RESUMO
The objective of the study was to evaluate the use of targeted multiplex Nanopore MinION amplicon re-sequencing of key Candida spp. from blood culture bottles to identify azole and echinocandin resistance associated SNPs. Targeted PCR amplification of azole (ERG11 and ERG3) and echinocandin (FKS) resistance-associated loci was performed on positive blood culture media. Sequencing was performed using MinION nanopore device with R9.4.1 Flow Cells. Twenty-eight spiked blood cultures (ATCC strains and clinical isolates) and 12 prospectively collected positive blood cultures with candidaemia were included. Isolate species included Candida albicans, Candida glabrata, Candida krusei, Candida parapsilosis, Candida tropicalis and Candida auris. SNPs that were identified on ERG and FKS genes using Snippy tool and CLC Genomic Workbench were correlated with phenotypic testing by broth microdilution (YeastOne™ Sensititre). Illumina whole-genome-sequencing and Sanger-sequencing were also performed as confirmatory testing of the mutations identified from nanopore sequencing data. There was a perfect agreement of the resistance-associated mutations detected by MinION-nanopore-sequencing compared to phenotypic testing for acquired resistance (16 with azole resistance; 3 with echinocandin resistance), and perfect concordance of the nanopore sequence mutations to Illumina and Sanger data. Mutations with no known association with phenotypic drug resistance and novel mutations were also detected.
Assuntos
Equinocandinas , Sequenciamento por Nanoporos , Antifúngicos/farmacologia , Antifúngicos/uso terapêutico , Azóis/farmacologia , Hemocultura , Candida/genética , Farmacorresistência Fúngica , Equinocandinas/farmacologia , Testes de Sensibilidade Microbiana , PichiaAssuntos
Farmacorresistência Bacteriana Múltipla , Plasmídeos/genética , Infecções por Salmonella/microbiologia , Salmonella enterica/isolamento & purificação , beta-Lactamases/genética , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Criança , Classe I de Fosfatidilinositol 3-Quinases , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Farmacorresistência Bacteriana Múltipla/genética , Humanos , Masculino , Testes de Sensibilidade Microbiana , Doenças da Imunodeficiência Primária/terapia , Infecções por Salmonella/diagnóstico , Infecções por Salmonella/tratamento farmacológico , Infecções por Salmonella/etiologia , Salmonella enterica/efeitos dos fármacos , Salmonella enterica/genética , Sorogrupo , Singapura , Transplante de Células-Tronco/efeitos adversos , Resultado do TratamentoAssuntos
Amoxicilina/farmacologia , Amoxicilina/uso terapêutico , Ceftriaxona/farmacologia , Ceftriaxona/uso terapêutico , Farmacorresistência Bacteriana Múltipla , Salmonella typhi/efeitos dos fármacos , Febre Tifoide/tratamento farmacológico , Amoxicilina/efeitos adversos , Antibacterianos/efeitos adversos , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Bactérias/efeitos dos fármacos , Bactérias/genética , Bactérias/isolamento & purificação , Ceftriaxona/efeitos adversos , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Farmacorresistência Bacteriana Múltipla/genética , Fezes/microbiologia , Genoma Bacteriano/genética , Humanos , Adesão à Medicação , Plasmídeos/genética , Recidiva , Salmonella typhi/genética , Salmonella typhi/isolamento & purificação , Febre Tifoide/microbiologiaAssuntos
Neoplasias Hematológicas/complicações , Infecções Fúngicas Invasivas/diagnóstico , Volvariella/isolamento & purificação , Antifúngicos/uso terapêutico , Humanos , Infecções Fúngicas Invasivas/tratamento farmacológico , Infecções Fúngicas Invasivas/patologia , Infecções Fúngicas Invasivas/fisiopatologia , Masculino , Técnicas Microbiológicas , Pessoa de Meia-Idade , Estudos Retrospectivos , Singapura , Resultado do TratamentoRESUMO
The global emergence of carbapenem-resistant Enterobacteriaceae (CRE) presents a significant clinical concern, prompting the WHO to prioritize CRE as a top priority pathogen in their 2017 global antibiotic-resistant bacteria priority list. Due to the fast-depleting antibiotic arsenal, clinicians are now resorting to using once-abandoned, highly toxic antibiotics such as the polymyxins and aminoglycosides, creating an urgent need for new antibiotics. Drug repurposing, the application of an approved drug for a new therapeutic indication, is deemed a plausible solution to this problem. A total of 1,163 FDA-approved drugs were screened for activity against a clinical carbapenem- and multidrug-resistant E. coli isolate using a single-point 10 µM assay. Hit compounds were then assessed for their suitability for repurposing. The lead candidate was then tested against a panel of clinical CREs, a bactericidal/static determination assay, a time-kill assay and a checkerboard assay to evaluate its suitability for use in combination with Tigecycline against CRE infections. Three drugs were identified. The lead candidate was determined to be Zidovudine (azidothymidine/AZT), an oral anti-viral drug used for HIV treatment. Zidovudine was shown to be the most promising candidate for use in combination with Tigecycline to treat systemic CRE infections. Further experiments should involve the use of animal infection models.
Assuntos
Antibacterianos/uso terapêutico , Enterobacteriáceas Resistentes a Carbapenêmicos/efeitos dos fármacos , Reposicionamento de Medicamentos , Infecções por Enterobacteriaceae/tratamento farmacológico , Escherichia coli/efeitos dos fármacos , Minociclina/análogos & derivados , Zidovudina/uso terapêutico , Animais , Farmacorresistência Bacteriana Múltipla , Infecções por Enterobacteriaceae/microbiologia , Humanos , Camundongos , Testes de Sensibilidade Microbiana , Minociclina/uso terapêutico , TigeciclinaRESUMO
The emergence of carbapenemase-producing Enterobacteriaceae is a rapidly evolving threat worldwide. Here, we report the molecular characterization of two Klebsiella pneumoniae isolates carrying both bla(OXA -181) and bla(NDM -1) or bla(NDM -5) isolated from epidemiologically unrelated patients in Singapore. The bla(OXA -181) genes were found existing in different genetic environments.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/enzimologia , Klebsiella pneumoniae/genética , beta-Lactamases/genética , beta-Lactamases/metabolismo , Adolescente , Técnicas de Tipagem Bacteriana , Pré-Escolar , Enterobacteriaceae/genética , Feminino , Humanos , Klebsiella pneumoniae/isolamento & purificação , Testes de Sensibilidade Microbiana , Tipagem de Sequências Multilocus , Estudos Prospectivos , SingapuraRESUMO
We report the emergence of OXA-232, a newly described OXA-48-like carbapenemase variant, in Southeast Asia. Molecular characterization of eight Klebsiella pneumoniae obtained from local and foreign patients reveals clonality of the isolates. bla OXA-232 was located on a non-conjugative plasmid of 6141 base pairs (GenBank accession number JX423831.1).
RESUMO
NDM-1 is a new metallo-beta-lactamase that readily hydrolyses carbapenems, penicillins and cephalosporins. Its rising incidence has been reported in many countries around the world, with many cases linked to a possible origin from the Indian subcontinent. Due to the lack of effective antibiotic regimes to treat these infections, the increased prevalence of NDM-1 is alarming. We describe a case of NDM-1 infection in an immunocompromised foreign patient, and discuss its implications.
Assuntos
Antibacterianos/uso terapêutico , Escherichia coli/metabolismo , Febre/diagnóstico , Neutropenia/diagnóstico , beta-Lactamases/biossíntese , Farmacorresistência Bacteriana , Resistência a Múltiplos Medicamentos , Feminino , Febre/etiologia , Humanos , Hospedeiro Imunocomprometido , Incidência , Turismo Médico , Pessoa de Meia-Idade , Neutropenia/etiologia , Resultado do Tratamento , beta-Lactamases/metabolismoRESUMO
AIMS: To accelerate the identification and differentiation of clinically relevant nontuberculous mycobacteria (NTM) with two sets of multiplex PCR (mPCR) targeting the 16S-23S rRNA internal transcribed spacer (ITS) region for timely patient management. METHODS AND RESULTS: Two mPCR assays were developed: Slow-Growers (SG) mPCR was used for the detection of slow-growing mycobacteria, which included Mycobacterium avium complex, Mycobacterium kansasii, Mycobacterium gordonae and Mycobacterium xenopi whilst the other mPCR assay labelled as Fast-Growers (FG) mPCR was used for the detection of Mycobacterium fortuitum complex, Mycobacterium abscessus and Mycobacterium chelonae. In these assays, a common forward primer based on a conserved section of the 16S rRNA region was used in conjunction with species-specific reverse primers. The mPCRs were tested against 247 clinical mycobacterial isolates and demonstrated 100% specificity and sensitivity. Identification of the mycobacterial species was also validated by DNA sequencing of the 16S-23S ITS region and when further confirmation was needed, hsp65 sequencing was performed. CONCLUSIONS: The mPCR assays could be a potentially useful diagnostic tool for the rapid and accurate identification of clinically relevant NTM. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, we looked at the frequency of hospital isolated NTM over the last 5 years (2005-2010), and an mPCR targeting the ITS region was developed for NTM species that appeared to be more prevalent in the context of Singapore.