scholarly journals Characterization of Pseudomonas aeruginosa isolates from patients with endophthalmitis using conventional microbiologic techniques and whole genome sequencing

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Jesse D. Sengillo ◽  
Jacob Duker ◽  
Maribel Hernandez ◽  
Jorge Maestre ◽  
Daniela Reyes-Capo ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Antibiotic Susceptibility ◽  
Virulence Genes ◽  
Susceptibility Testing ◽  
Secretion System ◽  
Whole Genome ◽  
Drug Resistant

Abstract Purpose To demonstrate antibiotic susceptibility and genomic virulence factor profiles of Pseudomonas aeruginosa isolates from patients with culture-confirmed endophthalmitis. Methods Clinical isolates from patients diagnosed with pseudomonas endophthalmitis were included. Laboratory antibiotic susceptibility testing and whole genome sequencing was performed on all isolates. Results In the current study, 8 patients had vitreous culture-confirmed endophthalmitis due to P. aeruginosa. All isolates were multi-drug resistant but sensitive to ceftazidime and each fluoroquinolone tested. Whole genome sequencing revealed a total of 179 unique genes. The most common type of virulence genes included those involved in adherence and the secretion system. Seven of 8 (88%) isolates were of the cytoinvasive phenotype (exoST) and no isolates contained exoU. Conclusions P. aeruginosa associated endophthalmitis is often multi-drug resistant and demonstrates a variety of virulence factors with those involved in adherence and the secretion system being the most common.

scholarly journals Whole Genome Sequencing Based Characterization of Extensively Drug-Resistant Mycobacterium tuberculosis Isolates from Pakistan

PLoS ONE ◽  
2015 ◽  
Vol 10 (2) ◽  
pp. e0117771 ◽  
Author(s):  
Asho Ali ◽  
Zahra Hasan ◽  
Ruth McNerney ◽  
Kim Mallard ◽  
Grant Hill-Cawthorne ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Whole Genome ◽  
Drug Resistant ◽  
Extensively Drug Resistant

scholarly journals Mortality of Drug-resistant Tuberculosis in High-burden Countries: Comparison of Routine Drug Susceptibility Testing with Whole-genome Sequencing

2020 ◽  
Author(s):  
Martina L. Reichmuth ◽  
Kathrin Zürcher ◽  
Marie Ballif ◽  
Chloé Loiseau ◽  
Sonia Borrell ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Susceptibility Testing ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
Whole Genome ◽  
Drug Resistant ◽  
Hiv Status ◽  
High Burden

AbstractBackgroundDrug-resistant Mycobacterium tuberculosis (Mtb) strains threaten tuberculosis (TB) control. We compared data on drug resistance obtained at clinics in seven high TB burden countries during routine care with whole-genome sequencing (WGS) carried out centrally.MethodsWe collected pulmonary Mtb isolates and clinical data from adult TB patients in Africa, Latin America, and Asia, stratified by HIV status and drug resistance, from 2013 to 2016. Participating sites performed drug susceptibility testing (DST) locally, using routinely available methods. WGS was done using Illumina HiSeq 2500 at laboratories in the USA and Switzerland. We used TBprofiler to analyse the genomes. We used multivariable logistic regression adjusted for sex, age, HIV-status, history of TB, sputum positivity, and Mtb-lineage to analyse mortality.FindingsWe included 582 TB patients. The median age was 32 years (interquartile range: 27-43 years), 225 (39%) were female, and 247 (42%) were HIV-positive. Based on WGS, 339 (58%) isolates were pan-susceptible, 35 (6%) monoresistant, 146 (25%) multidrug-resistant, and 24 (4%) pre-/ extensively drug-resistant (pre-XDR/XDR-TB). The local DST results were discordant compared to WGS results in 130/582 (22%) of patients. All testing methods identified isoniazid and rifampicin resistance with relatively high agreement (kappa 0.69 for isoniazid and 0.88 rifampicin). Resistance to ethambutol, pyrazinamide, and second-line drugs was rarely tested locally. Of 576 patients with known treatment, 86 (15%) patients received inadequate treatment according to WGS results and the World Health Organization treatment guidelines. The analysis of mortality was based on 530 patients; 63 patients (12%) died and 77 patients (15%) received inadequate treatment. Mortality ranged from 6% in patients with pan-susceptible Mtb (18/310) to 39% in patients with pre-XDR/XDR-TB (9/23). The adjusted odds ratio for mortality was 4.82 (95% CI 2.43-9.44) for under-treatment and 0.52 (95% CI 0.03-2.73) for over-treatment.InterpretationIn seven high-burden TB countries, we observed discrepancies between drug resistance patterns from local DST and WGS, which resulted in inadequate treatment and higher mortality. WGS can provide accurate and detailed drug resistance information, which is required to improve the outcomes of drug-resistant TB in high burden settings. Our results support the WHO’s call for point-of-care tests based on WGS.

scholarly journals Drug susceptibility testing of Mycobacterium tuberculosis using next generation sequencing and Mykrobe software

2022 ◽  
Vol 98 (6) ◽  
pp. 697-705
Author(s):  
V. Tolchkov ◽  
Y. Hodzhev ◽  
B. Tsafarova ◽  
E. Bachiyska ◽  
Yu. Atanasova ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Susceptibility Testing ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
Whole Genome ◽  
Drug Resistant ◽  
Molecular Tests ◽  
Bactec System ◽  
Probe Assay

Introduction. Mycobacterium tuberculosis is the causative agent of tuberculosis. Drug susceptibility testing is performed by phenotypic and molecular tests. Commonly used for phenotypic drug susceptibility testing is the automated BACTEC system in a liquid culture medium. Drug susceptibility by line probe molecular tests was introduced almost 15 years ago. Recently whole genome sequencing (WGS) analysis of M. tuberculosis strains demonstrated that genotyping of drug-resistance could be accurately performed. Several software tools were developed.Our study aimed to perform whole-genome sequencing on phenotypically confirmed multi-drug resistant (MDR) M. tuberculosis strains, to identify drug-resistant mutations and to compare whole-genome sequencing profiles with line probe assay and phenotypic results.Materials and methods. We performed analysis on 34 MDR M. tuberculosis Bulgarian strains. Phenotypic drug susceptibility testing was performed on the BACTEC system. For molecular testing of drug susceptibility to first- and second-line tuberculostatics, we applied line probe assay Geno Type MTBDR plus v.1.0 и Geno Type MTBDR sl v.1.0. Sequencing was performed on MiSeq. Generated FASTQ files were analyzed for known drugresistant mutations with the software platform Mykrobe v.0.8.1.Results. All three methods — phenotypic analysis using the BACTEC system, genetic analysis of strains applying the Geno Type test and Mykrobe software gave comparable sensitivity/resistance results for the studied strains. All phenotypically proven rifampicin and isoniazid-resistant strains were 100% confirmed using Mykrobe software. The C-15T mutation is a marker for isoniazid resistance in strains of the SIT41 spoligotype. We observed a 75% (21/28) agreement between BACTEC and Mykrobe for ethambutol resistance. Phenotypically, 87% (n = 27) of the strains are resistant to streptomycin, but only 59% (n = 19) are proven by Mykrobe software. Comparing phenotypic and genotypic resistance to ofloxacin, amikacin and kanamycin, we observed 100% coincidence of results.Conclusions. Whole-genome sequencing approach is relatively expensive and laborious but useful for detailed analysis such as epidemiological genotyping and molecular drug susceptibility testing.

scholarly journals Characterization of mcr-1-Harboring Plasmids from Pan Drug-Resistant Escherichia coli Strains Isolated from Retail Raw Chicken in South Korea

2019 ◽  
Vol 7 (9) ◽  
pp. 344 ◽  
Author(s):  
Jinshil Kim ◽  
Bo Kyoung Hwang ◽  
HyeLim Choi ◽  
Yang Wang ◽  
Sang Ho Choi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
South Korea ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Pathogenic Bacteria ◽  
Fluoroquinolone Resistance ◽  
Whole Genome ◽  
Drug Resistant ◽  
E Coli

A number of studies from different countries have characterized mcr-1-harboring plasmids isolated from food; however, nothing has been reported about it in South Korea. In this study, we report the characterization of mcr-1 plasmids from pan drug-resistant (PDR) Escherichia coli strains isolated from retail food in the country. Colistin-resistant E. coli strains were isolated from retail raw chicken, and PCR was carried out to detect the mcr-1 gene. Whole genome sequencing of the mcr-1-positive strains was performed for further characterization. The results of whole genome sequencing revealed that all mcr-1 plasmids belonged to the IncI2 type. In addition to the mcr-1 plasmids, all of the isolates also carried additional plasmids possessing multiple antibiotic resistance genes, and the PDR was mediated by resistant plasmids except for fluoroquinolone resistance resulting from mutations in gyrA and parC. Interestingly, the mcr-1 plasmids were transferred by conjugation to other pathogenic strains including enterohemorrhagic E. coli (EHEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), Salmonella, and Klebsiella at the frequencies of 10−3−10−6, 10−2−10−5, 10−4−10−5, 10−4−10−6, and 10−5−10−6, respectively. The results showed that mcr-1 plasmids can be easily transmitted to pathogenic bacteria by conjugation.

scholarly journals In Silico Genotyping of Escherichia coli Isolates for Extraintestinal Virulence Genes by Use of Whole-Genome Sequencing Data

2020 ◽  
Vol 58 (10) ◽  
Author(s):  
Anna Maria Malberg Tetzschner ◽  
James R. Johnson ◽  
Brian D. Johnston ◽  
Ole Lund ◽  
Flemming Scheutz
Keyword(s):  
Escherichia Coli ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
In Silico ◽  
Virulence Genes ◽  
Whole Genome Sequencing Data ◽  
Whole Genome ◽  
Content Type ◽  
E Coli

ABSTRACT Extraintestinal pathogenic Escherichia coli (ExPEC) is the leading cause in humans of urinary tract infection and bacteremia. The previously published web tool VirulenceFinder (http://cge.cbs.dtu.dk/services/VirulenceFinder/) uses whole-genome sequencing (WGS) data for in silico characterization of E. coli isolates and enables researchers and clinical health personnel to quickly extract and interpret virulence-relevant information from WGS data. In this study, 38 ExPEC-associated virulence genes were added to the existing E. coli VirulenceFinder database. In total, 14,441 alleles were downloaded. A total of 1,890 distinct alleles were added to the database after removal of redundant sequences and analysis of the remaining alleles for open reading frames (ORFs). The database now contains 139 genes—of which 44 are related to ExPEC—and 2,826 corresponding alleles. Construction of the database included validation against 27 primer pairs from previous studies, a search for serotype-specific P fimbriae papA alleles, and a BLASTn confirmation of seven genes (etsC, iucC, kpsE, neuC, sitA, tcpC, and terC) not covered by the primers. The augmented database was evaluated using (i) a panel of nine control strains and (ii) 288 human-source E. coli strains classified by PCR as ExPEC and non-ExPEC. We observed very high concordance (average, 93.4%) between PCR and WGS findings, but WGS identified more alleles. In conclusion, the addition of 38 ExPEC-associated genes and the associated alleles to the E. coli VirulenceFinder database allows for a more complete characterization of E. coli isolates based on WGS data, which has become increasingly important considering the plasticity of the E. coli genome.

scholarly journals Expanding U.S. Laboratory Capacity for Neisseria gonorrhoeae Antimicrobial Susceptibility Testing and Whole-Genome Sequencing through the CDC's Antibiotic Resistance Laboratory Network

2020 ◽  
Vol 58 (4) ◽  
Author(s):  
Ellen N. Kersh ◽  
Cau D. Pham ◽  
John R. Papp ◽  
Robert Myers ◽  
Richard Steece ◽  
...  
Keyword(s):  
Public Health ◽  
Antibiotic Resistance ◽  
Neisseria Gonorrhoeae ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Antimicrobial Susceptibility ◽  
Susceptibility Testing ◽  
Whole Genome ◽  
Content Type ◽  
Laboratory Capacity

ABSTRACT U.S. gonorrhea rates are rising, and antibiotic-resistant Neisseria gonorrhoeae (AR-Ng) is an urgent public health threat. Since implementation of nucleic acid amplification tests for N. gonorrhoeae identification, the capacity for culturing N. gonorrhoeae in the United States has declined, along with the ability to perform culture-based antimicrobial susceptibility testing (AST). Yet AST is critical for detecting and monitoring AR-Ng. In 2016, the CDC established the Antibiotic Resistance Laboratory Network (AR Lab Network) to shore up the national capacity for detecting several resistance threats including N. gonorrhoeae. AR-Ng testing, a subactivity of the CDC’s AR Lab Network, is performed in a tiered network of approximately 35 local laboratories, four regional laboratories (state public health laboratories in Maryland, Tennessee, Texas, and Washington), and the CDC’s national reference laboratory. Local laboratories receive specimens from approximately 60 clinics associated with the Gonococcal Isolate Surveillance Project (GISP), enhanced GISP (eGISP), and the program Strengthening the U.S. Response to Resistant Gonorrhea (SURRG). They isolate and ship up to 20,000 isolates to regional laboratories for culture-based agar dilution AST with seven antibiotics and for whole-genome sequencing of up to 5,000 isolates. The CDC further examines concerning isolates and monitors genetic AR markers. During 2017 and 2018, the network tested 8,214 and 8,628 N. gonorrhoeae isolates, respectively, and the CDC received 531 and 646 concerning isolates and 605 and 3,159 sequences, respectively. In summary, the AR Lab Network supported the laboratory capacity for N. gonorrhoeae AST and associated genetic marker detection, expanding preexisting notification and analysis systems for resistance detection. Continued, robust AST and genomic capacity can help inform national public health monitoring and intervention.

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