The Use of Next-Generation Sequencing in Fighting Antimicrobial Resistant Pathogens

Editors

Ibrahim Bitar

Charles University

Costas C Papagiannitsis

Faculty of Medicine, School of Health Sciences, University of Thessaly

Alberto Antonelli

University of Florence

Impact

Original Research

29 November 2022

Transcriptome profiling in response to Kanamycin B reveals its wider non-antibiotic cellular function in Escherichia coli

Yaowen Chang

, 2 more and

Dongrong Chen

2,526 views

2 citations

Original Research

06 October 2022

A resistome survey across hundreds of freshwater bacterial communities reveals the impacts of veterinary and human antibiotics use

Susanne A. Kraemer

, 3 more and

David A. Walsh

4,177 views

10 citations

Grape tree generated using cgMLST from genomic collection of Pseudomonas aeruginosa isolates from Africa (n = 22), Ethiopia, Nigeria, and South Africa, pubmlst database for microorganisms (https://pubmlst.org/bigsdb?db=pubmlst_paeruginosa_isolates&page=query&genomes=1) accessed on 29-11-2021. The numbers in circles/nodes indicate sequence types of the isolates, size of the nodes is proportional to the number of isolates in a particular sequence type and the numbers on the branches are the number of allelic differences between the two neighboring nodes. From 10 isolates sequenced in this study, eight were included in the tree, two of the sequences failed quality required to upload to the pubmlst database.

Original Research

20 September 2022

Molecular epidemiology and antimicrobial susceptibility of Pseudomonas spp. and Acinetobacter spp. from clinical samples at Jimma medical center, Ethiopia

Tsegaye Sewunet

, 3 more and

Christian G. Giske

4,150 views

13 citations

Very Short Patch repair system (VSP). Methylated cytosines spontaneously deaminate to Thymine creating T/G mismatches. The very short patch repair system (VSP) acts repairing these mismatches, always in favor of the Guanine (Drotschmann et al., 1998; Marinus, 2012). In this figure, the VSP system mechanism is divided into six steps: (A) CCWGG motifs are methylated on both strands by the Dcm enzyme. Methylated cytosines spontaneously deaminate to thymine, creating a mismatch with guanine; (B) MutS dimer recognizes the mismatch and binds MutL dimer which acts as a bridge with VSR protein; (C) VSR is activated by the MutSL complex and creates a nick in the strand containing the incorrect base, using as parental stand that containing the Guanosine; (D) the MutSL-VSR complex recruits DNA helicase, exonuclease, DNA polymerase, and DNA ligase to fix the mismatch; (E) CCWGG motif is restored and thus DCM methyltransferase can methylate it; (F) CCWGG is methylated on both strands.

Review

15 September 2022

The red thread between methylation and mutation in bacterial antibiotic resistance: How third-generation sequencing can help to unravel this relationship

Stella Papaleo

, 4 more and

Francesco Comandatore

5,633 views

6 citations

Original Research

09 September 2022

Clinical utility of target amplicon sequencing test for rapid diagnosis of drug-resistant Mycobacterium tuberculosis from respiratory specimens

Kenneth Siu-Sing Leung

, 9 more and

Wing-Cheong Yam

4,081 views

8 citations

Summary of most relevant features of studied strains (ST, virulence score, resistance score, K type and O type) and results obtained in the animal infection models. (A) Results of experiments in the G. mellonella model expressed as mortality rate (percentage of dead over the total of inoculated larvae). Means and SD of three replicate experiments are show at different time points for each strain (after 24, 48, and 72 h). (B) Results of virulence experiments, for representative HMV isolates, in the murine model expressed as Log10CFU/g organ of the studied strains, recovered from kidneys and livers. Each dot represents data obtained from one mouse specimen and the solid line represents the mean value. In both the models, the NTUH-K2044 virulent strain and the KKBO-1 low-virulence strain were included and used as reference for classification of studied isolates in three different virulence categories: low (green zone), intermediate (yellow) and high (red). Mortality rate at 72 h (A) and Log10 counts (B) for the three groups were compared. P-values < 0.05 were considered significant; *< 0.05, **<0.005, ****<0.0001.

Original Research

15 August 2022

Resistance and virulence features of hypermucoviscous Klebsiella pneumoniae from bloodstream infections: Results of a nationwide Italian surveillance study

Fabio Arena

, 7 more and

Gian Maria Rossolini

4,936 views

17 citations

$Characterization of the hospital microbiome and its AMR. (A) Detection of AMR genes and Staphylococcus aureus presence by real-time quantitative (qPCR) microarray. The results represent the mean values detected in the General Medicine wards of 12 Italian hospitals from 2015 to 2020, and are expressed as mean ± SE. Log10 fold change values for each gene compared to negative controls. In addition to AMR genes, S. aureus and two of its virulence factors (spa and lukF) are also identified by the microarray. (B) NGS characterization of the microbial communities residing on different types of surfaces in the wards of a pediatric hospital. The unifrac-based principal coordinates analysis (PCoA) plots show the clustering of bacterial communities according to the wards (A) and surfaces (B) investigated. Each dot represents a sample. PC, Pediatric Clinic; PS, Pediatric Surgery; NICU, Neonatal Intensive Care Unit; sNICU, sub-Intensive Care Unit; ICU, children’s Intensive Care Unit; SR, Surgical Rooms; DR, Delivery Room; PO, Pediatric Oncology. (C) Detection of AMR genes by qPCR microarray in the Pediatric Clinic ward of a children’s hospital. The results are expressed as log10 fold change values compared to negative controls. (D) Colonization of preterm infants by Neonatal Intensive Care Unit (NICU) environmental microorganisms; the AMR analysis was performed by qPCR microarray. The results are expressed as log10 fold change values compared to negative controls, relative to the NICU environment (NICU) and to the newborn’s nasal cavity after 2 weeks of hospitalization in NICU (newborns).$

Mini Review

28 July 2022

Next-generation sequencing and PCR technologies in monitoring the hospital microbiome and its drug resistance

Carolina Cason

, 4 more and

Elisabetta Caselli

8,933 views

26 citations

Clinical AMR class decision workflow. WGS-AST: whole-genome sequencing antimicrobial susceptibility testing; CR: carbapenem resistance; CRE: carbapenem-resistant Enterobacterales; 3GC: third-generation cephalosporin resistance, CP: carbapenemase; ESBL: extended-spectrum β-lactamase; pAmpC: plasmid AmpC; MBL: metallo-β-lactamase.

Original Research

08 August 2022

Automated antimicrobial susceptibility testing and antimicrobial resistance genotyping using Illumina and Oxford Nanopore Technologies sequencing data among Enterobacteriaceae

Rick Conzemius

, 6 more and

Patricia J. Simner

7,317 views

8 citations

Original Research

30 June 2022

CARB-ES-19 Multicenter Study of Carbapenemase-Producing Klebsiella pneumoniae and Escherichia coli From All Spanish Provinces Reveals Interregional Spread of High-Risk Clones Such as ST307/OXA-48 and ST512/KPC-3

Javier E. Cañada-García

, 105 more and

Jesús Rodríguez-Baño

Objectives: CARB-ES-19 is a comprehensive, multicenter, nationwide study integrating whole-genome sequencing (WGS) in the surveillance of carbapenemase-producing K. pneumoniae (CP-Kpn) and E. coli (CP-Eco) to determine their incidence, geographical distribution, phylogeny, and resistance mechanisms in Spain.

Methods: In total, 71 hospitals, representing all 50 Spanish provinces, collected the first 10 isolates per hospital (February to May 2019); CPE isolates were first identified according to EUCAST (meropenem MIC > 0.12 mg/L with immunochromatography, colorimetric tests, carbapenem inactivation, or carbapenem hydrolysis with MALDI-TOF). Prevalence and incidence were calculated according to population denominators. Antibiotic susceptibility testing was performed using the microdilution method (EUCAST). All 403 isolates collected were sequenced for high-resolution single-nucleotide polymorphism (SNP) typing, core genome multilocus sequence typing (cgMLST), and resistome analysis.

Results: In total, 377 (93.5%) CP-Kpn and 26 (6.5%) CP-Eco isolates were collected from 62 (87.3%) hospitals in 46 (92%) provinces. CP-Kpn was more prevalent in the blood (5.8%, 50/853) than in the urine (1.4%, 201/14,464). The cumulative incidence for both CP-Kpn and CP-Eco was 0.05 per 100 admitted patients. The main carbapenemase genes identified in CP-Kpn were bla_OXA–48 (263/377), bla_KPC–3 (62/377), bla_VIM–1 (28/377), and bla_NDM–1 (12/377). All isolates were susceptible to at least two antibiotics. Interregional dissemination of eight high-risk CP-Kpn clones was detected, mainly ST307/OXA-48 (16.4%), ST11/OXA-48 (16.4%), and ST512-ST258/KPC (13.8%). ST512/KPC and ST15/OXA-48 were the most frequent bacteremia-causative clones. The average number of acquired resistance genes was higher in CP-Kpn (7.9) than in CP-Eco (5.5).

Conclusion: This study serves as a first step toward WGS integration in the surveillance of carbapenemase-producing Enterobacterales in Spain. We detected important epidemiological changes, including increased CP-Kpn and CP-Eco prevalence and incidence compared to previous studies, wide interregional dissemination, and increased dissemination of high-risk clones, such as ST307/OXA-48 and ST512/KPC-3.

7,498 views

41 citations

Differential gene expression after 4 h of monensin treatment. Volcano plots showing differentially expressed genes in the monensin-resistant ETH-R strain (A) and the monensin-sensitive ETH-S strain (D) after 4 h of monensin treatment. Genes with an absolute fold change >2 and adjusted p < 0.01 were considered significantly differentially expressed (red dots). (B,E) Gene ontology enrichment of differentially expressed genes in ETH-R (B) and ETH-S (E) after two additional hours of monensin treatment (4 vs. 2 h). (C) Heatmap showing differentially expressed genes associated with ribosomal proteins in the monensin-resistant ETH-R strain after two additional hours of monensin treatment. (F,G) Heatmaps showing differentially expressed genes associated with calcium ion binding proteins (F) and oxidoreductive proteins (G) in the monensin-sensitive ETH-S strain after two additional hours of monensin treatment. For heatmap drawing, gene expression levels were transformed into normalized log2(TPM+1).

Original Research

04 July 2022

Early Transcriptional Response to Monensin in Sensitive and Resistant Strains of Eimeria tenella

Hongtao Zhang

, 4 more and

Dandan Hu

1,770 views

14 citations

(A) The CholerAegon pipeline is implemented in Nextflow, Download at: https://github.com/RaverJay/CholerAegon. For each input sample, CholerAegon produces a genome assembly, basic assembly statistics, the predicted AMR genes, and subsequent drug resistance, and a summary report. (B) For this specific study of Vibrio cholerae isolates, AMR susceptibility test results were compared to in silico resistance predictions with a custom Python script. CARD, the comprehensive antibiotic resistance database (Alcock et al., 2019); AST, antimicrobial susceptibility testing.

Original Research

22 June 2022

Prediction of Antibiotic Susceptibility Profiles of Vibrio cholerae Isolates From Whole Genome Illumina and Nanopore Sequencing Data: CholerAegon

Valeria Fuesslin

, 11 more and

Kathrin Schuldt

4,583 views

3 citations

Original Research

07 April 2022

Co-expression Mechanism Analysis of Different Tachyplesin I–Resistant Strains in Pseudomonas aeruginosa Based on Transcriptome Sequencing

Jun Hong

, 2 more and

Ruofei Hong

1,770 views

3 citations

Original Research

30 March 2022

Successful Intra- but Not Inter-species Recombination of msr(D) in Neisseria subflava

Tessa de Block

, 7 more and

Chris Kenyon

1,830 views

4 citations

Coverage of the reference hybrid assemblies throughout the Flongle sequencing run.

Original Research

17 March 2022

Ultrafast and Cost-Effective Pathogen Identification and Resistance Gene Detection in a Clinical Setting Using Nanopore Flongle Sequencing

Ekaterina Avershina

, 3 more and

Rafi Ahmad

Rapid bacterial identification and antimicrobial resistance gene (ARG) detection are crucial for fast optimization of antibiotic treatment, especially for septic patients where each hour of delayed antibiotic prescription might have lethal consequences. This work investigates whether the Oxford Nanopore Technology’s (ONT) Flongle sequencing platform is suitable for real-time sequencing directly from blood cultures to identify bacteria and detect resistance-encoding genes. For the analysis, we used pure bacterial cultures of four clinical isolates of Escherichia coli and Klebsiella pneumoniae and two blood samples spiked with either E. coli or K. pneumoniae that had been cultured overnight. We sequenced both the whole genome and plasmids isolated from these bacteria using two different sequencing kits. Generally, Flongle data allow rapid bacterial ID and resistome detection based on the first 1,000–3,000 generated sequences (10 min to 3 h from the sequencing start), albeit ARG variant identification did not always correspond to ONT MinION and Illumina sequencing-based data. Flongle data are sufficient for 99.9% genome coverage within at most 20,000 (clinical isolates) or 50,000 (positive blood cultures) sequences generated. The SQK-LSK110 Ligation kit resulted in higher genome coverage and more accurate bacterial identification than the SQK-RBK004 Rapid Barcode kit.

7,868 views

33 citations

Original Research

23 March 2022

Optimization of Early Antimicrobial Strategies for Lung Transplant Recipients Based on Metagenomic Next-Generation Sequencing

Xiao-qin Zhang

, 9 more and

Ling-ai Pan

1,861 views

2 citations

Scatter plot of MICs of cefoxitin (FOX) and oxacillin (OXA) and specific composition of OS-MRSA. On the left, the horizontal axis is the MICs of FOX for the isolates and the vertical axis is the MICs of OXA for the isolates. The figure depicts FOX MICs versus OXA MICs for Staphylococcus aureus. Green represents methicillin-susceptible Staphylococcus aureus (MSSA) and blue represents methicillin-resistant Staphylococcus aureus (MRSA) with Staphylococcal cassette chromosome mec (SCCmec) typing. Totally, 79 isolates were resistant to FOX and susceptible to OXA (OS-MRSA). The specific composition of these 79 isolates is shown in the table on the right; there were 56 ST59 isolates, accounting for the vast majority of OS-MRSA.

Original Research

02 March 2022

A Practical Approach for Predicting Antimicrobial Phenotype Resistance in Staphylococcus aureus Through Machine Learning Analysis of Genome Data

Shuyi Wang

, 4 more and

Hui Wang

With the reduction in sequencing price and acceleration of sequencing speed, it is particularly important to directly link the genotype and phenotype of bacteria. Here, we firstly predicted the minimum inhibitory concentrations of ten antimicrobial agents for Staphylococcus aureus using 466 isolates by directly extracting k-mer from whole genome sequencing data combined with three machine learning algorithms: random forest, support vector machine, and XGBoost. Considering one two-fold dilution, the essential agreement and the category agreement could reach >85% and >90% for most antimicrobial agents. For clindamycin, cefoxitin and trimethoprim-sulfamethoxazole, the essential agreement and the category agreement could reach >91% and >93%, providing important information for clinical treatment. The successful prediction of cefoxitin resistance showed that the model could identify methicillin-resistant S. aureus. The results suggest that small datasets available in large hospitals could bypass the existing basic research and known antimicrobial resistance genes and accurately predict the bacterial phenotype.

5,869 views

29 citations