scholarly journals Hybrid Assembly Provides Improved Resolution of Plasmids, Antimicrobial Resistance Genes, and Virulence Factors in Escherichia coli and Klebsiella pneumoniae Clinical Isolates

2021 ◽  
Vol 9 (12) ◽  
pp. 2560
Author(s):  
Abdolrahman Khezri ◽  
Ekaterina Avershina ◽  
Rafi Ahmad
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Resistance ◽  
Blood Culture ◽  
Klebsiella Pneumoniae ◽  
Virulence Factors ◽  
Whole Genome Sequence ◽  
Clinical Samples ◽  
Hybrid Assembly ◽  
Antimicrobial Resistance Genes ◽  
Quality Checks

Emerging new sequencing technologies have provided researchers with a unique opportunity to study factors related to microbial pathogenicity, such as antimicrobial resistance (AMR) genes and virulence factors. However, the use of whole-genome sequence (WGS) data requires good knowledge of the bioinformatics involved, as well as the necessary techniques. In this study, a total of nine Escherichia coli and Klebsiella pneumoniae isolates from Norwegian clinical samples were sequenced using both MinION and Illumina platforms. Three out of nine samples were sequenced directly from blood culture, and one sample was sequenced from a mixed-blood culture. For genome assembly, several long-read, (Canu, Flye, Unicycler, and Miniasm), short-read (ABySS, Unicycler and SPAdes) and hybrid assemblers (Unicycler, hybridSPAdes, and MaSurCa) were tested. Assembled genomes from the best-performing assemblers (according to quality checks using QUAST and BUSCO) were subjected to downstream analyses. Flye and Unicycler assemblers performed best for the assembly of long and short reads, respectively. For hybrid assembly, Unicycler was the top-performing assembler and produced more circularized and complete genome assemblies. Hybrid assembled genomes performed substantially better in downstream analyses to predict putative plasmids, AMR genes and β-lactamase gene variants, compared to MinION and Illumina assemblies. Thus, hybrid assembly has the potential to reveal factors related to microbial pathogenicity in clinical and mixed samples.

scholarly journals 1439. High Rates of Drug Resistance in Escherichia coli from a Pilot Antimicrobial Resistance Surveillance System in Cambodia

2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S723-S724
Author(s):  
Vandarith Nov ◽  
Darapheak Chau ◽  
Chhorvann Chhea
Keyword(s):  
Public Health ◽  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Antimicrobial Resistance ◽  
Blood Culture ◽  
Klebsiella Pneumoniae ◽  
Surveillance System ◽  
Methicillin Resistance ◽  
Salmonella Typhi ◽  
Blood Samples

Abstract Background Antimicrobial resistance (AMR) is a major and growing global public health problem. The Cambodia Ministry of Health established a pilot laboratory-based AMR surveillance system for blood specimens in 2017. The objective of this study is to characterize AMR among pathogenic isolates from blood samples. Methods A retrospective analysis was performed using one year of data from a pilot AMR Surveillance system in Cambodia. Four blood culture isolate pathogens were included: Escherichia coli, Klebsiella pneumoniae, Salmonella Typhi /Salmonella Paratyphi A and Staphylococcus aureus. Blood culture isolates that were referred from eleven sentinel sites were analyzed at the National Public Health Laboratory for identification. Antibiotic susceptibility testing (AST) was done using disk diffusion, minimum inhibitory concentration method following Clinical Laboratory Standard Institute (CLSI) guidelines. Results Among 214 pathogenic isolates from blood samples, E.coli was the most common (56.1%), followed by Salmonella Typhi/Salmonella Paratyphi A (18.7%), Staphylococcus aureus (13.5%), and Klebsiella pneumoniae (11.7%). Methicillin Resistance Staphylococcus aureus (MRSA) was detected in half of the isolates. E.coli was resistant to ampicillin (94.4%), trimethoprim-sulfamethoxazole (84.5%), and ceftriaxone (79.2%). Salmonella Typhi was resistant to ampicillin (73.3%) and trimethoprim-sulfamethoxazole (60.0%) and Salmonella Paratyphi A were resistant to fluoroquinolones (91.7%). For last resort antibiotics, E.coli was resistant to carbapenem groups (3.2% for imipenem, 4.9% for meropenem, and 5.0% for ertapenem). Klebsiella pneumoniae was not resistant to any groups. Conclusion E.coli was found at high rates in blood samples and was resistant to common antibiotics used in Cambodia. These pilot data show the importance of establishing a national AMR surveillance system in Cambodia to monitor AMR trends following GLASS guidelines. Disclosures All Authors: No reported disclosures

scholarly journals Virulence factors, antimicrobial resistance, and plasmid content of Escherichia coli isolated in swine commercial farms

2010 ◽  
Vol 62 (1) ◽  
pp. 30-36 ◽  
Author(s):  
M.M. Costa ◽  
G. Drescher ◽  
F Maboni ◽  
S.S. Weber ◽  
A. Schrank ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Resistance ◽  
Virulence Factors ◽  
Intestinal Content ◽  
Specific Pattern ◽  
Clinical Isolates ◽  
Clinical Samples ◽  
Plasmid Content ◽  
E Coli ◽  
Resistance Patterns

Virulence factors and antimicrobial resistance patterns of Escherichia coli isolates were evaluated. A total of 80 E. coli isolates were evaluated, being 64 from clinical samples (intestinal content and fragments of organs from diarrheic piglets), seven from feces of clinically healthy piglets and sows, and nine environmental samples (five from facilities, two from feed, one from insect, and one from waste). Molecular characterization was performed by PCR detection of fimbriae and toxin genes and plasmid content determination. The isolates were also characterized according to their resistance or sensitivity to the following drugs: ampicillin, trimethoprim:sulfamethoxazole, tetracycline, amikacine, colistin, norfloxacin, florfenicol, enrofloxacin, cefalexin, trimethoprim, neomycin, chloramphenicol, and gentamicin. From 80 E. coli isolates, 53.8% were classified as enterotoxigenic E. coli (ETEC), 2.5% were shiga toxin-producing E. coli (STEC), and 43.8% showed a non specific pattern and were unclassified. One fecal isolate from non-diarrheic piglet was classified as ETEC by PCR. Clinical isolates showed resistance mainly for tetracycline and trimethoprim:sulfamethoxazole. Plasmidial DNA was observed in 70 isolates, being 78.5% of clinical isolates, 8.57% of non-diarrheic feces, and 12.8% of environment.

scholarly journals Multidrug-Resistant Escherichia coli, Klebsiella pneumoniae and Staphylococcus spp. in Houseflies and Blowflies from Farms and Their Environmental Settings

2019 ◽  
Vol 16 (19) ◽  
pp. 3583 ◽  
Author(s):  
Anil Poudel ◽  
Terri Hathcock ◽  
Patrick Butaye ◽  
Yuan Kang ◽  
Stuart Price ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Resistance ◽  
Klebsiella Pneumoniae ◽  
Multidrug Resistant ◽  
Resistant Bacteria ◽  
Coagulase Negative Staphylococcus ◽  
Extended Spectrum Beta Lactamase ◽  
E Coli ◽  
Antimicrobial Resistance Genes ◽  
Staphylococcus Spp

Background: Antimicrobial resistance is rising globally at an alarming rate. While multiple active surveillance programs have been established to monitor the antimicrobial resistance, studies on the environmental link to antimicrobial spread are lacking. Methods: A total of 493 flies were trapped from a dairy unit, a dog kennel, a poultry farm, a beef cattle unit, an urban trash facility and an urban downtown area to isolate Escherichia coli, Klebsiella pneumoniae and Staphylococcus spp. for antimicrobial susceptibility testing and molecular characterization. Results: E. coli, K. pneumoniae and coagulase-negative Staphylococcus were recovered from 43.9%, 15.5% and 66.2% of the houseflies, and 26.0%, 19.2%, 37.0% of the blowflies, respectively. In total, 35.3% of flies were found to harbor antimicrobial-resistant bacteria and 9.0% contained multidrug-resistant isolates. Three Staphylococcus aureus isolates were recovered from blowflies while three extended spectrum beta lactamase (ESBL)-carrying E. coli and one ESBL-carrying K. pneumoniae were isolated from houseflies. Whole genome sequencing identified the antimicrobial resistance genes blaCMY-2 and blaCTXM-1 as ESBLs. Conclusion: Taken together, our data indicate that flies can be used as indicators for environmental contamination of antimicrobial resistance. More extensive studies are warranted to explore the sentinel role of flies for antimicrobial resistance.

scholarly journals Outcome of Different Sequencing and Assembly Approaches on the Detection of Plasmids and Localization of Antimicrobial Resistance Genes in Commensal Escherichia coli

2021 ◽  
Vol 9 (3) ◽  
pp. 598
Author(s):  
Katharina Juraschek ◽  
Maria Borowiak ◽  
Simon H. Tausch ◽  
Burkhard Malorny ◽  
Annemarie Käsbohrer ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Resistance ◽  
Mobile Genetic Elements ◽  
Hybrid Assembly ◽  
Small Plasmid ◽  
Short Read ◽  
Short Read Sequencing ◽  
Genetic Elements ◽  
Antimicrobial Resistance Genes ◽  
Long Read

Antimicrobial resistance (AMR) is a major threat to public health worldwide. Currently, AMR typing changes from phenotypic testing to whole-genome sequence (WGS)-based detection of resistance determinants for a better understanding of the isolate diversity and elements involved in gene transmission (e.g., plasmids, bacteriophages, transposons). However, the use of WGS data in monitoring purposes requires suitable techniques, standardized parameters and approved guidelines for reliable AMR gene detection and prediction of their association with mobile genetic elements (plasmids). In this study, different sequencing and assembly strategies were tested for their suitability in AMR monitoring in Escherichia coli in the routines of the German National Reference Laboratory for Antimicrobial Resistances. To assess the outcomes of the different approaches, results from in silico predictions were compared with conventional phenotypic- and genotypic-typing data. With the focus on (fluoro)quinolone-resistant E.coli, five qnrS-positive isolates with multiple extrachromosomal elements were subjected to WGS with NextSeq (Illumina), PacBio (Pacific BioSciences) and ONT (Oxford Nanopore) for in depth characterization of the qnrS1-carrying plasmids. Raw reads from short- and long-read sequencing were assembled individually by Unicycler or Flye or a combination of both (hybrid assembly). The generated contigs were subjected to bioinformatics analysis. Based on the generated data, assembly of long-read sequences are error prone and can yield in a loss of small plasmid genomes. In contrast, short-read sequencing was shown to be insufficient for the prediction of a linkage of AMR genes (e.g., qnrS1) to specific plasmid sequences. Furthermore, short-read sequencing failed to detect certain duplications and was unsuitable for genome finishing. Overall, the hybrid assembly led to the most comprehensive typing results, especially in predicting associations of AMR genes and mobile genetic elements. Thus, the use of different sequencing technologies and hybrid assemblies currently represents the best approach for reliable AMR typing and risk assessment.

scholarly journals Prophage elements function as reservoir for antibiotic resistance and virulence genes in nosocomial pathogens

2020 ◽  
Author(s):  
Kohei Kondo ◽  
Mitsuoki Kawano ◽  
Motoyuki Sugai
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Resistance ◽  
Klebsiella Pneumoniae ◽  
Resistance Genes ◽  
Complete Genome ◽  
Virulence Genes ◽  
Enterobacter Cloacae ◽  
Prophage Region ◽  
Antimicrobial Resistance Genes ◽  
Class 1

AbstractProphages are often involved in host survival strategies and contribute toward increasing the genetic diversity of the host genome. Prophages also drive horizontal propagation of various genes as vehicles. However, there are few retrospective studies contributing to the propagation of antimicrobial resistance (AMR) and virulence factor (VF) genes by prophage. In this study, we extracted complete genome sequences of seven pathogens, including ESKAPE bacteria and Escherichia coli deposited in a public database, and examined the distribution of both AMR and VF genes in certain genomic regions of prophage, including prophage-like element. We found that the ratios of AMR and VF genes greatly varied among the seven species. More than 55% of Enterobacter cloacae strains had VF genes, but only 0.8% of Klebsiella pneumoniae strains had VF genes from prophages. The prophage types carrying AMR genes were detected in a broad range of hosts, whereas prophages containing VF genes were conserved in only one or two species, suggesting that distribution patterns of prophages were different between prophages encoding AMR or VF genes. We also found that the prophage containing class 1 integrase possessed a significantly higher number of AMR genes than prophages with no class 1 integrase. Moreover, AMR genes in the prophage were located near transposase and integrase. The results of this study reveal a comprehensive picture of AMR and VF genes present in prophage elements and provide new insights into the horizontal transfer of genes associated with antimicrobial resistance and pathogenicity.ImportanceAlthough we believe phages play an important role in horizontal gene transfer in exchanging genetic material, we do not know the distribution of the antimicrobial resistance and/or virulence genes in prophages. We collected different prophage elements from the complete genome sequence of seven species – Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter cloacae, as well as Escherichia coli –, and characterized the distribution of antimicrobial resistance and virulence genes encoded in the prophage region. While virulence genes in prophage were found to be species-specific, antimicrobial resistance genes in prophages were highly conserved in various species. Integron structure was detected within prophage regions in almost all of the genera. Maximum of 11 antimicrobial resistance genes were found in a single prophage region, suggesting that prophages act as a reservoir for antimicrobial resistance genes. Our results highlight new insights on prophages as horizontal gene carriers.

scholarly journals A Multi-Center Clinical Study to Demonstrate the Diagnostic Accuracy of the GenMark Dx ePlex ® Blood Culture Identification Gram-Negative Panel

2021 ◽  
Author(s):  
Donna M. Wolk ◽  
Stephen Young ◽  
Natalie N. Whitfield ◽  
Jennifer L. Reid ◽  
Adam Thornberg ◽  
...  
Keyword(s):  
Clinical Study ◽  
Antimicrobial Resistance ◽  
Blood Culture ◽  
Resistance Genes ◽  
Limit Of Detection ◽  
Molecular Diagnostic ◽  
Clinical Samples ◽  
Gram Negative ◽  
Antimicrobial Resistance Genes ◽  
Culture Identification

Bacteremia can progress to septic shock and death without appropriate medical intervention. Increasing evidence supports the role of molecular diagnostic panels in reducing the clinical impact of these infections through rapid identification of the infecting organism and associated antimicrobial resistance genes. We report the results of a multicenter clinical study assessing the performance of the GenMark Dx ePlex ® Investigational Use Only Blood Culture Identification Gram-Negative Panel (BCID-GN), a rapid diagnostic assay for detection of bloodstream pathogens in positive blood culture (PBC) bottles. Prospective, retrospective, and contrived samples were tested. Results from the BCID-GN were compared to standard of care bacterial identification methods. Antimicrobial resistance genes (ARGs) were identified using PCR and sequence analysis. The final BCID-GN analysis included 2,444 PBC samples, of which 926 were clinical samples with gram-negative Gram stain results. Of these, 109 samples had false negative and/or positive results, resulting in an overall sample accuracy of 88.2% (817/926). After discordant resolution, overall sample accuracy increased to 92.9% (860/926). Pre- and post-discordant resolution sample accuracy excludes 37 gram-negative organisms representing 20 uncommon genera, 10 gram-positive organisms, and 1 Candida sp. present in 5% of samples that are not targeted by the BCID-GN. The overall weighted PPA, which averages the individual PPAs from the 27 targets (gram-negative and ARG), was 94.9%. The limit of detection ranged from 10 4 to 10 7 CFU/mL, except for one strain of Fusobacterium necrophorum at 10 8 CFU/mL.

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