2165. Helicobacter pylori Infections in the Bronx, New York: Whole-Genome Sequencing for Rapid Genotypic Susceptibility Testing

Abstract Background Susceptibility-guided treatment of H. pylori is superior to empiric therapy. We determined the accuracy of whole-genome sequencing (WGS) compared with phenotypic testing using CLSI/EUCAST breakpoints. Methods Thirty-three clinical isolates of H. pylori cultured from gastric biopsies were sequenced with a coverage range between 40x and 80x using Illumina Miseq platform and the reads were assembled and annotated with PATRIC. Phenotypic susceptibility tests were performed using E-test strips under microaerophilic conditions for 72 hours. Mutations associated with amoxicillin, tetracycline, clarithromycin, levofloxacin, metronidazole and rifampin resistance were examined. Results Of the 33 isolates, two were phenotypically resistant to amoxicillin: one carried a β-lactamase gene (blaTEM-116) and the other exhibited a point mutation pbp2 (A541T). All isolates were tetracycline susceptible phenotypically, but three isolates had point mutations in 16S rRNA that are associated with resistance (A926G). Clarithromycin results showed a good correlation between methods. Nine clarithromycin-resistant isolates demonstrated point mutations in 23S rRNA (A2142G/A2143G). Fifteen isolates were phenotypically resistant to levofloxacin, but resistance mutations were found in only 14 strains (N87I/N87K/D91Y/D91N/D91G/D99N in gyrA). We analyzed our strains for the presence of intact genes rdxA and frxA, either of which convert the prodrug form of metronidazole into the active form. Twenty-four of 33 isolates were tested phenotypically. We found 3 isolates with truncations in both genes. These isolates had metronidazole MICs >256. The presence of one or both intact genes did not always result in low MICs, indicating that there may be significant point mutations that contribute to resistance. Rifampin was not tested phenotypically, but no mutations in rpoB were found. In summary, the correlation of WGS and phenotypic testing was 100% for amoxicillin and clarithromycin, 97% for levofloxacin, 91% for tetracycline (n = 33), and 67% for metronidazole (n = 24). Conclusion WGS provides a detailed analysis of H. pylori resistance and a broader analysis of antimicrobials that may be of clinical value. Additional studies are needed for genotypic prediction of metronidazole resistance. Disclosures All authors: No reported disclosures.

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Helicobacter pylori Infections in the Bronx, New York: Surveying Antibiotic Susceptibility and Strain Lineage by Whole-Genome Sequencing

Journal of Clinical Microbiology ◽

10.1128/jcm.01591-19 ◽

2019 ◽

Vol 58 (3) ◽

Cited By ~ 2

Author(s):

Rajagopalan Saranathan ◽

Michael H. Levi ◽

Alice R. Wattam ◽

Adel Malek ◽

Emmanuel Asare ◽

...

Keyword(s):

Helicobacter Pylori ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Molecular Mechanisms ◽

Kappa Coefficient ◽

23S Rrna ◽

Whole Genome ◽

Content Type ◽

Phenotypic Resistance ◽

H Pylori

ABSTRACT The emergence of drug resistance in Helicobacter pylori has resulted in a greater need for susceptibility-guided treatment. While the alleles associated with resistance to clarithromycin and levofloxacin have been defined, there are limited data regarding the molecular mechanisms underlying resistance to other antimicrobials. Using H. pylori isolates from 42 clinical specimens, we compared phenotypic and whole-genome sequencing (WGS)-based detection of resistance. Phenotypic resistance correlated with the presence of alleles of 23S rRNA (A2142G/A2143G) for clarithromycin (kappa coefficient, 0.84; 95% confidence interval [CI], 0.67 to 1.0) and gyrA (N87I/N87K/D91Y/D91N/D91G/D99N) for levofloxacin (kappa coefficient, 0.90; 95% CI, 0.77 to 1.0). Phenotypic resistance to amoxicillin in three isolates correlated with mutations in pbp1, pbp2, and/or pbp3 within coding regions near known amoxicillin binding motifs. All isolates were phenotypically susceptible to tetracycline, although four bore a mutation in 16S rRNA (A926G). For metronidazole, nonsense mutations and R16H substitutions in rdxA correlated with phenotypic resistance (kappa coefficient, 0.76; 95% CI, 0.56 to 0.96). Previously identified mutations in the rpoB rifampin resistance-determining region (RRDR) were not present, but 14 novel mutations outside the RRDR were found in rifampin-resistant isolates. WGS also allowed for strain lineage determination, which may be important for future studies in associating precise MICs with specific resistance alleles. In summary, WGS allows for broad analyses of H. pylori isolates, and our findings support the use of WGS for the detection of clarithromycin and levofloxacin resistance. Additional studies are warranted to better define mutations conferring resistance to amoxicillin, tetracycline, and rifampin, but combinatorial analyses for rdxA gene truncations and R16H mutations have utility for determining metronidazole resistance.

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Phenotypic and Genotypic Antibiotic Resistance Patterns in Helicobacter pylori Strains From Ethnically Diverse Population in México

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2020.539115 ◽

2021 ◽

Vol 10 ◽

Author(s):

Margarita Camorlinga-Ponce ◽

Alejandro Gómez-Delgado ◽

Emmanuel Aguilar-Zamora ◽

Roberto C. Torres ◽

Silvia Giono-Cerezo ◽

...

Keyword(s):

Helicobacter Pylori ◽

Antibiotic Resistance ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Indigenous Communities ◽

Drug Susceptibility ◽

23S Rrna ◽

Whole Genome ◽

Phenotypic Resistance ◽

H Pylori

Helicobacter pylori strains carry a range of mutations in genes that confer antimicrobial resistance and restrict the available options to treat the infection. Latin America is a region that conserve a large number of indigenous communities relatively isolated that practice a traditional medicine without consumption of drugs. We hypothesized that rates of antibiotic resistance are lower in these communities. Recent progress in whole-genome sequencing has allowed the study of drug susceptibility by searching for the known mutations associated with antibiotic resistance. The aim of this work was to study trends of antibiotic resistance over a 20-year period in Mexican H. pylori strains and to compare susceptibility between strains from Mexican mestizos and from indigenous population; we also aimed to learn the prevalence of mutational patterns in genes gyrA, gyrB, rdxA, frxA, rpsU, omp11, dppA, and 23S rRNA and its association with phenotypic tests. Resistance to clarithromycin, metronidazole, amoxicillin and levofloxacin was determined in167 H. pylori isolates by E-test, and the occurrence of mutational patterns in specific genes was determined by whole genome sequencing (WGS). The trend of resistance over 20 years in mestizo isolates showed significant resistant increase for clarithromycin and levofloxacin to frequencies that banned its clinical use. Resistance in H. pylori isolates of native communities was lower for all antibiotics tested. Phenotypic resistance showed good to moderate correlation with genotypic tests. Genetic methods for characterizing antibiotic resistance require further validation in each population.

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Prediction of antimicrobial resistance in clinical Campylobacter jejuni isolates from whole-genome sequencing data

European Journal of Clinical Microbiology & Infectious Diseases ◽

10.1007/s10096-020-04043-y ◽

2020 ◽

Author(s):

Louise Gade Dahl ◽

Katrine Grimstrup Joensen ◽

Mark Thomas Østerlund ◽

Kristoffer Kiil ◽

Eva Møller Nielsen

Keyword(s):

Antimicrobial Resistance ◽

Whole Genome Sequencing ◽

Campylobacter Jejuni ◽

Genome Sequencing ◽

Resistance Genes ◽

Point Mutations ◽

23S Rrna ◽

Whole Genome Sequencing Data ◽

Whole Genome ◽

Sequencing Data

Abstract Campylobacter jejuni is recognised as the leading cause of bacterial gastroenteritis in industrialised countries. Although the majority of Campylobacter infections are self-limiting, antimicrobial treatment is necessary in severe cases. Therefore, the development of antimicrobial resistance (AMR) in Campylobacter is a growing public health challenge and surveillance of AMR is important for bacterial disease control. The aim of this study was to predict antimicrobial resistance in C. jejuni from whole-genome sequencing data. A total of 516 clinical C. jejuni isolates collected between 2014 and 2017 were subjected to WGS. Resistance phenotypes were determined by standard broth dilution, categorising isolates as either susceptible or resistant based on epidemiological cutoffs for six antimicrobials: ciprofloxacin, nalidixic acid, erythromycin, gentamicin, streptomycin, and tetracycline. Resistance genotypes were identified using an in-house database containing reference genes with known point mutations and the presence of resistance genes was determined using the ResFinder database and four bioinformatical methods (modified KMA, ABRicate, ARIBA, and ResFinder Batch Upload). We identified seven resistance genes including tet(O), tet(O/32/O), ant(6)-Ia, aph(2″)-If, blaOXA, aph(3′)-III, and cat as well as mutations in three genes: gyrA, 23S rRNA, and rpsL. There was a high correlation between phenotypic resistance and the presence of known resistance genes and/or point mutations. A correlation above 98% was seen for all antimicrobials except streptomycin with a correlation of 92%. In conclusion, we found that WGS can predict antimicrobial resistance with a high degree of accuracy and have the potential to be a powerful tool for AMR surveillance.

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Whole Genome Sequencing and Antimicrobial Resistance of Staphylococcus aureus from Surgical Site Infections in Ghana

Pathogens ◽

10.3390/pathogens10020196 ◽

2021 ◽

Vol 10 (2) ◽

pp. 196

Author(s):

Beverly Egyir ◽

Jeannette Bentum ◽

Naiki Attram ◽

Anne Fox ◽

Noah Obeng-Nkrumah ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Meca Gene ◽

Surgical Site Infections ◽

Illumina Miseq ◽

Multi Drug Resistance ◽

Toxin Gene ◽

Whole Genome ◽

Flight Mass Spectrometry

Staphylococcus aureus (S. aureus) is a common cause of surgical site infections (SSIs) globally. Data on the occurrence of methicillin-susceptible S. aureus (MSSA) as well as methicillin-resistant S. aureus (MRSA) among patients with surgical site infections (SSIs) in sub-Saharan African are scarce. We characterized S. aureus from SSIs in Ghana using molecular methods and antimicrobial susceptibility testing (AST). Wound swabs or aspirate samples were collected from subjects with SSIs. S. aureus was identified by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF-MS); AST was performed by Kirby-Bauer disk diffusion, and results were interpreted according to the Clinical and Laboratory Standards Institute (CLSI) guideline. Detection of spa, mecA, and pvl genes was performed by polymerase chain reaction (PCR). Whole-genome sequencing (WGS) was done using the Illumina MiSeq platform. Samples were collected from 112 subjects, with 13 S. aureus isolates recovered. Of these, 92% were sensitive to co-trimoxazole, 77% to clindamycin, and 54% to erythromycin. Multi-drug resistance was detected in 5 (38%) isolates. The four mecA gene-positive MRSA isolates detected belonged to ST152 (n = 3) and ST5 (n = 1). In total, 62% of the isolates were positive for the Panton-Valentine leukocidin (pvl) toxin gene. This study reports, for the first time, a pvl-positive ST152-t355 MRSA clone from SSIs in Ghana. The occurrence of multi-drug-resistant S. aureus epidemic clones suggests that continuous surveillance is required to monitor the spread and resistance trends of S. aureus in hospital settings in the country.

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Whole-Genome Sequencing and Comparative Genomics of Three Helicobacter pylori Strains Isolated from the Stomach of a Patient with Adenocarcinoma

Pathogens ◽

10.3390/pathogens10030331 ◽

2021 ◽

Vol 10 (3) ◽

pp. 331

Author(s):

Montserrat Palau ◽

Núria Piqué ◽

M. José Ramírez-Lázaro ◽

Sergio Lario ◽

Xavier Calvet ◽

...

Keyword(s):

Helicobacter Pylori ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Virulence Factors ◽

Outer Membrane Proteins ◽

Whole Genome ◽

Flagellar Biosynthesis ◽

H Pylori ◽

Restriction Modification

Helicobacter pylori is a common pathogen associated with several severe digestive diseases. Although multiple virulence factors have been described, it is still unclear the role of virulence factors on H. pylori pathogenesis and disease progression. Whole genome sequencing could help to find genetic markers of virulence strains. In this work, we analyzed three complete genomes from isolates obtained at the same point in time from a stomach of a patient with adenocarcinoma, using multiple available bioinformatics tools. The genome analysis of the strains B508A-S1, B508A-T2A and B508A-T4 revealed that they were cagA, babA and sabB/hopO negative. The differences among the three genomes were mainly related to outer membrane proteins, methylases, restriction modification systems and flagellar biosynthesis proteins. The strain B508A-T2A was the only one presenting the genotype vacA s1, and had the most distinct genome as it exhibited fewer shared genes, higher number of unique genes, and more polymorphisms were found in this genome. With all the accumulated information, no significant differences were found among the isolates regarding virulence and origin of the isolates. Nevertheless, some B508A-T2A genome characteristics could be linked to the pathogenicity of H. pylori.

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Generating 1200bp tiled Amplicons for SARS-CoV2 Whole Genome Sequencing v2

10.17504/protocols.io.b3viqn4e ◽

2022 ◽

Author(s):

jason.nguyen not provided ◽

Tracy Lee ◽

Rebecca Hickman ◽

Natalie Prystajecky ◽

John Tyson

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Illumina Miseq ◽

Whole Genome ◽

Oxford Nanopore ◽

Sequencing Platforms

This procedure provides instructions for how to generate amplicons across the entire SARS-CoV-2 genome to be used for downstream whole genome sequencing applications, including Illumina MiSeq/NextSeq or Oxford Nanopore MinION sequencing platforms. The steps involved in this protocol were derived from version 3 of Freed et al protocol nCoV-2019 sequencing protocol (RAPID barcoding, 1200bp amplicon)V.3 available at https://dx.doi.org/10.17504/protocols.io.bgggjttw

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Transmission of Multidrug/Rifampicin-Resistant Mycobacterium Tuberculosis in Chongqing, China: A Retrospective Observational Study Using Whole-Genome Sequencing

10.21203/rs.3.rs-717466/v1 ◽

2021 ◽

Author(s):

Bing Zhao ◽

Chunfa Liu ◽

Jiale Fan ◽

Aijing Ma ◽

Wencong He ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Whole Genome ◽

Nucleotide Polymorphisms ◽

Resistance Mutations ◽

Treatment Information ◽

Recent Transmission ◽

Drugs Analysis ◽

Lineage 2 ◽

And Cluster Analysis

Abstract Background: Multidrug/rifampicin-resistant tuberculosis (MDR/RR-TB) is a global barrel for ‘Stop TB plan’. China has the second highest MDR/RR-TB burden in whole world wide. Understanding the transmission dynamic is facilitated for disease control. Methods: Whole genome sequencing (WGS) data from patients of Chongqing tuberculosis control institute were used for phylogenetic classifications, resistance predictions, and cluster analysis as indicator for recent transmission (RT). Factors associated with MDR/RR-TB were defined by a logistic regression model. Results: A total of 223 cases of MDR/RR-TB were recorded between Jan 1, 2018 and Dec 31, 2020, and 200 cases obtained relevant treatment information. The patients who are older than 55 year old were more likely to suffering from death. 178 MDR/RR strains were obtained WGS data, 152 were classified as lineage 2 strains. 80 (44.9%, 80 of 178) strains were in 20 genomic clusters that differed by 12 or fewer single nucleotide polymorphisms (SNPs), indicating RT. Patients who were infected with lineage 2 strains is a significant factor driving the epidemic towards MDR/RR-TB. Resistance mutations of first-line tuberculosis drugs analysis found that 79 (98.8%) of all 80 strains defined as RT have same mutations among each clusters totally. 55% (44 of 80) of the MDR/RR-TB strains accumulated additional drug resistance mutations along the transmission chain, especially fluoroquinolones (FQs) (63.6%, 28 of 44). Conclusions: The age is the most significant factor that causes death of MDR/RR-TB patients. RT of MDR/RR strains is not only drove the MDR/RR-TB epidemic, but also accumulated more serious resistance along the transmission chains.

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Rapid, high throughput library preparation of SARS-CoV2 using Illumina’s DNA Prep Library Preparation Kit v2

10.17504/protocols.io.b3vgqn3w ◽

2022 ◽

Author(s):

Jason Nguyen ◽

Rebecca Hickman ◽

Tracy Lee ◽

Natalie Prystajecky ◽

John Tyson

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

High Throughput ◽

Illumina Miseq ◽

Whole Genome ◽

Library Preparation ◽

Dna Libraries ◽

Reaction Volumes ◽

Single Size ◽

Stop Buffer

This procedure provides instructions on how to prepare DNA libraries for whole genome sequencing on an Illumina MiSeq or NextSeq using Illumina’s DNA Prep Library Preparation Kit scaled to half reaction volumes with modifications to the post-PCR procedures; tagmentation stop buffer and associated washes are removed and libraries are pooled post PCR then a single size selection is performed. This protocol is used to sequence SARS-CoV-2 using the cDNA/PCR protocol: https://dx.doi.org/10.17504/protocols.io.b3viqn4e

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Genome Sequencing of Polydrug-, Multidrug-, and Extensively Drug-Resistant Mycobacterium tuberculosis Strains from South India

Microbiology Resource Announcements ◽

10.1128/mra.01388-18 ◽

2019 ◽

Vol 8 (12) ◽

Author(s):

Sivakumar Shanmugam ◽

Narender Kumar ◽

Dina Nair ◽

Mohan Natrajan ◽

Srikanth Prasad Tripathy ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

South India ◽

Sequence Data ◽

Whole Genome ◽

Drug Resistant ◽

Resistance Mutations ◽

Content Type ◽

Extensively Drug Resistant

The genomes of 16 clinical Mycobacterium tuberculosis isolates were subjected to whole-genome sequencing to identify mutations related to resistance to one or more anti-Mycobacterium drugs. The sequence data will help in understanding the genomic characteristics of M. tuberculosis isolates and their resistance mutations prevalent in South India.

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Recommendations To Address the Difficulties Encountered When Determining Linezolid Resistance from Whole-Genome Sequencing Data

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00613-18 ◽

2018 ◽

Vol 62 (8) ◽

Cited By ~ 2

Author(s):

Alicia G. Beukers ◽

Henrik Hasman ◽

Kristin Hegstad ◽

Sebastiaan J. van Hal

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

23S Rrna ◽

Whole Genome Sequencing Data ◽

Rrna Gene ◽

Whole Genome ◽

Sequencing Data ◽

Content Type ◽

Numbering System ◽

Linezolid Resistance

ABSTRACT Mutations associated with linezolid resistance within the V domain of 23S rRNA are annotated using an Escherichia coli numbering system. The 23S rRNA gene varies in length, nucleotide sequence, and copy number among bacterial species. Consequently, this numbering system is not intuitive and can lead to confusion when mutation sites are being located using whole-genome sequencing data. Using the mutation G2576T as an example, we demonstrate the difficulties associated with using the E. coli numbering system.

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