scholarly journals Evaluation of a Low-Density Hydrogel Microarray Technique for Mycobacterial Species Identification

2015 ◽  
Vol 53 (4) ◽  
pp. 1103-1114 ◽  
Author(s):  
Danila V. Zimenkov ◽  
Elena V. Kulagina ◽  
Olga V. Antonova ◽  
Maria A. Krasnova ◽  
Ekaterina N. Chernyaeva ◽  
...  
Keyword(s):  
Species Identification ◽  
Clinical Samples ◽  
Probe Design ◽  
Clinical Microbiology Laboratory ◽  
Low Density ◽  
Pathogenic Species ◽  
Mycobacterial Species ◽  
Accurate Identification ◽  
Content Type ◽  
Species Specific

In addition to the obligatory pathogenic species of theMycobacterium tuberculosiscomplex andMycobacterium leprae, the genusMycobacteriumalso includes conditionally pathogenic species that in rare cases can lead to the development of nontuberculous mycobacterial diseases. Because tuberculosis and mycobacteriosis have similar clinical signs, the accurate identification of the causative agent in a clinical microbiology laboratory is important for diagnostic verification and appropriate treatment. This report describes a low-density hydrogel-based microarray containing oligonucleotide probes based on the species-specific sequences of thegyrBgene fragment for mycobacterial species identification. The procedure included the amplification of a 352-nucleotide fragment of the gene and its hybridization on a microarray. The triple-species-specific probe design and the algorithm for hybridization profile recognition based on the calculation of Pearson correlation coefficients, followed by the construction of a profile database, allowed for the reliable and accurate identification of mycobacterial species, including mixed-DNA samples. The assay was used to evaluate 543 clinical isolates from two regions of Russia, demonstrating its ability to detect 35 mycobacterial species, with 99.8% sensitivity and 100% specificity when usinggyrB, 16S, and internal transcribed spacer (ITS) fragment sequencing as the standard. The testing of clinical samples showed that the sensitivity of the assay was 89% to 95% for smear-positive samples and 36% for smear-negative samples. The large number of identified species, the high level of sensitivity, the ability to detect mycobacteria in clinical samples, and the up-to-date profile database make the assay suitable for use in routine laboratory practice.

scholarly journals Mycobacterial DNA Extraction for Whole-Genome Sequencing from Early Positive Liquid (MGIT) Cultures

2015 ◽  
Vol 53 (4) ◽  
pp. 1137-1143 ◽  
Author(s):  
Antonina A. Votintseva ◽  
Louise J. Pankhurst ◽  
Luke W. Anson ◽  
Marcus R. Morgan ◽  
Deborah Gascoyne-Binzi ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Dna Extraction ◽  
Genome Sequencing ◽  
Solid Phase ◽  
Clinical Samples ◽  
Reference Laboratory ◽  
Whole Genome ◽  
Mycobacterial Species ◽  
Accurate Identification ◽  
Content Type

We developed a low-cost and reliable method of DNA extraction from as little as 1 ml of early positive mycobacterial growth indicator tube (MGIT) cultures that is suitable for whole-genome sequencing to identify mycobacterial species and predict antibiotic resistance in clinical samples. The DNA extraction method is based on ethanol precipitation supplemented by pretreatment steps with a MolYsis kit or saline wash for the removal of human DNA and a final DNA cleanup step with solid-phase reversible immobilization beads. The protocol yielded ≥0.2 ng/μl of DNA for 90% (MolYsis kit) and 83% (saline wash) of positive MGIT cultures. A total of 144 (94%) of the 154 samples sequenced on the MiSeq platform (Illumina) achieved the target of 1 million reads, with <5% of reads derived from human or nasopharyngeal flora for 88% and 91% of samples, respectively. A total of 59 (98%) of 60 samples that were identified by the national mycobacterial reference laboratory (NMRL) asMycobacterium tuberculosiswere successfully mapped to the H37Rv reference, with >90% coverage achieved. The DNA extraction protocol, therefore, will facilitate fast and accurate identification of mycobacterial species and resistance using a range of bioinformatics tools.

scholarly journals Precise Species Identification for Enterobacter: a Genome Sequence-Based Study with Reporting of Two Novel Species, Enterobacter quasiroggenkampii sp. nov. and Enterobacter quasimori sp. nov.

mSystems ◽  
2020 ◽  
Vol 5 (4) ◽  
Author(s):  
Wenjing Wu ◽  
Yu Feng ◽  
Zhiyong Zong
Keyword(s):  
Species Identification ◽  
Novel Species ◽  
Enterobacter Cloacae ◽  
Clinical Samples ◽  
Correct Identification ◽  
Content Type ◽  
A Genome ◽  
Enterobacter Hormaechei ◽  
Taxonomic Assignments ◽  
Genome Analyses

ABSTRACT The genus Enterobacter comprises common pathogens and has a complicated taxonomy. Precise taxonomic assignation lays a foundation for microbiology. In this study, we updated the Enterobacter taxonomy based on robust genome analyses. We found that all Enterobacter subspecies assignments were incorrect. Enterobacter cloacae subsp. dissolvens and Enterobacter hormaechei subsp. hoffmannii are species (Enterobacter dissolvens and Enterobacter hoffmannii, respectively) rather than subspecies. Enterobacter xiangfangensis, Enterobacter hormaechei subsp. oharae, and Enterobacter hormaechei subsp. steigerwaltii are not Enterobacter hormaechei subspecies but belong to the same species (Enterobacter xiangfangensis). Enterobacter timonensis should be removed to Pseudenterobacter, a novel genus. We then reported two novel species, Enterobacter quasiroggenkampii and Enterobacter quasimori, by genome- and phenotype-based characterization. We also applied the updated taxonomy to curate 1,997 Enterobacter genomes in GenBank. Species identification was changed following our updated taxonomy for the majority of publicly available strains (1,542, 77.2%). The most common Enterobacter species was E. xiangfangensis. We identified 14 novel tentative Enterobacter genomospecies. This study highlights that updated and curated taxonomic assignments are the premise of correct identification. IMPORTANCE Enterobacter species are major human pathogens. Precise species identification lays a foundation for microbiology, but the taxonomy of Enterobacter is complicated and confusing. In this study, first, we significantly updated the taxonomy of Enterobacter by rigorous genome analyses and found that all subspecies assignments of Enterobacter were incorrect. Second, we characterized and reported two novel Enterobacter species with clinical significance. Third, we curated 1,997 Enterobacter genome sequences deposited in GenBank and found that the species identification of most Enterobacter strains needed to be corrected. Fourth, we found that the most common Enterobacter species seen in clinical samples is Enterobacter xiangfangensis rather than Enterobacter cloacae. Fifth, we identified 14 tentative novel Enterobacter and 18 tentative novel non-Enterobacter species. This study highlights that updated and curated taxonomic assignments are the premise of correct species identification. We recommend that future Enterobacter studies need to use the updated taxonomy to avoid misleading information.

scholarly journals Evaluation of Whole-Genome Sequencing for Mycobacterial Species Identification and Drug Susceptibility Testing in a Clinical Setting: a Large-Scale Prospective Assessment of Performance against Line Probe Assays and Phenotyping

2017 ◽  
Vol 56 (2) ◽  
Author(s):  
T. Phuong Quan ◽  
Zharain Bawa ◽  
Dona Foster ◽  
Tim Walker ◽  
Carlos del Ojo Elias ◽  
...  
Keyword(s):  
Species Identification ◽  
Susceptibility Testing ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
Line Probe Assay ◽  
Mycobacterial Species ◽  
Sequencing Data ◽  
First Line ◽  
Content Type ◽  
Probe Assay

ABSTRACTUse of whole-genome sequencing (WGS) for routine mycobacterial species identification and drug susceptibility testing (DST) is becoming a reality. We compared the performances of WGS and standard laboratory workflows prospectively, by parallel processing at a major mycobacterial reference service over the course of 1 year, for species identification, first-lineMycobacterium tuberculosisresistance prediction, and turnaround time. Among 2,039 isolates with line probe assay results for species identification, 74 (3.6%) failed sequencing or WGS species identification. Excluding these isolates, clinically important species were identified for 1,902 isolates, of which 1,825 (96.0%) were identified as the same species by WGS and the line probe assay. A total of 2,157 line probe test results for detection of resistance to the first-line drugs isoniazid and rifampin were available for 728M. tuberculosiscomplex isolates. Excluding 216 (10.0%) cases where there were insufficient sequencing data for WGS to make a prediction, overall concordance was 99.3% (95% confidence interval [CI], 98.9 to 99.6%), sensitivity was 97.6% (91.7 to 99.7%), and specificity was 99.5% (99.0 to 99.7%). A total of 2,982 phenotypic DST results were available for 777M. tuberculosiscomplex isolates. Of these, 356 (11.9%) had no WGS comparator due to insufficient sequencing data, and in 154 (5.2%) cases the WGS prediction was indeterminate due to discovery of novel, previously uncharacterized mutations. Excluding these data, overall concordance was 99.2% (98.7 to 99.5%), sensitivity was 94.2% (88.4 to 97.6%), and specificity was 99.4% (99.0 to 99.7%). Median processing times for the routine laboratory tests versus WGS were similar overall, i.e., 20 days (interquartile range [IQR], 15 to 31 days) and 21 days (15 to 29 days), respectively (P= 0.41). In conclusion, WGS predicts species and drug susceptibility with great accuracy, but work is needed to increase the proportion of predictions made.

scholarly journals A New ESX-1 Substrate inMycobacterium marinumThat Is Required for Hemolysis but Not Host Cell Lysis

2019 ◽  
Vol 201 (14) ◽  
Author(s):  
Rachel E. Bosserman ◽  
Kathleen R. Nicholson ◽  
Matthew M. Champion ◽  
Patricia A. Champion
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Host Response ◽  
Secretion System ◽  
Mycobacterial Species ◽  
Macrophage Infection ◽  
Content Type ◽  
Family Protein ◽  
Species Specific ◽  
System 1 ◽  
Insight Into

ABSTRACTThe ESX-1 (ESAT-6 system 1) secretion system plays a conserved role in the virulence of diverse mycobacterial pathogens, including the human pathogenMycobacterium tuberculosisandM. marinum, an environmental mycobacterial species. The ESX-1 system promotes the secretion of protein virulence factors to the extracytoplasmic environment. The secretion of these proteins triggers the host response by lysing the phagosome during macrophage infection. Using proteomic analyses of theM. marinumsecretome in the presence and absence of a functional ESX-1 system, we and others have hypothesized that MMAR_2894, a PE family protein, is a potential ESX-1 substrate inM. marinum. We used genetic and quantitative proteomic approaches to determine if MMAR_2894 is secreted by the ESX-1 system, and we defined the requirement ofMMAR_2894for ESX-1-mediated secretion and virulence. We show that MMAR_2894 is secreted by the ESX-1 system inM. marinumand is itself required for the optimal secretion of the known ESX-1 substrates inM. marinum. Moreover, we found that MMAR_2894 was differentially required for hemolysis and cytolysis of macrophages, two lytic activities ascribed to theM. marinumESX-1 system.IMPORTANCEBothMycobacterium tuberculosis, the cause of human tuberculosis (TB), andMycobacterium marinum, a pathogen of ectotherms, use the ESX-1 secretion system to cause disease. There are many established similarities between the ESX-1 systems inM. tuberculosisand inM. marinum. Yet the two bacteria infect different hosts, hinting at species-specific functions of the ESX-1 system. Our findings demonstrate that MMAR_2894 is a PE protein secreted by the ESX-1 system ofM. marinum. We show that MMAR_2894 is required for the optimal secretion of mycobacterial proteins required for disease. Because theMMAR_2894gene is not conserved inM. tuberculosis, our findings demonstrate that MMAR_2894 may contribute to a species-specific function of the ESX-1 system inM. marinum, providing new insight into how theM. marinumandM. tuberculosissystems differ.

scholarly journals Aspergillus Collagen-Like Genes (acl): Identification, Sequence Polymorphism, and Assessment for PCR-Based Pathogen Detection

2013 ◽  
Vol 79 (24) ◽  
pp. 7882-7895 ◽  
Author(s):  
Kiril Tuntevski ◽  
Brandon C. Durney ◽  
Anna K. Snyder ◽  
P. Rocco LaSala ◽  
Ajay P. Nayak ◽  
...  
Keyword(s):  
Pathogen Detection ◽  
Morphological Characterization ◽  
Clinical Samples ◽  
Content Type ◽  
Bronchoalveolar Fluid ◽  
Immunosuppressed Patients ◽  
Amplicon Size ◽  
Species Specific ◽  
Ubiquitous Presence ◽  
Specific Conservation

ABSTRACTThe genusAspergillusis a burden to public health due to its ubiquitous presence in the environment, its production of allergens, and wide demographic susceptibility among cystic fibrosis, asthmatic, and immunosuppressed patients. Current methods of detection ofAspergilluscolonization and infection rely on lengthy morphological characterization or nonstandardized serological assays that are restricted to identifying a fungal etiology. Collagen-like genes have been shown to exhibit species-specific conservation across the noncollagenous regions as well as strain-specific polymorphism in the collagen-like regions. Here we assess the conserved region of theAspergilluscollagen-like (acl) genes and explore the application of PCR amplicon size-based discrimination among the five most common etiologic species of theAspergillusgenus, includingAspergillus fumigatus,A. flavus,A. nidulans,A. niger, andA. terreus. Genetic polymorphism and phylogenetic analysis of theaclF1gene were additionally examined among the available strains. Furthermore, the applicability of the PCR-based assay to identification of these five species in cultures derived from sputum and bronchoalveolar fluid from 19 clinical samples was explored. Application of capillary electrophoresis on nanogels was additionally demonstrated to improve the discrimination betweenAspergillusspecies. Overall, this study demonstrated thatAspergillusaclgenes could be used as PCR targets to discriminate between clinically relevantAspergillusspecies. Future studies aim to utilize the detection ofAspergillusaclgenes in PCR and microfluidic applications to determine the sensitivity and specificity for the identification ofAspergilluscolonization and invasive aspergillosis in immunocompromised subjects.

scholarly journals IP-MS Analysis of ESX-5 and ESX-1 Substrates Enables Mycobacterial Species Identification

2020 ◽  
Author(s):  
Qingbo Shu ◽  
Meena Rajagopal ◽  
Jia Fan ◽  
Lingpeng Zhan ◽  
Xiangxing Kong ◽  
...  
Keyword(s):  
Clinical Samples ◽  
Mycobacterial Species ◽  
The Past ◽  
Indicator Tube ◽  
Mgit Culture ◽  
Species Specific ◽  
Mycobacterial Growth ◽  
Slow Growing ◽  
Mycobacterial Growth Indicator Tube ◽  
Growth Indicator

AbstractPulmonary disease resulting from non-tuberculous mycobacteria (NTM) infection has emerged as an increasingly prevalent clinical entity in the past two to three decades, but there are no standardized, commercial assays available for the molecular diagnosis of NTM infections from clinical samples. Herein we discuss the development of an assay that employs immunoprecipitation coupled with mass spectrometry (IP-MS) to rapidly and accurately discriminate prevalent slow-growing mycobacterial species (i.e., M. avium and M. intracellulare, M. kansasii, M. gordonae, M. marinum and M. tuberculosis) during early growth in mycobacterial growth indicator tube (MGIT) cultures. This IP-MS assay employs antibodies specific for conserved tryptic peptides of M. tuberculosis EsxN (AQAASLEAEHQAIVR) and CFP-10 (TQIDQVESTAGSLQGQWR) to capture and identify specific mycobacterial species and allows species-specific mycobacterium identification at the first sign of MGIT culture positivity.

scholarly journals Cas12a/Guide RNA-Based Platform for Rapid and Accurate Identification of Major Mycobacterium Species

2019 ◽  
Vol 58 (2) ◽  
Author(s):  
Guohui Xiao ◽  
Xing He ◽  
Su Zhang ◽  
Yaya Liu ◽  
Zhihang Liang ◽  
...  
Keyword(s):  
Clinical Symptoms ◽  
Cost Effective ◽  
Cross Reactivity ◽  
Mycobacterium Abscessus ◽  
Mycobacterium Fortuitum ◽  
Accurate Identification ◽  
Content Type ◽  
Guide Rna ◽  
Promising Tool ◽  
Species Specific

ABSTRACT Mycobacterium tuberculosis infection and nontuberculous mycobacteria (NTM) infections exhibit similar clinical symptoms; however, the therapies for these two types of infections are different. Therefore, the rapid and accurate identification of M. tuberculosis and NTM species is very important for the control of tuberculosis and NTM infections. In the present study, a Cas12a/guide RNA (gRNA)-based platform was developed to identify M. tuberculosis and most NTM species. By designing species-specific gRNA probes targeting the rpoB sequence, a Cas12a/gRNA-based platform successfully identified M. tuberculosis and six major NTM species (Mycobacterium abscessus, Mycobacterium intracellulare, Mycobacterium avium, Mycobacterium kansasii, Mycobacterium gordonae, and Mycobacterium fortuitum) without cross-reactivity. In a blind assessment, a total of 72 out of 73 clinical Mycobacterium isolates were correctly identified, which is consistent with previous rpoB sequencing results. These results suggest that the Cas12a/gRNA-based platform is a promising tool for the rapid, accurate, and cost-effective identification of both M. tuberculosis and NTM species.

scholarly journals The Role of fosA in Challenges with Fosfomycin Susceptibility Testing of Multispecies Klebsiella pneumoniae Carbapenemase-Producing Clinical Isolates

2019 ◽  
Vol 57 (10) ◽  
Author(s):  
Zachary S. Elliott ◽  
Katie E. Barry ◽  
Heather L. Cox ◽  
Nicole Stoesser ◽  
Joanne Carroll ◽  
...  
Keyword(s):  
Susceptibility Testing ◽  
Clinical Laboratory ◽  
Multidrug Resistant ◽  
Clinical Isolates ◽  
Clinical Microbiology Laboratory ◽  
Content Type ◽  
Klebsiella Pneumoniae Carbapenemase ◽  
Agar Dilution ◽  
Species Specific ◽  
The Impact

ABSTRACT With multidrug-resistant (MDR) Enterobacterales on the rise, a nontoxic antimicrobial agent with a unique mechanism of action such as fosfomycin seems attractive. However, establishing accurate fosfomycin susceptibility testing for non-Escherichia coli isolates in a clinical microbiology laboratory remains problematic. We evaluated fosfomycin susceptibility by multiple methods with 96 KPC-producing clinical isolates of multiple strains and species collected at a single center between 2008 and 2016. In addition, we assessed the presence of fosfomycin resistance genes from whole-genome sequencing (WGS) data using NCBI’s AMRFinder and custom HMM search. Susceptibility testing was performed using a glucose-6-phosphate-supplemented fosfomycin Etest and Kirby-Bauer disk diffusion (DD) assays, and the results were compared to those obtained by agar dilution. Clinical Laboratory and Standards Institute (CLSI) breakpoints for E. coli were applied for interpretation. Overall, 63% (60/96) of isolates were susceptible by Etest, 70% (67/96) by DD, and 88% (84/96) by agar dilution. fosA was detected in 80% (70/88) of previously sequenced isolates, with species-specific associations and alleles, and fosA-positive isolates were associated with higher MIC distributions. Disk potentiation testing was performed using sodium phosphonoformate to inhibit fosA and showed significant increases in the zone diameter of DD testing for isolates that were fosA positive compared to those that were fosA negative. The addition of sodium phosphonoformate (PPF) corrected 10/14 (71%) major errors in categorical agreement with agar dilution. Our results indicate that fosA influences the inaccuracy of susceptibility testing by methods readily available in a clinical laboratory compared to agar dilution. Further research is needed to determine the impact of fosA on clinical outcomes.

scholarly journals Mutations in fbiD (Rv2983) as a Novel Determinant of Resistance to Pretomanid and Delamanid in Mycobacterium tuberculosis

2020 ◽  
Vol 65 (1) ◽  
pp. e01948-20
Author(s):  
Dalin Rifat ◽  
Si-Yang Li ◽  
Thomas Ioerger ◽  
Keshav Shah ◽  
Jean-Philippe Lanoix ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Clinical Evidence ◽  
Clinical Samples ◽  
Loss Of Function ◽  
Wild Type ◽  
Content Type ◽  
Solid Media ◽  
High Level ◽  
Level Resistance

ABSTRACTThe nitroimidazole prodrugs delamanid and pretomanid comprise one of only two new antimicrobial classes approved to treat tuberculosis (TB) in 50 years. Prior in vitro studies suggest a relatively low barrier to nitroimidazole resistance in Mycobacterium tuberculosis, but clinical evidence is limited to date. We selected pretomanid-resistant M. tuberculosis mutants in two mouse models of TB using a range of pretomanid doses. The frequency of spontaneous resistance was approximately 10−5 CFU. Whole-genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, of which 91% occurred in 1 of 5 genes previously associated with nitroimidazole activation and resistance, namely, fbiC (56%), fbiA (15%), ddn (12%), fgd (4%), and fbiB (4%). Nearly all mutations were unique to a single mouse and not previously identified. The remaining 9% of resistant mutants harbored mutations in Rv2983 (fbiD), a gene not previously associated with nitroimidazole resistance but recently shown to be a guanylyltransferase necessary for cofactor F420 synthesis. Most mutants exhibited high-level resistance to pretomanid and delamanid, although Rv2983 and fbiB mutants exhibited high-level pretomanid resistance but relatively small changes in delamanid susceptibility. Complementing an Rv2983 mutant with wild-type Rv2983 restored susceptibility to pretomanid and delamanid. By quantifying intracellular F420 and its precursor Fo in overexpressing and loss-of-function mutants, we provide further evidence that Rv2983 is necessary for F420 biosynthesis. Finally, Rv2983 mutants and other F420H2-deficient mutants displayed hypersusceptibility to some antibiotics and to concentrations of malachite green found in solid media used to isolate and propagate mycobacteria from clinical samples.

Collaborative design in public services: a wicked problem-reframing case

2020 ◽  
Vol ahead-of-print (ahead-of-print) ◽  
Author(s):  
Patrícia Moura e Sá ◽  
Catarina Frade ◽  
Fernanda Jesus ◽  
Mónica Lopes ◽  
Teresa Maneca Lima ◽  
...  
Keyword(s):  
Collaborative Design ◽  
Success Factors ◽  
Central Government ◽  
Wicked Problem ◽  
Low Density ◽  
Structured Interviews ◽  
Content Type ◽  
Critical Success ◽  
Definition Of ◽  
The Right

PurposeWicked problems require collaborative innovation approaches. Understanding the problem from the users' perspective is essential. Based on a complex and ill-defined case, the purpose of the current paper is to identify some critical success factors in defining the “right problem” to be addressed.Design/methodology/approachAn empirical research study was carried out in a low-density municipality (case study). Extensive data were collected from official databases, individual semi-structured interviews and a focus group involving citizens, local authorities, civil servants and other relevant stakeholders.FindingsAs defined by the central government, the problem to be addressed by the research team was to identify which justice services should be made available locally to a small- and low-density community. The problem was initially formulated using top-down reasoning. In-depth contact with citizens and key local players revealed that the lack of justice services was not “the issue” for that community. Mobility constraints and the shortage of economic opportunities had a considerable impact on the lack of demand for justice services. By using a bottom-up perspective, it was possible to reframe the problem to be addressed and suggest a new concept to be tested at later stages.Social implicationsThe approach followed called attention to the importance of listening to citizens and local organisations with a profound knowledge of the territory to effectively identify and circumscribe a local problem in the justice field.Originality/valueThe paper highlights the limitations of traditional rational problem-solving approaches and contributes to expanding the voice-of-the-customer principle showing how it can lead to a substantially new definition of the problem to be addressed.

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