scholarly journals Selective culture enrichment and sequencing of faeces to enhance detection of antimicrobial resistance genes

2019 ◽  
Author(s):  
Leon Peto ◽  
Nicola J. Fawcett ◽  
Derrick W. Crook ◽  
Tim E.A. Peto ◽  
Martin J. Llewelyn ◽  
...  
Keyword(s):  
Resistance Genes ◽  
Limit Of Detection ◽  
Broth Culture ◽  
Metagenomic Sequencing ◽  
Extended Spectrum Beta Lactamase ◽  
Antimicrobial Resistance Genes ◽  
Short Period ◽  
Culture Enrichment ◽  
Plasmid Extraction ◽  
Accurate Quantification

ABSTRACTMetagenomic sequencing of faecal DNA can usefully characterise an individual’s intestinal resistome but is limited by its inability to detect important pathogens that may be present at low abundance, such as carbapenemase or extended-spectrum beta-lactamase producingEnterobacteriaceae. Here we aimed to develop a hybrid protocol to improve detection of resistance genes inEnterobacteriaceaeby using a short period of culture enrichment prior to sequencing of DNA extracted directly from the enriched sample. Volunteer faeces were spiked with carbapenemase-producingEnterobacteriaceaeand incubated in selective broth culture for 6 hours before sequencing. Different DNA extraction methods were compared, including a plasmid extraction protocol to increase the detection of plasmid-associated resistance genes. Although enrichment prior to sequencing increased the detection of carbapenemase genes, the differing growth characteristics of the spike organisms precluded accurate quantification of their concentration prior to culture. Plasmid extraction protocols increased detection of resistance genes present on plasmids, but the effects were heterogeneous and dependent on plasmid size. Our results demonstrate methods of improving the limit of detection of selected resistance mechanisms in a faecal resistome assay, but they also highlight the difficulties in using these techniques for accurate quantification and should inform future efforts to achieve this goal.

scholarly journals Culture-Independent Genotyping, Virulence and Antimicrobial Resistance Gene Identification of Staphylococcus aureus from Orthopaedic Implant-Associated Infections

2021 ◽  
Vol 9 (4) ◽  
pp. 707
Author(s):  
J. Christopher Noone ◽  
Fabienne Antunes Ferreira ◽  
Hege Vangstein Aamot
Keyword(s):  
Staphylococcus Aureus ◽  
Antimicrobial Resistance ◽  
Resistance Genes ◽  
Virulence Gene ◽  
Orthopaedic Implant ◽  
Metagenomic Sequencing ◽  
Gene Detection ◽  
Shotgun Metagenomic Sequencing ◽  
Antimicrobial Resistance Genes ◽  
Culture Independent

Our culture-independent nanopore shotgun metagenomic sequencing protocol on biopsies has the potential for same-day diagnostics of orthopaedic implant-associated infections (OIAI). As OIAI are frequently caused by Staphylococcus aureus, we included S. aureus genotyping and virulence gene detection to exploit the protocol to its fullest. The aim was to evaluate S. aureus genotyping, virulence and antimicrobial resistance genes detection using the shotgun metagenomic sequencing protocol. This proof of concept study included six patients with S. aureus-associated OIAI at Akershus University Hospital, Norway. Five tissue biopsies from each patient were divided in two: (1) conventional microbiological diagnostics and genotyping, and whole genome sequencing (WGS) of S. aureus isolates; (2) shotgun metagenomic sequencing of DNA from the biopsies. Consensus sequences were analysed using spaTyper, MLST, VirulenceFinder, and ResFinder from the Center for Genomic Epidemiology (CGE). MLST was also compared using krocus. All spa-types, one CGE and four krocus MLST results matched Sanger sequencing results. Virulence gene detection matched between WGS and shotgun metagenomic sequencing. ResFinder results corresponded to resistance phenotype. S. aureus spa-typing, and identification of virulence and antimicrobial resistance genes are possible using our shotgun metagenomics protocol. MLST requires further optimization. The protocol has potential application to other species and infection types.

scholarly journals Merging Metagenomics and Spatial Epidemiology To Understand the Distribution of Antimicrobial Resistance Genes from Enterobacteriaceae in Wild Owls

2020 ◽  
Vol 86 (20) ◽  
Author(s):  
Elizabeth A. Miller ◽  
Julia B. Ponder ◽  
Michelle Willette ◽  
Timothy J. Johnson ◽  
Kimberly L. VanderWaal
Keyword(s):  
Antimicrobial Resistance ◽  
Resistance Genes ◽  
Spatial Epidemiology ◽  
Anthropogenic Sources ◽  
Resistant Bacteria ◽  
Metagenomic Sequencing ◽  
Wild Animals ◽  
Natural Ecosystems ◽  
Content Type ◽  
Antimicrobial Resistance Genes

ABSTRACT Antimicrobial resistance (AMR) is a well-documented phenomenon in bacteria from many natural ecosystems, including wild animals. However, the specific determinants and spatial distribution of resistant bacteria and antimicrobial resistance genes (ARGs) in the environment remain incompletely understood. In particular, information regarding the importance of anthropogenic sources of AMR relative to that of other biological and ecological influences is lacking. We conducted a cross-sectional study of AMR in great horned owls (Bubo virginianus) and barred owls (Strix varia) admitted to a rehabilitation center in the midwestern United States. A combination of selective culture enrichment and shotgun metagenomic sequencing was used to identify ARGs from Enterobacteriaceae. Overall, the prevalence of AMR was comparable to that in past studies of resistant Enterobacteriaceae in raptors, with acquired ARGs being identified in 23% of samples. Multimodel regression analyses identified seasonality and owl age to be important predictors of the likelihood of the presence of ARGs, with birds sampled during warmer months being more likely to harbor ARGs than those sampled during cooler months and with birds in their hatch year being more likely to harbor β-lactam ARGs than adults. Beyond host-specific determinants, ARG-positive owls were also more likely to be recovered from areas of high agricultural land cover. Spatial clustering analyses identified a significant high-risk cluster of tetracycline resistance gene-positive owls in the southern sampling range, but this could not be explained by any predictor variables. Taken together, these results highlight the complex distribution of AMR in natural environments and suggest that both biological and anthropogenic factors play important roles in determining the emergence and persistence of AMR in wildlife. IMPORTANCE Antimicrobial resistance (AMR) is a multifaceted problem that poses a worldwide threat to human and animal health. Recent reports suggest that wildlife may play an important role in the emergence, dissemination, and persistence of AMR. As such, there have been calls for better integration of wildlife into current research on AMR, including the use of wild animals as biosentinels of AMR contamination in the environment. A One Health approach can be used to gain a better understanding of all AMR sources and pathways, particularly those at the human-animal-environment interface. Our study focuses on this interface in order to assess the effect of human-impacted landscapes on AMR in a wild animal. This work highlights the value of wildlife rehabilitation centers for environmental AMR surveillance and demonstrates how metagenomic sequencing within a spatial epidemiology framework can be used to address questions surrounding AMR complexity in natural ecosystems.

scholarly journals Antibiotic resistance potential of the healthy preterm infant gut microbiome

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e2928 ◽  
Author(s):  
Graham Rose ◽  
Alexander G. Shaw ◽  
Kathleen Sim ◽  
David J. Wooldridge ◽  
Ming-Shi Li ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Preterm Infant ◽  
Resistance Genes ◽  
Gut Microbiome ◽  
Antibiotic Resistance Genes ◽  
Taxonomic Diversity ◽  
Metagenomic Sequencing ◽  
Important Species ◽  
Antimicrobial Resistance Genes ◽  
Infant Gut Microbiome

Background Few studies have investigated the gut microbiome of infants, fewer still preterm infants. In this study we sought to quantify and interrogate the resistome within a cohort of premature infants using shotgun metagenomic sequencing. We describe the gut microbiomes from preterm but healthy infants, characterising the taxonomic diversity identified and frequency of antibiotic resistance genes detected. Results Dominant clinically important species identified within the microbiomes included C. perfringens, K. pneumoniae and members of the Staphylococci and Enterobacter genera. Screening at the gene level we identified an average of 13 antimicrobial resistance genes per preterm infant, ranging across eight different antibiotic classes, including aminoglycosides and fluoroquinolones. Some antibiotic resistance genes were associated with clinically relevant bacteria, including the identification of mecA and high levels of Staphylococci within some infants. We were able to demonstrate that in a third of the infants the S. aureus identified was unrelated using MLST or metagenome assembly, but low abundance prevented such analysis within the remaining samples. Conclusions We found that the healthy preterm infant gut microbiomes in this study harboured a significant diversity of antibiotic resistance genes. This broad picture of resistances and the wider taxonomic diversity identified raises further caution to the use of antibiotics without consideration of the resident microbial communities.

scholarly journals Prevalence and Genetic Analysis of Chromosomal mcr-3/7 in Aeromonas From U.S. Animal-Derived Samples

2021 ◽  
Vol 12 ◽  
Author(s):  
Yan Wang ◽  
Naxin Hou ◽  
Reuven Rasooly ◽  
Yongqiang Gu ◽  
Xiaohua He
Keyword(s):  
Resistance Genes ◽  
Lipid A ◽  
Housekeeping Genes ◽  
Domestic Animal ◽  
Broth Culture ◽  
Transferase Gene ◽  
Antimicrobial Resistance Genes ◽  
Meat Samples ◽  
Inspection Service ◽  
Pcr Targeting

The prevalence of mcr-positive bacteria in 5,169 domestic animal-derived samples collected by USDA Food Safety and Inspection Service between October 2018 and May 2019 was investigated. A procedure including enriched broth culture and real-time PCR targeting mcr-1 to mcr-8 were used for the screening. Fifteen positive isolates were identified, including one plasmid-borne mcr-1-positive Escherichia coli strain, EC2492 (reported elsewhere) and 14 mcr-3/7-positive strains from poultry (1), catfish (2), and chicken rinse (11) samples, resulting in an overall prevalence of mcr-positive bacteria 0.29% in all meat samples tested. Analysis of 16S rRNA and whole genome sequences revealed that all 14 strains belonged to Aeromonas. Data from phylogenetic analysis of seven housekeeping genes, including gyrB, rpoD, gyrA, recA, dnaJ, dnaX, and atpD, indicated that nine strains belonged to Aeromonas hydrophila and five strains belonged to Aeromonas jandaei. Antimicrobial tests showed that almost all mcr-positive strains exhibited high resistance to colistin with MICs ≥ 128mg/L, except for one A. jandaei strain, which showed a borderline resistance with a MIC of 2 mg/L. A segment containing two adjacent mcr-3 and mcr-3-like genes was found in two A. hydrophila and one A. jandaei strains and a variety of IS-like elements were found in the flanking regions of this segment. A mcr-3-related lipid A phosphoethanolamine transferase gene was present in all 14 Aeromonas strains, while an additional mcr-7-related lipid A phosphoethanolamine transferase gene was found in 5 A. jandaei strains only. In addition to mcr genes, other antimicrobial resistance genes, including blaOXA–12/OXA–724, aqu-2, tru-1, cepS, cphA, imiH, ceph-A3, ant(3″)-IIa, aac(3)-Via, and sul1 were observed in chromosomes of some Aeromonas strains. The relative high prevalence of chromosome-borne mcr-3/7 genes and the close proximity of various IS elements to these genes highlights the need for continued vigilance to reduce the mobility of these colistin-resistance genes among food animals.

scholarly journals Intestinal microbiota domination under extreme selective pressures characterized by metagenomic read cloud sequencing and assembly

2019 ◽  
Vol 20 (S16) ◽  
Author(s):  
Joyce B. Kang ◽  
Benjamin A. Siranosian ◽  
Eli L. Moss ◽  
Niaz Banaei ◽  
Tessa M. Andermann ◽  
...  
Keyword(s):  
Resistance Genes ◽  
Gut Microbiome ◽  
Draft Genome ◽  
Strain Level ◽  
Comparative Genomic ◽  
Metagenomic Sequencing ◽  
Antibiotic Exposure ◽  
E Coli ◽  
Selective Pressures ◽  
Antimicrobial Resistance Genes

Abstract Background Low diversity of the gut microbiome, often progressing to the point of intestinal domination by a single species, has been linked to poor outcomes in patients undergoing hematopoietic cell transplantation (HCT). Our ability to understand how certain organisms attain intestinal domination over others has been restricted in part by current metagenomic sequencing technologies that are typically unable to reconstruct complete genomes for individual organisms present within a sequenced microbial community. We recently developed a metagenomic read cloud sequencing and assembly approach that generates improved draft genomes for individual organisms compared to conventional short-read sequencing and assembly methods. Herein, we applied metagenomic read cloud sequencing to four stool samples collected longitudinally from an HCT patient preceding treatment and over the course of heavy antibiotic exposure. Results Characterization of microbiome composition by taxonomic classification of reads reveals that that upon antibiotic exposure, the subject’s gut microbiome experienced a marked decrease in diversity and became dominated by Escherichia coli. While diversity is restored at the final time point, this occurs without recovery of the original species and strain-level composition. Draft genomes for individual organisms within each sample were generated using both read cloud and conventional assembly. Read clouds were found to improve the completeness and contiguity of genome assemblies compared to conventional assembly. Moreover, read clouds enabled the placement of antibiotic resistance genes present in multiple copies both within a single draft genome and across multiple organisms. The occurrence of resistance genes associates with the timing of antibiotics administered to the patient, and comparative genomic analysis of the various intestinal E. coli strains across time points as well as the bloodstream isolate showed that the subject’s E. coli bloodstream infection likely originated from the intestine. The E. coli genome from the initial pre-transplant stool sample harbors 46 known antimicrobial resistance genes, while all other species from the pre-transplant sample each contain at most 5 genes, consistent with a model of heavy antibiotic exposure resulting in selective outgrowth of the highly antibiotic-resistant E. coli. Conclusion This study demonstrates the application and utility of metagenomic read cloud sequencing and assembly to study the underlying strain-level genomic factors influencing gut microbiome dynamics under extreme selective pressures in the clinical context of HCT.

scholarly journals ResistoXplorer: a web-based tool for visual, statistical and exploratory data analysis of resistome data

2021 ◽  
Vol 3 (1) ◽  
Author(s):  
Achal Dhariwal ◽  
Roger Junges ◽  
Tsute Chen ◽  
Fernanda C Petersen
Keyword(s):  
Antimicrobial Resistance ◽  
Resistance Gene ◽  
High Throughput ◽  
Resistance Genes ◽  
Visual Analytics ◽  
Gene List ◽  
Metagenomic Sequencing ◽  
High Throughput Analysis ◽  
Antimicrobial Resistance Genes ◽  
Antimicrobial Resistance Gene

Abstract The study of resistomes using whole metagenomic sequencing enables high-throughput identification of resistance genes in complex microbial communities, such as the human microbiome. Over recent years, sophisticated and diverse pipelines have been established to facilitate raw data processing and annotation. Despite the progress, there are no easy-to-use tools for comprehensive visual, statistical and functional analysis of resistome data. Thus, exploration of the resulting large complex datasets remains a key bottleneck requiring robust computational resources and technical expertise, which creates a significant hurdle for advancements in the field. Here, we introduce ResistoXplorer, a user-friendly tool that integrates recent advancements in statistics and visualization, coupled with extensive functional annotations and phenotype collection, to enable high-throughput analysis of common outputs generated from metagenomic resistome studies. ResistoXplorer contains three modules—the ‘Antimicrobial Resistance Gene Table’ module offers various options for composition profiling, functional profiling and comparative analysis of resistome data; the ‘Integration’ module supports integrative exploratory analysis of resistome and microbiome abundance profiles derived from metagenomic samples; finally, the ‘Antimicrobial Resistance Gene List’ module enables users to intuitively explore the associations between antimicrobial resistance genes and the microbial hosts using network visual analytics to gain biological insights. ResistoXplorer is publicly available at http://www.resistoxplorer.no.

scholarly journals PHENOTYPIC AND GENOTYPIC CHARACTERIZATION OF FIVE ANTIMICROBIAL RESISTANCE GENES ASSOCIATED WITH KLEBSIELLA PNEUMONIAE ISOLATED FROM BURN INFECTION PATIENTS

2021 ◽  
Vol 9 (3) ◽  
pp. 378-387
Author(s):  
Mohsin Razzaq Azeez ◽  
◽  
Ahmed Abduljabbar Jaloob Aljanaby ◽  
Ilkay Corak Ocal ◽  
◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Klebsiella Pneumoniae ◽  
Resistance Genes ◽  
Beta Lactamase ◽  
Extended Spectrum Beta Lactamase ◽  
Extended Spectrum ◽  
Genotypic Characterization ◽  
Antimicrobial Resistance Genes ◽  
Phenotypic And Genotypic Characterization

The current study was carried out for the phenotypic and genotypic characterization of five antimicrobial resistance-associated genes in Klebsiella pneumoniae isolated from burn infection patients. Total one hundred three (103) bacterial samples (strains) were isolated from the 103 burn infection patients admitted at Middle Euphrates Burns Center of AL-Kufa City Iraq. Out of total isolated bacterial samples (103), there were 31 isolates (30%) identified as Pseudomonas sp., 23 isolates (22.3%) as K. pneumonia, 22 isolates as Staphylococcus sps. (21.4%), 11 isolates as E. coli (10.6%), 8 isolates as Acinetobacter sps. (7.8%), 5 isolates as Enterobacter sps. (4.9%), while the lowest prevalence (3 isolates) was reported for the Proteus spp. (3%). The antimicrobial sensitivity test indicated that all isolated K. pneumoniae have resistant (100%) against standard antibiotic Amoxicillin. While Imipenem is the only antibiotic that can inhibit the growth of all 23 isolates. Further, according to the phenotypic detection method, there were 14 isolates (61%) capable of production of extended spectrum beta lactamase (ESBL). Genotypic method to detect the presence of five antibiotic resistance genes by polymerase chain reaction proved that 13 isolates (56.5%) were Tem gene, 18 isolates (78.2%) were positive for Shv gene, 8 isolates (34.7%) were positive Ctxm gene, three isolates (13%) were positive for Oxa gene and 10 isolates (43.7%) positive for AmpC gene. Results of the study can be concluded that K. pneumoniae is the second causative agent that causes burn infection and has higher antibiotics resistance. Extended spectrum beta lactamase of K. pneumoniae was higher prevalence in burn infection and harbored many beta lactamase genes.

scholarly journals 129. Antimicrobial Resistance Genes Were Reduced Following Administration of Investigational Microbiota-Based Live Biotherapeutic RBX2660 to Individuals with Recurrent Clostridioides difficile Infection

2021 ◽  
Vol 8 (Supplement_1) ◽  
pp. S79-S79
Author(s):  
Heidi Hau ◽  
Dana M Walsh ◽  
Carlos Gonzalez ◽  
Bill Shannon ◽  
Ken Blount
Keyword(s):  
Resistance Genes ◽  
Mixed Model ◽  
Linear Mixed Model ◽  
Fluoroquinolone Resistance ◽  
Sequence Coverage ◽  
Phase 3 ◽  
Extended Spectrum Beta Lactamase ◽  
Clostridioides Difficile ◽  
Gut Colonization ◽  
Antimicrobial Resistance Genes

Abstract Background Intestinal colonization by antimicrobial resistant (AMR) pathogens is a known health and infection risk, and is common among individuals with recurrent Clostridioides difficile infections (rCDI). Accordingly, therapeutic approaches that decolonize the gut of AMR pathogens could be valuable to patients to reduce risk of associated illnesses. Herein, we assessed gut colonization with AMR bacteria before and after treatment with RBX2660—a microbiota-based investigational live biotherapeutic—in the PUNCH CD3 Phase 3 trial for reducing CDI recurrence. Methods rCDI participants enrolled in PUNCH CD3 received a blinded single dose of RBX2660 or placebo within 24 to 72 hours after completing antibiotic treatment for the most recent rCDI episode. Clinical response was the absence of CDI recurrence at eight weeks after treatment, and participants were asked to submit stool samples prior to RBX2660 or placebo treatment (baseline) and 1, 4 and 8 weeks, 3 and 6 months after study treatment. Samples were extracted and sequenced using a shallow shotgun method. The presence and number of AMR genes was determined for 175 participant samples and 116 RBX2660 samples using 90% K-mer sequence coverage based on the MEGARes database. AMR gene count data were collapsed into count columns to adjust for sparseness in the matrices and were analyzed using a Generalized Linear Mixed Model. Results Clinically, RBX2660 demonstrated superior efficacy versus placebo (70.4% and 58.1%, respectively), and the total AMR genes per participant decreased significantly from before to after treatment in RBX2660-treated responders (p< .05, Figure 1) and remained low to at least 6 months. Among genes that decreased in RBX2660 responders were clinically important extended-spectrum beta-lactamase (blaTEM, blaSHV, blaCTX-M), vancomycin resistance (vanA, vanB), and fluoroquinolone resistance genes (gyrA, parC). Total AMR genes per PUNCH CD3 participant among RBX2660-treated responders at the indicated time points and in the RBX2660 investigational product. The red lines indicate timepoint group medians. Conclusion In the PUNCH CD3 Phase 3 trial of RBX2660 for rCDI, AMR gene content decreased after RBX2660 treatment and remained low to at least 6 months, consistent with prior RBX2660 trials. This underscores the potential of microbiota-based biotherapeutics for decolonizing AMR bacteria from gut microbiota and thereby reducing AMR infection risks. Disclosures Heidi Hau, PhD, Rebiotix Inc. (Employee) Dana M. Walsh, PhD, Rebiotix (Employee) Ken Blount, PhD, Rebiotix Inc., a Ferring Company (Employee)

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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