Preparation of functional metagenomic libraries from low biomass samples using METa assembly and their application to capture antibiotic resistance genes

mSystems · 2025-09-10

ABSTRACT A significant challenge in the field of microbiology is the functional annotation of novel genes from microbiomes. The increasing pace of sequencing technology development has made solving this challenge in a high-throughput manner even more important. Functional metagenomics offers a sequence-naive and cultivation-independent solution. Unfortunately, most methods for constructing functional metagenomic libraries require large input masses of metagenomic DNA, putting many sample types out of reach. Here, we show that our functional metagenomic library preparation method, METa assembly, can be used to prepare useful libraries from much lower input DNA quantities. Standard methods of functional metagenomic library preparation generally call for 5–60 µg of input metagenomic DNA. We demonstrate that the threshold for input DNA mass can be lowered at least to 30.5 ng, a 3-log decrease from prior art. We prepared functional metagenomic libraries using between 30.5 ng and 100 ng of metagenomic DNA and found that despite their limited input mass, they were sufficient to link MFS transporters lacking substrate-specific annotations to tetracycline resistance and capture a gene encoding a novel GNAT family acetyltransferase that represents a new streptothricin acetyltransferase, satB . Our preparation of functional metagenomic libraries from aquatic samples and a human stool swab demonstrates that METa assembly can be used to prepare functional metagenomic libraries from microbiomes that were previously incompatible with this approach. IMPORTANCE Bacterial genes in microbial communities, including those that give resistance to antibiotics, are often so novel that sequencing-based approaches cannot predict their functions. Functional metagenomic libraries offer a high-throughput, sequence-naive solution to this problem, but their use is often held back due to their need for large quantities of metagenomic DNA. We demonstrate that our functional metagenomic library preparation method, METa assembly, can prepare these libraries using as little as ~30 ng of DNA, approximately 1,000-fold less than other methods. We use METa assembly to prepare functional metagenomic libraries from low-biomass aquatic and fecal swab microbiomes and show that they are home to novel tetracycline efflux pumps and a new family of streptothricin resistance gene, respectively. The efficiency of the METa assembly library preparation method makes many otherwise off-limits, low-biomass microbiome samples compatible with functional metagenomics.

A functionally selected <i>Acinetobacter</i> sp. phosphoethanolamine transferase gene from the goose fecal microbiome confers colistin resistance in <i>E. coli</i>

bioRxiv (Cold Spring Harbor Laboratory) · 2025-12-10

ABSTRACT Polymyxins are last-resort antibiotics for infections caused by multidrug resistant Gram-negative bacteria such as Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii . This makes the rise of bacteria exhibiting polymyxin E (colistin) resistance, largely through modification of lipid A moieties, concerning and suggests that it is important to document potential sources of the corresponding resistance genes. This study searched for potential emerging colistin-resistance genes from the environment by investigating a previously performed functional metagenomic selection for colistin resistance of a goose fecal microbiome. We found that the selection captured Acinetobacter sp. DNA fragments which all contained eptA genes. We confirmed their ability to confer significant colistin resistance in E. coli via modification of lipid A in the outer membrane. Furthermore, we found evidence for mobilization of closely related eptA genes in Acinetobacter strains, marking them as potential mcr genes or their precursors. This study highlights the goose fecal microbiome as a potential source for colistin resistance in the environment.

History of the streptothricin antibiotics and evidence for the neglect of the streptothricin resistome

npj Antimicrobials and Resistance · 2024-02-07 · 22 citations

The streptothricin antibiotics were among the first antibiotics to be discovered from the environment and remain some of the most recovered antimicrobials in natural product screens. Increasing rates of antibiotic resistance and recognition that streptothricin antibiotics may play a role in countering so-called super-bugs has led to the re-evaluation of their clinical potential. Here we will review the current state of knowledge of streptothricins and their resistance in bacteria, with a focus on the potential for new resistance mechanisms and determinants to emerge in the context of potential widespread clinical adoption of this antibiotic class.

Preparation of functional metagenomic libraries from low biomass samples using METa assembly and their application to capture antibiotic resistance genes

bioRxiv (Cold Spring Harbor Laboratory) · 2024-06-29

ABSTRACT A significant challenge in the field of microbiology is the functional annotation of sequence novel genes from microbiomes. The increasing pace of sequencing technology development has made solving this challenge in a high-throughput manner even more important. Functional metagenomics offer a sequence-naïve and cultivation-independent solution. Unfortunately, most methods for constructing functional metagenomic libraries require large input masses of metagenomic DNA, putting many sample types out of reach. Here, we show that our functional metagenomic library preparation method, METa assembly, can be used to prepare useful libraries from much lower input DNA quantities. Standard methods of functional metagenomic library preparation generally call for 5 μg to 60 μg of input metagenomic DNA. We demonstrate that the threshold for input DNA mass can be lowered at least to 30.5 ng, a three-log decrease from prior art. We prepared functional metagenomic libraries using between 30.5 ng and 100 ng of metagenomic DNA, and found that, despite their limited input mass, they were sufficient to link MFS transporters lacking substrate-specific annotations to tetracycline resistance and to capture a gene encoding a sequence novel GNAT family acetyltransferase that represents a new streptothricin acetyltransferase, satB . Our preparation of functional metagenomic libraries from aquatic samples and a human stool swab demonstrate that METa assembly can be used to prepare functional metagenomic libraries from microbiomes that were previously incompatible with this approach.

Functional and Structural Characterization of Diverse NfsB Chloramphenicol Reductase Enzymes from Human Pathogens

Microbiology Spectrum · 2022-02-23 · 12 citations

strains to discover that expression of chloramphenicol reductases is sufficient to confer chloramphenicol resistance to Es. coli, confirming that chloramphenicol reductase activity is widespread across this nitroreductase family. By solving the high-resolution crystal structures of active chloramphenicol reductases, we identified residues important for this activity. Our work supports the hypothesis that housekeeping proteins possessing multiple activities can evolve into antibiotic resistance enzymes.

#24: Death is Antibiotic-Microbiota Dependent in a Humanized Mouse Model of Late-Onset Neonatal Sepsis

Journal of the Pediatric Infectious Diseases Society · 2021-03-01

Abstract Background Premature infants receive antibiotics frequently for culture-negative sepsis, which diminishes gut microbial diversity and increases susceptibility to infections by antibiotic-resistant pathogens. Neonates with decreased gut microbiota diversity, termed dysbiotic, have dysregulated immune systems marked by increased concentrations of circulating activated T cells and decreased concentrations of circulating neutrophils and dendritic cells. We hypothesize that antibiotics (1) enrich for pathobionts within the gut, (2) promote a systemic, proinflammatory host response, and (3) cause death in an antibiotic specific manner in a gnotobiotic model of preterm gut microbiota development. Methods We colonized germ-free (GF) dams and sires with stools from preterm infants. Mouse pups acquire this neonatal microbiota, and at 10 days of life (DOL), we treat them with clinically relevant doses of antibiotics subcutaneously for 3 days. We use metagenomic shotgun sequencing of individual pup fecal samples longitudinally to ascertain phylogenetic composition, and use flow cytometry and multiplex cytokine arrays to determine the local and peripheral immune response. Results Using two representative microbiota from human neonates (hereafter referred to as microbiota A or B), we show that 94% of pups given microbiota A survive vs. 64% given microbiota B after meropenem/probenecid treatment (Figure 1; P &amp;lt; 0.05; n = 18–28 mice in &amp;gt; independent experiments). 40% of pups given microbiota A treated with ampicillin/gentamicin/probenecid survived (Figure 1; P &amp;lt; 0.01 relative to meropenem/probenecid or probenecid). Klebsiella species dominated the gut microbiota of microbiota A-humanized pups who succumbed and were found in the lung, liver, and spleen of one animal at necropsy. Enterococci dominated the gut microbiota of microbiota B-humanized pups who died during treatment. Pups colonized with microbiota B had increased peripheral CD4+ T cells at sacrifice after treatment compared with microbiota A-humanized pups (61% vs. 44% of circulating T cells, P &amp;lt; 0.0005). Conclusions Our model of preterm microbiota development and perturbation by antibiotics demonstrates potential bacterial translocation, proinflammatory immune response, and death dependent on the microbiota–antibiotic combination. Our transgenerational humanized-microbiota mouse model can be utilized to determine antibiotic by microbiota perturbation and examine risks of late-onset sepsis from antimicrobials.

#8: Microbiome and immune disruption accompany mouse death in a gnotobiotic mouse model of neonatal sepsis

Journal of the Pediatric Infectious Diseases Society · 2021-06-01

Abstract Background Premature infants frequently receive antibiotics, which diminishes gut microbial diversity and increases susceptibility to infections by antibiotic resistant pathogens. Neonates with decreased gut microbiota diversity, termed dysbiotic, have dysregulated immune systems marked by increased concentrations of circulating activated T cells and decreased concentrations of circulating neutrophils and dendritic cells. We hypothesize that antibiotics (1) enrich for pathogens within the gut, 2) promote a systemic, proinflammatory host response, and 3) cause death in an antibiotic- and microbiome-specific manner in a gnotobiotic model of preterm gut microbiota disruption. Methods We colonized germ free (GF) dams with stools from preterm infants. Mouse pups acquire this neonatal microbiota, and at 10 days of life (DOL), we treat them with clinically-relevant doses of antibiotics subcutaneously for 3 days. We determined serum concentrations of antibiotics in 10 DOL pups using tandem mass spectrometry to achieve approximate pharmacokinetics as observed in the neonatal intensive care unit (NICU). We ascertained phylogenetic composition using metagenomic shotgun sequencing of individual pup fecal samples longitudinally. We performed flow cytometry on peripheral blood and gut permeability assays to determine the local and peripheral immune response. Results We found adding probenecid prolonged the half-life of ampicillin and meropenem allowing for an approximation of serum levels observed in the NICU with an every 8 hour dosing regimen. Using two representative microbiomes from human neonates (hereafter referred to as microbiota A or B), we show that 95% of pups given microbiota A survive versus 54% given microbiota B after meropenem/probenecid treatment (Fig. 1A; p&amp;lt;0.01; n= 18–42 mice in 3–6 independent experiments). Conversely, only 28% of microbiota-A humanized pups survive during ampicillin/probenecid treatment (Fig. 1; p&amp;lt;0.0001). Ampicillin-resistant Klebsiella species and E. coli dominated the gut of microbiota A-humanized pups who succumbed during ampicillin/probenecid treatment whereas Enterococci dominated the gut of microbiota B-humanized pups who died during treatment. To test the reproducibility of this phenotype, we colonized mice with 2 additional preterm neonatal microbiomes with similar compositions to microbiota A and B (D and C, respectively). We found that microbiota-C humanized pups were similarly dominated by Enterococcus faecalis resulting in 42% mortality during meropenem/probenecid treatment (Fig. 1). Pups colonized with microbiota B had decreased circulating granulocytes, B cells, and CD8+ T cells at sacrifice after treatment compared to microbiota A-humanized pups. We next assessed gut permeability after antibiotic treatment by measuring 4kDa FITC-Dextran in mouse serum after oral gavage. Microbiota-A humanized pups treated with ampicillin/probenecid and microbiota B-humanized pups treated with meropenem/probenecid had elevated serum levels of FITC-Dextran (p&amp;lt;0.05 relative to vehicle control, one way ANOVA), indicative of increased gut permeability. Conclusions Our model of preterm microbiota perturbation by antibiotics demonstrates increased gut permeability, proinflammatory immune response, and death dependent on the microbiota-antibiotic combination. Our transgenerational humanized-microbiota mouse model can be utilized to determine antibiotic by microbiota perturbation and examine risks of late onset sepsis from specific antimicrobial administration. This research can lead to a personalized medicine approach of antibiotic treatment in the NICU to limit antibiotic side effects and mortality.

Mosaic Ends Tagmentation (METa) Assembly for Highly Efficient Construction of Functional Metagenomic Libraries

mSystems · 2021-06-29 · 16 citations

into an expression host to produce a functional metagenomic library, directly connecting genes to functions, is a sequence-naive and cultivation-independent method to discover novel genes. The process of preparing these libraries is DNA greedy and inefficient, however. Here, we describe a library preparation method that is an order of magnitude more efficient and less DNA greedy. This method is consistently efficient across libraries prepared from cultures, a soil microbiome, and a goose fecal microbiome and allowed us to discover new antibiotic resistance genes and mechanisms. This library preparation method will potentially allow the functional metagenomic exploration of microbiomes that were previously off limits due to their rarity or low microbial biomass, such as biomedical swabs or exotic samples.

Crystal structure of putative NAD(P)H-flavin oxidoreductase from Haemophilus influenzae R2846

2021-01-13

Mosaic Ends Tagmentation (METa) assembly for extremely efficient construction of functional metagenomic libraries

bioRxiv (Cold Spring Harbor Laboratory) · 2021-02-02 · 3 citations

ABSTRACT Functional metagenomic libraries, physical bacterial libraries which allow the high-throughput capture and expression of microbiome genes, have been instrumental in the sequence-naïve and cultivation-independent discovery of novel genes from microbial communities. Preparation of these libraries is limited by their high DNA input requirement and their low cloning efficiency. Here, we describe a new method, METa assembly, for extremely efficient functional metagenomic library preparation. We apply tagmentation to metagenomic DNA from soil and gut microbiomes to prepare DNA inserts for high-throughput cloning into functional metagenomic libraries. The presence of mosaic end sequences in the resulting DNA fragments synergizes with homology-based assembly cloning to result in a 300-fold increase in library size compared to traditional blunt cloning based protocols. Compared to published libraries prepared by state-of-the-art protocols we show that METa assembly is on average 23- to 270-fold more efficient and can be effectively used to prepare gigabase-sized libraries with as little as 200 ng of input DNA. We demonstrate the utility of METa assembly to capture novel genes based on their function by discovering novel aminoglycoside (26% amino acid identity) and colistin (36% amino acid identity) resistance genes in soil and goose gut microbiomes. METa assembly provides a streamlined, flexible, and efficient method for preparing functional metagenomic libraries, enabling new avenues of genetic and biochemical research into low biomass or scarce microbiomes. IMPORTANCE Medically and industrially important genes can be recovered from microbial communities by high-throughput sequencing but are limited to previously sequenced genes and their relatives. Cloning a metagenome en masse into an expression host to produce a functional metagenomic library is a sequence-naïve and cultivation-independent method to discover novel genes. This directly connects genes to functions, but the process of preparing these libraries is DNA greedy and inefficient. Here we describe a library preparation method that is an order of magnitude more efficient and less DNA greedy. This method is consistently efficient across libraries prepared from cultures, a soil microbiome, and from a goose fecal microbiome and allowed us to discover novel antibiotic resistance genes. This new library preparation method will potentially allow for the functional metagenomic exploration of microbiomes that were previously off limits due to their rarity or low microbial biomass, such biomedical swabs or exotic samples.