scholarly journals Tracking Reservoirs of Antimicrobial Resistance Genes in a Complex Microbial Community Using Metagenomic Hi-C: The Case of Bovine Digital Dermatitis

Antibiotics ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 221
Author(s):  
Ashenafi F. Beyi ◽  
Alan Hassall ◽  
Gregory J. Phillips ◽  
Paul J. Plummer
Keyword(s):  
Antimicrobial Resistance ◽  
Microbial Communities ◽  
Resistance Genes ◽  
Bacterial Species ◽  
Tetracycline Resistance ◽  
Digital Dermatitis ◽  
Proximity Ligation ◽  
Tetracycline Resistance Genes ◽  
Complex Microbial Community ◽  
Antimicrobial Resistance Genes

Bovine digital dermatitis (DD) is a contagious infectious cause of lameness in cattle with unknown definitive etiologies. Many of the bacterial species detected in metagenomic analyses of DD lesions are difficult to culture, and their antimicrobial resistance status is largely unknown. Recently, a novel proximity ligation-guided metagenomic approach (Hi-C ProxiMeta) has been used to identify bacterial reservoirs of antimicrobial resistance genes (ARGs) directly from microbial communities, without the need to culture individual bacteria. The objective of this study was to track tetracycline resistance determinants in bacteria involved in DD pathogenesis using Hi-C. A pooled sample of macerated tissues from clinical DD lesions was used for this purpose. Metagenome deconvolution using ProxiMeta resulted in the creation of 40 metagenome-assembled genomes with ≥80% complete genomes, classified into five phyla. Further, 1959 tetracycline resistance genes and ARGs conferring resistance to aminoglycoside, beta-lactams, sulfonamide, phenicol, lincosamide, and erythromycin were identified along with their bacterial hosts. In conclusion, the widespread distribution of genes conferring resistance against tetracycline and other antimicrobials in bacteria of DD lesions is reported for the first time. Use of proximity ligation to identify microorganisms hosting specific ARGs holds promise for tracking ARGs transmission in complex microbial communities.

scholarly journals Genotypic antimicrobial resistance characterization of E. coli from dairy calves at high risk of respiratory disease administered enrofloxacin or tulathromycin

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
R. V. Pereira ◽  
C. Foditsch ◽  
J. D. Siler ◽  
S. C. Dulièpre ◽  
C. Altier ◽  
...  
Keyword(s):  
High Risk ◽  
Antimicrobial Resistance ◽  
Respiratory Disease ◽  
Resistance Genes ◽  
Tetracycline Resistance ◽  
Control Group ◽  
E Coli ◽  
Tetracycline Resistance Genes ◽  
Antimicrobial Resistance Genes

Abstract The objective of this study was to evaluate the longitudinal effect of enrofloxacin or tulathromycin use in calves at high risk of bovine respiratory disease (BRD) on antimicrobial resistance genes and mutation in quinolone resistance-determining regions (QRDR) in fecal E. coli. Calves at high risk of developing BRD were randomly enrolled in one of three groups receiving: (1) enrofloxacin (ENR; n = 22); (2) tulathromycin (TUL; n = 24); or (3) no treatment (CTL; n = 21). Fecal samples were collected at enrollment and at 7, 28, and 56 days after beginning treatment, cultured for Escherichiacoli (EC) and DNA extracted. Isolates were screened for cephalosporin, quinolone and tetracycline resistance genes using PCR. QRDR screening was conducted using Sanger sequencing. The only resistance genes detected were aac(6′)Ib-cr (n = 13), bla-CTX-M (n = 51), bla-TEM (n = 117), tetA (n = 142) and tetB (n = 101). A significantly higher detection of gyrA mutated at position 248 at time points 7 (OR = 11.5; P value = 0.03) and 28 (OR = 9.0; P value = 0.05) was observed in the ENR group when compared to calves in the control group. Our findings support a better understanding of the potential impacts from the use of enrofloxacin in calves on the selection and persistence of resistance.

scholarly journals Metagenomic Quantification of Genes with Internal Standards

mBio ◽  
2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Emily Crossette ◽  
Jordan Gumm ◽  
Kathryn Langenfeld ◽  
Lutgarde Raskin ◽  
Melissa Duhaime ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Resistance Genes ◽  
Tetracycline Resistance ◽  
Dairy Manure ◽  
Molecular Techniques ◽  
Gene Copy ◽  
Gene Quantification ◽  
Gene Copies ◽  
Antimicrobial Resistance Genes ◽  
Metagenomic Approach

ABSTRACT We demonstrate that an assembly-independent and spike-in facilitated metagenomic quantification approach can be used to screen and quantify over 2,000 genes simultaneously, while delivering absolute gene concentrations comparable to those for quantitative PCR (qPCR). DNA extracted from dairy manure slurry, digestate, and compost was spiked with genomic DNA from a marine bacterium and sequenced using the Illumina HiSeq4000. We compared gene copy concentrations, in gene copies per mass of sample, of five antimicrobial resistance genes (ARGs) generated with (i) our quantitative metagenomic approach, (ii) targeted qPCR, and (iii) a hybrid quantification approach involving metagenomics and qPCR-based 16S rRNA gene quantification. Although qPCR achieved lower quantification limits, the metagenomic method avoided biases caused by primer specificity inherent to qPCR-based methods and was able to detect orders of magnitude more genes than is possible with qPCR assays. We used the approach to simultaneously quantify ARGs in the Comprehensive Antimicrobial Resistance Database (CARD). We observed that the total abundance of tetracycline resistance genes was consistent across different stages of manure treatment on three farms, but different samples were dominated by different tetracycline resistance gene families. IMPORTANCE qPCR and metagenomics are central molecular techniques that have offered insights into biological processes for decades, from monitoring spatial and temporal gene dynamics to tracking ARGs or pathogens. Still needed is a tool that can quantify thousands of relevant genes in a sample as gene copies per sample mass or volume. We compare a quantitative metagenomic approach with traditional qPCR approaches in the quantification of ARG targets in dairy manure samples. By leveraging the benefits of nontargeted community genomics, we demonstrate high-throughput absolute gene quantification of all known ARG sequences in environmental samples.

scholarly journals Antimicrobial Susceptibility Testing and Tentative Epidemiological Cutoff Values for FiveBacillusSpecies Relevant for Use as Animal Feed Additives or for Plant Protection

2018 ◽  
Vol 84 (19) ◽  
Author(s):  
Yvonne Agersø ◽  
Birgitte Stuer-Lauridsen ◽  
Karin Bjerre ◽  
Michelle Geervliet Jensen ◽  
Eric Johansen ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Resistance Genes ◽  
Pathogenic Bacteria ◽  
Animal Feed ◽  
Bacterial Species ◽  
Feed Additives ◽  
Content Type ◽  
Cutoff Values ◽  
Antimicrobial Resistance Genes ◽  
Species Specific

ABSTRACTBacillus megaterium(n= 29),Bacillus velezensis(n= 26),Bacillus amyloliquefaciens(n= 6),Bacillus paralicheniformis(n= 28), andBacillus licheniformis(n= 35) strains from different sources, origins, and time periods were tested for the MICs for nine antimicrobial agents by the CLSI-recommended method (Mueller-Hinton broth, 35°C, for 18 to 20 h), as well as with a modified CLSI method (Iso-Sensitest [IST] broth, 37°C [35°C forB. megaterium], 24 h). This allows a proposal of species-specific epidemiological cutoff values (ECOFFs) for the interpretation of antimicrobial resistance in these species. MICs determined by the modified CLSI method were 2- to 16-fold higher than with the CLSI-recommended method for several antimicrobials. The MIC distributions differed between species for five of the nine antimicrobials. Consequently, use of the modified CLSI method and interpretation of resistance by use of species-specific ECOFFs is recommended. The genome sequences of all strains were determined and used for screening for resistance genes against the ResFinder database and for multilocus sequence typing. A putative chloramphenicol acetyltransferase (cat) gene was found in oneB. megateriumstrain with an elevated chloramphenicol MIC compared to the otherB. megateriumstrains. InB. velezensisandB. amyloliquefaciens, a putative tetracycline efflux gene,tet(L), was found in all strains (n= 27) with reduced tetracycline susceptibility but was absent in susceptible strains. AllB. paralicheniformisand 23% ofB. licheniformisstrains had elevated MICs for erythromycin and harboredermD. The presence of these resistance genes follows taxonomy suggesting they may be intrinsic rather than horizontally acquired. Reduced susceptibility to chloramphenicol, streptomycin, and clindamycin could not be explained in all species.IMPORTANCEWhen commercializing bacterial strains, likeBacillusspp., for feed applications or plant bioprotection, it is required that the strains are free of acquired antimicrobial resistance genes that could potentially spread to pathogenic bacteria, thereby adding to the pool of resistance genes that may cause treatment failures in humans or animals. Conversely, if antimicrobial resistance is intrinsic to a bacterial species, the risk of spreading horizontally to other bacteria is considered very low. Reliable susceptibility test methods and interpretation criteria at the species level are needed to accurately assess antimicrobial resistance levels. In the present study, tentative ECOFFs for fiveBacillusspecies were determined, and the results showed that the variation in MICs followed the respective species. Moreover, putative resistance genes, which were detected by whole-genome sequencing and suggested to be intrinsic rather that acquired, could explain the resistance phenotypes in most cases.

scholarly journals Detection of a Novel, and Likely Ancestral, Tn916-Like Element from a Human Saliva Metagenomic Library

Genes ◽  
2020 ◽  
Vol 11 (5) ◽  
pp. 548
Author(s):  
Liam J. Reynolds ◽  
Muna F. Anjum ◽  
Adam P. Roberts
Keyword(s):  
Resistance Genes ◽  
Metagenomic Library ◽  
Tetracycline Resistance ◽  
Human Saliva ◽  
Fusion Event ◽  
Tetracycline Resistance Genes ◽  
Antimicrobial Resistance Genes ◽  
Genes Encoding ◽  
Gene Fusion Event ◽  
Metagenomic Approach

Tn916 is a conjugative transposon (CTn) and the first reported and most well characterised of the Tn916/Tn1545 family of CTns. Tn916-like elements have a characteristic modular structure and different members of this family have been identified based on similarities and variations in these modules. In addition to carrying genes encoding proteins required for their conjugation, Tn916-like elements also carry accessory, antimicrobial resistance genes; most commonly the tetracycline resistance gene, tet(M). Our study aimed to identify and characterise tetracycline resistance genes from the human saliva metagenome using a functional metagenomic approach. We identified a tetracycline-resistant clone, TT31, the sequencing of which revealed it to encode both tet(M) and tet(L). Comparison of the TT31 sequence with the accessory, regulation, and recombination modules of other Tn916-like elements indicated that a partial Tn916-like element encoding a truncated orf9 was cloned in TT31. Analysis indicated that a previous insertion within the truncated orf9 created the full length orf9 found in most Tn916-like transposons; demonstrating that orf9 is, in fact, the result of a gene fusion event. Thus, we hypothesise that the Tn916-like element cloned in TT31 likely represents an ancestral Tn916.

Related Antimicrobial Resistance Genes Detected in Different Bacterial Species Co-isolated from Swine Fecal Samples

2011 ◽  
Vol 8 (6) ◽  
pp. 663-679 ◽  
Author(s):  
Jonathan G. Frye ◽  
Rebecca L. Lindsey ◽  
Richard J. Meinersmann ◽  
Mark E. Berrang ◽  
Charlene R. Jackson ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Resistance Genes ◽  
Bacterial Species ◽  
Fecal Samples ◽  
Antimicrobial Resistance Genes

scholarly journals Antimicrobial Resistance Genes in Porcine Pasteurella multocida Are Not Associated with Its Antimicrobial Susceptibility Pattern

Antibiotics ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 614
Author(s):  
Máximo Petrocchi-Rilo ◽  
César-B. Gutiérrez-Martín ◽  
Esther Pérez-Fernández ◽  
Anna Vilaró ◽  
Lorenzo Fraile ◽  
...  
Keyword(s):  
Resistance Genes ◽  
Antimicrobial Susceptibility ◽  
Pasteurella Multocida ◽  
Antimicrobial Agents ◽  
Tetracycline Resistance ◽  
Susceptibility Pattern ◽  
Antimicrobial Susceptibility Pattern ◽  
Tetracycline Resistance Genes ◽  
Antimicrobial Resistance Genes ◽  
Western Spain

Forty-eight Pasteurella multocida isolates were recovered from porcine pneumonic lungs collected from farms in “Castilla y León” (north-western Spain) in 2017–2019. These isolates were characterized for their minimal inhibition concentrations to twelve antimicrobial agents and for the appearance of eight resistance genes: tetA, tetB, blaROB1, blaTEM, ermA, ermC, mphE and msrE. Relevant resistance percentages were shown against tetracyclines (52.1% for doxycycline, 68.7% for oxytetracycline), sulphamethoxazole/trimethoprim (43.7%) and tiamulin (25.0%), thus suggesting that P. multocida isolates were mostly susceptible to amoxicillin, ceftiofur, enrofloxacin, florfenicol, marbofloxacin and macrolides. Overall, 29.2% of isolates were resistant to more than two antimicrobials. The tetracycline resistance genes (tetA and tetB) were detected in 22.9% of the isolates, but none were positive to both simultaneously; blaROB1 and blaTEM genes were found in one third of isolates but both genes were detected simultaneously in only one isolate. The ermC gene was observed in 41.7% of isolates, a percentage that decreased to 22.9% for msrE; finally, ermA was harbored by 16.7% and mphE was not found in any of them. Six clusters were established based on hierarchical clustering analysis on antimicrobial susceptibility for the twelve antimicrobials. Generally, it was unable to foresee the antimicrobial susceptibility pattern for each family and the association of each particular isolate inside the clusters established from the presence or absence of the resistance genes analyzed.

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