WO2023187041A1 - Banques métagénomiques - Google Patents

Banques métagénomiques Download PDF

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WO2023187041A1
WO2023187041A1 PCT/EP2023/058268 EP2023058268W WO2023187041A1 WO 2023187041 A1 WO2023187041 A1 WO 2023187041A1 EP 2023058268 W EP2023058268 W EP 2023058268W WO 2023187041 A1 WO2023187041 A1 WO 2023187041A1
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library
phage
bacterial
metagenomic
host
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Balint KINTSES
Gábor APJOK
Mónika SZAMEL
Tóbiás SARI
Orsolya Katinka MEHI
Ehud Qimron
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Biological Research Centre
Hcemm Nonprofit Kft.
Ramot At Tel-Aviv University Ltd.
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Publication of WO2023187041A1 publication Critical patent/WO2023187041A1/fr

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    • C12Q2521/00—Reaction characterised by the enzymatic activity
    • C12Q2521/30—Phosphoric diester hydrolysing, i.e. nuclease
    • C12Q2521/301—Endonuclease

Definitions

  • the invention refers to methods of producing a metagenomic bacterial library incorporating a library of fragmented genomic DNA using T7 bacteriophage transducing particles which are engineered to have an altered host range in order to deliver a wide range of fragmented genomic or metagenomic DNA into one or more species or strains of interest (SOI).
  • SOI species or strains of interest
  • the invention further refers to use of such metagenomic bacterial library in functional screens employing one or more SOI, and predicting the risk of horizontal gene transfer for heterologous(s) genes, such as genes conferring antibiotic resistance (antibiotic resistance genes, ARGs), if functional in multiple host bacteria. Broadly functional ARGs are more likely to be horizontally transferable.
  • Functional metagenomics is a powerful experimental tool that can be used to identify antibiotic resistance genes (ARGs) in the environment, but the range of suitable host bacterial species is limited. This limitation affects both the scope of the identified ARGs in the environment and the interpretation of their clinical relevance.
  • Metagenomics allows exhaustive analysis of microbial communities, including species which cannot be cultivated in laboratory conditions. By extracting genomic data from environmental samples, researchers gain knowledge on the species compositions and functionality of the microbiome in a range of natural environments. In particular, functional metagenomics is devoted to screening metagenomic DNA for the presence of genes that encode specific molecular functions.
  • WO2018/002940A1 discloses bacteriophage variants having extended host- range.
  • Yosef et al. describe bacteriophages with extended host range for DNA transduction.
  • Torres-Cortés Gloria et al. disclose identification of antibiotic resistance genes by functional metagenomics on soil samples.
  • Metagenomic libraries were constructed using a lambdaZAP-expressing phagemid system. Restriction analysis of selected clones showed a mean insert size to be 6.5-7 kb. Zhang Keya et al.
  • T7 phage display can be employed to clone biosynthetic genes from metagenomic DNA libraries.
  • Hiroki Ando et al. (Cell Systems 2015, 1(3): 187-196) disclose engineering modular viral scaffolds for targeted bacterial population editing. Synthetic phages are described with modulated host ranges to achieve killing of new target bacteria.
  • US2015064770 A1 discloses phage engineering to produce recombinant bacteriophages with tunable host ranges for controlling phage specificity.
  • US10953052 B2 discloses modifying lytic bacteriophages to express different host range determinants or tail fiber proteins.
  • Genomic DNA fragments are of different lengths from very small to very large (5.000 bp or larger). Metagenomic libraries will need to cover the full-size range, avoiding a bias towards the smaller fragments, which are more readily packaged into bacteriophages and transduced into target cells. Therefore, there is a need for improved methods to produce a metagenomic bacterial library using bacteriophage transducing particles, to allow identifying relevant functional genes within the full-size range.
  • HA003P SUMMARY OF THE INVENTION It is the objective of the invention to provide methods for producing a metagenomic bacterial library, covering a diversity of DNA fragments originating from biological sources with a broad size range.
  • the objective is solved by the subject matter as claimed and as further described herein.
  • the invention provides for a method of producing a metagenomic bacterial library incorporating a library of fragmented genomic DNA, which method comprises the steps: a) providing a library of fragmented genomic DNA originating from a biological source material, comprising a size range of at least 1.5 to 5 kb; b) incorporating said library of fragmented genomic DNA into transducing T7 bacteriophage particles which are engineered for altering the host range, thereby obtaining a transducing phage particle library comprising fragmented genomic DNA comprising a size range of at least 1.5 to 5 kb; and c) transducing the phage particle library into bacterial target cells, thereby obtaining a metagenomic bacterial library comprising said library of fragmented genomic DNA.
  • the invention provides for a method of producing a metagenomic bacterial library incorporating a library of fragmented genomic DNA, which method comprises the steps: a) providing a library of fragmented genomic DNA originating from a biological source material with a size range coverage of at least 1.5 to 5 kb; b) incorporating said library of fragmented genomic DNA into transducing T7 bacteriophage particles which are engineered for altering the host range, thereby obtaining a transducing phage particle library comprising fragmented genomic DNA comprising a size range to cover at least sizes between 1.5 and 5 kb; and c) transducing the phage particle library into bacterial target cells, thereby obtaining a metagenomic bacterial library comprising said library of fragmented genomic DNA.
  • the metagenomic bacterial library comprises said library of fragmented genomic DNA comprising the size range of at least 1.5 to 5 kb.
  • the size range covers genomic DNA fragments of at least any one of 0.5, 1, 1.5 kb size, and at least any one of 5, 6, 7, 8, 9, or 10 kb size.
  • the average size of the genomic DNA fragments is at least 1.5 kb, preferably within 1.5-5 kb, or 2-5 kb, or 3-5 kb, 1-4 kb, or 2-4 kb, or 2-3 kb.
  • the library of fragmented genomic DNA comprises small DNA fragments with a size below 5 kb, or up to 5kb, e.g., a size ranging between 1.5 and 5 kb.
  • the size coverage of the library of fragmented genomic DNA is understood to comprise at least the small sized DNA fragments, but may also comprise larger fragments.
  • the library of fragmented genomic DNA is characterized by a wide size coverage which at least includes the small sized DNA fragments.
  • the library of fragmented genomic DNA comprises small DNA fragments with a size below or up to 5kb; and large DNA fragments with a size of at least 5kb, or more than 5kb such as e.g., at least 6kb.
  • the library comprises DNA fragments with a size ranging between 1.5 and 5 kb, and DNA fragments with a size ranging between 5 and 10 kb e.g., 6-10 kb.
  • the size range of DNA fragments comprised in the library of fragmented genomic DNA covers genomic DNA fragments with a size of at least any one of 0.5, 1, 1.5 kb, and with a size of at least any one of 5, 6, 7, 8, 9, or 10 kb.
  • the average size of the genomic DNA fragments in the library of fragmented genomic DNA is at least 1.5 kb, preferably within 1.5-5 kb, or 2-5 kb, or 3-5 kb, 1-4 kb, or 2-4 kb, or 2-3 kb. It was surprising that T7 bacteriophage particles which are engineered for altering the host range can be used to produce a phage particle library comprising fragmented genomic DNA comprising a size range to cover a wide size coverage which at least includes the small sized DNA fragments such as a size below 5 kb, or up to 5kb, e.g., a size ranging between 1.5 and 5 kb, without a bias towards smaller DNA fragments.
  • the transducing phage particle library as used in the method described herein, comprises fragmented genomic DNA with about the same size coverage as the library of fragmented genomic DNA originating from a biological source material.
  • the transducing phage particle library comprises small DNA fragments with a size below 5 kb, or up to 5kb, e.g., a size ranging between 1.5 and 5 kb.
  • the size coverage of the transducing phage particle library is understood to comprise at least the small sized DNA fragments, but may also comprise larger fragments.
  • the transducing phage particle library is characterized by a wide size coverage of fragmented genomic DNA, which at least includes the small sized DNA fragments.
  • the transducing phage particle library comprises fragmented genomic DNA including small DNA fragments with a size below or up to 5kb; and large DNA fragments with a size of at least 5kb, or more than 5kb such as e.g., at least 6kb.
  • the transducing phage particle library comprises fragmented genomic DNA including DNA fragments with a size ranging between 1.5 and 5 kb, and DNA fragments with a size ranging between 5 and 10 kb e.g., 6-10 kb.
  • the size range of DNA fragments comprised in the phage library covers genomic DNA fragments with a size of at least any one of 0.5, 1, 1.5 kb, and with a size of at least any one of 5, 6, 7, 8, 9, or 10 kb.
  • the average size of the genomic DNA fragments in the phage library is at least 1.5 kb, preferably within 1.5-5 kb, or 2-5 kb, or 3-5 kb, 1-4 kb, or 2-4 kb, or 2- 3 kb.
  • the size range of the fragmented genomic DNA comprised in the library of fragmented genomic DNA originating from a biological source material, and comprised in the transducing phage particle library covers at least sizes between 1.5 and 5 kb.
  • the size range of fragmented genomic DNA comprised in both libraries is about the same.
  • the difference of the size range of DNA fragments comprised in the phage library compared to the library of fragmented genomic DNA originating from the biological source is less than 20%, or less than 10%, in particular when comparing the smallest fragments and the largest fragments.
  • the size range of both libraries covers genomic DNA fragments with a size of at least any one of 0.5, 1, 1.5 kb, and with a size of at least any one of 5, 6, 7, 8, 9, or 10 kb.
  • the average size of the genomic DNA fragments in both libraries is at least 1.5 kb, preferably within 1.5-5 kb, or 2-5 kb, or 3-5 kb, 1-4 kb, or 2-4 kb, or 2-3 kb.
  • the library of fragmented genomic DNA comprises a gene repertoire originating from prokaryotes or eukaryotes, preferably wherein the biological source material is an environmental source, preferably a water, soil, or plant source, or a biological sample of human or non-human animals.
  • the biological source may be a soil, wetland, river, volcano, tidal mudflat, salt pan, fresh water, or a seawater sample, or a sample of body fluid, such as urine, or feces.
  • the fragmented genomic DNA originates from microorganisms present in environmental sources or animals.
  • the fragmented genomic DNA originates from a microbiome.
  • the prokaryotes are bacteria or archaea.
  • the eukaryotes are yeast, filamentous fungi, plants or animals, including human or non-human animals.
  • the biological sample of human or non-human animals are samples of bodily tissue or fluids, such as e.g., feces, gut and skin.
  • a biological sample comprises colonizing microorganisms.
  • the gene repertoire is obtained from the microbiome of biological samples.
  • the metagenomic library may originate and be derived from any one or more, or all of: i) bacterial strains of environmental samples collected from anthropogenic soil, such as soil or river sediment at antibiotic polluted sites, e.g., in close vicinity of an antibiotic production plant; ii) bacterial strains of fecal samples from humans or animals; and/or iii) bacterial strains isolated in healthcare facilities.
  • the metagenomic bacterial library comprises a) a diversity of fragmented genomic DNA to cover the genome of at least 3, 4, or 5 x10E3 different organisms; and/or b) a diversity of fragmented genomic DNA of at least 3, 4, 5, or 6 x10E6 different DNA fragments.
  • said T7 bacteriophage particles comprise a heterologous phage tail protein for reprogramming host specificity, in particular to alter the host specificity, such as to broaden (i.e., to extend), shift (i.e., to change for a different one), and/or to limit to a part of the host range (i.e., to reduce).
  • the T7 bacteriophage particles are engineered for an altered host specificity with a preference to recognize the target cells over the phage particle-producing bacterial host cells.
  • the heterologous phage tail protein may be heterologous insofar that it originates from a bacteriophage that is any other than an Escherichia T7 phage, such as e.g., originating from a Salmonella phage ⁇ SG-JL2, Salmonella phage Vi06, or Klebsiella phage KP11 bacteriophage.
  • a bacteriophage that is any other than an Escherichia T7 phage, such as e.g., originating from a Salmonella phage ⁇ SG-JL2, Salmonella phage Vi06, or Klebsiella phage KP11 bacteriophage.
  • the heterologous phage tail protein may be heterologous, because being an artificial or recombinant gp17 protein (i.e., a gp17 that is not naturally-occurring), e.g., a gp17 protein different from a wild-type (wt) gp17 protein, in particular a recombinant T7 gp17 protein that comprises a mutation compared to the wt T7 gp17 protein.
  • the heterologous phage tail protein may be comprised in a heterologous phage tail.
  • Exemplary heterologous phage tail proteins may comprise the amino acid sequence of a gp17 protein originating from a T7, ⁇ SG-JL2, Vi06, or KP11 bacteriophage, which is modified, such as by one or more point mutations for altering the host range and/or altering the transduction efficiency to modulate, e.g., improve or otherwise change the transduction efficiency.
  • the heterologous phage tail protein is the tail fiber protein gp17 that is engineered to comprise one or more point mutations e.g., in the host-range- determining regions (HRDRs), to alter the transduction efficiency, such as e.g., to improve the transduction into a target host, and to reduce the transduction into a producing host.
  • HRDRs host-range- determining regions
  • the library of fragmented genomic DNA is cloned into vectors, specifically plasmids, such as broad host range plasmids.
  • any such plasmid carries a packaging signal, allowing translocation of the plasmid into the phage particles.
  • each plasmid carries one nucleic acid fragment derived from the metagenomic library.
  • the T7 phages are converted to transducing phage particles as described earlier (Yosef et al 2017). The transducing phage particles are generated inside transducing phage particle-producing bacterial host cells.
  • the T7 phages package the plasmids, comprising the specific packaging signals and the fragmented genomic DNA into a fraction of the T7 phage protein shells (capsid).
  • T7 phages protect their genetic material by packaging the nucleic acid in concentric layers to an almost crystalline concentration inside the capsid.
  • said T7 bacteriophage is a transducing phage particle, in particular a phage particle without the phage genome but comprising a plasmid for transduction into a host.
  • the fraction of the T7 phage capsids with a plasmid inside instead of the T7 genome becomes a transducing phage particle.
  • the remaining fraction of the T7 phage capsids packages the T7 genome that can replicate itself. These T7 phages become replicative phage contamination within the transducing phage particle preparation.
  • the transducing T7 phage particles can transduce said library of fragmented genomic DNA in the form of the plasmid into the SOI. By transduction, the packaged genetic material is injected into the host bacterium in a process named ejection.
  • Bacteriophage T7 belongs to the Podoviridae family and has a short, non- contractile tail formed by a tubular structure surrounded by fibers. T7 phages use their tail to deliver the genetic material without disrupting cell integrity. Specifically, T7 transducing phage particles can be generated in E.
  • E. coli transducing phage particle generating host cells such as E. coli BW25113, as described earlier (Yosef et al 2017).
  • E. coli BW25113 has been used to generate T7 transducing phage particles containing a plasmid library with uniform plasmid size.
  • this plasmid library contained point mutant versions of a phage tail gene (insert in the plasmid) and all library members had the same size of the plasmid insert.
  • the plasmid insert consists of the part of HA003P the plasmid comprising or consisting of the genomic fragmented DNA.
  • such insert is incorporated into the plasmid by fusion to the plasmid backbone.
  • the plasmid backbone is typically designed as a template for genetic manipulation. At a minimum, it contains a selectable marker, so that the presence of the plasmid can be selected for in the bacterial host, and a cloning site region to allow easy integration of foreign DNA into the plasmid.
  • the present invention is based on the finding that T7 phage with wild type T7 phage tail in combination with E. coli BW25113 transducing phage particle generating host cells) was not sufficient to generate high quality transducing phage particles containing a fragmented genomic DNA plasmid library with a wide size range.
  • T7 phages with wild type T7 phage tail were also found to generate replicative phage contamination at a concentration that abolishes the generation of metagenomic bacterial cells.
  • Transducing a large metagenomic library into a SOI requires a highly concentrated transducing phage particle preparation. At this concentration, however, HA003P replicative phage contamination becomes critical and kills a large fraction of the SOI. Such killing lowers the transduction efficiency of the fragmented genomic DNA plasmid library.
  • the transducing phage particle preparation is a preparation of T7 bacteriophage transducing particles comprising the metagenomic library as further described herein.
  • the transducing phage particle preparation is characterized by a low content of propagating (or replicative) phages, which is less than any one of 50%, 40%, 30%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than1% of the phage particles.
  • the amount of contaminating propagating (or replicative) phages in a transducing phage particle preparation or a respective library of phage particles can be determined by suitable methods known in the art, such as e.g., plaque assays. Comparing the number of plaque formation units to that of transduction formation units, provides the relative prevalence of the replicative phage contamination in the transducing phage particle preparation.
  • the phage particles described herein are engineered to comprise at least one host recognition element compatible with said target cells to confer DNA transduction ability with tail compatibility to said target cells.
  • the phage particles described herein are engineered to comprise a host recognition element that preferably recognizes the target cells, and recognizes the phage particle-producing bacterial host cells to a lesser extent. For example, less than 10%, or less than 1%, or less than 0.1%, of the number of transduced colonies (tfu; Yosef et al. 2017) per mL are obtained with phage articles comprising the engineered host recognition element as compared to phage particles comprising the wild type T7 host recognition element.
  • the phage particles described herein are engineered to comprise a host recognition element that recognizes the target cells, but does not recognize the phage particle-producing bacterial host cells with high efficiency (i.e., the producing host bacteria).
  • High efficiency is >10E9 tfu/ml (tfu/mL, number of transduced colonies per mL; Yosef et al. 2017) or >10E8 tfu/ml, or >10E7 tfu/ml.
  • the phage particles described herein are engineered to not comprise an efficient host recognition element towards the transducing phage particle-producing bacterial host cells (for example, E. coli BW25113).
  • transducing phage particle generated with the wild type T7 phage tail can transduce the E. coli BW25113 cell with an efficiency of >10E9 tfu/ml (Yosef et al.2017).
  • Such highly efficient recognition of the transducing phage particle-producing host cells was fond to be disadvantageous, because it favors the accumulation of propagating phages over transducing phage particles.
  • Reinfection of the transducing phage particle-producing bacterial host cells would continuously generate phages that package the T7 genome instead of the fragmented genomic DNA containing plasmid. This process would increase the concentration of propagating phage contamination in the transducing phage particle preparation.
  • T7 phage particles are used for the purpose described herein, which are able to transduce the fragmented genomic DNA into the target host bacteria with improved transduction efficiency, compared to a wt T7 phage e.g., at least 10, 100, or 1000 fold improved.
  • T7 phage particles are used for the purpose described herein, which are selected for recognizing target host bacteria other than E. coli, and/or a series of different hosts. Specifically, phages are used with tails that are compatible with desired hosts.
  • the target host cells are bacterial cells of one or more strains or one or more species. Host cells incorporating the metagenomic library are herein also referred to as “target cells”. The target cells are specifically understood as “target host bacteria”. The phage particle-producing bacterial host cells are specifically understood as “producing host bacteria”.
  • the target host bacteria are of a SOI.
  • SOI shall refer to either a “strain of interest” or “species of interest”, as the case may be.
  • the target cells are gram-negative bacteria of one or more different species, preferably of pathogen species.
  • the T7 spectrum of target hosts is HA003P extended for several species of Klebsiella, Salmonella, Escherichia, Shigella, and Enterobacter.
  • the target cells are human pathogens, or Clinically Relevant Bacterial Pathogens (CRBP), preferably selected from the Enterobacteriaceae family, such as the species Salmonella enterica, Klebsiella pneumoniae or Shigella sonnei.
  • the target cells are of at least two different bacterial species of interest (SOI).
  • the T7 phage are used which are particles that deliver only the desired genomic DNA, whereas a replication-competent phage DNA is not transduced.
  • the target cells are a strain which is not supporting T7 phage propagation, in particular propagation of the wt T7 phage.
  • the present method does not require phage propagation in the host cells. Therefore, the host cells can be selected form those that do not support phage propagation. For example, Shigella sonnei is not supporting T7 phage propagation.
  • the invention provides for a metagenomic bacterial library obtained by a method as further described herein.
  • Such metagenomic library specifically comprises or consists of bacterial host cells incorporating the library of fragmented genomic DNA that has been transduced by the phages, and the respective phage packaging signals.
  • the bacterial target cells comprise T7 phage DNA encoding a packaging signal incorporated into the plasmid.
  • the metagenomic bacterial library described herein is specifically characterized by the library of fragmented genomic DNA which have a size range as further described herein. Specifically, the sizes of genomic material or genes are distributed throughout the size range, as occurring upon sourcing from the biological source material, in particular without a bias towards to smaller sized DNA fragments (e.g., below 500 or 1.000 bases).
  • the metagenomic bacterial library described herein is specifically characterized by the coverage of functional genes of larger sizes (e.g., at least 1.5 kb or higher), or functional fragments thereof.
  • the invention provides for the use of the metagenomic bacterial library described herein in a functional screening method to identify one or more heterologous genes which are functional in bacterial cells. Specifically, genes are identified which are functional in the target bacterial cells.
  • the invention provides for a method of identifying one or more heterologous genes which are functional in bacterial cells by functional screening of a metagenomic bacterial library described herein, comprising: a) culturing the metagenomic bacterial library under selection conditions; b) selecting from said bacterial library a bacterial repertoire which is functional under the selection conditions; and c) identifying said one or more heterologous genes in said bacterial repertoire which confer functionality.
  • the target cells can be cultured under selection conditions, to identify those genes of the metagenomic library which are functional under the selection conditions.
  • one or more ARGs can be identified which were transferred by the metagenomic library, are heterologous to the host cell, and functional in the host cell.
  • the selection conditions employ an antibiotic agent, and said functionality is antibiotic resistance.
  • the bacterial repertoire comprises a resistome consisting of cells resistant to said antibiotic agent, and a repertoire of antibiotic-resistance genes (ARGs) is identified from said heterologous bacterial genes.
  • any one or more of said ARGs encode antibiotic inactivating enzymes, such as a beta-lactamase or acetyltransferase.
  • said functional screening is performed in a metagenomic bacterial library of more than one bacterial species of interest (SOI), to identify one or more functional heterologous bacterial genes in said bacterial repertoire which are shared by more than one SOI, thereby determining the risk of horizontal gene transfer of said heterologous bacterial genes.
  • SOI bacterial species of interest
  • the invention provides for a method of predicting the risk of a horizontally-transferable heterologous gene in bacterial cells, by identifying functionality of heterologous genes in a functional screening method using a metagenomic bacterial library described herein, and determining the risk of horizontal transfer of a heterologous gene, where said heterologous gene is functional in more than one bacterial SOI.
  • said heterologous gene is an antibiotic-resistance gene (ARG) which is predicted to bear a risk of horizontal gene transfer.
  • ARG antibiotic-resistance gene
  • the method provided herein provides for predicting broadly functional antibiotic-resistance genes (ARGs) conferring resistance to an antibiotic agent in target cells of at least two different bacterial species of interest (SOI).
  • the method comprises selecting from said target cells a resistome consisting of cells resistant to said antibiotic agent.
  • Antibiotic resistant bacteria and a respective resistome can be identified and further characterized to determine antibiotic-resistance genes (ARGs) which are functional in said SOI.
  • the ARGs are heterologous to said target cells, and were originating form said library of fragmented genomic DNA of a biological source material.
  • the invention provides for the use of a T7 bacteriophage that is engineered for reprogramming host specificity comprising a heterologous phage tail fiber protein gp17 in a method of preparing a metagenomic bacterial library, such as the metagenomic bacterial library as further described herein.
  • the T7 bacteriophage is engineered for extending the host range, as further described herein.
  • the host range can be extended by modulating the host cell recognition range of the phage, typically by changing the host cell recognition to include the original recognition range as in a wild-type phage (which has not been engineered for extending the host range), and to further include an additional recognition range beyond the original recognition range, to cover the recognition of not only the original (permissive) host, but also further hosts that were not recognized by the wild-type phage; or to only include the additional recognition range, without coverage of the original recognition range.
  • the T7 bacteriophage is engineered for improved DNA transduction efficiency, as further described herein.
  • the T7 bacteriophage comprises a T7 phage tail fiber protein gp17 comprising SEQ ID NO:1 that is modified by an amino acid substitution at position V544, preferably V544G or V544A.
  • HA003P provides for metagenomic phage library of T7 bacteriophage transducing particles that are engineered for reprogramming host specificity comprising a heterologous phage tail fiber protein gp17, which phage library comprises a diversity of phage particles incorporating a library of genomic DNA fragments.
  • the engineered T7 phage transducing particle has improved DNA transduction efficiency, to transduce the metagenomic library into bacterial host cells, such as to produce a metagenomic library with a broad size range of the genetic material.
  • the T7 phage transducing particles are engineered to comprise a heterologous tail, or a heterologous (such as an artificial or recombinant) gp17 protein originating from a T7, ⁇ SG-JL2, Vi06, or KP11 bacteriophage, which comprises one or more modifications for improved transduction efficiency into a host cell.
  • the T7 bacteriophage transducing particle comprises a heterologous phage tail protein which is an artificial gp17, such as gp17 that is comprises a wt gp17 sequence which is modified by one or more point mutations e.g., in the host-range- determining regions (HRDRs).
  • said gp17 originates from a T7 bacteriophage comprising the amino acid sequence of SEQ ID NO:1, which comprises one or more of the amino acid substitutions, preferably 1, 2, 3, 4, up to 5 amino acid substitutions, e.g., substitutions in the HRDRs.
  • the engineered T7 phage transducing particles have a differential DNA transduction efficiency for a target cell and a producing cell.
  • the DNA transduction efficiency can be determined by suitable methods known in the art, such as e.g., transduction assay (Yosef et al. 2017). Specifically, a plasmid containing an antibiotic selection marker, such as an antibiotic resistance gene, is transduced into a population of cells and selected on the antibiotic-containing plates. The efficiency of transduction is then determined by counting the surviving colonies. According to a specific aspect, the engineered T7 phage transducing particles have an improved DNA transduction efficiency e.g., by at least 1.5 or 2-fold into the target cells, in particular a SOI.
  • the T7 bacteriophage transducing particle comprises a heterologous phage tail protein that is modified to have a lowered transduction efficiency for the transducing phage particle producing host cell e.g., with a HA003P DNA transduction efficiency that is reduced by at least 90%, or at least any one of 95%, 96%, 97%, 98%, 99%, or even 100% reduced.
  • the reduced DNA transduction efficiency will result in the respective reduced or no transduction of the phage particle producing host cell.
  • the engineered T7 phage transducing particles described herein comprise a T7 phage tail fiber protein gp17 comprising or consisting of SEQ ID NO:1, which is modified by one or more point mutations, and comprises at least 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO:1.
  • said one or more point mutations comprise one or more amino acid substitutions at positions 500-560 of SEQ ID NO:1.
  • said one or more amino acid substitutions are within aa510 to aa550 of SEQ ID NO:1, preferably at position(s) selected from the group consisting of position 511, 521, 524, 536, 537, 539, 540, 541, 544, 545, 548, 549.
  • amino acid substitutions are selected from the group consisting of: V544X, wherein X is any one of G, A; N537X; wherein X is any one of T, I; D540X, wherein X is any one of G, A, N, H, E, S; S541R; M549I; G521R; P545T; I548L.
  • the host recognition element of the T7 phage may comprise at least one mutated T7 gp17 protein, specifically, the mutated T7 gp17 carrying a mutation in position 540, specifically, substituting aspartic acid (D, or Asp) with Glycine (G, or Gly), also referred to as the D540G mutant of SEQ ID NO:1, identified as SEQ ID NO:4.
  • the engineered T7 phage comprises gp17 comprising or consisting of SEQ ID NO:1, which is modified by only one of the amino acid substitution V544G or V544A, or by only the D540G substitution.
  • SEQ ID NO:4 identifies the T7 phage gp17 protein which comprises the D540G substitution as compared to SEQ ID NO:1.
  • the mutated T7 gp17 protein may comprise any one of the V544G or V544A substitution.
  • SEQ ID NO:5 identifies the T7 phage gp17 protein which comprises the V544G substitution as compared to SEQ ID NO:1.
  • SEQ ID NO:6 identifies the T7 phage gp17 protein which comprises the D540G substitution as compared to SEQ ID NO:1.
  • the V544G mutant of SEQ ID NO:1 is identified as SEQ ID NO:5.
  • the V544A mutant of SEQ ID NO:1 is identified as SEQ ID NO:6.
  • said gp17 comprises or consists of SEQ ID NO:1 which is modified by any one of the following, in particular, by any one of the following as the only mutation compared to SEQ ID NO:1: i. V544G, ii. V544A, iii. D540G, iv. N537T, D540A, and M549I, v. G521R, L524L, D540N, and A540V, vi. D540N, and S541R, vii. D540H, and S541R, viii. N537I, D540E, S541R, and P545T, ix. D540S, and S541R, x.
  • SEQ ID NO:1 which is modified by any one of the following, in particular, by any one of the following as the only mutation compared to SEQ ID NO:1: i. V544G, ii. V544A, iii. D540G, iv. N5
  • said gp17 originates from a ⁇ SG-JL2 bacteriophage comprising the amino acid sequence of SEQ ID NO:2, which comprises one or more of the amino acid substitutions, preferably 1, 2, 3, 4, up to 5 amino acid substitutions, e.g., one or more substitutions in the HRDRs.
  • the engineered T7 phage transducing particles described herein comprise a ⁇ SG-JL2 phage tail fiber protein gp17 comprising or consisting of SEQ ID NO:2, which is modified by one or more point mutations, and comprises at least 95, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO:2.
  • said one or more point mutations comprise one or more amino acid substitutions at positions 340-460 of SEQ ID NO:2.
  • said one or more amino acid substitutions are within aa350 to aa440 of SEQ ID NO:2, preferably at position(s) selected from the group consisting of position 346, 349, 355, 360, 361, 363, 364, 366, 379, 383, 384, 385, 389, 396, 398, 400, 401, 402, 404, 405, 408, 413, 434, 459.
  • amino acid substitutions are selected from the group consisting of: S346R; F349Y; V355M; V355A; A361S; G362R; G363A; E364G; R366G; F379L; I383F; I383M; I383Y; Q384R; N385K; A389S; A389G; G396R; G398R; N400K; N400T; P401L; N402K; P404A; Q405R; R408K; G413A; G434C; I459M.
  • said gp17 comprises or consists of SEQ ID NO:2 which is modified by any one of the following, in particular, by any one of the following as the only mutation compared to SEQ ID NO:2: HA003P i. N385K, and A389S; ii. R366G, F379L, and N400K; iii. N400K, N402K, and G413A; iv. G396R, N402K, and Q405R; v. N400K, N402K, P404A, and I459M; vi. V355M, and G398R; vii. I383F, and Q384R; viii.
  • said gp17 originates from a Vi06 bacteriophage comprising the amino acid sequence of SEQ ID NO:3, comprising one or more of the amino acid substitutions, preferably 1, 2, 3, 4, up to 5 amino acid substitutions, e.g., one or more substitutions in the HRDRs.
  • the engineered T7 phage transducing particles described herein comprise a Vi06 phage tail fiber protein gp17 comprising or consisting of SEQ ID NO:3, which is modified by one or more point mutations, and comprises at least 95, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO:3.
  • said one or more point mutations comprise one or more amino acid substitutions at positions 200 to 350 of SEQ ID NO:3.
  • said one or more amino acid substitutions are at position(s) selected from the group consisting of position 205, 216, 217, 217, 277, 331, 333, 347.
  • the amino acid substitutions are selected from the group consisting of: N205K, P216L, A217V, D277G, G331R, A333G, K347T.
  • said gp17 is identified by SEQ ID NO:3 which is modified by any one of the following, in particular, by any one of the following as the only mutation compared to SEQ ID NO:3: HA003P i.
  • the metagenomic phage library of T7 bacteriophage transducing particles is characterized by a low content of replicative phages.
  • the phage library described herein comprises a percentage of replicative phages which is less than 10%, or less than any one of 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or preferably less than 1%, or even less than any one of 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or even less than any one of 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, or even less than any one of 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%.
  • Replicative phages are herein specifically understood as being “replication competent”, in particular phages comprising genomic elements as necessary to replicate in a permissive host cell.
  • the phage library described herein comprises the T7 bacteriophage transducing particles incorporating the library of genomic DNA fragments as further described herein, in particular comprising a DNA size range of at least 1.5 to 5 kb.
  • FIGURES Figure 1 Functional metagenomic plasmid library delivery by reprogrammed hybrid bacteriophage particles a, Schematic overview of functional metagenomic plasmid library delivery using DEEPMINE.
  • c, and d Delivered metagenomic DNA fragment lengths and diversities, respectively, determined by using long-read deep- sequencing right after electroporation and transduction. Dashed lines and solid lines represent the average and median sizes of the DNA fragments, respectively.
  • the figure shows the transduction efficiency of the T7 transducing phage particle harboring wild type T7 phage tail (black line) on Shigella sonnei HNCMB 25021 at different dilutions. With the increasing phage concentration, transduction efficiency is not growing as would be expected, but declines. Dashed line shows the expected increase in transduction efficiency without any detectable killing effect of replicative phages. Replicative phage contamination measured by plaque formation.
  • Figure 3 Directed evolution of the phage tail fibers optimizes functional metagenomic library delivery. a, Schematic overview of the directed evolution experiment consisting of the following steps. (1) Phage tail mutagenesis in E. coli using DIVERGE.
  • DIVERGE is a recombineering technique that incorporates soft-randomised single-stranded DNA oligonucleotides into multiple target sites.
  • Phage tails are encoded on packagable plasmids.
  • TFU/mL Transduction efficiencies (number of transduced cells in 1 mL (TFU/mL)) of the most efficient mutant tail fibers as compared to the parental wild type tails.
  • T7 Mut1 V544G; T7 Mut2: V544A, T7 Mut3: D540H, S541R; ⁇ SG-JL2 Mut1: N400K, P401L, N402K, ⁇ SG-JL2 Mut2: G398R, N400K, R408K, ⁇ SG-JL2 Mut3: G398R, N400K, ⁇ SG-JL2 Mut4: I383M, G398R, N400T; Vi06 Mut1: V13, Vi06 Mut2: A217V, D277G, G331R, A333G, K347T, Vi06 Mut3: P216L, N205K, Vi06 Mut4: P216L, N218N.
  • Figure 4
  • T7 V544G mutation Effect of T7 V544G mutation on the transduction efficiency of metagenomic libraries and replicative phage contamination.
  • e-f The assumed mechanism for the absence of replicative phage contamination. Specifically, while wild type T7 phage tail recognises the transducing HA003P phage particle generating E.
  • each band on the gel picture is a PCR-amplified DNA fragment insert from single clones of Shigella sonnei HNCBM 25021 after fragmented genomic DNA delivery. While wild type T7 delivered DNA fragments with a size smaller than 0,5 kb into the cells in 14 cases out of 20 (upper image), the same figure for T7 V544G is 3 cases out 10 (lower image).
  • FIG. 1 Venn diagram showing the distribution of the isolated ARGs across the four examined host bacterial species.
  • c Heatmap showing gene families of the identified ARGs using the four host species. Colour code quantifies the number of identified ARGs belonging to the gene family.
  • d Number of ARGs identified in the four hosts across the three used resistomes.
  • Figure 8. ARG mobility associates with broad functional compatibility. a, The more hosts an ARG is functional in, the higher the propensity of that ARG to be involved in HGT. Figure shows the percentage of mobile and non-mobile ARGs as a function of the number of hosts in which the ARG is functional.
  • Figure 9 Resistance threats to novel antibiotics.
  • the pipeline resembles to a previously published workflow (Dual Barcoded Shotgun Expression Library Sequencing pipeline (Mutalik et al., 2019)) with a modification that avoids PCR amplification of resistance-conferring metagenomic DNA fragments, and therefore, preserves the original composition of the samples.
  • the workflow consists of the following steps. First, all the functional metagenomic plasmids obtained from the screens were pooled and then linearized using SrfI restriction endonuclease. SrfI has an 8 base-pair-long recognition sequence to minimize the digestion of the metagenomic insert. The linearized plasmids are then subjected to Nanopore long-read sequencing.
  • Long-read sequencing identifies the metagenomic DNA fragment (insert) and the two 10 nucleotide long random barcodes pre-cloned up- and down-stream (Uptag and Downtag, respectively) of each metagenomic DNA fragment.
  • a multiplexed short-read deep-sequencing was applied to read out the plasmid-encoded unique barcodes on each side of the metagenomic fragments in each functional metagenomic screen.
  • the Uptag and Downtag sequences were PCR amplified with barcoded Illumina sequencing compatible primers (BC).
  • HA003P Cell culture”, or “culturing”, as used herein shall refer to the growth and propagation of cells in vitro, i.e. outside of a higher organism or tissue. It is particularly understood that the term shall not apply to transgenic animals or human beings. Suitable culture conditions for individual types of cells are known in the art, such as taught in Cell Culture Technology for Pharmaceutical and Cell-Based Therapies (2005).
  • Cells may be cultured in a cell culture medium, in particular in suspension or while attached to a solid substrate.
  • the present disclosure provides a method for identifying, optimizing, improving and/or isolating genes that are functional in a target cell.
  • identifying it means at least one of determining, classifying, finding, optimizing and/or selecting a protein, or gene encoding such that is functional in a target cell.
  • isolated and “characterizing” in the context of the nucleic acid sequence encoding the at least one functional protein means the separation of the nucleic acid sequence(s) encoding the at least one functional protein from their natural milieu and determining the nucleic acid sequence thereof, thereby characterizing it.
  • phage is herein understood as a bacteriophage which is a phage or a phage particle.
  • modified bacteriophages and hybrid bacteriophages as described herein may be used as delivery vehicles to deliver a metagenomic library into target cells. Therefore, the terms “phage”, “phage particle”, “delivery vehicle” and “nucleic acid delivery vehicle” are herein synonymously used.
  • modified bacteriophages as described herein which may be used for the delivery of specific cargo, such as genes from a diversity of genomes to target cells.
  • a phage particle is composed of a protein capsid, protecting and encapsulating genetic material, such as DNA or RNA.
  • the phage particle further comprises a host recognition element, typically a tail (made of proteins), that enables the specific recognition of a receptor at the surface of the host cell.
  • the term “phage particle”, as used herein, refers to a viral particle, which is derived from a bacteriophage and comprises a host recognition element as described herein, such as a tail fiber protein (such as further described herein) and an encapsulated nucleic acid molecule comprising a packaging signal.
  • HA003P Phages are composed of proteins that may encapsulate a DNA or RNA genome, which may encode only a few or hundreds of genes thereby producing virions with relatively simple or elaborate structures. Phages are classified according to the International Committee on Taxonomy of Viruses (ICTV) considering morphology and the type of nucleic acid (DNA or RNA, single- or double-stranded, linear or circular).
  • phage families have been recognized so far that infect bacteria and/or archaea (a prokaryotic domain previously classified as archaebacteria). Many bacteriophages are specific to a particular genus or species or strain of cell.
  • the phages used for the purpose described herein are Escherichia coli phage T7.
  • the T7 phage is a member of the Podoviridae family of the Caudovirales (tailed phages) order.
  • T7 is composed of an icosahedral capsid with a 20-nm short tail at one of the vertices.
  • the capsid is formed by the shell protein gene product (gp) 10 and encloses a DNA of 40 kb.
  • a cylindrical structure composed of gp14, gp15, and gp16 is present inside the capsid, attached to the special vertex formed by the connector, a circular dodecamer of gp8.
  • the proteins gp11 and gp12 form the tail; gp13, gp6.7, and gp7.3 have also been shown to be part of the virion and to be necessary for infection, although their location has not been established.
  • the main portion of the tail is composed of gp12, a large protein of which six copies are present; the small gp11 protein is also located in the tail. Attached to the tail are six fibers, each containing three copies of the gp17 protein.
  • T7 phages typically recognize only Escherichia coli as a host organism.
  • T7 phages which are engineered with an altered (e.g., extended) host specificity range may recognize bacteria belonging to one or more of the following genera: Escherichia, Pseudomonas, Streptococcus, Staphylococcus, Salmonella, Shigella, Clostridium, Enterococcus, Klebsiella Acinetobacter and Enterobacter.
  • bacteria belonging to one or more of the following genera: Escherichia, Pseudomonas, Streptococcus, Staphylococcus, Salmonella, Shigella, Clostridium, Enterococcus, Klebsiella Acinetobacter and Enterobacter.
  • bacteriophages that specifically target any of the “ESKAPE” pathogens.
  • these pathogens include but are not limited to Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, and Enterobacter.
  • the term “host-recognition element”, or simpler “recognition element”, as used herein, encompasses any vehicle component associated with vehicle-host recognition, namely an element mediating the interaction between the delivery vehicle and the host.
  • the term host recognition element refers to any bacteriophage component localized at the tail-end of the bacteriophage.
  • host recognition element may HA003P be interpreted herein in its broadest meaning, and therefore, in some embodiments, may encompass any element of the delivery vehicle that participates, facilitates, improves or enables at least one of host recognition, attachment to the host, penetration, injection of the nucleic acid molecules (or any other transduced material), and/or stability of the injected material within the host (e.g., resistance to the host restriction enzymes, and the like), or any element that participates at any stage of any of the processes described herein, or any combinations thereof.
  • the T7 phages which are engineered for reprogramming host specificity comprise modified host recognition elements such as a hybrid or modified tail, or a mutated tail protein.
  • the recognition elements applicable for the methods and modified bacteriophages described herein may be derived from any other bacteriophages, or obtained by recombination techniques, such as to produce hybrids, mutants, variants or orthologs thereof.
  • the host recognition element comprises at least one protein, at least two proteins, at least three proteins or more, specifically, structural bacteriophage protein/s that interact with the host receptor.
  • structural bacteriophage protein may be a protein residing in the tail region of a bacteriophage.
  • the tail is a protein complex present in the majority of the phages and is involved in host recognition and genome delivery.
  • tails have a central tubular structure that forms the channel for DNA ejection, which is surrounded by fibers or spikes that are essential in the initial steps of host recognition.
  • the tail of T7 phage is assembled from a dodecamer (i.e., 12 copies) of gp11 (the adaptor) and a hexamer (i.e., 6 copies) of gp12 (the nozzle), onto which six trimers of gp17 attach.
  • T7 s six tail fibers attach at the interface between the adaptor and nozzle, thus making contacts with both proteins.
  • the adaptor ring is responsible for the attachment of the preformed tail to the prohead via interactions with the portal composed of 12 subunits of gp8.
  • Bacteriophage components localized at the tail-end of the bacteriophage may be classified as “tail proteins” or “tail-tube proteins” (e.g., referring to gp11 and gp12) and tail fiber (e.g., referring to gp17).
  • the host recognition element described herein may comprise at least one of these proteins, derived from any of the bacteriophages described herein that may comprise any combination of mutations, specifically, combinations of any of the mutations described herein.
  • bacteriophage components localized at the tail-end of the bacteriophage may be classified as tail proteins (e.g., referring to gp11 and gp12) and tail fiber (e.g., referring to gp17).
  • the host-recognition element described herein may comprise at least one tail fiber or at least one tail protein.
  • the host-recognition element described herein may comprise at least one of gp11, gp12 and gp17, or any combinations thereof.
  • these proteins may be, but not limited to, T7 gp17, gp11 or gp12, any mutant thereof as described herein or any native or mutated heterologous variants as explained below, or any combination thereof.
  • Any protein residing in the tail region of any naturally occurring bacteriophage that infects target cells as herein defined may be used for reprogramming host specificity to target said cells.
  • the present disclosure relates to proteins residing in the tail region of T7-like bacteriophages (e.g., "tail proteins” or "tail-tube proteins” as herein defined), either mutated or not, for use to target the host cells of the metagenomic library.
  • the present disclosure provides for modified host-recognition elements.
  • modified or “mutated” it is meant that the native nucleic acid sequences encoding at least one host recognition element are altered, revised or mutated.
  • Any procedure known in the art for mutating a nucleic acid sequence may be used for obtaining mutated nucleic acid sequences, in particular, the methods exemplified herein, for example Ethyl methanesulfonate (EMS), the use of mutator plasmid, such as MP6, or any other mutagen or use of low-fidelity protein(s) associated with DNA synthesis or repair.
  • EMS Ethyl methanesulfonate
  • MP6 mutator plasmid
  • any other mutagen or use of low-fidelity protein(s) associated with DNA synthesis or repair.
  • the host recognition element may be mutated by mutagenesis, which is either spontaneously or by the use of at least one mutagen as disclosed above.
  • the resulting mutagenized nucleic acid sequences encode a host recognition element that may comprise at least one mutation.
  • a specific and non-limiting example of phages used as delivery vehicle includes those comprising host recognition elements that are mutagenized using a directed evolution technique, called DIVERGE, such as disclosed in WO2018108987A1.
  • DIVERGE is a recombineering technique that incorporates soft-randomized single- stranded DNA oligonucleotides into multiple target sites.
  • the technique comprises in vivo mutagenesis of a preselected target region (PTR) using a pool of partially overlapping single stranded DNA (ssDNA) oligonucleotides which upon alignment form a continuous sequence that is complementary to the sequence of HA003P interest, wherein the pool contains a diversity of mutagenizing oligonucleotides covering nucleobase mismatches at every position of said sequence of interest and combinations of said nucleobase mismatches.
  • PTR preselected target region
  • ssDNA partially overlapping single stranded DNA
  • the tail and fiber proteins as herein defined may be either of the same bacteriophage; or, tail or fiber proteins comprised within the recognition elements may be derived from a different bacteriophage as herein defined and therefore may be considered as a protein/s which are “heterologous” to the bacteriophage that comprises the host-recognition element being used. It should be appreciated that the tail and fiber proteins may be derived from any of the bacteriophages disclosed herein. It should be further appreciated that the host recognition element described herein may comprise any mutant, specifically any mutants disclosed herein or any combinations of mutations disclosed herein. Such mutants are herein referred to as being “artificial”. Any of the artificial elements are herein also understood as being “heterologous”.
  • the host recognition element described herein may comprise any of the proteins disclosed herein, specifically, any of gp17, gp11, or gp12 of any bacteriophage, specifically, any of gp17, gp11, or gp12 disclosed herein or any homologues thereof.
  • homologues is used to define amino acid sequences (polypeptide) which maintain a minimal homology to certain amino acid sequences, e.g., specifically have at least about any one of 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity any of the parent amino acid sequences, such as a parent sequence which is of a wild-type recognition element gp17, gp11, or gp12, or of any of the mutants disclosed herein. Specifically, the homologue differs from the parent sequence by only one or more point mutations, preferably up to 10, 9, 8, 7, 6, 5, 4, 3, 2, or only one point mutations.
  • a point mutation is the substitution, deletion or insertion of only one amino acid.
  • antimicrobial agent refers to any entity with antimicrobial activity (either bactericidal or bacteriostatic) i.e., the ability to inhibit the growth and/or kill bacterium, for example Gram positive- and/or Gram negative bacteria.
  • An antimicrobial agent may be any agent which results in inhibition of growth or reduction of viability of bacteria by at least about 10%, 20%, 30% or at least about 40%, or at least about 50% or at least about 60% or at least about 70% or more than 70%, for example, 75%, 80%, 85%, 90%, 95%, 100% or any integer between 30% and 70% or more, as compared to in the absence of the antimicrobial agent.
  • an HA003P antimicrobial agent is any agent which reduces a population of microbial cells, such as bacteria by at least about 30% or at least about 40%, or at least about 50% or at least about 60% or at least about 70% or more than 70%, or any integer between 30% and 70% as compared to in the absence of the antimicrobial agent.
  • an antimicrobial agent is an agent which specifically targets a bacteria cell.
  • an antimicrobial agent modifies (i.e., inhibits or activates or increases) a pathway which is specifically expressed in bacterial cells.
  • An antimicrobial agent can include any chemical, peptide (i.e., an antimicrobial peptide), peptidomimetic, entity or moiety, or analogues of hybrids thereof, including without limitation synthetic and naturally occurring non-proteinaceous entities.
  • an antimicrobial agent is a small molecule having a chemical moiety.
  • chemical moieties include unsubstituted or substituted alkyl, aromatic or heterocyclic moieties including macrolides, leptomycins and related natural products or analogues thereof.
  • Antimicrobial agents can be any entity known to have a desired activity and/or property, or can be selected from a library of diverse compounds.
  • antibiotic resistance genes refers to genes that confer resistance to antibiotics, for example by coding for enzymes which destroy said antibiotic compound, by coding for surface proteins which prevent the entrance of an antibiotic compound to the microorganism, actively exports it, or by being a mutated form of the antibiotic's target thereby preventing its antibiotic function.
  • the resistance gene confers resistance to a narrow- spectrum beta-lactam antibiotic of the penicillin class of antibiotics.
  • the resistance gene confers resistance to methicillin (e.g., methicillin or oxacillin), or flucloxacillin, or dicloxacillin, or some or all of these antibiotics.
  • vancomycin resistant S. aureus may also be resistant to at least one of linezolid, daptomycin, and quinupristin/dalfopristin.
  • Additional antibiotic resistant genes include but are not limited to fosfomycin resistance gene fosB, tetracycline resistance gene tetM, kanamycin nucleotidyltransferase aadD, bifunctional aminoglycoside modifying enzyme genes aacA-aphD, chloramphenicol acetyltransferase cat, mupirocin-resistance gene ileS2, vancomycin resistance genes vanX, vanR, vanH, vraE, vraD, methicillin resistance factor femA, fmtA, mec1, streptomycin adenylyltransferase spc1, spc2, anti, ant2, pectinomycin adenyltransferase spd,
  • the pathogenic or undesired gene may be a gene encoding any gene conferring resistance to any ⁇ -lactam antibiotic compound.
  • such gene may encode at least one ⁇ -lactamase.
  • ⁇ -lactamase denotes a protein capable of catalyzing cleavage of a ⁇ - lactamase substrate such as a ⁇ -lactam containing molecule (such as a ⁇ -lactam antibiotic) or derivative thereof.
  • ⁇ -lactamases are organized into four molecular classes (A, B, C and D) based on their amino acid sequences.
  • Class A enzymes have a molecular weight of about 29 kDa and preferentially hydrolyze penicillins.
  • Examples of class A enzymes include RTEM and the ⁇ -lactamase of Staphylococcus aureus.
  • Class B enzymes include metalloenzymes that have a broader substrate profile than the other classes of ⁇ - lactamases.
  • Class C enzymes have molecular weights of approximately 39 kDa and include the chromosomal cephalosporinases of gram-negative bacteria, which are responsible for the resistance of gram-negative bacteria to a variety of both traditional and newly designed antibiotics.
  • class C enzymes also include the lactamase of P99 Enterobacter cloacae, which is responsible for making this Enterobacter species one of the most widely spread bacterial agents in United States hospitals.
  • the class D enzymes are serine hydrolases, which exhibit a unique substrate profile.
  • the kits and systems described herein may be directed against any gene that may confer resistance to any ⁇ lactam antibiotics.
  • ⁇ -lactam or " ⁇ lactam antibiotics” as used herein refers to any antibiotic agent which contains a b-lactam ring in its molecular structure
  • ⁇ -lactam antibiotics are a broad group of antibiotics that include different classes such as natural and semi-synthetic penicillins, clavulanic acid, carbapenems, penicillin derivatives (penams), cephalosporins (cephems), cephamycins and monobactams, that is, any antibiotic agent that contains a ⁇ -lactam ring in its molecular structure. They are the most widely-used group of antibiotics.
  • ⁇ -lactam antibiotics are analogues of D-alanyl-D-alanine the terminal amino acid residues on the precursor NAM/NAG-peptide subunits of the nascent peptidoglycan layer.
  • the structural similarity between ⁇ -lactam antibiotics and D-alanyl-D-alanine prevents the final crosslinking (transpeptidation) of the nascent peptidoglycan layer, disrupting cell wall synthesis.
  • peptidoglycan precursors signal a reorganisation of the bacterial cell wall and, as a consequence, trigger the activation of autolytic cell wall hydrolases.
  • ⁇ -lactams are classified and grouped according to their core ring structures, where each group may be divided into different categories.
  • penam is used to describe the core skeleton of a member of a penicillin antibiotic, i.e. a ⁇ -lactam containing a thiazolidine ring.
  • Penicillins contain a ⁇ -lactam ring fused to a 5-membered ring, where one of the atoms in the ring is sulfur and the ring is fully saturated.
  • Penicillins may include narrow-spectrum penicillins, such as benzathine penicillin, benzylpenicillin (penicillin G), phenoxymethylpenicillin (penicillin V), procaine penicillin and oxacillin.
  • Narrow spectrum penicillinase-resistant penicillins include methicillin, dicloxacillin and flucioxacillin.
  • the narrow spectrum ⁇ -lactamase-resistant penicillins may include temocillin.
  • the moderate-spectrum penicillins include for example, amoxicillin and ampicillin.
  • the broad-spectrum penicillins include the co-amoxiclav (amoxicillin+clavulanic acid).
  • the penicillin group also includes the extended spectrum penicillins, for example, azlocillin, carbenicillin, ticarcillin, mezlocillin and piperacillin.
  • Other members of this class include pivampicillin, hetacillin, bacampicillin, metampicillin, talampicillin, epicillin, carbenicillin, carindacillin, ticarcillin, azlocillin, piperacillin, mezlocillin, mecillinam, pivmecillinam, sulbenicillin, clometocillin, procaine benzylpenicillin, azidocillin, penamecillin, propicillin, pheneticillin, cioxacillin and nafcillin.
  • ⁇ - lactams containing pyrrolidine rings are named carbapenams.
  • a carbapenam is a ⁇ - lactam compound that is a saturated carbapenem.
  • Carbapenems exist primarily as biosynthetic intermediates on the way to the carbapenem antibiotics.
  • Carbapenems have a structure that renders them highly resistant to ⁇ -lactamases and therefore are considered as the broadest spectrum of ⁇ -lactam antibiotics.
  • the carbapenems are structurally very similar to the penicillins, but the sulfur atom in position 1 of the structure has been replaced with a carbon atom, and hence the name of the group, the carbapenems.
  • Carbapenem antibiotics were originally developed from thienamycin, a naturally-derived product of Streptomyces cattleya.
  • the carbapenems group includes: biapenem, doripenem, ertapenem, imipenem, meropenem, panipenem and PZ-601.
  • ⁇ -lactams containing 2, 3- dihydrothiazole rings are named penems.
  • Penems are similar in structure to carbapenems. However, where penems have a sulfur, carbapenems have another carbon. There are no naturally occurring penems; all of them are synthetically made. An example for penems is faropenem.
  • ⁇ - lactams containing 3, 6-dihydro-2H-l, 3-thiazine HA003P rings are named cephems.
  • Cephems are a subgroup of b-lactam antibiotics and include cephalosporins and cephamycins.
  • the cephalosporins are broad-spectrum, semisynthetic antibiotics, which share a nucleus of 7-aminocephalosporanic acid.
  • First generation cephalosporins, also considered as the moderate spectrum includes cephalexin, cephalothin and cefazolin.
  • Second generation cephalosporins that are considered as having moderate spectrum with anb-Haemophilus activity may include cefaclor, cefuroxime and cefamandole.
  • Second generation cephamycins that exhibit moderate spectrum with anti-anaerobic activity include cefotetan and cefoxitin.
  • Third generation cephalosporins considered as having broad spectrum of activity includes cefotaxime and cefpodoxime.
  • the fourth generation cephalosporins considered as broad spectrum with enhanced activity against Gram positive bacteria and ⁇ -lactamase stability include the cefepime and cefpirome.
  • the cephalosporin class may further include: cefadroxil, cefixime, cefprozil, cephalexin, cephalothin, cefuroxime, cefamandole, cefepime and cefpirome.
  • Cephamycins are very similar to cephalosporins and are sometimes classified as cephalosporins. Like cephalosporins, cephamycins are based upon the cephem nucleus.
  • Cephamycins were originally produced by Streptomyces, but synthetic ones have been produced as well. Cephamycins possess a methoxy group at the 7-alpha position and include: cefoxitin, cefotetan, cefmetazole and flomoxef. ⁇ -lactams containing 1, 2, 3, 4-tetrahydropyridine rings are named carbacephems. Carbacephems are synthetically made antibiotics, based on the structure of cephalosporin, a cephem. Carbacephems are similar to cephems but with a carbon substituted for the sulfur. An example of carbacephems is loracarbef.
  • Monobactams are b-lactam compounds wherein the ⁇ -lactam ring is alone and not fused to another ring (in contrast to most other ⁇ -lactams, which have two rings). They work only against Gram negative bacteria. Other examples of monobactams are tigemonam, nocardicin A and tabtoxin. ⁇ -lactams containing 3, 6-dihydro-2H-l, 3-oxazine rings are named oxacephems or clavams. Oxacephems are molecules similar to cephems, but with oxygen substituting for the sulfur. Thus, they are also known as oxapenams. An example for oxapenams is clavulanic acid.
  • oxacephems include moxalactam and flomoxef.
  • HA003P Another group of ⁇ -lactam antibiotics is the ⁇ -lactamase inhibitors, for example, clavulanic acid. Although they exhibit negligible antimicrobial activity, they contain the ⁇ - lactam ring.
  • a "target cell” as used herein refers to any cell known in the art which can be recombinantly transformed, transduced or transfected with naked DNA or the delivery vehicle as herein defined using procedures known in the art.
  • Transformation and “transfection” mean the introduction of a nucleic acid, e.g., in the form of a plasmid or delivery vehicle, into a recipient cell (“host cell”) by nucleic acid-mediated gene transfer.
  • the target cells described herein, and particularly, the target cell of a species of interest (SOI) are prokaryotic (i.e., single-celled organisms that lack a membrane-bound nucleus or any other membrane-bound organelle), in particular example bacteria.
  • bacteria in singular, a "bacterium”
  • microbe encompasses bacteria belonging to general classes according to their basic shapes, such as spherical (cocci), rod (bacilli), spiral (spirilla), comma (vibrios) or corkscrew (spirochaetes), as well as bacteria that exist as single cells, in pairs, chains or clusters.
  • the bacterial species of interest may be any bacteria involved in nosocomial infections or any mixture of such bacteria.
  • nosocomial Infections refers to hospital-acquired infections, namely, an infection whose development is favoured by a hospital environment, such as surfaces and/or medical personnel, and is acquired by a patient during hospitalization.
  • Nosocomial infections are infections that are potentially caused by organisms resistant to antibiotics. Nosocomial infections have an impact on morbidity and mortality, and pose a significant economic burden. In view of the rising levels of antibiotic resistance and the increasing severity of illness of hospital in-patients, this problem needs an urgent solution.
  • Clostridium difficile methicillin-resistant Staphylococcus aureus, coagulase-negative Staphylococci, vancomycin-resistant Enteroccocci, resistant Enterobacteriaceae, Pseudomonas aeruginosa, Acinetobacter and Stenotrophomonas maltophilia.
  • the nosocomial-infection pathogens could be subdivided into Gram-positive bacteria (Staphylococcus aureus, Coagulase-negative staphylococci), Gram-positive HA003P cocci (Enterococcus faecalis and Enterococcus faecium), Gram-negative rod-shaped organisms (Klebsiella pneumonia, Klebsiella oxytoca, Escherichia coli, Proteus aeruginosa, Serratia spp.), Gram-negative bacilli (Enterobacter aerogenes, Enterobacter cloacae), aerobic Gram-negative coccobacilli (Acinetobacter baumanii, Stenotrophomonas maltophilia) and Gram-negative aerobic bacillus (Stenotrophomonas maltophilia, previously known as Pseudomonas maltophilia).
  • Gram-positive bacteria Staphylococcus aureus, Coa
  • a target cell may be an antibiotic- resistant target cell, or any mixture or population comprising said cells.
  • ESKAPE antibiotic- resistant target cell
  • these pathogens include but are not limited to Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, and Enterobacter.
  • the target cell may be bacteria of any strain of at least one of E.
  • the bacterial species of interest may include Yersinia enterocolitica, Yersinia pseudotuberculosis, Salmonella typhi, Pseudomonas aeruginosa, Vibrio cholerae, Shigella sonnei, Bordetella Pertussis, Plasmodium falciparum, Chlamydia trachomatis, Bacillus anthracis, Helicobacter pylori and Listeria monocytogens.
  • the bacterial species of interest may be any E.coli strain, specifically, any one of O157:H7, enteroaggregative (EAEC), enterohemorrhagic (EHEC), enteroinvasive (EIEC), enteropathogenic (EPEC), enterotoxigenic (ETEC) and diffuse adherent (DAEC) E. coli.
  • EAEC enteroaggregative
  • EHEC enterohemorrhagic
  • EIEC enteroinvasive
  • EPEC enteropathogenic
  • ETEC enterotoxigenic
  • DAEC diffuse adherent
  • the bacteriophage variant comprises a mutation and is infecting permissive target cells which are also recognized by the bacteriophage without such mutation (i.e., the wild-type bacterohage that has not been reprogrammed for extending the target specificity) and the phage is capable of transducing genetic material (such as plasmids) into said non-restrictive host. Still, the bacteriophage variant may exhibit an improved transduction efficiency through said mutation for transduction into a target cell. It surprisingly turned out that mutagenesis of phages to reprogram the HA003P bacteriophage for extended host specificity even improves transduction upon infecting the phage’s natural target cell.
  • the bacterial species of interest may be a restrictive host and the bacteriophage variant comprises a mutation to infect said cells, where the bacteriophage without such mutation would not (naturally) be capable of infecting, transduction and/or propagating in said restrictive (“non-permissive”) host.
  • restrictive or “non-permissive”, as used herein refers to a host that does not permit or allow infection or penetration by a specific bacteriophage (or more generally, a nucleic acid delivery vehicle) and/or propagation thereof.
  • Restrictive host cells are such cells into which the permeation of certain nucleic acid delivery vehicles, certain bacteriophages, is prohibited.
  • variants of delivery vehicles are described herein, in particular bacteriophage variants, having an extended host range.
  • altering in particular “extending”, in the context of host range as used herein shall mean modulating e.g., affecting, varying, enhancing, tuning or changing the ability of a specific nucleic acid delivery vehicle to recognize and transduce nucleic acids of interest to a specific host cell that is not considered a natural (“permissive”) host thereof.
  • the nucleic acid delivery vehicles described herein may recognize a host that is not a natural host thereof, upon replacing or mutagenizing of at least one of its host-recognition element/s or any protein or fragment thereof, by at least one of the host-recognition element/s, any proteins thereof or any combinations thereof, as described herein.
  • any host recognition element obtained either from other naturally occurring delivery vehicle/s (e.g., heterologous host recognition element or proteins thereof), or alternatively, by replacing with a mutated or otherwise altered host recognition element, e.g. such as the modified host recognition elements described herein.
  • hybrid vehicles that may carry at least one mutation or a combination of at least two mutations either in the coding or the non-coding regions thereof.
  • phages need to adsorb to the host surfaces, penetrate cell walls and inject genetic materials into the host.
  • Mechanisms used to initiate the connection to bacterial hosts prior to phage genome injection are referred to as tails and the adsorption machinery dedicated for HA003P specific host recognition is localized at the tail-end.
  • the interactions between phages and hosts occur between phage tail proteins and bacterial receptors. These interactions determine host specificity and host range of the bacteriophages.
  • the methods can be employed which are based on replacing at least one host-recognition element or any protein or parts thereof, of a given bacteriophage with a host recognition element compatible with, and therefore allowing recognition of, a target cell of a species of interest.
  • the nucleic acid delivery vehicles described herein may be modified for reprogramming to comprise one or more heterologous parts, such as any hybrid or mutagenized tails or respective heterologous tail proteins, and may further comprise a deletion of one or more of the respective endogenous parts, such as to replace the endogenous parts by the heterologous parts.
  • a nucleic acid delivery vehicle may be made "compatible with” or may recognize a host cell that is not naturally permissive (a “restrictive” or non-permissive host) upon replacing at least one host-recognition element in the delivery vehicle, specifically, bacteriophage, by a host-recognition element obtained from other naturally occurring delivery vehicle. Namely replacing at least one host-recognition element by a heterologous element which may be either native or modified.
  • these host-recognition elements may be elements derived from any bacteriophage. Specifically, these recognition elements may be used either in their native form (such as in case of heterologous elements originating from other phages that have a different host recognition or range), or alternatively, in an altered, for example, mutated form as described herein (such as in case of heterologous, artificial elements which are e.g., homologous to the native form), or any combinations thereof.
  • the host recognition element comprised within the delivery vehicle prepared by the methods described herein may comprise gp17, gp11, or gp12 or any combination thereof.
  • specific non limiting examples for such combinations may include gp17, gp11, or gp12; one of gp11, gp12 and gp17, gp11 and gp12; gp11 and gp17; gp12 and gp17 derived from any HA003P of the bacteriophages disclosed herein, and/or comprising any of the mutations disclosed herein and any combinations thereof.
  • a method of producing the phages and the respective metagenomic phage library comprising: In a first step (a), providing a plurality of nucleic acid molecule of interest, in particular the genomic DNA of the metagenomic library, such as the library of fragmented genomic DNA described herein, in the form of plasmids comprising a packaging signal. Step (b) involves providing phages that have been reprogrammed for modulated host specificity, comprising at least one host recognition element compatible for a target cell of interest.
  • the next step (c) involves introducing into transducing phage particle producing bacterial host cell/s the nucleic acid molecules of (a) and the phages of (b) to infect the host cells and to recover from the infected host cell phages comprising the nucleic acid molecules packaged therein.
  • Specific producing bacterial cells can be used which are selected for high-titer phage particle production.
  • Selected producing bacteria are e.g., E. coli BW25113, or E. coli BL21.
  • Bacteriophage T7 can be grown with the producing bacteria as host organism to yield T7 phage particles in the culture lysates.
  • High titers of e.g., 10E7, 10E8, 10E9 , or 10E10 tfu/ml can be obtained.
  • the phage particles can be purified e.g., by chloroform treatment and centrifugation (Yosef et al.2017).
  • the plurality of phages comprising the packaged library of fragmented genomic DNA of the metagenomic library is herein understood as a metagenomic phage library.
  • phages are introducing DNA into said host cells.
  • the term "introducing into host cells" as used herein, refers to incorporating exogenous DNA into these cells.
  • transducing a nucleic acid molecule of interest into a target host cell of interest comprises contacting the target cell of interest with said delivery vehicle comprising the nucleic acid molecule of interest, thereby transducing said nucleic acid molecule into the target host cell.
  • library shall refer to a collection of library members which are nucleic acid fragments (e.g., a library of fragmented genome(s), or polynucleotides) or a collection of phages, such as transducing phage particles, or a collection of cells (e.g., a bacterial cell library).
  • the library members share common features but differ in at least one mutation and/or phenotype.
  • a library typically contains library members which are diverse, besides those that have common features.
  • HA003P Metagenomic libraries typically comprise a plurality of nucleic acid molecules which differ in the respective nucleic acid sequence.
  • a metagenomic library comprising a plurality of fragmented genomic DNA form one or more (multiple) genomes, comprises library members comprising a diversity to cover the whole or parts of the genome(s), at least the repertoire of relevant gene(s) within said genome(s).
  • the plurality of fragmented genomic DNA typically is composed such that, upon combining the DNA fragments, considering overlaps and gaps, the whole or parts of the genome(s) can be recombined.
  • the present disclosure refers to a library of phage particles comprising a variety of encapsulated nucleic acid fragments (e.g., the plurality of fragmented genomic DNA), or a library of host cells.
  • Target cells transduced with nucleic acid molecules by the phage particles described herein may be selected by determining the desired function of the cell with a phenotype-based selection or screening method or (genetic or functional) single cell analysis, allowing the identification of host cells or a host cell repertoire that includes said phage particles or a respective selection marker, among a large population of cells.
  • Exemplary methods for phenotype-based selection or screening of cells from a library are based on viability of cells or survival of a microorganism library or repertoire of variant microorganisms under selective conditions, for example in the presence of a toxin or drug, such as an antibiotic.
  • selection is based on growth differences where the growth is quantified with an optical measurement or growth over time is used to enrich clones with improved growth capacity.
  • the enrichment or dilution of particular genotypes, originating from the growth differences may be quantified by determining the frequency of certain genotypes in the population by one of the below-mentioned DNA sequencing-based quantification techniques.
  • the subject of DNA sequencing may be a selected polynucleotide region included within the nucleic acid of interest or the packaged nucleic acid material, or an identification tag or barcode that is a short DNA sequence which labels each cell in the population.
  • phenotype-based selection or screening is based on the growth differences, which originate from the improved utilization of a nutrient, for example of a carbon source.
  • the improved utilization of the nutrient or chemical substance is quantified with an analytical technique, for example by the measurement of intracellular metabolite concentration, the increase of which is the improvement in the desired phenotype.
  • the phenotype-based HA003P selection and screening may involve the quantification of the catalysis of a chemical reaction, which is based on optical quantification of the reaction product or the reactants for example by detecting the signal from a fluorescence, absorbance or colorimetric assay or using mass spectrometry.
  • the phenotype-based screening or selection involves a differentiation in the binding capacity of a protein to a target molecule, for example using a binding assay to enrich variants with improved affinity to a specific ligand or using an optical assay based on for example fluorescence, absorbance or colorimetric assays.
  • exemplary methods for sequencing-based screening of cells within a library are the following: SNP genotyping methods, including hybridization-based methods (e.g.
  • molecular beacons SNP microarrays, restriction fragment length polymorphism, PCR- based methods, including Allele-specific PCR, primer extension-, 5’-nuclease or Oligonucleotide Ligation Assay, Single strand conformation polymorphism, Temperature gradient gel electrophoresis, Denaturing high performance liquid chromatography, High- resolution Melting of the entire amplicon (HRM), SNPlex and surveyor nuclease assay; Sequencing based mutation analysis, including capillary sequencing or high-throughput sequencing of an entire PCR amplicon of the PTR (amplicon sequencing).
  • Such high- throughput (HT) amplicon sequencing methods include, but are not restricted to polony sequencing, pyrosequencing, Illumina (Solexa) sequencing, SOLiD sequencing, semiconductor sequencing, DNA nanoball sequencing, Heliscope single molecule sequencing, Single molecule real time (SMRT) sequencing, Nanopore DNA sequencing, tunneling currents DNA sequencing, sequencing by hybridization, sequencing with mass spectrometry, Microfluidic Sanger sequencing, Microscopy-based sequencing, RNAP sequencing.
  • the libraries described herein are specifically characterized by a size (which is understood as the number of diverse library members, i.e., the library diversity) which is at least 10 5 , 10 6 , 10 7 , 10 8 or 10 9 , library members, which are each characterized by different nucleic acid sequences.
  • Each library member may be individually characterized and marked by a selectable marker or a DNA sequence tag or barcode, to facilitate the selection of a library member in the library or the identification of a library member in the library. It may be desirable to locate the library members in separate containers.
  • the library is provided in an array e.g., a cell chip, wherein the array comprises a series of spots on a solid carrier.
  • HA003P Metagenomic libraries may be used to study the function of genes originating from different sources e.g., from genome(s) isolated from environmental sources or microbiomes, in host cells other than the host cell of origin. If a certain gene is found to be functional in one or more different host cells other than the host cell of origin e.g., in a series of SOIs, such gene has an increased risk of a horizontal gene transfer, being transferred from one species to another species. The risk of such horizontal gene transfer is of critical importance if the host cell is a pathogen SOI. Libraries may be used to select specific library members to study the interaction with a predefined substance e.g., a chemical or biological, such as a drug, specifically antibiotics.
  • a predefined substance e.g., a chemical or biological, such as a drug, specifically antibiotics.
  • a library typically refers to genomes or genetic material, such as nucleic acid fragments, derived from a biological source such as an environmental or clinical sample.
  • the genetic material is specifically characterized by genomic fragments, such as fragments containing gene(s), part of gene(s) and/or non-coding genomic parts.
  • the metagenomic library may be obtained by collecting microorganism populations present in a natural or specific area, extracting the genome directly, and introducing it into a vector.
  • the vector may be a plasmid, introduced in a phage particle as described herein.
  • the metagenomic library may be provided in the form of a library of phage particles, each particle comprising a nucleic acid fragment derived from the biological source. Obtaining the genetic material from microorganisms present in a given sample of a biological source material can be directly or indirectly.
  • Indirect DNA extraction consists of the separation and collection of cells from the matrix and its subsequent lysis and purification of genetic material. This technique generally has lower yields compared to the direct extraction techniques, which consist of the prior cell lysis in the sample matrix and the subsequent collection and purification of genetic material.
  • Direct extraction methodologies are preferably performed for soil and sediments as they provide greater concentrations of DNA. Indirect techniques have the advantage of maintaining diversity, gene integrity, and purity of the genomic material.
  • HA003P Cell lysis can be performed by physical, enzymatic, chemical methods, or a combination thereof. The most effective physical treatments for cell disruption generally consist of the use of beads in a cell disruptor or by vortexing.
  • Soil for example, is estimated to contain 10 9 prokaryotes and more than 2,000 genome types per gram of sample.
  • the methodology for DNA extraction from soil samples can vary according to sample content and all factors to be considered, as well-known in the art.
  • isolation and purification of genetic material are followed by the construction of a metagenomic library.
  • the construction of the library typically consists of the cloning of DNA fragments at specific vectors to be packaged into phage particles, typically using production bacterial cell strains, followed by screening for the genes and/or functions of interest.
  • the metagenomic libraries can be constructed from large DNA fragments (25 to 200 Kb) extracted from environmental samples and cloned into specific vectors.
  • the choice of vector will depend on the size of the insert to be cloned.
  • the bacterial artificial chromosome (BAC) supports DNA fragments from 100–200 Kb, cosmids from 25–35 Kb, fosmids from 25–40 Kb, and yeast artificial chromosome (YAC) over 40 Kb. Libraries can be classified into two groups according to the size of their inserts: small ones (less than 15 Kb) are constructed using plasmids, and large inserts are constructed in vectors like fosmids, cosmids, and BAC.
  • the screening based on sequence may have several objectives, ranging from the analysis of microbial ecology in a given environment, to the search for new catabolic genes and mobile genetic elements in bacteria.
  • PCR-based approach is useful in environmental communities ’analysis, where the primers can be designed for screening-specific characteristics of biotechnological interest, such as enzymes, antibiotics, or resistant genes.
  • This approach is applied for the screening of metagenomic libraries, which can be obtained in clones screened in pools of 100 clones (e.g., for different genes such as rRNA, recA, radA, nif, and phenol hydroxylase), allowing the identification of members of a particular environment and their phylogenetic relationships.
  • probes are constructed from homologous sequences present in online databases.
  • probes have targeted gene- encoding enzymes such as dioxygenases, nitrite reductases, hydrogenases, hydrazine oxidoreductases, chitinases, and glycerol dehydratase; enzymes involved in pollutant compound degradation; genes for different antibiotics; or taxonomic groups.
  • enzymes such as dioxygenases, nitrite reductases, hydrogenases, hydrazine oxidoreductases, chitinases, and glycerol dehydratase
  • enzymes involved in pollutant compound degradation genes for different antibiotics
  • taxonomic groups genes for different antibiotics
  • Functional analysis detects the interest activity, and knowledge about the sequence or similarity to known genes is not necessary.
  • Function-based metagenomic analyses can be performed using different strategies: i) direct detection of gene products in individual clones, normally using fluorescent catabolic products to evaluate the enzymatic reaction; ii) heterologous complementation of host strains or mutants, allowing the growth of clones having some supplementation in the insert, that allows the clone to grow in selective conditions; iii) induced gene expression, iv) enzymatic assay.
  • phage particles are provided herein which display extended host range and are capable of transducing nucleic acid HA003P fragments into bacteria with greater transduction efficiency e.g., including different, preferably multiple, bacterial species of interest.
  • a further aspect described herein relates to a kit comprising: (a) a plurality of nucleic acid molecules, such as a metagenomic library as described herein.
  • the nucleic acid molecules further comprise at least one packaging signal, and optionally a selectable element, (b) at least one delivery vehicle (bacteriophage, in particular a T7 phage) that has been engineered for extending the host range; and optionally, (c) at least one compound for selecting cells that carry said selectable element.
  • bacteriophage in particular a T7 phage
  • Such kit can be used in a method of producing the metagenomic phage library, and in a method of producing a metagenomic bacterial cell library, as further described herein.
  • microbiome refers to the ecological community of commensal, symbiotic, or pathogenic microorganisms in a sample.
  • microbiomes that can be used with the present disclosure include but are not limited to skin microbiome, umbilical microbiome, vaginal microbiome, conjunctival microbiome, intestinal microbiome, stomach microbiome, gut microbiome and oral microbiome, nasal microbiome, gastrointestinal tract microbiome, and urogenital tract microbiome.
  • gut microbiome in the colloquial “gut flora” encompasses a complex community of microorganism species that live in the digestive tracts of animals (in this case mammals).
  • gut is synonymous with intestinal and flora with microbiota and microflora.
  • the gut microbiome refers to the genomes of the gut microbiota. Although the mammalian host can most probably survive without the gut flora, the relationship between the two is not merely commensal (a non-harmful coexistence), but rather mutualistic.
  • the mammalian gut microflora fulfills a variety of useful functions, including digestion of unutilized energy substrates, stimulating cell growth, repressing the growth of harmful microorganisms, training the immune system to respond only to pathogens and defending against some diseases. In certain conditions, however, some species are capable of causing disease by producing infection or increasing risk for cancer.
  • the methods described herein may be applicable in manipulating the gut microbiome in a subject.
  • a therapeutic tailor-made tool is described herein for modulating conditions caused by certain microorganisms that are part of the gut microbiome.
  • HA003P Composition of the mammalian gut microbiome consists predominantly of bacteria, for the most part anaerobic Gram-positive and Gram-negative strains, and to a lesser extent of fungi, protozoa, and archaea.
  • composition or content of the human microbiome, or microbiota is meant a composition with respect to the four predominant phyla of bacteria, namely Firmicutes, Bacteroidetes, Actinobacteria and Proteobacteria, or alternatively with respect to the predominant bacterial genera, namely Bacteroides, Clostridium, Fusobacterium, Eubacterium, Ruminococcus, Peptococcus, Peptostreptococcus and Bifidobacterium. Particularly the Bacteroides, which are the most predominant, may be important for host functioning. Other genera, such as Escherichia and Lactobacillus, although present to a lesser extent, were shown to contribute to host functioning.
  • methods described herein pertain to the entire range of bacterial species constituting the mammalian gut microbiome, including qualitative as well as quantitative aspects thereof. They further pertain to less ubiquitous microbiome components, such as of fungi, the known genera include Candida, Saccharomyces, Aspergillus and Penicillium, as well as microorganisms belonging to the domain of Archaea (also Archaebacteria), and further yet unclassified species that cannot be cultured.
  • heterologous as used herein with respect to a nucleotide sequence or an amino acid sequence or protein, refers to a compound which is either foreign, i.e.
  • exogenous such as not found in nature, in a given host (e.g., a host phage or host cell); or that is naturally found in a given host, e.g., is “endogenous”, but “artificial” such as e.g., comprising a mutated sequence that is not naturally-occurring in a wild-type host, or is “mutated”, or in the context of a heterologous construct, e.g. employing a heterologous nucleic acid.
  • the heterologous nucleotide sequence as found endogenously may also be produced in an unnatural e.g., greater than expected or greater than naturally found, amount in the host.
  • heterologous nucleotide sequence or a nucleic acid comprising the heterologous nucleotide sequence, possibly differs in sequence from the endogenous nucleotide sequence but encodes the same protein as found endogenously.
  • heterologous nucleotide sequences are those not found in the same relationship to a host in nature. Any recombinant or artificial nucleotide sequence engineered to transform a particular host is understood to be heterologous to the host cell.
  • HA003P "Percent (%) identity" with respect to a nucleotide sequence is defined as the percentage of nucleotides in a candidate DNA sequence that is identical with the nucleotides in the DNA sequence, after aligning the sequence and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleotide sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • point mutation or nucleobase alterations as used herein shall refer to a mutation event altering a nucleic acid or amino acid sequence at a certain location, such as by introducing or exchanging single nucleobases or amino acids or introducing gaps.
  • a point mutation or nucleobase alterations may involve a change in one or more single or adjacent or consecutive nucleobases or amino acid residues in a sequence, such as to delete, insert or substitute one amino acid within an amino acid sequence.
  • random as used herein regarding mutagenesis shall mean a method wherein DNA mutations are randomly introduced to produce mutant genes or genomic sequences, which are also referred to as “randomized”.
  • Randomized DNA sequences may encode a series of amino acid sequences, which are termed “randomized” as well.
  • a multitude of randomized nucleotide sequences, cells comprising such nucleotide sequences, and their expression products are conveniently compiled into a library, herein referred to as randomized library or randomized library members.
  • each residue, or each 2 or each 3 consecutive residues is mutated to a limited extent e.g., the occurrence of the original nucleobase (the nucleobase which is in the sequence of interest “SEQOI”at a given position on the oligonucleotides in the pool (herein understood as “frequency”) is bigger than the occurrence or frequency of each mismatching nucleobases.
  • the nucleobase composition in the oligo pool at each position is highly biased toward the original sequence.
  • the theoretical frequency of an amino acid substitution at any position depends on the quantitative ratios between the nucleotides added at particular steps during the synthesis of the oligonucleotides.
  • the overall frequency of the mismatching nucleobases is preferably less than 50%, 25%, 10%, 5%, or 1%, or 0.5% or in a ratio which is lower or equal to about 0.1%.
  • Soft randomized oligonucleotides are specifically characterized by a limited extent (frequency) of nucleobase mismatches, such that the overall sequence identity to the corresponding region within the SEQOI remains high e.g., at least 80% or at least 90% or at least 95%.
  • a method of producing a metagenomic bacterial library incorporating a library of fragmented genomic DNA comprises the steps: a) providing a library of fragmented genomic DNA originating from a biological source material, comprising a size range of at least 1.5 to 5 kb; b) incorporating said library of fragmented genomic DNA into transducing T7 bacteriophage particles which are engineered for altering the host range, thereby obtaining a transducing phage particle library comprising fragmented genomic DNA comprising a size range of at least 1.5 to 5 kb; and c) transducing the phage particle library into bacterial target cells, thereby obtaining a metagenomic bacterial library comprising said library of fragmented genomic DNA.
  • the size range covers genomic DNA fragments of at least any one of 0.5, 1, 1.5 kb size, and at least any one of 5, 6, 7, 8, 9, or 10 kb size.
  • the library of fragmented genomic DNA comprises a gene repertoire originating from prokaryotes or eukaryotes, preferably wherein the biological source material is an environmental source, preferably a water, soil, or plant source, or a biological sample of human or non-human animals. 4.
  • the metagenomic bacterial library comprises a) a diversity of fragmented genomic DNA to cover the genome of at least 3, 4, or 5 x10E3 different organisms; and/or b) a diversity of fragmented genomic DNA of at least 3, 4, 5, or 6 x10E6 different DNA fragments.
  • said T7 bacteriophage transducing particles comprise a heterologous phage tail protein for reprogramming host specificity.
  • said heterologous phage tail protein is originating from a ⁇ SG-JL2, Vi06, or KP11 bacteriophage. HA003P 7.
  • said heterologous phage tail protein is the tail fiber protein gp17 that is engineered to comprise one or more point mutations to alter the transduction efficiency.
  • said bacterial target cells are gram-negative bacteria of one or more different species, preferably of pathogen species.
  • the bacterial target cells are Clinically Relevant Bacterial Pathogens, preferably selected from the Enterobacteriaceae family, such as the species Salmonella enterica, Klebsiella pneumoniae or Shigella sonnei. 10.
  • a metagenomic bacterial library obtained by a method of any one of items 1 to 9. 11.
  • a metagenomic bacterial library of item 10 in a functional screening method to identify one or more heterologous genes which are functional in bacterial cells.
  • the bacterial repertoire comprises a resistome consisting of cells resistant to said antibiotic agent, and a repertoire of antibiotic- resistance genes (ARGs) is identified from said heterologous bacterial genes.
  • ARGs antibiotic- resistance genes
  • any one or more of said ARGs encode antibiotic inactivating enzymes, such as a beta-lactamase or acetyltransferase.
  • said functional screening is performed in a metagenomic bacterial library of more than one bacterial species of interest (SOI), to identify one or more functional heterologous bacterial genes in said bacterial repertoire which are shared by more than one SOI, thereby determining the risk of horizontal gene transfer of said heterologous bacterial genes.
  • SOI bacterial species of interest
  • a method of predicting the risk of a horizontally-transferable heterologous gene in bacterial cells by identifying functionality of heterologous genes in a functional screening method using a metagenomic bacterial library of item 10 or 11, and determining the risk of horizontal transfer of a heterologous gene, where said heterologous gene is functional in more than one bacterial species of interest (SOI). 18.
  • SOI bacterial species of interest
  • said heterologous gene is an antibiotic- resistance gene (ARG) which is predicted to bear a risk of horizontal gene transfer.
  • ATG antibiotic- resistance gene
  • the T7 bacteriophage transducing particle comprises a T7 phage tail fiber protein gp17 comprising SEQ ID NO:1 that is modified by an amino acid substitution at position V544, preferably V544G or V544A.
  • the phage library of item 21, comprising less than 1% replicative phages. 23.
  • the foregoing description will be more fully understood with reference to the following examples. Such examples are, however, merely representative of methods of practicing one or more embodiments of the present invention and should not be read as limiting the scope of invention.
  • EXAMPLES Introduction of metagenomic libraries by phage transduction is currently limited by the narrow host ranges of bacteriophage particles used for this purpose.
  • DEEPMINE Reprogrammed Bacteriophage Particle Assisted Multi-species Functional Metagenomics
  • Phage engineering was used to reprogram T7 bacteriophage host specificity through modification of the phage-tail and the corresponding molecular apparatus.
  • two complementary approaches were uniquely combined.
  • hybrid transducing bacteriophage particles were generated by exchanging the tails between different types of bacteriophages according to Yosef et al., 2017. These hybrid HA003P bacteriophage particles were used to deliver plasmid DNA into bacterial species.
  • the phage tails of these hybrid bacteriophage particles were modified by using directed evolution to improve phage transduction efficiency further.
  • DEEPMINE employs these reprogrammed bacteriophage transducing particles with altered bacterial host specificity to deliver large metagenomic plasmid libraries into a range of bacterial species.
  • DEEPMINE metagenomic screens were carried out in previously untapped clinically relevant bacterial pathogen species from the Enterobacteriaceae family.
  • Mobile ARGs were identified, which are functionally compatible in multiple bacterial species, and it could be demonstrated that these ARGs are frequently subjected to horizontal gene transfer in nature.
  • DEEPMINE to study a set of antibiotics which have only recently been approved for clinical usage or are in late-stage clinical development showed that these new antibiotics are exceptionally prone to resistance formation by mobile ARGs.
  • metagenomic libraries were created from environmental and clinical resistomes, including (i) river sediment and soil samples from 7 antibiotic polluted industrial sites in close vicinity of antibiotic production plants in India (that is, anthropogenic soil), (ii) the fecal samples of 10 European individuals who had not taken any antibiotics for at least one year before sample donation, and (iii) samples from a pool of 68 multi-drug resistant bacteria isolated in healthcare facilities or obtained from strain collections. DNA fragments ranging from 1.5 to 5 kb in size were shotgun cloned into a broad host range plasmid. The plasmid DNA carries a packaging signal sequence that allows translocation of the plasmid into the T7 bacteriophage ( Figure 1A., see Methods).
  • Each constructed library contained 3 to 5 million DNA fragments, corresponding to a total coverage of 25 Gb (i.e. the size of ⁇ 5,000 bacterial genomes).
  • the resulting plasmid libraries were packaged into two, previously characterized hybrid T7 phage particles that HA003P display tail fiber proteins from Salmonella phage ⁇ SG-JL2 and Klebsiella phage KP11 (Yosef et al., 2017).
  • the three metagenomic libraries were transduced into Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 and Klebsiella pneumoniae NCTC 9131, both of which are known bacterial targets of the hybrid T7 bacteriophage particles.
  • the average and the median sizes of the genomic DNA fragments are between 1.5 kb and 2kb both in the libraries that were delivered by hybrid T7 phage particles into their respective bacterial hosts and in the case of libraries delivered by electroporation into E coli ( Figure 1C).
  • the genomic DNA fragments have a size within the range of 1.5-5 kb. This indicates that transduction by reprogrammed bacteriophage particles has no significant distorting effect on the size and diversity of the delivered metagenomics libraries as compared to that of electroporation ( Figure 1C and 1D).
  • the T7 transducing phage particles were used with the wild type T7 phage tail to transduce E. coli BW25133 cells with the libraries.
  • This experiment was carried out to compare the size distribution of the delivered DNA fragments in the case of transducing phage particles with wild type T7 phage tail and heterologous phage tails.
  • the fragmented genomic DNA insert plasmid content of 20 isolated individual bacterial clones was PCR amplified after phage transduction of Klebsiella pneumoniae NCTC 9131, Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 and E.
  • Transduction efficiencies of most hybrid phage particles are well below the threshold (>10 7 transductants / ml) required for the delivery of entire functional metagenomic libraries into the target bacterial cells (Yosef et al., 2017). Moreover, the delivery of such libraries requires the application of high concentration of the transducing phage particles. In such cases, replicative phage contamination — a common issue of transducing bacteriophage particle generation — kills a significant fraction of the target cells ( Figure 2). Additionally, the size distribution of the delivered fragments with the T7 wild type phage tail displaying transducing phage particles was biased towards smaller DNA fragment sizes.
  • a directed evolution experiment was set up to genetically modify the tail fiber regions in the transducing phage particles. Specifically, it was aimed to select for host specificity altering point mutations in the host-range- determining regions (HRDRs) of the phage tail fibers (Yehl et al., 2019). To this end, three tail fibers (Escherichia phage T7, Salmonella phage ⁇ SG-JL2 and Salmonella phage Vi06) with especially broad host ranges were selected (Yosef et al., 2017).
  • DIvERGE has the advantage over other mutagenesis protocols that it introduces random mutations HA003P along multiple DNA sites simultaneously, and can cover relatively long DNA segments, potentially beyond the predicted HRDR regions.
  • phage tail variants with improved capacity to deliver plasmid libraries into three rationally chosen pathogen bacterial strains of Enterobacter cloaceae, Shigella sonnei HNCMB 25021 and Escherichia coli were selected next (Methods, Figure 3B).
  • the T7 phage tail library was selected with the same protocol in the presence of a phage resistant E.
  • the adaptive mutations increased transduction efficiency (Figure 3B, Figure 4), and at least in one case (T7 Mut1 with Shigella sonnei HNCMB 25021), it also minimized replicative phage contamination (Figure 5). Reassuringly, transduction of the three metagenomic libraries into Shigella sonnei HNCMB 25021 by this T7 phage tail variant resulted in functional metagenomic libraries that are as large and diverse as the library achieved by electroporation in Escherichia coli K-12 strain BW25113.
  • the average and median sizes of the genomic DNA fragments in the library of fragmented genomic DNA is 1.5 kb both in the case of electroporation into E. coli and in the case of transduction by T7 V544G phage tail displaying T7 transducing phage particles into Shigella sonnei HNCMB 25021.
  • 50% of the genomic DNA fragments have a size within the range of 1.5-5 kb.
  • transduction with wild type T7 tail HA003P displaying particles resulted in DNA fragments that are smaller than 1.5 kb in 70% of the cases (14 out of 20), and only 40% of the samples contained a DNA fragment in the size range between 1.5 kb and 5 kb.
  • 50% (5 out of 10) of the clones contained a DNA fragment that is smaller than 1.5 kb.
  • the T7 V544G phage tail has substantially lower transduction efficiency into the transducing phage particle generating host cell as compared to the wild type T7 phage tail ( Figure 6E-F).
  • This lower transduction efficiency avoids the re-infection of the transducing phage particle generating host cells in the case of the T7 V544G tail displaying particles, while the same is not true for the wild type T7 tail.
  • This phenomenon results in increasing concentration of propagating phage contamination in the transducing phage particles preparation. Fragments with small sizes may be enriched in the case of wild type T7 phage for a similar reason. Specifically, if a DNA insert is small, the cell may generate more copies of it that results in more assembled phage particles.
  • the screens were performed on solid agar in the presence of one HA003P of 13 selected antibiotics covering 5 major antibiotic classes (aminoglycoside, carbapenem, cephalosporin, gyrase inhibitors and tetracycline), at concentrations where the wild-type host strain is susceptible.
  • the list includes 6 antibiotics with long clinical history (“old”) and 7 others that have recently been developed or are currently in clinical development (“new”, as of 04/2020, Table 1).
  • the obtained resistance-conferring plasmids were pooled and sequenced with a modified dual barcoded shotgun expression library sequencing pipeline. The protocol avoids PCR amplification of resistance-conferring DNA fragments, and therefore, preserves the original composition of the samples.
  • DEEPMINE By screening ARGs in multiple bacterial hosts, DEEPMINE could identify such ARGs. To address this hypothesis, it was first examined which of the identified 114 ARGs have been horizontally transferred in nature. For this purpose, a mobile gene catalogue was generated based on the identification of nearly identical genes that are shared by distantly related bacterial genomes. Specifically, the pairwise alignment of 2794 genomes of phylogenetically diverse human-associated bacterial species was carried out. This dataset was extended with a sequence database of 27,939 natural plasmids derived from diverse environments. As might be expected, ARGs found on plasmids were especially likely to be transferred between bacterial species, with a 91% agreement between the two datasets on mobile ARGs.
  • ARGs functionally compatible in multiple bacterial hosts are more frequently subjected to horizontal gene transfer in nature as compared to those ARGs that are functional in a single host ( Figure 8A).
  • Multiple regression analysis has revealed that this pattern is independent of potential cofounding factors such as antibiotic classes, the associated bacterial hosts, the phylogenetic origin or the biochemical mechanisms of the isolated ARGs.
  • Broad functional compatibility is rather linked to widespread prevalence.
  • ARGs functional in multiple hosts are present in more human-associated bacterial species ( Figure 8B, Methods) and show higher sequence identities to known ARGs as compared to those that are functional in a single host ( Figure 8C). Importantly, these trends are evident even within the clinical resistome (Figure 8D).
  • Anthropogenic soil is increasingly recognized as a rich source of clinically relevant ARGs, even though ARGs do not transfer between soil bacteria as readily as is observed between human-associated bacteria in the gut microbiome or among clinical pathogens.
  • ARGs do not transfer between soil bacteria as readily as is observed between human-associated bacteria in the gut microbiome or among clinical pathogens.
  • Figure 8F-G the number of ARGs with broad functional compatibilities in the anthropogenic soil is not lower than in the human- associated environments.
  • functional compatibility with multiple bacterial hosts is a key characteristic of widespread mobile ARGs in human-associated bacteria.
  • the anthropogenic soil microbiome is a rich source of ARGs which could be functionally compatible with a range of bacterial pathogens.
  • Ceftobiprole a fifth-generation cephalosporin that has been recently approved for the treatment of hospital-acquired pneumonia highlights this point.
  • ARGs e.g., beta-lactamases
  • the broadly functional mobile ARGs in the human-associated bacteria were exceptionally high compared to that observed against “old” beta-lactam antibiotics with decades of clinical usage (Figure 9D).
  • a notable exception to this trend is apramycin sulfate. Only a single ARG against this antibiotic was detected in the human-associated resistomes ( Figure 9D). However, in agreement with extensive usage of this antibiotic in veterinary medicine for decades, multiple ARGs against apramycin sulfate were detected in the soil microbiome ( Figure 9D).
  • ARGs are aminoglycoside acetyltransferases are functionally HA003P compatible in multiple pathogenic hosts ( Figure 9D). This suggests that these genes can be of potential clinical risk in the future. In agreement with this expectation, one of these aminoglycoside acetyltransferases, AAC(3)-IV has already been detected in apramycin resistant clinical bacteria. Overall, DEEPMINE is a useful tool to predict ARGs with potential health hazards currently detectable in non-human associated microbiomes only.
  • DEEPMINE a custom plasmid was created from pZE21 expression vector (EMBL, Heidelberg, Germany) to fulfil the following requirements: (1) compatibility with T7 phage transduction, (2) compatibility with the sequencing pipeline.
  • the origin of replication of the vector was switched from ColE1 to p15A to make the plasmid compatible with the phage tail encoding plasmids (see Transducing hybrid bacteriophage particle preparation).
  • the medium copy number of p15A resembles more to the copy number of natural antibiotic resistance plasmids as compared to the previously used high copy plasmids in functional metagenomic studies.
  • the vector was PCR amplified using the primers pZE_orisw_fseI_F and pZE_orisw_fseI_R.
  • the p15A replication origin was amplified from the pACYC184 vector by using the primers p15A_fseI_F and p15A_fseI_R. Both pair of primers contained the recognition site of the FseI restriction enzyme as an overhang for sticky end ligation.
  • PCR products were digested by FseI (NEB) and ligated by T4 ligase (Thermo Fisher Scientific) according to the manufacturer’s instructions.
  • T7 phage packaging signal region into the vector.
  • the vector was amplified using the primers T7_pZE_SpeI_Fw and T7_pZE_SpeI_Rev.
  • the T7 packaging region was amplified from the vector pIYPE41 using the primers T7PS+SpeI_Fw and T7PS+SpeI_Rev (Yosef et al 2017). Both pairs of primers contained the recognition site of the SpeI restriction enzyme as an overhang for sticky end ligation.
  • PCR products were digested by SpeI (NEB) and ligated by T4 ligase (Thermo Fisher Scientific) according to the manufacturer’s instructions. Positive clones were identified by PCR using the primers T7_ch_F2 and T7_ch_R (see Table 3). Then, the pZE21_p15A vector was amplified by PCR using a mixture of forward and reverse oligonucleotides each containing 10-10 nt HA003P long hard randomized barcodes (pZET_bc_F_SrfI_v2 and pZET_bc_R, see Table 3).
  • PCR product was self-ligated followed by Fast Digest restriction endonuclease EcoRI treatment (Thermo Fisher Scientific) in the following settings: for every 1 ⁇ g of DNA 3 ⁇ L of enzyme was added then incubated for 100 min at 37 0C.
  • Sample collection and construction of metagenomic libraries Metagenomic libraries were built from three types of environmental samples that represent the gut, anthropogenic soil and clinical resistomes, respectively: the human gut microbiome of 10 individuals, 7 anthropogenic soil samples from highly polluted industrial areas in India, and an in-house strain collection of clinical bacterial isolates, consisting of 68 samples.
  • Gut resistome faecal samples were collected from 10 unrelated, healthy individuals, with no history of taking antibiotics in the year prior to sample donation.
  • Genomic DNA was extracted from all sediments using Qiagen DNeasy PowerSoil Kit according to the manufacturer’s recommendations. Eventually, 7 non-fragmented samples were chosen after quality control for further processing.
  • Clinical resistome Genomic DNA was isolated from 68 different Gram- positive as well as Gram-negative clinical isolates using the Sigma GenElute Bacterial Genomic DNA Kit. Next, 40 ⁇ g of extracted DNA from each environmental source was treated with MluCI enzyme. MluCI digestion involved 10 minutes of incubation time at 37 0C, followed by 20 minutes of inactivation at 85 °C temperature. The quantity of the MluCI enzyme was varied to maximize the yield in the target size range of between 1 and 5 kb.
  • DNA in the target size range was isolated with pulsed field gel electrophoresis using BluePippin DNA Size Selection System (Sage Science) with a 0.75% agarose gel cassette and low voltage 1-6 kb marker S1 cassette definition.
  • HA003P For cloning the metagenomic DNA fragments, the above-described pZE21_p15A vector was EcoRI digested and separated from non-digested plasmid DNA by electrophoresis through a 0.75% agarose gel in Tris-acetate-EDTA (TAE) buffer stained with ECO Safe Nucleic Acid Staining Solution (Pacific Image Electronics).
  • a gel slice corresponding to 3500 bp was excised and DNA was extracted with the Monarch DNA Gel Extraction Kit (NEB) following suggested protocols after which pure vector DNA was dephosphorylated with FastAP Thermosensitive Alkaline Phosphatase (Thermo Fisher Scientific) enzyme at 37 0C for 20 h where 4 ⁇ L of enzyme was added for every 1 ⁇ g of DNA before inactivation at 75 0C for 10 min. It was followed by another cleaning step.
  • NEB Monarch DNA Gel Extraction Kit
  • Metagenomic DNA was ligated into p15A plasmid vectors at the EcoRI site using a 3:1 mass ratio of insert:vector, after which pure ligation mixture was electroporated into 40 ⁇ L of electrocompetent cells: either E.coli MegaX (Invitrogen) or E. cloni 10G ELITE Electrocompetent Cells (Lucigen). Electroporation was conducted in a chilled 1 mm cuvette at 1,800 V after which cells were immediately transferred into 1 mL of preheated (37 0C) recovery medium.
  • transformants were plated onto kanamycin (50 ⁇ g/mL) containing Luria Bertani (LB) agar plates in 10 1 x, 10 2 x and 10 3 x dilutions for CFU determination.
  • CFUs were counted and insert size distribution was estimated after PCR amplification and gel electrophoresis of 10-20 randomly selected clones. PCR was performed using a primer pair flanking the EcoRI site of the multiple cloning site of the p15A plasmid. The average insert size was determined to be 2-3 kb which was then multiplied by the number of total CFUs.
  • Transducing hybrid bacteriophage particle preparation Transducing hybrid bacteriophage preparation is adapted from Yosef et al., 2017. In brief, E. coli BW25113 cells containing the plasmids encoding the phage tail genes were grown to ⁇ OD600nm 0.7 (250 rpm at 37 o C). After growth, cells were placed on ice for 15 minutes.
  • T7 bacteriophages in which genes HA003P encoding the tail fiber regions were deleted from the genome (T7 ⁇ gp11-12,17) (Yosef et al 2017), were used to infect cells at multiplicity of infection (MOI) 2-3, unless otherwise noted.
  • phages attack bacterial cells, assemble the expressed tail proteins onto their original structure and, if packaging signal region is present, encapsulate plasmids. Following 2 h of incubation (aerating cells on 100 rpm at 37 o C), cells were treated with chloroform (2% final concentration) and were exposed to a vigorous vortex treatment, to eliminate remaining bacteria. This mixture was then centrifuged with the same parameters as above. Finally, supernatant that contained phage particles was collected. Number of transduced colonies per mL (TFU/mL) in the generated stock was calculated by transducing eligible bacterial cells (see ‘Transducing assay’). This value will be different with each different bacterial strain investigated.
  • Transduction efficiency of the hybrid bacteriophage transducing particles was measured as before (Yosef et al., 2017).
  • target bacterial cells were grown to ⁇ OD600nm 0.5 (250 rpm at 37 o C) in LB or TB, followed by 15 minutes long incubation on ice.
  • dilutions of the transducing phage particles were prepared in 96- well plates using LB with 10-fold dilution steps in a range between 1x and 10 7 x dilution. Then, 100 ⁇ l of target cells were mixed with 100 ⁇ l of phage particles from each dilution.
  • Phage tail encoding plasmids were first electroporated into Escherichia coli K-12 BW 25113 strain. Next, the strains were made electrocompetent and 30 ng of each plasmid library was electroporated (BTX, Harvard Apparatus, CM-630 Exponential Decay Wave Electroporation System) with 1 mm gap electroporation cuvettes. Each library was electroporated in 5 parallels to achieve suitable colony numbers. Right after electroporation cells were resuspended in 1 mL of SOC medium and incubated by shaking at 230 rpm at 37°C for 1 hour.
  • E. coli BW25113 were provided with both plasmid that carried the tail encoding genes to be mutated, also carrying a packaging signal, and the mutagenesis plasmid (pORTMAGE313B unless noted otherwise, encoding recombinase and mutated MutL under the control of a chemically inducible promoter).
  • Cells were grown to ⁇ OD600nm 0.4 in TB (250 rpm at 37 o C) supplied with HA003P appropriate antibiotics.
  • m-toluic acid was added (1mM final concentration) to induce gene expression and after 1 h of incubation, cells were transferred to ice for at least 15 minutes. Afterwards, cell culture was washed and centrifuged (4500 rpm, 4 o C, 10 minutes and washed with distilled water, 3 times) to make it electrocompetent. Electroporation was done on a BTX (Harvard Apparatus) CM-630 Exponential Decay Wave Electroporation System in 1 mm gap electroporation cuvettes. Oligos were added to the electroporation mixture at a final concentration of 2.5 ⁇ M.
  • Generating bacteriophage particle library E. coli BW25113 cells provided with mutagenized plasmids were grown to ⁇ OD600nm 0.7 (250 rpm at 37 o C). After growth, cells were placed on ice for 15 minutes. Subsequently cultures were centrifuged (4500 rpm, 4 o C, 10 minutes). Supernatant was removed, along with antibiotic present during cell growth. Cells were resuspended in the same amount of medium (either Luria-Bertani (LB) or Terrific Broth (TB) unless noted otherwise). Afterwards, T7 ⁇ gp11-12,17 bacteriophage was used to infect cells at multiplicity of infection (MOI) 2-3, unless otherwise noted.
  • MOI multiplicity of infection
  • target bacterial cells were grown to ⁇ OD600nm 0.5 (250 rpm at 37 o C) in LB. After growth, cells were placed on ice for 15 minutes. In the meantime, 2 ml of the phage particle libraries were HA003P transferred to test tubes and were mixed in a a 1:1 volume ratio with the target cells. The mixture then was placed to 37 o C, 100 rpm for 1 h. After incubation, mixture was plated out to 13.5 mm agar plates supplied with appropriate antibiotics and were placed at 37 o C to grow O/N. As a control experiment, the same protocol was carried out with the non-mutagenized corresponding wild type phage tail carrying particles.
  • the lawn of cells were washed together in 4 ml of LB and 250 ⁇ l of them were transferred into 40 ml TB supplied with appropriate antibiotics, and were grown to ⁇ OD600nm 0.7 (250 rpm at 37 o C). After growth, cells were placed on ice for 15 minutes subsequently were centrifuged (4500 rpm, 4 o C, 10 minutes) and resuspended in the same amount of fresh medium. Afterwards, cell cultures were infected with T7 ⁇ gp11-12,17 bacteriophages. Following 2 h of incubation (100 rpm, at 37 o C), cells were treated with chloroform (2% of the volume of medium) and were exposed to a vigorous vortex treatment, to eliminate remaining bacteria.
  • MGP4240 and MGP4240_gp17 V544G plasmids encoding the T7 gp17 WT and T7 gp17 V544G , respectively, were transformed by electroporation into E. coli BW25113 harbouring the pZE21_p15A.
  • the resulting E. coli cells were infected by T7 ⁇ (gp11-12-17) phage to package the pZE21_p15A plasmid into phage particles.
  • the resulting phage particles were used to generate phage lysates in E. coli BW25113 and S. sonnei HNCMB25021 (Fois et al.
  • long-read sequencing identifies the metagenomic DNA fragments (insert) and the two 10 nucleotide long random barcodes pre-cloned up- and down-stream (Uptag and Downtag, respectively) of each metagenomic DNA fragment.
  • an aliquot from all the functional metagenomic plasmid DNA preparations obtained from the screens were pooled in an equimolar ratio.
  • Genomic DNA contamination was removed from the mixture by Lambda-exonuclease and Exonuclease-I double digestion, by using 2.5 U of each enzyme for every 1 ⁇ g of plasmid DNA at 37°C for 30 minutes, followed by 10 minutes inactivation at 80°C.
  • the resulting sample was cleaned (DNA Clean & Concentrator TM- 5 Zymo Research Kit) and quantified.
  • the plasmid mixture was linearized by adding 5U of SrfI restriction endonuclease (NEB) for every 1 ⁇ g of plasmid HA003P DNA. The reaction was performed for 1 hour at 37°C following by inactivation at 65°C for 20 minutes. DNA concentrations were quantified by using Qubit dsDNA Broad-Range Assay Kit (Thermo Scientific) before applying to Oxford Nanopore long-read sequencing.
  • PCR was performed using Phusion High-Fidelity DNA Polymerase (Thermo Scientific) using the following reaction mixture: 15 ng of template plasmid DNA, 4 ⁇ L 5x GC buffer, 0.2 ⁇ L Phusion High-Fidelity DNA polymerase, 0.6 ⁇ L DMSO, 0.2 mM dNTPs, 0.5-0.5 ⁇ M forward and reverse primers and water in a final volume of 20 ⁇ L.
  • the following thermocycler conditions were used: 95°C for 5 mins, 30 cycles of 95°C for 30 sec + 59°C for 30 sec + 72°C for 5 sec, 72°C for 7 mins. Following concentration measurement of each PCR reaction the samples were mixed in 1:1 mass ratio.
  • Nanopore sequencing Library preparation was carried out using Ligation Sequencing Kit (Oxford Nanopore Technologies, Oxford, UK) and 1 ⁇ g plasmid DNA was inputted to the library.
  • the DNA was end-prepped with the NEB Next FFPE Repair and Ultra II End Prep Kit and purified using Agencourt AMPure XP (Beckman Coulter Inc., Brea, CA, USA) then the adapter ligation was performed using NEBNext Quick T4 DNA Ligase.
  • the adapted library was purified by Agencourt AMPure and the concentration was determined using Qubit 3.0.
  • the pipeline contained the following steps: 1) mapping reads to the reference sequence with minimap2 (ver HA003P 2.17); 2) converting SAM files to sorted BAMs; 3) extracting the insert sequences and identifying barcodes which added to the read/insert names applying samtools tview (1.11-9-ga53817f) subcommand; 4) creating individual FASTQ files using SEQTK (v0.13.2); 5) generating consensus sequences using SPOA (v4.0.2) with the following parameters: -l 0 -r 0 -g -2. Finally, the raw consensus inserts were polished using the relevant set of insert sequences by minimap2 and racon (v1.4.19) to create the final consensus inserts.
  • Denatured sequencing pools were loaded to MiSeq Reagent kit V2-300 (Illumina) and 2 ⁇ 70 bp sequence reads were generated with an Illumina MiSeq instrument with custom read 1, read 2 and index 1 sequencing primers spiked in the appropriate cartridge positions (12, 14, and 13, respectively) at a final concentration of 0.5 ⁇ M. Sequencing data analysis and functional annotation of ARGs Each consensus insert sequence from nanopore sequencing was associated with screening samples (host, resistome, antibiotic) by combining the nanopore and Illumina datasets through the unique Uptag and Downtag barcodes with a custom R script.
  • metagenomic DNA fragments supported by less than 9 consensus insert sequences in the nanopore dataset and less than 8 reads in the Illumina Uptag and Downtag barcode dataset were filtered out. Thresholds were chosen to minimize the number of the out-filtered data points and maximaize reproducibility based on the two replicate screens with K. pneumoniae.
  • Each cluster was represented by the closest hit to known ARGs in the Card and ResFinder databases.
  • a nucleotide sequence similarity search was carried out for the entire DNA contigs as query sequences against the NCBI Reference Prokaryotic (RefProk) database with a threshold of evalue 10E-10.
  • RefProk NCBI Reference Prokaryotic
  • the parameters for filtering the blast results were the following: minimum percentage of identity: 99%, minimum alignment length: 500, maximum alignment length: 20000.
  • the blast hits were clustered by cd-hit-est 4.8.1 with sequence identity threshold 99%.
  • the predicted ORFs were clustered by cd-hit-est 4.8.1 with minimum similarity 90%.
  • natural plasmid sequences were identified by downloading 27,939 complete plasmid sequences from the PLSDB database (version 2020-11-19). Then, representative sequences of the isolated 114 ARG clusters were BLASTN searched both in the mobile gene catalogue and in natural plasmid sequences with an identity and coverage threshold of 90%.
  • Those ARGs were considered as mobile which were present in the mobile gene catalogue and/or in natural plasmid sequences.
  • HA003P REFERENCES Fois B., Skok, Z., Tomasic, T., Ilas, J., Zidar, N., Zega, A., Masic, L.P., Szili, P., Draskovits, G., Nyerges, A., Pal, C. & Kikelj, D. (2019). Dual Escherichia coli DNA Gyrase A and B Inhibitors with Antibacterial Activity. ChemMedChem, 5;15(3):265-269. https://doi: 10.1002/cmdc.201900607.

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Abstract

Procédé de production d'une banque bactérienne métagénomique incorporant une banque d'ADN génomique fragmenté, ledit procédé comprenant les étapes suivantes : a) présenter une banque d'ADN génomique fragmenté provenant d'un matériau de source biologique avec une couverture d'intervalle de taille d'au moins 1,5 à 5 kb ; b) incorporation de ladite banque d'ADN génomique fragmenté dans des particules de bactériophage T7 transductrices conçues pour modifier la gamme d'hôtes, ce qui permet d'obtenir une banque de particules de phage transductrices comprenant de l'ADN génomique fragmenté dont la gamme de tailles couvre au moins les tailles comprises entre 1,5 et 5 kb ; et c) transduction de la banque de particules de phage dans des cellules cibles bactériennes, ce qui permet d'obtenir une banque bactérienne métagénomique comprenant ladite banque d'ADN génomique fragmenté.
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