Metagenomic Quantification of Genes with Internal Standards

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.

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

Genes ◽

10.3390/genes11050548 ◽

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.

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Open Waste Canals as Potential Sources of Antimicrobial Resistance Genes in Aerosols in Urban Kanpur, India

American Journal of Tropical Medicine and Hygiene ◽

10.4269/ajtmh.20-1222 ◽

2021 ◽

Author(s):

Olivia Ginn ◽

David Berendes ◽

Anna Wood ◽

Aaron Bivins ◽

Lucas Rocha-Melogno ◽

...

Keyword(s):

Antimicrobial Resistance ◽

Resistance Genes ◽

Ambient Air ◽

Control Site ◽

Urban Environments ◽

Air Samples ◽

Gene Copies ◽

Antimicrobial Resistance Genes ◽

Class 1 ◽

Control Samples

Understanding the movement of antimicrobial resistance genes (ARGs) in the environment is critical to managing their spread. To assess potential ARG transport through the air via urban bioaerosols in cities with poor sanitation, we quantified ARGs and a mobile integron (MI) in ambient air over periods spanning rainy and dry seasons in Kanpur, India (n = 53), where open wastewater canals (OCWs) are prevalent. Gene targets represented major antibiotic groups—tetracyclines (tetA), fluoroquinolines (qnrB), and beta-lactams (blaTEM)—and a class 1 mobile integron (intI1). Over half of air samples located near, and up to 1 km from OCWs with fecal contamination (n = 45) in Kanpur had detectable targets above the experimentally determined limits of detection (LOD): most commonly intI1 and tetA (56% and 51% of samples, respectively), followed by blaTEM (8.9%) and qnrB (0%). ARG and MI densities in these positive air samples ranged from 6.9 × 101 to 5.2 × 103 gene copies/m3 air. Most (7/8) control samples collected 1 km away from OCWs were negative for any targets. In comparing experimental samples with control samples, we found that intI1 and tetA densities in air are significantly higher (P = 0.04 and P = 0.01, respectively, alpha = 0.05) near laboratory-confirmed fecal contaminated waters than at the control site. These data suggest increased densities of ARGs and MIs in bioaerosols in urban environments with inadequate sanitation. In such settings, aerosols may play a role in the spread of AR.

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

Scientific Reports ◽

10.1038/s41598-020-76232-w ◽

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.

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Genes on the Move: In Vitro Transduction of Antimicrobial Resistance Genes between Human and Canine Staphylococcal Pathogens

Microorganisms ◽

10.3390/microorganisms8122031 ◽

2020 ◽

Vol 8 (12) ◽

pp. 2031

Author(s):

Sian Marie Frosini ◽

Ross Bond ◽

Alex J. McCarthy ◽

Claudia Feudi ◽

Stefan Schwarz ◽

...

Keyword(s):

Antimicrobial Resistance ◽

Resistance Genes ◽

One Health ◽

Methicillin Resistant Staphylococcus Aureus ◽

Tetracycline Resistance ◽

Methicillin Resistant ◽

Antimicrobial Resistance Genes ◽

Resistance Transfer ◽

Coagulase Positive Staphylococci

Transmission of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus pseudintermedius (MRSP) between people and pets, and their co-carriage, are well-described. Potential exchange of antimicrobial resistance (AMR) genes amongst these staphylococci was investigated in vitro through endogenous bacteriophage-mediated transduction. Bacteriophages were UV-induced from seven donor isolates of canine (MRSP) and human (MRSA) origin, containing tet(M), tet(K), fusB or fusC, and lysates filtered. Twenty-seven tetracycline- and fusidic acid- (FA-) susceptible recipients were used in 122 donor-recipient combinations (22 tetracycline, 100 FA) across 415 assays (115 tetracycline, 300 FA). Bacteriophage lysates were incubated with recipients and presumed transductants quantified on antimicrobial-supplemented agar plates. Tetracycline resistance transduction from MRSP and MRSA to methicillin-susceptible S. pseudintermedius (MSSP) was confirmed by PCR in 15/115 assays. No FA-resistance transfer occurred, confirmed by negative fusB/fusC PCR, but colonies resulting from FA assays had high MICs (≥32 mg/L) and showed mutations in fusA, two at a novel position (F88L), nine at H457[Y/N/L]. Horizontal gene transfer of tetracycline-resistance confirms that resistance genes can be shared between coagulase-positive staphylococci from different hosts. Cross-species AMR transmission highlights the importance of good antimicrobial stewardship across humans and veterinary species to support One Health.

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

Antibiotics ◽

10.3390/antibiotics10020221 ◽

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.

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Bacteriophages Isolated from Chicken Meat and the Horizontal Transfer of Antimicrobial Resistance Genes

Applied and Environmental Microbiology ◽

10.1128/aem.00872-15 ◽

2015 ◽

Vol 81 (14) ◽

pp. 4600-4606 ◽

Cited By ~ 40

Author(s):

Amira Shousha ◽

Nattakarn Awaiwanont ◽

Dmitrij Sofka ◽

Frans J. M. Smulders ◽

Peter Paulsen ◽

...

Keyword(s):

Antimicrobial Resistance ◽

Resistance Genes ◽

Horizontal Transfer ◽

Tetracycline Resistance ◽

Kanamycin Resistance ◽

Chicken Meat ◽

Resistant Bacteria ◽

Content Type ◽

E Coli ◽

Antimicrobial Resistance Genes

ABSTRACTAntimicrobial resistance in microbes poses a global and increasing threat to public health. The horizontal transfer of antimicrobial resistance genes was thought to be due largely to conjugative plasmids or transposons, with only a minor part being played by transduction through bacteriophages. However, whole-genome sequencing has recently shown that the latter mechanism could be highly important in the exchange of antimicrobial resistance genes between microorganisms and environments. The transfer of antimicrobial resistance genes by phages could underlie the origin of resistant bacteria found in food. We show that chicken meat carries a number of phages capable of transferring antimicrobial resistance. Of 243 phages randomly isolated from chicken meat, about a quarter (24.7%) were able to transduce resistance to one or more of the five antimicrobials tested intoEscherichia coliATCC 13706 (DSM 12242). Resistance to kanamycin was transduced the most often, followed by that to chloramphenicol, with four phages transducing tetracycline resistance and three transducing ampicillin resistance. Phages able to transduce antimicrobial resistance were isolated from 44% of the samples of chicken meat that we tested. The statistically significant (P= 0.01) relationship between the presence of phages transducing kanamycin resistance andE. coliisolates resistant to this antibiotic suggests that transduction may be an important mechanism for transferring kanamycin resistance toE. coli. It appears that the transduction of resistance to certain antimicrobials, e.g., kanamycin, not only is widely distributed inE. coliisolates found on meat but also could represent a major mechanism for resistance transfer. The result is of high importance for animal and human health.

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Phylogenetic Groups and Antimicrobial Resistance Genes in Escherichia coli from Different Meat Species

Antibiotics ◽

10.3390/antibiotics10121543 ◽

2021 ◽

Vol 10 (12) ◽

pp. 1543

Author(s):

Angelika Sacher-Pirkelbauer ◽

Daniela Klein-Jöbstl ◽

Dmitrij Sofka ◽

Anne-Béatrice Blanc-Potard ◽

Friederike Hilbert

Keyword(s):

Escherichia Coli ◽

Antimicrobial Resistance ◽

Resistance Genes ◽

Tetracycline Resistance ◽

Kanamycin Resistance ◽

Phylogenetic Group ◽

Phylogenetic Groups ◽

E Coli ◽

Antimicrobial Resistance Genes ◽

Meat Species

Escherichia coli isolated from meat of different animal species may harbour antimicrobial resistance genes and may thus be a threat to human health. The objectives of this study were to define antimicrobial resistance genes in E. coli isolates from pork, beef, chicken- and turkey meat and analyse whether their resistance genotypes associated with phylogenetic groups or meat species. A total number of 313 E. coli samples were isolated using standard cultural techniques. In 98% of resistant isolates, a dedicated resistance gene could be identified by PCR. Resistance genes detected were tet(A) and tet(B) for tetracycline resistance, strA and aadA1 for streptomycin resistance, sulI and sulII for resistance against sulphonamides, dfr and aphA for kanamycin resistance and blaTEM for ampicillin resistance. One stx1 harbouring E. coli isolated from pork harboured the tet(A) gene and belonged to phylogenetic group B2, whilst another stx1 positive isolate from beef was multi-resistant and tested positive for blaTEM,aphA, strA–B, sulII, and tet(A) and belonged to phylogenetic group A. In conclusion, the distribution of resistance elements was almost identical and statistically indifferent in isolates of different meat species. Phylogenetic groups did not associate with the distribution of resistance genes and a rather low number of diverse resistance genes were detected. Most E. coli populations with different resistance genes against one drug often revealed statistically significant different MIC values.

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Genomics of Environmental Salmonella: Engaging Students in the Microbiology and Bioinformatics of Foodborne Pathogens

Frontiers in Microbiology ◽

10.3389/fmicb.2021.592422 ◽

2021 ◽

Vol 12 ◽

Author(s):

Noah A. Greenman ◽

Sophie K. Jurgensen ◽

Charles P. Holmes ◽

Curtis J. Kapsak ◽

Raechel E. Davis ◽

...

Keyword(s):

Heavy Metal ◽

Antimicrobial Resistance ◽

Resistance Genes ◽

Sequence Data ◽

Antibiotic Resistance Genes ◽

Metal Resistance ◽

Molecular Techniques ◽

Microbiological Methods ◽

Antimicrobial Resistance Genes ◽

Field Samples

We have developed and implemented an undergraduate microbiology course in which students isolate, characterize, and perform whole genome assembly and analysis of Salmonella enterica from stream sediments and poultry litter. In the development of the course and over three semesters, successive teams of undergraduate students collected field samples and performed enrichment and isolation techniques specific for the detection of S. enterica. Eighty-eight strains were confirmed using standard microbiological methods and PCR of the invA gene. The isolates’ genomes were Illumina-sequenced by the Center for Food Safety and Applied Nutrition at the FDA and the Virginia state Division of Consolidated Laboratory Services as part of the GenomeTrakr program. Students used GalaxyTrakr and other web- and non-web-based platforms and tools to perform quality control on raw and assembled sequence data, assemble, and annotate genomes, identify antimicrobial resistance and virulence genes, putative plasmids, and other mobile genetic elements. Strains with putative plasmid-borne antimicrobial resistance genes were further sequenced by students in our research lab using the Oxford Nanopore MinIONTM platform. Strains of Salmonella that were isolated include human infectious serotypes such as Typhimurium and Infantis. Over 31 of the isolates possessed antibiotic resistance genes, some of which were located on large, multidrug resistance plasmids. Plasmid pHJ-38, identified in a Typhimurium isolate, is an apparently self-transmissible 183 kb IncA/C2 plasmid that possesses multiple antimicrobial resistance and heavy-metal resistance genes. Plasmid pFHS-02, identified in an Infantis isolate, is an apparently self-transmissible 303 kb IncF1B plasmid that also possesses numerous heavy-metal and antimicrobial resistance genes. Using direct and indirect measures to assess student outcomes, results indicate that course participation contributed to cognitive gains in relevant content knowledge and research skills such as field sampling, molecular techniques, and computational analysis. Furthermore, participants self-reported a deeper interest in scientific research and careers as well as psychosocial outcomes (e.g., sense of belonging and self-efficacy) commonly associated with student success and persistence in STEM. Overall, this course provided a powerful combination of field, wet lab, and computational biology experiences for students, while also providing data potentially useful in pathogen surveillance, epidemiological tracking, and for the further study of environmental reservoirs of S. enterica.

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

Microorganisms ◽

10.3390/microorganisms9040707 ◽

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.

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