ABSTRACT
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.
Subject(s)
Echinocandins , Nanopore Sequencing , Antifungal Agents/pharmacology , Antifungal Agents/therapeutic use , Azoles/pharmacology , Blood Culture , Candida/genetics , Drug Resistance, Fungal , Echinocandins/pharmacology , Microbial Sensitivity Tests , PichiaSubject(s)
Drug Resistance, Multiple, Bacterial , Plasmids/genetics , Salmonella Infections/microbiology , Salmonella enterica/isolation & purification , beta-Lactamases/genetics , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , Child , Class I Phosphatidylinositol 3-Kinases , Drug Resistance, Multiple, Bacterial/drug effects , Drug Resistance, Multiple, Bacterial/genetics , Humans , Male , Microbial Sensitivity Tests , Primary Immunodeficiency Diseases/therapy , Salmonella Infections/diagnosis , Salmonella Infections/drug therapy , Salmonella Infections/etiology , Salmonella enterica/drug effects , Salmonella enterica/genetics , Serogroup , Singapore , Stem Cell Transplantation/adverse effects , Treatment OutcomeSubject(s)
Amoxicillin/pharmacology , Amoxicillin/therapeutic use , Ceftriaxone/pharmacology , Ceftriaxone/therapeutic use , Drug Resistance, Multiple, Bacterial , Salmonella typhi/drug effects , Typhoid Fever/drug therapy , Amoxicillin/adverse effects , Anti-Bacterial Agents/adverse effects , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , Bacteria/drug effects , Bacteria/genetics , Bacteria/isolation & purification , Ceftriaxone/adverse effects , Drug Resistance, Multiple, Bacterial/drug effects , Drug Resistance, Multiple, Bacterial/genetics , Feces/microbiology , Genome, Bacterial/genetics , Humans , Medication Adherence , Plasmids/genetics , Recurrence , Salmonella typhi/genetics , Salmonella typhi/isolation & purification , Typhoid Fever/microbiologySubject(s)
Hematologic Neoplasms/complications , Invasive Fungal Infections/diagnosis , Volvariella/isolation & purification , Antifungal Agents/therapeutic use , Humans , Invasive Fungal Infections/drug therapy , Invasive Fungal Infections/pathology , Invasive Fungal Infections/physiopathology , Male , Microbiological Techniques , Middle Aged , Retrospective Studies , Singapore , Treatment OutcomeABSTRACT
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.
Subject(s)
Anti-Bacterial Agents/therapeutic use , Carbapenem-Resistant Enterobacteriaceae/drug effects , Drug Repositioning , Enterobacteriaceae Infections/drug therapy , Escherichia coli/drug effects , Minocycline/analogs & derivatives , Zidovudine/therapeutic use , Animals , Drug Resistance, Multiple, Bacterial , Enterobacteriaceae Infections/microbiology , Humans , Mice , Microbial Sensitivity Tests , Minocycline/therapeutic use , TigecyclineABSTRACT
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.
Subject(s)
Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Klebsiella Infections/microbiology , Klebsiella pneumoniae/enzymology , Klebsiella pneumoniae/genetics , beta-Lactamases/genetics , beta-Lactamases/metabolism , Adolescent , Bacterial Typing Techniques , Child, Preschool , Enterobacteriaceae/genetics , Female , Humans , Klebsiella pneumoniae/isolation & purification , Microbial Sensitivity Tests , Multilocus Sequence Typing , Prospective Studies , SingaporeABSTRACT
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).
ABSTRACT
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.
Subject(s)
Anti-Bacterial Agents/therapeutic use , Escherichia coli/metabolism , Fever/diagnosis , Neutropenia/diagnosis , beta-Lactamases/biosynthesis , Drug Resistance, Bacterial , Drug Resistance, Multiple , Female , Fever/etiology , Humans , Immunocompromised Host , Incidence , Medical Tourism , Middle Aged , Neutropenia/etiology , Treatment Outcome , beta-Lactamases/metabolismABSTRACT
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.