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.

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 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 Development, Validation, and Application of PCR Primers for Detection of Tetracycline Efflux Genes of Gram-Negative Bacteria

2002 ◽  
Vol 68 (4) ◽  
pp. 1786-1793 ◽  
Author(s):  
R. I. Aminov ◽  
J. C. Chee-Sanford ◽  
N. Garrigues ◽  
B. Teferedegne ◽  
I. J. Krapac ◽  
...  
Keyword(s):  
Resistance Genes ◽  
Production Systems ◽  
Animal Feed ◽  
Antibiotic Resistance Gene ◽  
Tetracycline Resistance ◽  
Pcr Primers ◽  
Tetracycline Resistance Genes ◽  
Common Structure ◽  
Waste Lagoons ◽  
Genes Encoding

ABSTRACT Phylogenetic analysis of tetracycline resistance genes, which confer resistance due to the efflux of tetracycline from the cell catalyzed by drug:H+ antiport and share a common structure with 12 transmembrane segments (12-TMS), suggested the monophyletic origin of these genes. With a high degree of confidence, this tet subcluster unifies 11 genes encoding tet efflux pumps and includes tet(A), tet(B), tet(C), tet(D), tet(E), tet(G), tet(H), tet(J), tet(Y), tet(Z), and tet(30). Phylogeny-aided alignments were used to design a set of PCR primers for detection, retrieval, and sequence analysis of the corresponding gene fragments from a variety of bacterial and environmental sources. After rigorous validation with the characterized control tet templates, this primer set was used to determine the genotype of the corresponding tetracycline resistance genes in total DNA of swine feed and feces and in the lagoons and groundwater underlying two large swine production facilities known to be impacted by waste seepage. The compounded tet fingerprint of animal feed was found to be tetCDEHZ, while the corresponding fingerprint of total intestinal microbiota was tetBCGHYZ. Interestingly, the tet fingerprints in geographically distant waste lagoons were identical (tetBCEHYZ) and were similar to the fecal fingerprint at the third location mentioned above. Despite the sporadic detection of chlortetracycline in waste lagoons, no auxiliary diversity of tet genes in comparison with the fecal diversity could be detected, suggesting that the tet pool is generated mainly in the gut of tetracycline-fed animals, with a negligible contribution from selection imposed by tetracycline that is released into the environment. The tet efflux genes were found to be percolating into the underlying groundwater and could be detected as far as 250 m downstream from the lagoons. With yet another family of tet genes, this study confirmed our earlier findings that the antibiotic resistance gene pool generated in animal production systems may be mobile and persistent in the environment with the potential to enter the food chain.

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.

scholarly journals Mosaic Tetracycline Resistance Genes Are Widespread in Human and Animal Fecal Samples

2006 ◽  
Vol 51 (3) ◽  
pp. 1115-1118 ◽  
Author(s):  
Andrea J. Patterson ◽  
Marco T. Rincon ◽  
Harry J. Flint ◽  
Karen P. Scott
Keyword(s):  
Resistance Genes ◽  
Tetracycline Resistance ◽  
Human Saliva ◽  
Fecal Samples ◽  
Tetracycline Resistance Genes ◽  
Two Samples

ABSTRACT Mosaic tetracycline resistance genes comprising tet(O), tet(W), and tet(32) sequences were abundant in DNA extracted from pig and human fecal samples, accounting for 78% (50/64) and 46% (37/80) of genes amplified with a tet(O) primer set, respectively, in two samples. The nonmosaic tet(32) gene was isolated from a human saliva bacterium.

scholarly journals Tetracycline Resistome of the Organic Pig Gut

2009 ◽  
Vol 75 (6) ◽  
pp. 1717-1722 ◽  
Author(s):  
Katarzyna A. Kazimierczak ◽  
Karen P. Scott ◽  
Denise Kelly ◽  
Rustam I. Aminov
Keyword(s):  
Resistance Genes ◽  
Metagenomic Library ◽  
Antibiotic Resistance Genes ◽  
Artificial Chromosome ◽  
Mobile Genetic Elements ◽  
Structure Characteristic ◽  
Genetic Elements ◽  
Genes Encoding ◽  
Flanking Regions ◽  
Metagenomic Approach

ABSTRACT The occurrence of genes conferring resistance to tetracyclines in the organic pig gut was assessed through the metagenomic approach. Of 9,000 bacterial artificial chromosome clones analyzed, 10 were identified as carrying the known tet(C), tet(W), and tet(40) genes, as well as novel genes encoding resistance to the tetracyclines minocycline and doxycycline. The latter are different from the known tet genes and are homologous to genes encoding UDP-glucose 4-epimerases, with the domain structure characteristic for these enzymes. The majority of the resistance genes were associated with putative mobile genetic elements. The sequence of a novel 9.7-kb plasmid carrying tet(W) and tet(40) was also identified. Conserved flanking regions identified around the tet(W) and tet(40) genes in our metagenomic library may play a role in genetic exchange of these genes. This is the first report describing the occurrence of tet(40) outside the human intestine. The maintenance of antibiotic resistance genes in apparently antibiotic-free animals is probably due to their presence on mobile genetic elements, the fitness cost of which for the cell is ameliorated during the previous antibiotic selection.

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 Antimicrobial Resistance Genes in Porcine Pasteurella Multocida are not Associated with its Antimicrobial Susceptibility Pattern

2020 ◽  
Author(s):  
Máximo Patrocchi-Rilo ◽  
César-B. Gutiérrez-Martín ◽  
Esther Pérez-Fernández ◽  
Anna Vilaró ◽  
Lorenzo Fraile ◽  
...  
Keyword(s):  
Resistance Genes ◽  
Antimicrobial Susceptibility ◽  
Clustering Analysis ◽  
Pasteurella Multocida ◽  
Antimicrobial Agents ◽  
Tetracycline Resistance ◽  
Susceptibility Pattern ◽  
Antimicrobial Susceptibility Pattern ◽  
Tetracycline Resistance Genes ◽  
Antimicrobial Resistance Genes

Forty-eight Pasteurella multocida isolates were recovered from porcine pneumonic lungs collected in Norwestern Spain (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 to teracyclines, sulphamethoxazole/trimethoprim and tiamulin, thus suggesting that P. multocida isolates were mostly susceptible to amoxicillin, ceftiofur, enrofloxacin, florfenicol, marbofloxacin and macrolides. 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 was 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. ermC gene was observed in 41.7% of isolates, a percentage that decreased until 22.9% for msrE; finally, ermA was harboured 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.

scholarly journals Prevalence of Tetracycline Resistance Genes in Oral Bacteria

2003 ◽  
Vol 47 (3) ◽  
pp. 878-882 ◽  
Author(s):  
A. Villedieu ◽  
M. L. Diaz-Torres ◽  
N. Hunt ◽  
R. McNab ◽  
D. A. Spratt ◽  
...  
Keyword(s):  
Resistance Genes ◽  
Tetracycline Resistance ◽  
Oral Bacteria ◽  
Healthy Adults ◽  
Resistant Bacteria ◽  
First Report ◽  
Tetracycline Resistance Genes ◽  
Oral Microflora ◽  
Genes Encoding ◽  
Ribosomal Protection Protein

ABSTRACT Tetracycline is a broad-spectrum antibiotic used in humans, animals, and aquaculture; therefore, many bacteria from different ecosystems are exposed to this antibiotic. In order to determine the genetic basis for resistance to tetracycline in bacteria from the oral cavity, saliva and dental plaque samples were obtained from 20 healthy adults who had not taken antibiotics during the previous 3 months. The samples were screened for the presence of bacteria resistant to tetracycline, and the tetracycline resistance genes in these isolates were identified by multiplex PCR and DNA sequencing. Tetracycline-resistant bacteria constituted an average of 11% of the total cultivable oral microflora. A representative 105 tetracycline-resistant isolates from the 20 samples were investigated; most of the isolates carried tetracycline resistance genes encoding a ribosomal protection protein. The most common tet gene identified was tet(M), which was found in 79% of all the isolates. The second most common gene identified was tet(W), which was found in 21% of all the isolates, followed by tet(O) and tet(Q) (10.5 and 9.5% of the isolates, respectively) and then tet(S) (2.8% of the isolates). Tetracycline resistance genes encoding an efflux protein were detected in 4.8% of all the tetracycline-resistant isolates; 2.8% of the isolates had tet(L) and 1% carried tet(A) and tet(K) each. The results have shown that a variety of tetracycline resistance genes are present in the oral microflora of healthy adults. This is the first report of tet(W) in oral bacteria and the first report to show that tet(O), tet(Q), tet(A), and tet(S) can be found in some oral species.

scholarly journals Manure Compost Is a Potential Source of Tetracycline-Resistant Escherichia coli and Tetracycline Resistance Genes in Japanese Farms

Antibiotics ◽  
2020 ◽  
Vol 9 (2) ◽  
pp. 76 ◽  
Author(s):  
Nobuki Yoshizawa ◽  
Masaru Usui ◽  
Akira Fukuda ◽  
Tetsuo Asai ◽  
Hidetoshi Higuchi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Resistance Genes ◽  
Tetracycline Resistance ◽  
Resistant Bacteria ◽  
E Coli ◽  
Tetracycline Resistance Genes ◽  
Manure Compost ◽  
Potential Source ◽  
Antimicrobial Resistance Genes ◽  
Route Of Transmission

Manure compost has been thought of as a potential important route of transmission of antimicrobial-resistant bacteria (ARB) and antimicrobial resistance genes (ARGs) from livestock to humans. To clarify the abundance of ARB and ARGs, ARB and ARGs were quantitatively determined in tetracycline-resistant Escherichia coli (harboring the tetA gene)-spiked feces in simulated composts. In the simulated composts, the concentration of spiked E. coli decreased below the detection limit at day 7. The tetA gene remained in manure compost for 20 days, although the levels of the gene decreased. Next, to clarify the field conditions of manure compost in Japan, the quantities of tetracycline-resistant bacteria, tetracycline resistance genes, and residual tetracyclines were determined using field-manure-matured composts in livestock farms. Tetracycline-resistant bacteria were detected in 54.5% of tested matured compost (6/11 farms). The copy number of the tetA gene and the concentrations of residual tetracyclines in field manure compost were significantly correlated. These results suggest that the use of antimicrobials in livestock constitutes a selective pressure, not only in livestock feces but also in manure compost. The appropriate use of antimicrobials in livestock and treatment of manure compost are important for avoiding the spread of ARB and ARGs.

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