WO2025076345A1

WO2025076345A1 - Compositions and methods for identifying bacterial characteristics

Info

Publication number: WO2025076345A1
Application number: PCT/US2024/049954
Authority: WO
Inventors: Timothy Wesley HAND; Darryl A. ABBOTT; William H. DEPAS; Christine TIN
Original assignee: University of Pittsburgh
Current assignee: University of Pittsburgh
Priority date: 2023-10-06
Filing date: 2024-10-04
Publication date: 2025-04-10
Anticipated expiration: 2026-04-06

Abstract

The present disclosure relates to a method of identifying two or more characteristics of bacterium in a testing sample comprising use of fluorescent labels and hybridization chain reaction.

Description

COMPOSITIONS AND METHODS FOR IDENTIFYING BACTERIAL CHARACTERISTICS

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to, and the benefit of, U.S. Provisional Patent Application No. 63/588,491, filed October 6, 2023, which is incorporated by reference herein in its entirety.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with Government support under grant number DK120697 awarded by the National Institutes of Health. The government has certain rights in the invention.

REFERENCE TO SEQUENCE LISTING

The sequence listing submitted on October 4, 2024 as an .XML file entitled “10504- 091WOl_ST26” created on October 4, 2024 and having a file size of 69,679 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5).

BACKGROUND OF THE INVENTION

The intestine is colonized by a large and diverse microbiota that is important in the development of various intestinal infections and conditions ranging from inflammatory bowel disease to necrotizing enterocolitis. For example, both Crohn’s Disease and ulcerative colitis are associated with shifts in the intestinal microbiota. Current methods for measuring the microbiota, based upon sequencing, provide data too slowly and of too high a complexity for most clinical use. Thus, there is a critical need for a faster, more quantitative, and more accurate method for measuring both the structure of the intestinal microbiota and the presence of specific bacteria at the single cell level that may contribute to disease.

SUMMARY

In one aspect disclosed herein is a is a method for identifying two or more characteristics of a bacterium in a testing sample. In some embodiments the method further includes an absolute quantification. In some embodiments the method further includes a relative quantification. In some embodiments, the method comprises the following steps 1) obtaining the testing sample comprising the bacterium and a first ligand; 2) contacting the bacterium and the first ligand with a second ligand that is specific for the first ligand and comprising a first fluorescent label, wherein binding of the second ligand to the first ligand creates a labeled ligand conjugate comprising the first fluorescent label; 3) cross linking the labeled ligand conjugate that is bound to the bacterium to obtain a fluorescently labeled bacterium; 4) fixing the bacterium; 5) permeabilizing the bacterium; 6) obtaining an identification sample by performing a hybridization chain reaction (HCR) using the permeabilized bacterium, wherein the identification sample comprises the fluorescently labeled permeabilized bacterium comprising the first fluorescent label and one or more fluorescently labeled bacterial polynucleotide HCR products comprising at least a second fluorescent label, and wherein the first and second fluorescent labels are different; and 7) isolating the bacterium from any other bacteria in the identification sample and identifying a first ligand binding characteristic represented by the first fluorescent label and second characteristic represented by the second fluorescent label. In some embodiments, the ligand is an antibody.

In some embodiments, the method further comprises determining the total number of bacteria in the identification sample. In some embodiments, the first antibody is an IgA antibody, and the first characteristic of the bacteria is an ability to bind IgA. In some embodiments, the first antibody is an IgM antibody, and the first characteristic of the bacteria is an ability to bind IgM. In some embodiments, the first antibody is an IgG antibody, and the first characteristic of the bacteria is an ability to bind IgG.

In some embodiments, the second bacterial characteristic is a bacterial domain. In some embodiments, the second bacterial characteristic is a bacterial phylum. In some embodiments, the second bacterial characteristic is a bacterial class. In some embodiments, the second bacterial characteristic is a bacterial order. In some embodiments, the second bacterial characteristic is a bacterial family. In some embodiments, the second bacterial characteristic is abacterial genus. In some embodiments, the second bacterial characteristic is a bacterial species.

In some embodiments, prior to contacting the bacterium in the testing sample with the first ligand, one or more contaminants are removed from the testing sample using a PERCOLL solution. In some embodiments, the fluorescently labeled ligand comprises a red fluorescent label. In some embodiments, the labeled ligand conjugate is crosslinked to the bacterium using a crosslinking agent comprising bis-(sulfosuccinimidyl)-subcratc.

In some embodiments, the bacterium is fixed using a fixing agent comprising paraformaldehyde. In some embodiments, the bacterium is permeabilized using a permeabilizing agent comprising lysozyme and lysostaphin. isolating the fluorescently labeled permeabilized bacterium and the one or more fluorescently labeled bacterial polynucleotide HCR products in the identification sample can be performed using fluorescence activated sorting. In some embodiments, the hybridization chain reaction (HCR) comprises use of an initiator probe selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40 and SEQ ID NO:41.

BRIEF DESCRIPTION OF FIGURES

Figure l(A-C) shows a schematic of the hybridization chain reaction. Figure 1A shows the probes and initiator, Figure IB shows the probes’ alignment with the target mRNA, and Figure 1C shows the polymerization reactions.

Figure 2 shows a schematic of a method of determining the identity of the absolute amount of and antibody binding to bacteria in a sample using hybridization chain reaction and flow cytometry.

Figure 3(A-B) shows that the present method can distinguish a defined bacteria community of 50% S. aureus and 50% E. coli grown and combined in cell culture. Figure 3A shows a flow cytometry analysis of the mixed sample, and Figure 3B shows confocal imaging of the mixed sample.

Figure 4 shows flow cytometry analyses demonstrating identification and quantification of Clostridium difficile and C. difficile toxin expression in human patient samples that are either infected with C. difficile (left two panels) or an uninfected control (right panel).

Figure 5 shows that the present method discriminates IgA-bound bacteria in stool from a maternal milk-fed preterm infant. Flow cytometric analysis shows the method detects bacteria- specific IgA to subpopulations of different taxa in the same preterm stool sample. Isotype antibody control is for mixed longitudinal samples from the same patient. Enterobacteriaceae and Bifidobacterium gated on Eubacteria+ cells.

Figure 6 shows that the present method quantifies total population changes (Enterobacteriaceae and Bifidobacterium in a human microbiota sample. Longitudinal samples compared between a formula-fed (left) and a breast milk-fed (right) patient across days of life 10 to 17. Total bacterial cells normalized to mg of stool.

Figure 7(A-B) shows changes in relative abundances do not reflect true population changes in longitudinal stool. Figure 7A shows longitudinal analysis of stool from an infant at days of life 10 to 16. Absolute abundance shows stabilization of Bacilli and Gammaproteobacteria communities as Bifidobacterium increases. Figure 7B shows longitudinal analysis of stool from an infant at days of life 15 to 17. IgA-bound bacteria were measured. Dashed line shows three distinct trends of absolute population changes despite similar changes in relative abundance. Relative & absolute abundances were measured with the present method. Figure 8 shows that the present method retains IgA antibodies bound to bacteria. IgA was purified from breast milk and incubated with pure culture E. coll. Flow cytometric analysis of samples run before and after the present method shows that anti-bacteria antibodies remain on bacteria surface throughout the protocol.

Figure 9(A-C) shows that the present method quantifies all members of a pediatric microbial consortia (PedsCom: Staphylococcus xylosus, Staphylococcus sciuri, Enterococcus faecalis, Lactobacillus johnsonii, Ligolactobacillus murinus, Anaerostipes sp., Clostridium intestinale, Kosakonia cowanii, Parabacteroides distastonis) in gnotobiotic mice. Figure 9A shows flow cytometric plots showing identification of all PedsCom members at the family level in fecal samples from gnotobiotic PedsCom mice. Figure 9B shows that the present method (MicFly) is comparable to qPCR to measure relative abundances. Figure 9C shows that the present method is comparable to qPCR for absolute abundance quantitation.

Figure 10(A-B) shows how the present method measures gene expression of arabinose- inducible GFP in E. coli. Figure 10A shows flow cytometric plots of GFP mRNA measured by the present method and GFP reporter protein expression during a time course. Figure 10B shows the mean fluorescence intensity of GFP reporter expression or GFP mRNA expression over time under different arabinose concentrations.

Figure 11 shows a sample absolute abundance calculation.

DETAILED DESCRIPTION

Provided herein is a method for identifying two or more characteristics of a bacterium in a testing sample. In some embodiments, the final identifications can be made simultaneously or near simultaneously. The present method is a significant improvement upon the prior art methods at least because, in some embodiments, it allows for a faster, more quantitative, and more accurate method for measuring both the structure of intestinal microbiota and the presence of specific bacteria that may contribute to disease at the single cell, or single bacterium, level. In some embodiments, it also allows for concurrent measurement of intestinal bacterial identities, combined with detection of surface protein expression, including mammalian proteins, allowing, for example, the identification of mammalian antibodies bound to the bacteria.

Terminology

Terms used throughout this application are to be construed with ordinary and typical meaning to those of ordinary skill in the art. However, Applicant desires that the following terms be given the particular definition as provided below. As used in the specification and claims, the singular form "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a cell" includes a plurality of cells, including mixtures thereof.

The term "antibody" is used in the broadest sense, and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, and multi-specific antibodies (e.g., bispecific antibodies). Antibodies (Abs) and immunoglobulins (Igs) are glycoproteins having the same structural characteristics. While antibodies exhibit binding specificity to a specific target, immunoglobulins include both antibodies and other antibody-like molecules which lack target specificity. Native antibodies and immunoglobulins are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end.

The term "antibody fragment" refers to a portion of a full-length antibody, generally the target binding or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2 and Fv fragments. The phrase "functional fragment or analog" of an antibody is a compound having qualitative biological activity in common with a full-length antibody. For example, a functional fragment or analog of an anti-IgE antibody is one which can bind to an IgE immunoglobulin in such a manner so as to prevent or substantially reduce the ability of such molecule from having the ability to bind to the high affinity receptor, FcsRI. As used herein, "functional fragment" with respect to antibodies, refers to Fv, F(ab) and F(ab')2 fragments. An "Fv" fragment is the minimum antibody fragment which contains a complete target recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in a tight, non-covalent association (VH-VL dimer). It is in this configuration that the three CDRs of each variable domain interact to define a target binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer target binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for a target) has the ability to recognize and bind target, although at a lower affinity than the entire binding site. "Single-chain Fv" or "sFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the sFv to form the desired structure for target binding. The Fab fragment contains the constant domain of the light chain and the first constant domain (CHI) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CHI domain including one or more cysteines from the antibody hinge region. F(ab') fragments are produced by cleavage of the disulfide bond at the hinge cysteines of the F(ab')2 pepsin digestion product. Additional chemical couplings of antibody fragments are known to those of ordinary skill in the art.

The term “monoclonal antibody” as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies within the population are identical except for possible naturally occurring mutations that may be present in a small subset of the antibody molecules.

A "control" is an alternative subject or sample used in an experiment for comparison purpose. A control can be "positive" or "negative."

The term “hybridization chain reaction” or “HCR” refers to a polynucleotide polymerization method that employs two labeled hairpin polynucleotides and an initiator polynucleotide. The initiator polynucleotide can also be referred to as an initiator probe. A sequence of the initiator probe is complementary to a sequence of the target polynucleotide. Here, the target polynucleotide is bacterial DNA or RNA. The single stranded initiator polynucleotide opens one of the hairpins, which exposes a single stranded region in that hairpin, which in turn opens the second hairpin. Therefore, HCR creates labeled polynucleotide products that correlate with the target polynucleotide. These HCR created polynucleotides are referred to herein as “labeled polynucleotide HCR products.”

The term "identity" shall be constmed to mean the percentage of nucleotide bases or amino acid residues in the candidate sequence that are identical with the bases or residues of a corresponding sequence to which it is compared, after aligning the sequences and introducing gaps, if necessary to achieve the maximum percent identity for the entire sequence, and not considering any conservative substitutions as part of the sequence identity. Neither N- nor C-terminal extensions nor insertions shall be construed as reducing identity or homology. A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) that has a certain percentage (for example, 80%, 85%, 90%, or 95%) of "sequence identity" to another sequence means that, when aligned over their full lengths, that percentage of bases (or amino acids) are the same in comparing the two sequences. This alignment and the percent homology or sequence identity can be determined using software programs known in the art. In one embodiment, default parameters are used for alignment. In one embodiment a BLAST program is used with default parameters. In one embodiment, BLAST programs BLASTN and BLASTP are used with the following default parameters: Genetic code=standard; filtei-none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR.

As used herein the term “isolating” as in when “isolating the bacterium from any other bacteria in the identification sample,” refers to separation of the bacterium from other bacteria in the sample for a sufficient amount of time to detect one or more characteristics of the single bacterium. In some embodiments, the isolating step is achieved using fluorescence activated cell sorting (FACS).

The word "label" when used herein refers to a detectable compound or composition which can be conjugated directly or indirectly to a molecule or protein, c.g., a ligand. The label may itself be detectable (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable.

“Ligand” refers to a molecule that binds to another molecule. In some embodiments, the ligand is a polypeptide. An antibody is one non-limiting example of a ligand. An antibody fragment is another non-limiting example of a ligand.

As used herein, the word “near” refers to close in time. In some embodiments, the phrase “near simultaneously” means within up to ten minutes. In other embodiments, the phrase “near simultaneously” means within up to five minutes. In some embodiments, the phrase “near simultaneously” means within two minutes. In some embodiments, the phrase “near simultaneously" refers to within a single run of an assay, and in some embodiments, the assay is a flow cytometry assay.

The terms “specific binding,” “specifically binds,” “selective binding,” and “selectively binds” mean that a ligand exhibits appreciable affinity for its binding partner. Appreciable binding affinity includes binding with an affinity of at least 10⁶ M'¹, specifically at least 10⁷ M'¹, more specifically at least 10⁸ M’¹, yet more specifically at least 10⁹ M’¹, or even yet more specifically at least IO¹⁰ M’¹. A binding affinity can also be indicated as a range of affinities, for example, 10⁶ M'¹ to IO¹⁰ M'¹, specifically 10⁷ M'¹ to IO¹⁰ M'¹, more specifically 10⁸ M^-1to IO¹⁰ M'¹. Specific binding can be determined according to any art-recognized means for determining such binding. In some embodiments, specific binding is determined according to Scatchard analysis and/or competitive binding assays.

The terms "polynucleotide" and "oligonucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: a gene or gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, DNA of any sequence, RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components.

Compositions and Methods

Provided herein is a method for identifying two or more characteristics of a bacterium in a testing sample. In some embodiments the method further includes an absolute quantification. In some embodiments the method further includes a relative quantification. In some embodiments, the method comprises the following steps 1) obtaining the testing sample comprising the bacterium and a first ligand; 2) contacting the bacterium and the first ligand with a second ligand that is specific for the first ligand and comprising a first fluorescent label, wherein binding of the second ligand to the first ligand creates a labeled ligand conjugate comprising the first fluorescent label; 3) cross linking the labeled ligand conjugate that is bound to the bacterium to obtain a fluorescently labeled bacterium; 4) fixing the bacterium; 5) permeabilizing the bacterium; 6) obtaining an identification sample by performing a hybridization chain reaction (HCR) using the permeabilized bacterium, wherein the identification sample comprises the fluorescently labeled permeabilized bacterium comprising the first fluorescent label and one or more fluorescently labeled bacterial polynucleotide HCR products comprising at least a second fluorescent label, and wherein the first and second fluorescent labels are different; and 7) isolating the bacterium from any other bacteria in the identification sample and identifying a first ligand binding characteristic represented by the first fluorescent label and second characteristic represented by the second fluorescent label. Certain embodiments of the present method are sometimes referred to herein as the MICFLY of MicFly method or test.

As noted above, one of the benefits of the method is that it allows for a faster identification and/or quantification of multiple characteristics of a bacterium or a group of bacteria, and those identifications or quantifications can, in some embodiments, further allow for defining or characterizing subgroups of bacteria within the larger group of bacteria. That “faster identification” can be achieved by a step 8) identification of the two or more characteristics within a single assay run, and without the need for obtaining the identifications in two or more assays or performing a sequencing step. For example, in some embodiments, the two or more identifications can be obtained in the same flow cytometry assay. The hybridization chain reaction (HCR) comprises contacting bacterial DNA and/or RNA with one or more initiator probes that are specific for a bacterial DNA or RNA sequence and two labeled hairpin sequences, that when used with the initiator probe, create polynucleotide HCR products that incorporate the label and that correspond to the bacterial DNA or RNA sequence. Accordingly, included herein are initiator probes, hairpin sequences, and labels for use in HCR. In some embodiments, the one or more initiator probes are selected from those listed in Table 1 and/or Table 2: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40 and SEQ ID NO:41. Accordingly, included herein are methods wherein an initiator probe comprises SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40 or SEQ ID NO:41. In some embodiments, the two labeled hairpin sequences are selected from those listed in Table 3: SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ

ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ

ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ

ID NO:30, SEQ ID NO:31, SEQ ID NO:32 and SEQ ID NO:33. In some aspects, the two hairpin sequences comprise SEQ ID NO: 12 and SEQ ID NO: 13, respectively. In other aspects, the two hairpin sequences comprise SEQ ID NO: 14 and SEQ ID NO: 15, respectively. In other aspects, the two hairpin sequences comprise SEQ ID NO: 16 and SEQ ID NO: 17, respectively. In other aspects, the two hairpin sequences comprise SEQ ID NO: 18 and SEQ ID NO: 19, respectively. In other aspects, the two hairpin sequences comprise SEQ ID NO:20 and SEQ ID NO:21, respectively. In other aspects, the two hairpin sequences comprise SEQ ID NO:22and SEQ ID NO:23, respectively. In other aspects, the two hairpin sequences comprise SEQ ID NO:24 and SEQ ID NO:25, respectively. In other aspects, the two hairpin sequences comprise SEQ ID NO:26 and SEQ ID NO:27, respectively. In other aspects, the two hairpin sequences comprise SEQ ID NO:28 and SEQ ID NO:29, respectively. In other aspects, the two hairpin sequences comprise SEQ ID NO:30 and SEQ ID NO: 31, respectively. In other aspects, the two hairpin sequences comprise SEQ ID NO:32 and SEQ ID NO:33, respectively.

In some embodiments, the two hairpin sequences are labeled with a fluorophore. In some embodiments, the fluorophore used as a label for the two hairpin sequences is selected from the group consisting of Alexa Fluor 647, Alexa Fluor 594, Alexa Fluor 546, Alexa Fluor 488, Alexa Fluor 514, Alexa Fluor 660, Alexa Fluor 405, ATTO 490LS, ATTO 647N, ATTO 620, and ATTO 565.

In some embodiments, the ligand is an antibody or antibody fragment. Accordingly, one of the characteristics that can be determined and quantified using the present method is antibody binding, or whether an antibody binds to a group or subgroup of bacteria. In some embodiments, the first characteristic of the bacteria is an ability to bind IgA, and accordingly, the first antibody is an IgA antibody. In other or further embodiments, a characteristic of the bacteria is an ability to bind IgM and in step 1), the bacteria is contacted with an IgM antibody and in step 2), a fluorescently labeled antibody is specific for the IgM antibody. In other or further embodiments, a characteristic of the bacteria is an ability to bind IgG and in step 1), the bacteria is contacted with an IgG antibody and in step 2), a fluorescently labeled antibody is specific for the IgG antibody. The present method can be used to quantify the bacterial subgroups within a sample on these bases.

In some embodiments, further characteristics that can be determined and quantified using the present method are bacterial phylum, bacterial class, bacterial order, bacterial family, bacterial genus and/or bacterial species. The present method can be used to quantify the bacterial subgroups within a sample on these bases. Quantification of the subgroups on these bases is achieved using phylum-, class-, order-, family-, genus- or species- specific initiator probes in the hybridization chain reaction. Tables 1 and 2 provide exemplary, and non-limiting, lists of initiator probes for use in the hybridization chain reaction.

Accordingly, in some embodiments, the one or more fluorescently labeled bacterial polynucleotide amplification products represent a second characteristic that is the bacterial domain, bacterial phylum, bacterial class, bacterial order, bacterial family, bacterial genus or bacterial species. In these embodiments, the fluorescently labeled bacterial polynucleotide amplification products depend on or correlate with the initiator probe. For example, an initiator probe that can identify an Actinobacteria phylum characteristic of a bacteria comprises SEQ ID NO: 11 or a sequence having about at least 80%, 85%, 90% or 95% identity with SEQ ID NO: 11. In other embodiments, an initiator probe that can identify a Bifidobacterium species characteristic of a bacteria comprises SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NOTO or a sequence having about at least 80%, 85%, 90% or 95% identity with SEQ ID NO:9 or SEQ ID NO: 10. In other embodiments, an initiator probe that can identify a Bacilli class characteristic of a bacteria comprises SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7 or a sequence having about at least 80%, 85%, 90% or 95% identity with SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NOT. In other embodiments, an initiator probe that can identify a Enterobacteriaceae family characteristic of a bacteria comprises SEQ ID NO:4 or SEQ ID NO:34 or a sequence having about at least 80%, 85%, 90% or 95% identity with SEQ ID NO:4 or SEQ ID NO:34. In some embodiments, the initiator probe that can identify a Enterobacteriaceae family characteristic of a bacteria comprises a sequence that binds to SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47 or SEQ ID NO:48. In some embodiments, an initiator probe that can identify a Lactobacillaceae family characteristic of a bacteria comprises SEQ ID NO: 35 or a sequence having about at least 80%, 85%, 90% or 95% identity with SEQ ID NO:35. In some embodiments, the initiator probe that can identify a Lactobacillaceae family characteristic of a bacteria comprises a sequence that binds to SEQ ID NO:49 or SEQ ID NO:50. In some embodiments, an initiator probe that can identify an Enterococacceae family characteristic of a bacteria comprises SEQ ID NO:36 or a sequence having about at least 80%, 85%, 90% or 95% identity with SEQ ID NO:36. In some embodiments, the initiator probe that can identify an Enterococacceae family characteristic of a bacteria comprises a sequence that binds to SEQ ID NO:52 or SEQ ID NO:53. In some embodiments, an initiator probe that can identify a Clostridiaceae family characteristic of a bacteria comprises SEQ ID NO:37 or a sequence having about at least 80%, 85%, 90% or 95% identity with SEQ ID NO:37. In some embodiments, the initiator probe that can identify a Clostridiaceae family characteristic of a bacteria comprises a sequence that binds to SEQ ID NO:56 or SEQ ID NO:57. In some embodiments, an initiator probe that can identify a Bacteroidales family characteristic of a bacteria comprises SEQ ID NO:38 or a sequence having about at least 80%, 85%, 90% or 95% identity with SEQ ID NO:38. In some embodiments, the initiator probe that can identify a Bacteroidales family characteristic of a bacteria comprises a sequence that binds to SEQ ID NO:61 or SEQ ID NO:62. In some embodiments, an initiator probe that can identify a Staphylococacceae family characteristic of a bacteria comprises SEQ ID NO:39 or a sequence having about at least 80%, 85%, 90% or 95% identity with SEQ ID NO:39. In some embodiments, the initiator probe that can identify a Staphylococacceae family characteristic of a bacteria comprises a sequence that binds to SEQ ID NO:63, SEQ ID NO:64 or SEQ ID NO:65. In some embodiments, an initiator probe that can identify a Lachnospiraceae family characteristic of a bacteria comprises SEQ ID NO:40 or a sequence having about at least 80%, 85%, 90% or 95% identity with SEQ ID NO:40. In some embodiments, the initiator probe that can identify a Lachnospiraceae family characteristic of a bacteria comprises a sequence that binds to SEQ ID NO:67, SEQ ID NO:68 or SEQ ID NO:69. In some embodiments, an initiator probe that can identify a Eubacteria domain characteristic of a bacteria comprises SEQ ID NO:41 or a sequence having about at least 80%, 85%, 90% or 95% identity with SEQ ID NO:41. In some embodiments, the initiator probe that can identify a Eubacteria domain characteristic of a bacteria comprises a sequence that binds to SEQ ID NO:70, SEQ ID N0:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75 or SEQ ID NO:76.

Therefore, included herein are methods wherein the second bacterial characteristic is a bacterial domain. In some embodiments, the bacterial domain is Eubacteria and the HCR comprises use of an initiator probe comprising SEQ ID NO:41 or a polynucleotide sequence having about at least 80%, 85%, 90% or 95% identity with SEQ ID NO:41.

Also included herein arc methods wherein the second bacterial characteristic is a bacterial phylum. In some embodiments, the bacterial phylum is Actinobacteria. In some embodiments, the bacterial phylum is Actinobacteria and the HCR comprises use of an initiator probe comprising SEQ ID NO: 11 or a polynucleotide sequence having about at least 80%, 85%, 90% or 95% identity with SEQ ID NO: 11. Also included herein are methods where the second bacterial characteristic is a a bacterial genus or a bacterial order. In some embodiments, the second bacterial characteristic is a bacterial class. In some embodiments, the bacterial class is Bacilli and the HCR comprises use of an initiator probe comprising SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7 or a sequence having about at least 80%, 85%, 90% or 95% identity with SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7.

In some embodiments, the second bacterial characteristic is a bacterial family, and in some embodiments, the bacterial family is an Enterobacteriaceae family, a Lactobacillaceae family, an Enterococacceae family, a Clostridiaceae family, a Bacteroidales family, a Staphylococacceae family or a Lachnospiraceae family. In some embodiments, the bacterial family is Enterobacteriaceae and the HCR comprises use of an initiator probe comprising SEQ ID NO:4, SEQ ID NO:34 or a polynucleotide sequence having about at least 80%, 85%, 90% or 95% identity with SEQ ID NO:4 or SEQ ID NO:34. In some embodiments, the bacterial family is Lactobacillaceae and the HCR comprises use of an initiator probe comprising SEQ ID NO:35 or a polynucleotide sequence having about at least 80%, 85%, 90% or 95% identity with SEQ ID NO:35. In some embodiments, the bacterial family is Enterococacceae and the HCR comprises use of an initiator probe comprising SEQ ID NO:36 or a polynucleotide sequence having about at least 80%, 85%, 90% or 95% identity with SEQ ID NO:36. In some embodiments, the bacterial family is Clostridiaceae and the HCR comprises use of an initiator probe comprising SEQ ID NO:37 or a polynucleotide sequence having about at least 80%, 85%, 90% or 95% identity with SEQ ID NO:37. In some embodiments, the bacterial family is Bacteroidales and the HCR comprises use of an initiator probe comprising SEQ ID NO:38 or a polynucleotide sequence having about at least 80%, 85%, 90% or 95% identity with SEQ ID NO:38. In some embodiments, the bacterial family is Staphylococacceae and the HCR comprises use of an initiator probe comprising SEQ ID NO:39 or a polynucleotide sequence having about at least 80%, 85%, 90% or 95% identity with SEQ ID NO:39. In some embodiments, the bacterial family is Lachnospiraceae and the HCR comprises use of an initiator probe comprising SEQ ID NQ:40 or a polynucleotide sequence having about at least 80%, 85%, 90% or 95% identity with SEQ ID NO:40.

In some aspects, the second bacterial characteristic is a bacterial species, and in some embodiments the bacterial species is a Bifidobacterium species. Included herein are methods wherein the bacterial species is a Bifidobacterium species and the HCR comprises use of an initiator probe comprising SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO: 10 or a sequence having about at least 80%, 85%, 90% or 95% identity with SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO: 10.

In some embodiments, the method further comprises determining a total, or absolute, amount of bacteria in the testing sample. This quantification can be made via any means known to one of ordinary skill in the art, and in some embodiments, absolute counting beads are added to the sample prior to step 7).

In certain aspects of the method, prior to contacting the group of bacteria in the testing sample with the first ligand, one or more contaminants are removed from the testing sample using a PERCOLL solution. In some embodiments, the PERCOLL solution is an about 20% PERCOLL solution. In some embodiments, the PERCOLL solution is between an about 5% and about 25% PERCOLL solution. In some embodiments, the PERCOLL solution is about 5%, about 10%, about 15%, about 20% or about 25%. In some embodiments, the PERCOLL solution is about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24% or about 25%. In some embodiments, the PERCOLL solution comprises PERCOLL and sodium chloride.

In certain aspects of the method, the fluorescently labeled second ligand comprises a red fluorescent label. In some embodiments, the red fluorescent label is a CF633 label (Biotium).

In certain aspects of the method, the labeled ligand conjugate is crosslinked to the bacteria using a crosslinking agent comprising bis-(sulfosuccinimidyl)-suberate. In some embodiments the bis-(sulfosuccinimidyl)-suberate is at a concentration of about 1 mM.

In certain aspects of the method, the bacteria are fixed using a fixing agent comprising paraformaldehyde. In some embodiments the fixing agent comprises about 4% paraformaldehyde. In some embodiments, the fixing agent comprises about 0.5% to about 8% paraformaldehyde. In some embodiments the fixing agent comprises about 0.5% paraformaldehyde, about 1% paraformaldehyde, about 2% paraformaldehyde, about 3% paraformaldehyde, about 4% paraformaldehyde, about 5% paraformaldehyde, about 6% paraformaldehyde, about 7% paraformaldehyde, or about 8% paraformaldehyde.

In certain aspects of the method, the bacteria are permeabilized using a permeabilizing agent comprising lysozyme. In other or further embodiments, the permeabilizing agent comprises lysostaphin.

It should be understood that the foregoing relates to preferred embodiments of the present invention and that numerous changes may be made therein without departing from the scope of the invention. The invention is further illustrated by the following examples, which are not to be construed in any way as imposing limitations upon the scope thereof. On the contrary, it is to be clearly understood that resort may be had to various other embodiments, modifications, and equivalents thereof, which, after reading the description herein, may suggest themselves to those skilled in the art without departing from the spirit of the present invention and/or the scope of the appended claims. All patents, patent applications, and publications referenced herein are incorporated by reference in their entirety for all purposes.

EXAMPLES

Example 1

Isolation of microbiota from human stool

Bacteria were isolated using a modified protocol for patient fecal microbiota transplantation. Approximately 50 to 400 mg of stool was weighed in a 50 mL Falcon tube, and the exact sample mass was recorded. Fecal material was homogenized by resuspending in 10 mL phosphate buffered saline (PBS) and hard vortexing for 1 minute. The fecal solution was centrifuged at low speed (50 x g) for 10 minutes at 4°C to remove larger cells and debris. Supernatant was collected and filtered through a 40 pm cell strainer and washed with an additional 10 mL PBS. To pellet bacteria, a high-speed spin (4700 x g) was performed on a benchtop centrifuge for 10 minutes at 4°C. Supernatant was discarded, and the pellet was resuspended in 2 mL of a 20% PERCOLL solution in 0.15 M NaCl. The Percoll resuspension was spun at 10,000 x g for 10 minutes at 4°C, allowing non-microbial intestinal matter to settle above the supernatant and bacterial cells to pellet at the bottom. Supernatant is discarded, and the pellet was washed in 2 mL PBS and centrifuged for 10 minutes at 9000 x g at 4°C. The final pellet was resuspended in exactly 2 mL PBS and filtered through a 20 pm cell strainer. This resuspension was the isolated fecal microbiota used in the present methods, and the final volume was recorded for absolute abundance calculations. Example 2

Cross-linking and fixation of isolated fecal microbiota

All centrifugation steps were performed for 10 minutes at 9000 x g. The 2 mL isolated microbiota resuspension was split equally (500 pL each) into four separate 1.5 mL conical microcentrifuge tubes (allowing duplicate tests with matched isotype controls). Tubes were centrifuged, and supernatant was discarded. Two tubes were incubated with goat anti-human IgA CF633 (Biotium 20427) (1:10), and the other two tubes were incubated with goat anti-chicken IgY CF633 as an isotype control (Biotium 20126 ) (1:10) in 50 pL of PBS with 10% normal goat serum (ThermoFisher) in the dark for 1 hour on ice. CF633 was used because it was found that it is stable to both BS3 cross-linking and PFA fixation in downstream steps. All steps going forward using CF633- labeled bacteria were performed in the dark whenever possible. Bacteria were washed three times with PBS at pH 8.0, which is slightly basic to increase the cross-linking efficiency of bis- (sulfosuccinimidyl)-suberate (BS3) (ThermoFisher) to stabilize human IgA and the fluorophore- conjugated secondary antibody on the bacterial surface. After the last wash, the supernatant was removed, and each sample was resuspended with 100 uL of PBS at pH 8.0. To crosslink bacteria at a final concentration of 1 mM BS3, 100 pL of 2 mM BS3 (1:1 dilution) was added to 100 pL of bacteria in each tube then incubated for 1 hour on ice. Cross-linked bacteria were centrifuged, washed with 500 pL of PBS pH 7.4, then fixed with 4% PFA at room temperature for 1 hour to preserve the integrity of bacterial RNA for testing. Bacteria were centrifuged and washed twice with PBS pH 7.4, then stored in equal volume of PBS as initial sample volume prior to cross-linking/fixation (500 pL per tube) at 4°C in the dark for future testing.

Example 3

Hybridization chain reaction

All centrifugation steps were performed for 10 minutes at 9000 x g. 500 pL of cross- linked/fixed bacteria were used for hybridization chain reaction (HCR). Bacteria were permeabilized by incubating with 0.1 mg/mL lysozyme and 0.01 mg/mL lysostaphin in 10 mM Tris HC1 at 37°C for 1 hour. Cells were centrifuged and washed with PBS with 0.1% Tween (PBST) once. Supernatant was discarded and the pellet was resuspended with 400 pL of probe hybridization buffer (Molecular Instruments Inc.) to pre-hybridize for 1 hour at 37°C. 100 pL of each initiator probe (Table 1 and Table 2) was pooled at a concentration of 16 nM and directly added to pre-hybridized samples. Samples were incubated overnight to hybridize at 37 °C on a thermoshaker. After hybridization, cells were centrifuged and washed three times with probe wash buffer (Molecular Instruments, Inc.), with 10-minute incubation steps at 37°C during each wash. The supernatant was discarded, and the pellet was resuspended in 75 pL of amplification buffer (Molecular Instruments, Inc.). Fluorescent hairpin amplifiers (Table 3) were incubated on a dry heat block at 95°C for 90 seconds, then cooled for 30 minutes at room temperature. To tag the initiator-labeled bacteria, snap-cooled hairpins were added at a 80 nM concentration in 50 pL of amplification buffer to bacteria samples with 75 pL of amplification buffer (final volume 125 pL) then incubated overnight at room temperature. Samples were centrifuged and washed three times with 5X saline-sodium citrate buffer with 0.1% Tween (SSCT). Samples were stored in equal volume of 5X SSCT as initial sample volume used for HCR (500 pL) at 4°C in the dark until flow cytometric testing.

Example 4

Spectral Flow cytometry

Prior to flow cytometry, 500 pL HCR samples were filtered through a 20 pm cell strainer into a 5 mL round-bottom FACS tube. Pooled microbiota samples were used for single color spectral unmixing controls. To detect bacterial cells, a low threshold on the flow cytometer (Cytek Aurora) was set at 1500 for FSC-A and 1500 for SSC-A, with a gain of 400 for FSC-A, 300 for SSC-A, and 300 for SSC-B. To eliminate detection of debris and other particulate matter, a threshold of 500 was set for the channel that detects the Eubacteria probe. All parameters were acquired on a logarithmic scale. For all samples, the stopping gate was placed on Eubacteria-positive cells, with at least 20,000 events recorded. Data were analyzed using FlowJo vl0.9. Absolute abundance was quantified using the volumetric function provided from the spectral cytometer. Absolute abundance can also be quantified using counting beads if the flow cytometer does not have a volumetric counting function.

Example 5

Absolute abundance calculation

Absolute count of bacteria cells per mg stool was calculated as follows:

A sample calculation is provided in Figure 11.

Example 6

Use ofMicFly to Determine Maternal IgA Modulates the Preterm Gut Microbiome Microbiota research is primarily sequencing-based, subjected to PCR amplification bias, and restricted to relative abundances. Further, the lack of single cell methods obscures having a granular understanding of the microbiota that could be used for clinical and diagnostic purposes.

Secretory IgA regulates gut bacterial colonization, but its role in the assembly of the early life microbiome is unclear. Differential IgA binding of specific bacterial taxa has been implicated in several intestinal inflammatory and enteric diseases. Investigating how IgA alters the temporal development of specific bacterial populations is challenging with sequencing-based analyses of relative abundance. Disclosed herein is MicFly (Microbiome Flow Cytometry): a novel single-cell technology combining spectral flow cytometry, in situ RNA hybridization chain reaction, and surface protein-targeted antibodies to quantitate taxa-specific, absolute population changes of IgA-bound bacteria. This approach allows for single cell analysis of the microbiota, which circumvents the biases associated with sequencing technologies that prevent accurate quantification. It was found that mlgA binding influences the growth and interactions of different bacterial communities in several breastfed preterm infants. Using an in-house curated 16s and 23s rRNA database, comprehensive probe sets spanning all major taxa in the infant gut microbiome for use in a 14-color spectral panel, were designed. MicFly ’s ability to distinguish bacterial species bound by IgA was validated both in vitro culture using breast milk-derived IgA and ex vivo human stool. To investigate the effects of maternal IgA on bacterial population dynamics in the early life microbiome, longitudinal analyses were performed on a prospective cohort of preterm infants. MicFly was used to measure IgA-bound bacteria for each major stage of early life microbiome development (Bacilli, Enterobacteriaceae, Bifidobacterium, Clostridiales, and Bacteroidota). Comparing breast milk-fed to formula-fed infants demonstrates that maternal IgA influences the growth of several gut bacterial communities. Lastly, detection of population fluctuations at the species-level (K. pneumoniae and K. oxytoca, strongly implicated in necrotizing enterocolitis development) reveals the clinical applicability of MicFly to identify disease-associated bacteria with high granularity. For example, the clinical application of MicFly was tested on stool from C. difficile infected adult and pediatric patients who received fecal microbiota transplantation (FMT). MicFly is a tool that can be used in clinical settings (for example, during evaluation of infant gut ecosystem) that require precise information on both bacterial identity and function to predict or diagnose microbiome-related disease. TABLES

Table 1. Oligonucleotides used as initiator probes for hybridization chain reaction.

Bolded nucleotides are specific to the target bacteria, underlined nucleotides are specific to the hairpin amplifier, and nucleotides that are neither bolded nor underlined are used as spacers.

Table 2.

Table 3. Oligonucleotides and associated fluorophore-conjugates, used as hairpin amplifiers hybridization chain reaction.

Claims

CLAIMS What is claimed is:

1. A method of identifying two or more characteristics of a bacterium in a testing sample comprising: a. Obtaining the testing sample comprising the bacterium and a first ligand; b. Contacting the bacterium and the first ligand with a second ligand that is specific for the first ligand and comprising a first fluorescent label, wherein binding of the second ligand to the first ligand creates a labeled ligand conjugate comprising the first fluorescent label; c. Cross linking the labeled ligand conjugate that is bound to the bacterium to obtain a fluorescently labeled bacterium; d. Fixing the bacterium; e. Permeabilizing the bacterium; f. Obtaining an identification sample by performing a hybridization chain reaction (HCR) using the permeabilized bacterium, wherein the identification sample comprises the fluorescently labeled permeabilized bacterium comprising the first fluorescent label and one or more fluorescently labeled bacterial polynucleotide HCR products comprising at least a second fluorescent label, and wherein the first and second fluorescent labels are different; and g. Isolating the bacterium from any other bacteria in the identification sample and identifying a first ligand binding characteristic represented by the first fluorescent label and second characteristic represented by the second fluorescent label.

2. The method of claim 1, further comprising determining a total number of bacteria in the quantification sample.

3. The method of claim 1 or claim 2, wherein the first ligand is an antibody.

4. The method of any one of claims 1-3, wherein the first ligand is an IgA antibody, and the first characteristic of the bacteria is an ability to bind IgA.

5. The method of any one of claims 1-3, wherein the first ligand is an IgM antibody, and the first characteristic of the bacteria is an ability to bind IgM.

6. The method of any one of claims 1-3, wherein the first ligand is an IgG antibody, and the first characteristic of the bacteria is an ability to bind IgG.

7. The method of any one of claims 1-6, wherein the second characteristic is a bacterial phylum.

8. The method of claim 7, wherein the bacterial phylum is an Actinobacteria phylum.

9. The method of claim 8, wherein the HCR comprises use of an initiator probe comprising SEQ ID NO: 11 or a polynucleotide sequence having about at least 80%, 85%, 90% or 95% identity with SEQ ID NO:11.

10. The method of any one of claims 1-6, wherein the second characteristic is a bacterial class.

11. The method of claim 10, wherein the bacterial class is a Bacilli class.

12. The method of claim 10, wherein the HCR comprises use of an initiator probe comprising SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7 or a sequence having about at least 80%, 85%, 90% or 95% identity with SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7.

13. The method of any one of claims 1-6, wherein the second characteristic is a bacterial order.

14. The method of any one of claims 1-6, wherein the second characteristic is a bacterial family.

15. The method of claim 14, wherein the bacterial family is an Enterobacteriaceae family, a Lactobacillaceae family, an Enterococacceae family, a Clostridiaceae family, a Bacteroidales family, a Staphylococacceae family or a Lachnospiraceae family.

16. The method of claim 15, wherein the bacterial family is the Enterobacteriaceae family and the HCR comprises use of an initiator probe comprising SEQ ID NO:4 or SEQ ID NO:34 or a polynucleotide sequence having about at least 80%, 85%, 90% or 95% identity with SEQ ID NO:4 or SEQ ID NO:34.

17. The method of claim 15, wherein the bacterial family is the Lactobacillaceae family and the HCR comprises use of an initiator probe comprising SEQ ID NO:35 or a polynucleotide sequence having about at least 80%, 85%, 90% or 95% identity with SEQ ID NO:35.

18. The method of claim 15, wherein the bacterial family is the Enterococacceae family and the HCR comprises use of an initiator probe comprising SEQ ID NO:36 or a polynucleotide sequence having about at least 80%, 85%, 90% or 95% identity with SEQ ID NO:36.

19. The method of claim 15, wherein the bacterial family is the Clostridiaceae family and the HCR comprises use of an initiator probe comprising SEQ ID NO:37 or a polynucleotide sequence having about at least 80%, 85%, 90% or 95% identity with SEQ ID NO:37.

20. The method of claim 15, wherein the bacterial family is the Bacteroidales family and the HCR comprises use of an initiator probe comprising SEQ ID NO:38 or a polynucleotide sequence having about at least 80%, 85%, 90% or 95% identity with SEQ ID NO:38.

21. The method of claim 15, wherein the bacterial family is the Staphylococacceae family and the HCR comprises use of an initiator probe comprising SEQ ID NO:39 or a polynucleotide sequence having about at least 80%, 85%, 90% or 95% identity with SEQ ID NO:39.

22. The method of claim 15, wherein the bacterial family is the Lachnospiraceae family and the HCR comprises use of an initiator probe comprising SEQ ID NO:40 or a polynucleotide sequence having about at least 80%, 85%, 90% or 95% identity with SEQ ID NO:40.

23. The method of any one of claims 1-6, wherein the second characteristic is a bacterial genus.

24. The method of any one of claims 1-6, wherein the second characteristic is a bacterial species.

25. The method of claim 24, wherein the bacterial species is a Bifidobacterium species.

26. The method of claim 25, wherein the HCR comprises use of an initiator probe comprising SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO: 10 or a sequence having about at least 80%, 85%, 90% or 95% identity with SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO: 10.

27. The method of any one of claims 1-26, wherein prior to contacting the group of bacteria in the testing sample with the first antibody, one or more contaminants are removed from the testing sample using a PERCOLL solution.

28. The method of any one of claims 1-27, wherein the fluorescently labeled second ligand comprises a red fluorescent label.

29. The method of any one of claims 1-28, wherein the labeled antibody conjugate is crosslinked to the bacteria using a crosslinking agent comprising bis-(sulfosuccinimidyl)-suberate.

30. The method of any one of claims 1-29, wherein the bacteria are fixed using a fixing agent comprising paraformaldehyde.

31. The method of any one of claims 1-30, wherein the bacteria are permeabilized using a permeabilizing agent comprising lysozyme and lysostaphin.

32. The method of any one of claims 1-31, wherein step (g) is performed using fluorescence activated sorting.

33. The method of any one of claims 1-6, wherein the hybridization chain reaction (HCR) comprises use of an initiator probe selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40 and SEQ ID NO:41.

34. The method of any one of claims 1-33, wherein the hybridization chain reaction (HCR) comprises use of two hairpin sequences selected from a group consisting of SEQ ID NO: 12 and SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:23, SEQ ID NO:24 and SEQ ID NO:25, SEQ ID NO:26 and SEQ ID NO:27, SEQ ID NO:28 and SEQ ID NO:29, SEQ ID NO:30 and SEQ ID NO:31, and SEQ ID NO:32 and SEQ ID NO:23.

35. The method of claim 34, wherein the two hairpin sequences are labeled with a fluorophore selected from the group consisting of Alexa Fluor 647, Alexa Fluor 594, Alexa Fluor 546, Alexa Fluor 488, Alexa Fluor 514, Alexa Fluor 660, Alexa Fluor 405, ATTO 490LS, ATTO 647N, ATTO 620, and ATTO 565.

36. A composition comprising SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40 or SEQ ID NO:41.