por Pseudomonas , Pseudomonas aeruginosa/efeitos dos fármacos , Humanos , Infecções por Pseudomonas/microbiologia
2.
Nat Rev Microbiol ; 18(5): 299-311, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32055026

RESUMO

Antimicrobial resistance (AMR) is a major threat to human health worldwide, and the rapid detection and quantification of resistance, combined with antimicrobial stewardship, are key interventions to combat the spread and emergence of AMR. Antimicrobial susceptibility testing (AST) systems are the collective set of diagnostic processes that facilitate the phenotypic and genotypic assessment of AMR and antibiotic susceptibility. Over the past 30 years, only a few high-throughput AST methods have been developed and widely implemented. By contrast, several studies have established proof of principle for various innovative AST methods, including both molecular-based and genome-based methods, which await clinical trials and regulatory review. In this Review, we discuss the current state of AST systems in the broadest technical, translational and implementation-related scope.


Assuntos
Anti-Infecciosos/farmacologia , Ensaios de Triagem em Larga Escala/tendências , Testes de Sensibilidade Microbiana/métodos , Testes de Sensibilidade Microbiana/tendências
3.
Mymensingh Med J ; 29(1): 37-42, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31915333

RESUMO

Uropathogenic Escherichia coli is frequently resistant to different antibiotic leading to a critical condition of the patients. The purpose of the present study was to see antibiotic resistance pattern and genetic characteristics of ESBL and Carbapenemase-producing Escherichia coli. This cross sectional study was conducted in the Department of Microbiology at Mymensingh Medical College, Mymensingh, Bangladesh from October 2014 to December 2015. Patients presented with clinically diagnosed urinary tract infection at any age with both sexes who attended in the OPD of Mymensingh Medical College Hospital and the Doctors Diagnostic Centre in Mymensingh, Bangladesh was selected as study population. Non duplicate clinical isolates from urine were collected in full aseptic precaution for culture of bacteria. Escherichia coli were confirmed by PCR Stargetingadk. Antimicrobial susceptibility was measured by broth microdilution test. Minimum inhibitory concentrations against 18 antimicrobial agents were measured. Beta-lactamase genes were detected by multiplex PCR. For all the isolates showing resistance to imipenem and/or meropenem, presence of carbapenemase genes was confirmed by multiplex/uniplex PCR using primers. A total of 233 non-duplicate clinical isolates of Escherichia coli were collected from patients of which dominant phylogenetic group was B2 which was 78(33.5%) isolates of which 71 isolates were B2a and 7 isolates were B2b. Furthermore, Group A was in 29.6% isolates and Group D was in 26.6% isolates. E. coli showed significantly higher resistance rates to piperacillin, cephalosporins, and some other antimicrobials. Meropenem-resistance was detected in 8.2% of E. coli. The detection rate of blaTEM was 41.6% in E. coli. Carbapenemase genes were detected in 9(3.9%) isolates of E. coli and identified as genes encoding NDM-1, -5, and 7 and OXA-181. All the blaNDM-positive E. coli isolates carried also blaCTX-M-15, except for a group B1 isolate. E. coli is significantly higher resistance rates to piperacillin, cephalosporins, and some other antimicrobials and possesses different ESBL and carbapenemase genes.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana/genética , Infecções por Escherichia coli/tratamento farmacológico , Escherichia coli/genética , beta-Lactamases/genética , Bangladesh , Estudos Transversais , Escherichia coli/classificação , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Infecções por Escherichia coli/microbiologia , Feminino , Genes Bacterianos , Humanos , Masculino , Testes de Sensibilidade Microbiana/métodos , Filogenia , Reação em Cadeia da Polimerase , Centros de Atenção Terciária , beta-Lactamases/metabolismo
4.
PLoS One ; 15(1): e0227215, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31910223

RESUMO

Rapid diagnosis of tuberculosis (TB) and antibiotic resistances are imperative to initiate effective treatment and to stop transmission of the disease. A new generation of more sensitive, automated molecular TB diagnostic tests has been recently launched giving microbiologists more choice between several assays with the potential to detect resistance markers for rifampicin and isoniazid. In this study, we determined analytical sensitivities as 95% limits of detection (LoD95) for Xpert MTB/Rif Ultra (XP-Ultra) and BD-MAX MDR-TB (BD-MAX) as two representatives of the new test generation, in comparison to the conventional FluoroType MTB (FT-MTB). Test matrices used were physiological saline solution, human and a mucin-based artificial sputum (MUCAS) each spiked with Mycobacterium tuberculosis in declining culture- and qPCR-controlled concentrations. With BD-MAX, XP-Ultra, and FT-MTB, we measured LoD95TB values of 2.1 cfu/ml (CI95%: 0.9-23.3), 3.1 cfu/ml (CI95%: 1.2-88.9), and 52.1 cfu/ml (CI95%: 16.7-664.4) in human sputum; of 6.3 cfu/ml (CI95%: 2.9-31.8), 1.5 cfu/ml (CI95%: 0.7-5.0), and 30.4 cfu/ml (CI95%: 17.4-60.7) in MUCAS; and of 2.3 cfu/ml (CI95%: 1.1-12.0), 11.5 cfu/ml (CI95%: 5.6-47.3), and 129.1 cfu/ml (CI95%: 82.8-273.8) in saline solution, respectively. LoD95 of resistance markers were 9 to 48 times higher compared to LoD95TB. BD-MAX and XP-Ultra have an equal and significantly increased analytical sensitivity compared to conventional tests. MUCAS resembled human sputum, while both yielded significantly different results than normal saline. MUCAS proved to be suitable for quality control of PCR assays for TB diagnostics.


Assuntos
Técnicas de Diagnóstico Molecular/instrumentação , Mycobacterium tuberculosis/isolamento & purificação , Kit de Reagentes para Diagnóstico/normas , Tuberculose Resistente a Múltiplos Medicamentos/diagnóstico , Tuberculose Pulmonar/diagnóstico , Antibióticos Antituberculose/farmacologia , Antibióticos Antituberculose/uso terapêutico , Farmacorresistência Bacteriana Múltipla , Humanos , Limite de Detecção , Testes de Sensibilidade Microbiana/instrumentação , Testes de Sensibilidade Microbiana/métodos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Diagnóstico Molecular/normas , Mycobacterium tuberculosis/fisiologia , Controle de Qualidade , Reação em Cadeia da Polimerase em Tempo Real , Escarro/microbiologia , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Tuberculose Pulmonar/tratamento farmacológico , Tuberculose Pulmonar/microbiologia
5.
Biosci Biotechnol Biochem ; 84(1): 143-153, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31549575

RESUMO

Brevinin-GR23 (B-GR23) was a brevinin-2 like antimicrobial peptide, which had antimicrobial activity against Staphylococcus aureus with minimum inhibitory concentration (MIC) of 16 µM. B-GR23 increased the bacterial membrane permeation, leading to the damage of membrane integrity and the leakage of genomic DNA, then causing the cell death. The peptide nearly inhibited all plantonic bacteria to start the initial attachment of biofilm at the concentration of 1 × MIC. Whereas the disruption rates on immature and mature biofilm decreased from 60% to 20%. B-GR23 reduced the production of extracellular polysaccharides (EPS) in the planktonic growth of S. aureus, which is a crucial structure of biofilm formation. B-GR23 with the concentration of ½ × MIC inhibited 50% water-soluble EPS, and 48% water-insoluble EPS, which contributed to the antibiofilm activity. B-GR23 had no significant toxicity to human blood cells under-tested concentration (200 µM), making it a potential template for designing antimicrobial peptides.


Assuntos
Proteínas de Anfíbios/farmacologia , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Biofilmes/efeitos dos fármacos , Staphylococcus aureus/fisiologia , Animais , Antibacterianos/síntese química , Peptídeos Catiônicos Antimicrobianos/síntese química , Peptídeos Catiônicos Antimicrobianos/química , Permeabilidade da Membrana Celular/efeitos dos fármacos , DNA Bacteriano/efeitos dos fármacos , DNA Bacteriano/metabolismo , Eritrócitos/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Temperatura Alta , Humanos , Concentração de Íons de Hidrogênio , Testes de Sensibilidade Microbiana/métodos , Polissacarídeos Bacterianos/antagonistas & inibidores , Conformação Proteica em alfa-Hélice , Estabilidade Proteica/efeitos da radiação , Ranidae , Infecções Estafilocócicas/tratamento farmacológico
7.
J Med Microbiol ; 69(1): 52-62, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31846419

RESUMO

Introduction. The alarming rise in urinary tract infection (UTI) antimicrobial resistance has resulted from a combination of high prevalence, low specificity and the lack of a rapid, point-of-care (POC) antibiotic susceptibility test (AST), which has led to the overuse/inappropriate use of antibiotics.Aim. This study aimed to evaluate the performance of a rapid POC phenotypic AST device in reporting susceptibility information within 2 h.Methodology. Instrument calibration was performed with model bacteria and fluorescent microbeads to determine the dynamic range and limit of detection for quantifying concentrations of bacteria and demonstrate the ability to rapidly differentiate susceptible and resistant model bacteria. We then evaluated 30 presumptive UTI-positive patient urine samples in a clinical pilot study using a panel of 5 common UTI antibiotics plus a growth control and compared our results to the hospital standard of care AST.Results. Our device was able to robustly detect and quantify bacteria concentrations from 50 to 105 colony-forming units (c.f.u.) ml-1. The high sensitivity of this measurement technique enabled the device to differentiate between susceptible and resistant model bacteria with 100 % specificity over a 2 h growth period. In the clinical pilot study, an overall categorical agreement (CA) of 90.7 % was observed (sensitivity=91.4 %, specificity=88.9 %, n=97) with performance for individual drugs ranging from 85 % CA (ceftazidime) to 100 % (nitrofurantoin).Conclusions. By reducing the typical timeframe for susceptibility testing from 2-3 days to 2 h, our POC phenotypic AST can provide critical information to clinicians prior to the administration of antibiotic therapy.


Assuntos
Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Testes de Sensibilidade Microbiana/métodos , Sistemas Automatizados de Assistência Junto ao Leito , Infecções Urinárias/microbiologia , Bactérias/isolamento & purificação , Humanos , Projetos Piloto , Sensibilidade e Especificidade , Fatores de Tempo , Urina/microbiologia
8.
Ann Lab Med ; 40(1): 57-62, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31432640

RESUMO

As various linezolid resistance mechanisms have been identified in methicillin-resistant Staphylococcus aureus (MRSA), we investigated the molecular characteristics of MRSA with elevated linezolid minimum inhibitory concentrations (MICs), using the VITEK 2 system (bioMérieux, Marcy-l'Étoile, France). Twenty-seven MRSA isolates from 14 patients exhibiting linezolid MICs ≥8 µg/mL were examined by broth microdilution (BMD) test as well as by sequencing for mutations in the 23S rRNA gene or ribosomal proteins (L3, L4, and L22) and the presence of the optrA, cfr, and cfr(B) genes. Of the 27 isolates, four (14.8%) from one patient were confirmed as linezolid resistant by BMD and harbored a 23S rRNA T2500A mutation. The remaining 23 were confirmed as linezolid susceptible, indicating that the linezolid-resistant results were major errors generated by VITEK 2. The most commonly detected mutation (19/27, 70.4%), L3 Gly152Asp, was detected in only linezolid-susceptible isolates. No isolates contained optrA, cfr, or cfr(B) or any L4 or L22 protein alterations. Our results show that the 23S rRNA T2500A mutation was mainly associated with linezolid resistance, while the L3 Gly152Asp mutation was not related to linezolid resistance. A confirmatory test is recommended for VITEK 2 linezolid-resistant results owing to the high probability of false resistant results.


Assuntos
Farmacorresistência Bacteriana/genética , Linezolida/farmacologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Testes de Sensibilidade Microbiana/métodos , Reações Falso-Positivas , Humanos , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Mutação Puntual , RNA Ribossômico 23S/genética , Kit de Reagentes para Diagnóstico , República da Coreia , Proteínas Ribossômicas/genética
9.
Zhonghua Jie He He Hu Xi Za Zhi ; 42(12): 901-906, 2019 Dec 12.
Artigo em Chinês | MEDLINE | ID: mdl-31826533

RESUMO

Objective: To investigate the clinical characteristics and drug susceptibility test (DST) of patients infected with different nontuberculous mycobacteria (NTM). Methods: The patients with nontuberculous mycobacterial lung disease (NMLD) in Shanghai Pulmonary Hospital from March 2014 to March 2015 were studied retrospectively by analyzing the clinical characteristics, radiological features and DST results. A total of 201 NMLD patients [male 108, age(58±15) yrs] were enrolled into this study including 48 cases of M. Kansasii [male 13, age (52±16) yrs],46 cases of M. Abscess[male 46, age (57±16) yrs], 92 cases of M. Intracellulare [male 43, age (61±13) yrs], and 15 cases of M. Avium [male 6, age (67±10) yrs]. Clinical data were collected when the diagnosis was made and Chi-square test was used to compare the differences among 4 groups of patients. Bonferroni method was used for further pairwise comparisons. Results: There were significant differences among the 4 groups in the age(χ(2)=6.42, P<0.001) and the gender(χ(2)=49.18, P<0.001) of the patients. The history of bronchiectasis in the groups of M. Kansasii, M. Abscess, M. Intracellulare and M. Avium were 2/48, 31/46, 39/92 and 4/15 cases respectively(χ(2)=41.84, P<0.001). For the Gamma-interferon release assays (ELISA) (IGRA), the positive rate of IGRA in the groups of M. Kansasii, M. Abscess, M. Intracellulare and M. Avium were 83%(40/48), 30%(14/46), 23%(21/92) and 33% (5/15) respectively(χ(2)=50.96, P<0.001). The radiological features were significantly different in tree-in-bud(8/48, 35/46, 36/92 and 4/15 cases respectively, χ(2)=36.48, P<0.001), pleural thickness or mild effusion (21/48, 36/46, 69/92 and 7/15 cases, χ(2)=19.54, P<0.001), bronchiectasis (20/48, 39/46, 78/92 and 10/15 cases, P<0.001) and cavities (38/48, 21/46, 63/92 and 10/15 cases, χ(2)=12.38, P<0.001) among the 4 groups(M. Kansasii, M. Abscess, M. Intracellulare and M. Avium). The drug resistance rates of M. Kansasii to rifampin, ethambutanol and ofloxacin were 10%(5/48), 8%(4/48) and 15%(7/48) respectively; the resistance rates of M.Intracellulare to ethambutanol was 45%(41/92), and the resistance rates of M.Abscess were all over 80% to all anti-TB drugs. The results of pairwise comparisons showed that the male proportion(46/48) and IGRAs positive rate(40/48) of patients with M. Kansasii were higher than those of other groups, and the incidence of bronchiectasis(20/48) and pleural changes(21/48) was lower than those of other groups. The female ratio(33/46), history of bronchiectasis (31/46) and tree-in-bud sign of patients(35/46) with M. Abscess were higher than those of other groups. Conclusions: There were differences in the clinical manifestations and imaging features of 4 common NMLD diseases, which were helpful for clinical differentiation. The patients with M. Kansasii infection were mainly male, with a high IGRA positive rate and fewer lesions of bronchiectasis or pleural changes. Most of the patients with M. Abscess were female, with a previous history of bronchiectasis, and with most of the lesions showing tree-in-bud signs. The NTM species had a high rate of resistance to anti-TB drugs except M. Kansasii.


Assuntos
Antibacterianos/farmacologia , Pneumopatias/tratamento farmacológico , Infecções por Mycobacterium não Tuberculosas/tratamento farmacológico , Micobactérias não Tuberculosas/efeitos dos fármacos , Adulto , Idoso , Idoso de 80 Anos ou mais , China , Farmacorresistência Bacteriana , Etambutol/farmacologia , Feminino , Humanos , Pneumopatias/microbiologia , Masculino , Testes de Sensibilidade Microbiana/métodos , Pessoa de Meia-Idade , Infecções por Mycobacterium não Tuberculosas/microbiologia , Micobactérias não Tuberculosas/isolamento & purificação , Ofloxacino/farmacologia , Estudos Retrospectivos , Rifampina/farmacologia , Especificidade da Espécie
10.
Int. microbiol ; 22(4): 479-490, dic. 2019. ilus, tab
Artigo em Inglês | IBECS | ID: ibc-185066

RESUMO

Bacterial diseases are the main cause of high economic loss in aquaculture, particularly gram-negative bacteria. This study was conducted for the isolation and identification of Aeromonas and Pseudomonas spp. from diseased fish. Twenty-two Aeromonas and sixteen Pseudomonas isolates were recovered from diseased Nile tilapia (Oreochromis niloticus) raised in eight earthen ponds in Elhox, Metoubes, Kafrelsheikh, Egypt. The recovered isolates were further identified using PCR as 22 Aeromonas hydrophila, 11 Pseudomonas aeruginosa, and 5 Pseudomonas fluorescens isolates. The 22 A. hydrophila isolates were screened for the presence of four virulence genes. Sixteen of the isolates (72.72%) were positive for the aerolysin gene (aer); 4 (18.18%) harbored the cytotoxic enterotoxin gene (act); and 2 (9.09%) carried the hemolysin A gene (hylA) while the cytotonic heat-stable enterotoxin gene (ast) was absent from all the tested isolates. The pathogenicity test indicated the direct relationship between the mortality percentage and the genotype of the tested A. hydrophila isolates as the mortality rates were 63.3 and 73.3% for isolates with two virulence genes (aer+ & act+, and aer+ and hylA+, respectively), followed by 40, 53.3, and 56.6% for isolates with only one virulence gene (hylA, act, and aer, respectively) and 20% for isolates lacking virulence genes. Based on the sensitivity test, the multi-antibiotic resistance profiles were as follows: 90.9% of the A. hydrophila isolates were sensitive to florfenicol and doxycycline; then 68.18% were susceptible to oxytetracycline, norfloxacin, and ciprofloxacin; and 63.63% were susceptible to sulfamethoxazole-trimethoprim, while only 27.27 and 4.5% were sensitive to erythromycin and cephradine, respectively, and all the isolates were resistant to amoxicillin and ampicillin


No disponible


Assuntos
Animais , Virulência/genética , Aeromonas hydrophila/patogenicidade , Ciclídeos/microbiologia , Aeromonas hydrophila/genética , Aeromonas hydrophila/isolamento & purificação , Norfloxacino/isolamento & purificação , Testes de Sensibilidade Microbiana/métodos , Reação em Cadeia da Polimerase
11.
Clin Lab ; 65(12)2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31850708

RESUMO

BACKGROUND: KPC-producing Klebsiella pneumoniae (KPC-Kp) has become a serious threat to patients worldwide, as the treatment options are limited. The combination of fosfomycin with other antibiotics has been reported but with inconsistent results. Thus, we performed synergy testing of fosfomycin combined with tigecycline by E-test, which was easy to perform and to determine results, compared with microdilution checkerboard, which is considered to be the "gold standard", to evaluate the agreement between the two methods. METHODS: Thirty non-repetitive KPC-Kp isolates from different patients were included in this study. Bacterial identification and routine antibiotic susceptibility testing were performed by a VITEK 2 Compact automated system. The KPC producing isolates were identified by modified Carbapenem Inhibitory Method (mCIM) and PCR amplification of carbapenemase genes. Synergy testing of fosfomycin combined with tigecycline was performed by E-test (E-test stripes were placed at 90° angle), with microdilution chequerboard performed in parallel. Fractional inhibitory concentration index (FICI) was calculated. Statistical analyses were performed by SPSS 18.0 software. RESULTS: All 30 KPC-Kp were mCIM test positive and KPC-2 producing. The susceptibility rates of fosfomycin and tigecycline were 36.7% (11/30) and 63.3% (19/30), respectively. Both checkerboard and E-test results showed that most MICs of fosfomycin and tigecycline decreased in the combination group. FICI showed 13.3% - 16.7% isolates were synergistic, 30.0% - 36.7% were additive, and 50.0% - 53.3% were indifferent. No antagonism was found. There was no significant difference between the two groups (p > 0.05), and the overall agreement (with FICI difference ≤ 0.25 in each isolate) between the two methods was 76.7% (23/30). CONCLUSIONS: The synergy testing results determined by E-test correlated well with microdilution checkerboard. Thus E-test synergy testing has the potential to be used in routine clinical laboratory.


Assuntos
Proteínas de Bactérias/biossíntese , Fosfomicina/farmacologia , Klebsiella pneumoniae/efeitos dos fármacos , Tigeciclina/farmacologia , beta-Lactamases/biossíntese , Antibacterianos/farmacologia , Sinergismo Farmacológico , Humanos , Klebsiella pneumoniae/metabolismo , Testes de Sensibilidade Microbiana/métodos , Testes de Sensibilidade Microbiana/estatística & dados numéricos , Reprodutibilidade dos Testes
12.
Int J Mol Sci ; 21(1)2019 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-31877837

RESUMO

The effects of trans-cinnamaldehyde (TC) on transcriptional profiles of biofilm-associated genes and the metabolic activity of two methicillin-resistant Staphylococcus aureus (MRSA) strains showing a different degree of adherence to polystyrene, were evaluated. Metabolic activity of S. aureus in biofilm was significantly decreased in the presence of TC at 1/2 minimum biofilm inhibition concentration (MBIC). Expression levels of the genes encoding laminin binding protein (eno), elastin binding protein (ebps) and fibrinogen binding protein (fib) in the presence of TC at 1/2 MBIC were lower than in untreated biofilm in both the weakly and strongly adhering strain. The highest decrease of expression level was observed in case of fib in the strongly adhering strain, in which the amount of fib transcript was 10-fold lower compared to biofilm without TC. In the presence of TC at 1/2 MBIC after 3, 6, 8 and 12 h, the expression level of icaA and icaD, that are involved in the biosynthesis of polysaccharide intercellular adhesin, was above half lower in the weakly adhering strain compared to biofilm without TC. In the strongly adhering strain the highest decrease in expression of these genes was observed after 3 and 6 h. This study showed that TC is a promising anti-biofilm agent for use in MRSA biofilm-related infections.


Assuntos
Acroleína/análogos & derivados , Biofilmes/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Genes Bacterianos/genética , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Acroleína/química , Acroleína/farmacologia , Antibacterianos/química , Antibacterianos/farmacologia , Biofilmes/crescimento & desenvolvimento , Humanos , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/fisiologia , Testes de Sensibilidade Microbiana/métodos , Polissacarídeos Bacterianos/genética , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/prevenção & controle
13.
PLoS Negl Trop Dis ; 13(12): e0007946, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31881061

RESUMO

BACKGROUND: Although leprosy is efficiently treated by multidrug therapy, resistance to first-line (dapsone, rifampin) and second-line (fluoroquinolones) drugs has been described worldwide. However, the characteristics of drug resistance in Southwest China remain unknown. Furthermore, the sensitivity of polymerase chain reaction (PCR)/sequencing for resistance detection is limited, especially for paucibacillary (PB) leprosy patients. The current study aimed to develop a nested PCR/sequencing and TaqMan SNP Genotyping Assay to increase the sensitivity of the method used to detect drug resistance in Mycobacterium leprae and to reveal the nature of M. leprae drug resistance in Southwest China. METHODOLOGY/PRINCIPAL FINDINGS: Seventy-six specimens, including skin biopsy (n = 64), formalin-fixed paraffin-embedded (FFPE) (n = 11) and skin-slit smear (SSS) (n = 1) samples from multibacillary (MB, n = 70) and PB (n = 6) leprosy patients from Southwest China, were included in this study. The presence of mutations in drug resistance-determining regions (DRDRs) of the rpoB, folP1, and gyrA genes, which are associated with rifampicin, dapsone, and quinolone resistance, respectively, was detected by PCR/sequencing, as recommended by the WHO, and the nested PCR and TaqMan SNP Genotyping Assay developed in this study. Mutations in the folP gene were detected in 19 (25.00%) samples, indicating dapsone-resistant M. leprae, with one (1.31%) sample showing mutations in two genes, folP and gyrA, reflecting multidrug-resistant strains to dapsone and ofloxacin. However, no rpoB mutation was detected. Compared with PCR/sequencing, nested PCR increased the sensitivity of detecting rpoB (from 51.39% to 78.94% for leprosy patients and from 0.00% to 50.00% for PB), gyrA (from 75.00% to 80.26% for leprosy patients and from 50.00% to 66.67% for PB), and folP1 (from 5.26% to 84.21% for leprosy patients and from 0.00% to 66.67% for PB). Moreover, the TaqMan SNP Genotyping Assay showed greater sensitivity for folP1 detection (from 5.26% to 78.94-86.84% for leprosy patients and from 0.00% to 33.33%-83.33% for PB patients) than the PCR/sequencing method. In addition, the latter method was able to more easily distinguish heterozygous genotypes and mutant homozygous genotypes from homozygous genotypes. CONCLUSIONS/SIGNIFICANCE: Nested PCR/sequencing and the TaqMan SNP Genotyping Assay are rapid and highly sensitive methods for detecting drug resistance in leprosy cases. The current study revealed that diamino-diphenylsulfone (DDS; also known as dapsone) resistance in M. leprae, as indicated by folP1 gene detection, is still the most concerning form of drug resistance in leprosy patients from Southwest China.


Assuntos
Farmacorresistência Bacteriana , Técnicas de Genotipagem/métodos , Hanseníase/microbiologia , Testes de Sensibilidade Microbiana/métodos , Mycobacterium leprae/efeitos dos fármacos , Mycobacterium leprae/genética , Reação em Cadeia da Polimerase/métodos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , China , Feminino , Genes Bacterianos , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Mycobacterium leprae/isolamento & purificação , Sensibilidade e Especificidade , Análise de Sequência de DNA/métodos , Adulto Jovem
14.
PLoS One ; 14(12): e0227071, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31887220

RESUMO

PURPOSE: Prompt clinical diagnosis and initiation of treatment are critical in the management of infectious endophthalmitis. Current methods used to identify causative agents of infectious endophthalmitis are mostly inefficient, owing to suboptimal sensitivity, length, and cost. Matrix Assisted Laser Desorption-Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) can be used to rapidly identity pathogens without a need for culture. Similarly, automated antimicrobial susceptibility test systems (AST, VITEK 2) provide accurate antimicrobial susceptibility profiles. In this proof-of-concept study, we apply these technologies for the direct identification and characterization of pathogens in vitreous samples, without culture, as an in vitro model of infectious endophthalmitis. METHODS: Vitreous humor aspirated from freshly enucleated porcine eyes was inoculated with different inocula of Staphylococcus aureus (S. aureus) and incubated at 37°C. Vitreous endophthalmitis samples were centrifuged and pellets were directly analyzed with MALDI-TOF MS and VITEK 2 without prior culture. S. aureus colonies that were conventionally grown on culture medium were used as control samples. Time-to-identification, minimum concentration of bacteria required for identification, and accuracy of results compared to standard methods were determined. RESULTS: MALDI-TOF MS achieved accurate pathogen identification from direct analysis of intraocular samples with confidence values of up to 99.9%. Time from sample processing to pathogen identification was <30 minutes. The minimum number of bacteria needed for positive identification was 7.889x106 colony forming units (cfu/µl). Direct analysis of intraocular samples with VITEK 2 gave AST profiles that were up to 94.4% identical to the positive control S. aureus analyzed per standard protocol. CONCLUSION: Our findings demonstrate that the direct analysis of vitreous samples with MALDI-TOF MS and VITEK 2 without prior culture could serve as new, improved methods for rapid, accurate pathogen identification and targeted treatment design in infectious endophthalmitis. In vivo models and standardized comparisons against other microbiological methods are needed to determine the value of direct analysis of intraocular samples from infectious endophthalmitis with MALDI-TOF MS and VITEK 2.


Assuntos
Antibacterianos/farmacologia , Endoftalmite/diagnóstico , Testes de Sensibilidade Microbiana/instrumentação , Infecções Estafilocócicas/diagnóstico , Staphylococcus aureus/isolamento & purificação , Animais , Antibacterianos/uso terapêutico , Modelos Animais de Doenças , Endoftalmite/tratamento farmacológico , Endoftalmite/microbiologia , Estudos de Viabilidade , Humanos , Testes de Sensibilidade Microbiana/métodos , Estudo de Prova de Conceito , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/efeitos dos fármacos , Suínos , Fatores de Tempo , Corpo Vítreo/microbiologia
15.
BMC Infect Dis ; 19(1): 989, 2019 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-31752735

RESUMO

BACKGROUND: A bacteremia diagnosis with speeded-up identification and antimicrobial susceptibility testing (AST) is mandatory to adjust empirical broad-spectrum antibiotherapy and avoid the emergence of multi-resistant bacteria. Alfred 60AST (Alifax, Polverara, PD, Italy) is an innovative automated system based on light scattering measurements allowing direct AST from positive blood cultures with rapid results. In this study we aimed to evaluate the system's performances and turnaround time (TAT) compared to routine AST. METHODS: The study was conducted during 2 non-consecutive 3-month periods at the microbiology laboratory of the Cliniques universitaires Saint-Luc. All blood cultures detected positive in the 0 AM-10 AM time frame with a pure Gram-positive cocci or Gram-negative bacilli stain were included for Alfred 60AST testing. Two customized EUCAST antibiotic panels were set up composed of 1) a "Gram-negative" panel including cefuroxime, ceftazidime Enterobacteriaceae, piperacillin-tazobactam Enterobacteriaceae, ciprofloxacine, and ceftazidime Pseudomonas 2) a "Gram-positive" panel including cefoxitin Staphylococcus aureus, cefoxitin coagulase-negative (CNS) Staphylococci and ampicillin Enterococci. Categorical agreement (CA), very major errors (VME), major errors (ME), minor errors (mE) and TAT to Alfred 60AST results were calculated in comparison with AST results obtained from direct testing on positive blood cultures with the Phoenix system (Becton Dickinson, Franklin Lakes, NJ, USA). RESULTS: Five hundred seventy and one hundred nine antibiotics were evaluated on respectively 166 Gram-negative bacilli and 109 Gram-positive cocci included in the studied population. During the first study period regarding Gram-negative strains a CA of 89.5% was obtained with a high rate of VME (19 and 15.4% respectively) for cefuroxime and piperacillin-tazobactam Enterobacteriaceae. Considering this, Alifax reviewed these antibiotics' formulations improving Gram-negative bacilli total CA to 92.2% with no VME during the second study period. For Gram-positive cocci, total CA was 88.1% with 2.3% VME, 13.8% ME (mainly cefoxitin CNS) and 12% mE rates both study periods combined. Median TAT to AST results was 5 h with Alfred versus 12 h34 with Phoenix. CONCLUSION: The Alfred 60AST system shows correct yet improvable microbiological performances and a major TAT reduction compared to direct automated AST testing. Clinical studies measuring the impact of the approach on antibiotic management of patients with bacteremia are recommended.


Assuntos
Antibacterianos/farmacologia , Bacteriemia/diagnóstico , Bactérias/efeitos dos fármacos , Difusão Dinâmica da Luz/métodos , Testes de Sensibilidade Microbiana/métodos , Bacteriemia/sangue , Bacteriemia/tratamento farmacológico , Bacteriemia/microbiologia , Bactérias/genética , Bactérias/isolamento & purificação , Hemocultura , Testes Diagnósticos de Rotina , Difusão Dinâmica da Luz/instrumentação , Humanos , Itália , Testes de Sensibilidade Microbiana/instrumentação
16.
J Photochem Photobiol B ; 199: 111632, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31610431

RESUMO

The development of eco-benign experimental procedures for the synthesis of nanomaterials is a fundamental developing branch of green nanotechnology. In this paper, green synthetic route was followed to synthesize novel Au@Fe2O3nanocomposite using Citrus sinensis fruit extract as a reducing and stabilizing agent. The as synthesized Au@Fe2O3nanocomposite was successfully characterized by UV-visible spectroscopy, X-ray diffraction (XRD), Scanning electron microscope (SEM), Energy-dispersive X-ray (EDX), Fourier transform infrared (FT1R) spectrophotometry and Zeta potential. UV-vis spectroscopy showed two SPR peaks for Fe2O3 and coated Au at 290 and 520 nm respectively. XRD confirmed the crystallinity of Au@Fe2O3. Au@Fe2O3 nanocomposite showed better antioxidant activity to effectively scavenge DPPH. The Au@Fe2O3 has been also tested for antibacterial activity which showed an effective antibacterial activity against multi drug resistant E.coli and Bacillus subtilis. Furthermore, Au@Fe2O3 also demonstrated better photo catalytic activity for methylene blue (MB) degradation. We proposed that the existence of organic acids (citric acids) also played a significant role in the stabilization of Au@Fe2O3, and plant (Citrus sinensis Var Kozan yerly) containing such component may be more effective for the green synthesis of Au@Fe2O3 nanocomposite. The findings of this study prove the overwhelming therapeutic and photocatalytic potential of bio-inspired Au@Fe2O3nanocomposite which can be a novel candidate for the effective remediation of microbes and toxic organic pollutants.


Assuntos
Antibacterianos/síntese química , Antioxidantes/síntese química , Citrus sinensis/química , Compostos Férricos/química , Ouro/química , Nanocompostos/química , Processos Fotoquímicos , Extratos Vegetais/química , Bacillus subtilis/efeitos dos fármacos , Catálise , Ácido Cítrico/química , Escherichia coli/efeitos dos fármacos , Química Verde/métodos , Luz , Azul de Metileno/química , Testes de Sensibilidade Microbiana/métodos , Oxirredução , Propriedades de Superfície , Poluição Química da Água
17.
Microb Drug Resist ; 25(8): 1155-1163, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31613200

RESUMO

Nosocomial infections caused by Klebsiella pneumoniae are primarily characterized by a high prevalence of extended-spectrum ß-lactamases (ESBL's) and a soaring pace of carbapenemase dissemination. Availability of limited antimicrobial agents as a therapeutic option for multidrug-resistant bacteria raises an alarming concern. This study aimed at the molecular characterization of multidrug-resistant K. pneumoniae clinical isolates and studied the role of efflux pumps in ß-lactam resistance. Thirty-three isolates confirmed as ESBL-positive K. pneumoniae that harbored resistance genes to major classes of antibiotics. The results showed that CTX-M15 was the preeminent ß-lactamase along with carbapenemases in ESBL-positive isolates. However, the efficacy of different antibiotics varied in the presence of lactamase inhibitors and efflux pump inhibitors (EPIs). Those showing increased efficacy of antibiotics with EPI were further explored for the expression of efflux pump genes and expressed a significantly different level of efflux pumps. We found that an isolate had higher expression of kpnF (SMR family) and kdeA (MATE family) pump genes relative to RND family pump genes. No mutations were observed in the genes for porins. Together, the findings suggest that ß-lactamases are not the only single factor responsible for providing resistance against the existing ß-lactam drugs. Resistance may increase many folds by simultaneous expression of RND family (the most prominent family in Gram-negative bacteria) and other efflux pump family.


Assuntos
Proteínas de Bactérias/genética , Farmacorresistência Bacteriana Múltipla/genética , Klebsiella pneumoniae/genética , beta-Lactamases/genética , Antibacterianos/farmacologia , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Humanos , Infecções por Klebsiella/tratamento farmacológico , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/efeitos dos fármacos , Testes de Sensibilidade Microbiana/métodos , Porinas/genética , Resistência beta-Lactâmica/efeitos dos fármacos , Resistência beta-Lactâmica/genética
18.
PLoS Negl Trop Dis ; 13(9): e0007729, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31568511

RESUMO

INTRODUCTION: The prevalence of bacteremia caused by Gram negative non-fermentative (GNNF) bacteria has been increasing globally over the past decade. Many studies have investigated their epidemiology but focus on the common GNNF including Pseudomonas aeruginosa and Acinetobacter baumannii. Knowledge of the uncommon GNNF bacteremias is very limited. This study explores invasive bloodstream infection GNNF isolates that were initially unidentified after testing with standard microbiological techniques. All isolations were made during laboratory-based surveillance activities in two rural provinces of Thailand between 2006 and 2014. METHODS: A subset of GNNF clinical isolates (204/947), not identified by standard manual biochemical methodologies were run on the BD Phoenix automated identification and susceptibility testing system. If an organism was not identified (12/204) DNA was extracted for whole genome sequencing (WGS) on a MiSeq platform and data analysis performed using 3 web-based platforms: Taxonomer, CGE KmerFinder and One Codex. RESULTS: The BD Phoenix automated identification system recognized 92% (187/204) of the GNNF isolates, and because of their taxonomic complexity and high phenotypic similarity 37% (69/187) were only identified to the genus level. Five isolates grew too slowly for identification. Antimicrobial sensitivity (AST) data was not obtained for 93/187 (50%) identified isolates either because of their slow growth or their taxa were not in the AST database associated with the instrument. WGS identified the 12 remaining unknowns, four to genus level only. CONCLUSION: The GNNF bacteria are of increasing concern in the clinical setting, and our inability to identify these organisms and determine their AST profiles will impede treatment. Databases for automated identification systems and sequencing annotation need to be improved so that opportunistic organisms are better covered.


Assuntos
Bacteriemia/microbiologia , Técnicas de Tipagem Bacteriana/métodos , Bactérias Gram-Negativas/isolamento & purificação , DNA Bacteriano/genética , Bactérias Gram-Negativas/classificação , Bactérias Gram-Negativas/genética , Humanos , Testes de Sensibilidade Microbiana/métodos , Tailândia , Sequenciamento Completo do Genoma/métodos
19.
BMJ ; 367: l5894, 2019 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-31649017

RESUMO

OBJECTIVE: To measure the association between phenotypic drug resistance and the risk of tuberculosis infection and disease among household contacts of patients with pulmonary tuberculosis. SETTING: 106 district health centers in Lima, Peru between September 2009 and September 2012. DESIGN: Prospective cohort study. PARTICIPANTS: 10 160 household contacts of 3339 index patients with tuberculosis were classified on the basis of the drug resistance profile of the patient: 6189 were exposed to drug susceptible strains of Mycobacterium tuberculosis, 1659 to strains resistant to isoniazid or rifampicin, and 1541 to strains that were multidrug resistant (resistant to isoniazid and rifampicin). MAIN OUTCOME MEASURES: Tuberculosis infection (positive tuberculin skin test) and the incidence of active disease (diagnosed by positive sputum smear or chest radiograph) after 12 months of follow-up. RESULTS: Household contacts exposed to patients with multidrug resistant tuberculosis had an 8% (95% confidence interval 4% to 13%) higher risk of infection by the end of follow-up compared with household contacts of patients with drug sensitive tuberculosis. The relative hazard of incident tuberculosis disease did not differ among household contacts exposed to multidrug resistant tuberculosis and those exposed to drug sensitive tuberculosis (adjusted hazard ratio 1.28, 95% confidence interval 0.9 to 1.83). CONCLUSION: Household contacts of patients with multidrug resistant tuberculosis were at higher risk of tuberculosis infection than contacts exposed to drug sensitive tuberculosis. The risk of developing tuberculosis disease did not differ among contacts in both groups. The evidence invites guideline producers to take action by targeting drug resistant and drug sensitive tuberculosis, such as early detection and effective treatment of infection and disease. TRIAL REGISTRATION: ClinicalTrials.gov NCT00676754.


Assuntos
Tuberculose Resistente a Múltiplos Medicamentos/transmissão , Tuberculose Pulmonar/transmissão , Adolescente , Adulto , Idoso , Antituberculosos/farmacologia , Antituberculosos/uso terapêutico , Criança , Pré-Escolar , Busca de Comunicante/métodos , Progressão da Doença , Feminino , Seguimentos , Humanos , Incidência , Lactente , Recém-Nascido , Isoniazida/farmacologia , Isoniazida/uso terapêutico , Estimativa de Kaplan-Meier , Masculino , Testes de Sensibilidade Microbiana/métodos , Pessoa de Meia-Idade , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/isolamento & purificação , Peru/epidemiologia , Estudos Prospectivos , Rifampina/farmacologia , Rifampina/uso terapêutico , Escarro/microbiologia , Teste Tuberculínico , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Resistente a Múltiplos Medicamentos/epidemiologia , Tuberculose Pulmonar/tratamento farmacológico , Tuberculose Pulmonar/epidemiologia , Tuberculose Pulmonar/microbiologia , Adulto Jovem
20.
World J Gastroenterol ; 25(39): 5961-5972, 2019 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-31660033

RESUMO

BACKGROUND: Previously, we have successfully constructed replication-competent hepatitis B virus (HBV) vectors by uncoupling the P open reading frame (ORF) from the preC/C ORF to carefully design the transgene insertion site to overcome the compact organization of the HBV genome and maintain HBV replication competence. Consequently, the replication-competent HBV vectors carrying foreign genes, including pCH-BsdR, carrying blasticidin resistance gene (399 bp), and pCH-hrGFP, carrying humanized renilla green fluorescent protein gene (720 bp), were successfully obtained. However, the replication efficiency of the former is higher but it is tedious to use, while that of the latter is poor and cannot be quantified. Hence, we need to search for a new reporter gene that is convenient and quantifiable for further research. AIM: To establish a helpful tool for intracellular HBV replication and anti-viral drugs screening studies. METHODS: We utilized the replication-competent HBV viral vectors constructed by our laboratory, combined with the secreted luciferase reporter gene, to construct replication-competent HBV vectors expressing the reporter gene secretory Nanoluc Luciferase (SecNluc). HepG2.TA2-7 cells were transfected with this vector to obtain cell lines with stably secreted HBV particles carrying secNluc reporter gene. RESULTS: The replication-competent HBV vector carrying the SecNluc reporter gene pCH-sNLuc could produce all major viral RNAs and a full set of envelope proteins and achieve high-level secreted luciferase expression. HBV replication intermediates could be produced from this vector. Via transfection with pTRE-sNLuc and selection by hygromycin, we obtained isolated cell clones, named HBV-NLuc-35 cells, which could secrete secNLuc recombinant viruses, and were sensitive to existing anti-HBV drugs. Using differentiated HepaRG cells, it was verified that recombinant HBV possessed infectivity. CONCLUSION: Our research demonstrated that a replication-competent HBV vector carrying a secreted luciferase transgene possesses replication and expression ability, and the established HBV replication and expression cell lines could stably secrete viral particles carrying secNluc reporter gene. More importantly, the cell line and the secreted recombinant viral particles could be used to trace HBV replication or infection.


Assuntos
Antivirais/farmacologia , Vetores Genéticos/genética , Vírus da Hepatite B/genética , Luciferases/genética , Replicação Viral/efeitos dos fármacos , Linhagem Celular , Genes Reporter/genética , Células Hep G2 , Humanos , Lamivudina/farmacologia , Testes de Sensibilidade Microbiana/métodos , Plasmídeos/genética , RNA/genética , RNA Viral/genética , Transfecção/métodos , Transgenes/genética , Replicação Viral/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA