Bacteriophages as biomarkers

Abstract

The present invention relates to the use of fluorescently labeled phages as biomarkers to facilitate an analysis of phage-bacteria interactions. An objective of present invention is to make an assay suitable to screen for strains with altered susceptibility to phages.

Description

[0001] Bacteriophages as biomarkers

[0002] FIELD OF INVENTION

[0003] The present invention relates to the use of fluorescently labeled phages as biomarkers to facilitate an analysis of phage-bacteria interactions.

[0004] An objective of present invention is to make an assay suitable to screen for strains where e.g. the phage receptor, such as e.g. glycan (e.g. EPS / CPS or RGP) is altered to inhibit phage attachment when compared to a corresponding parent or wild-type (WT) strain.

[0005] BACKGROUND OF INVENTION

[0006] Fermented milk products, such as fermented milk drinks, lactic acid bacteria beverages, yoghurt, cultured milks and cheese, are often produced by providing milk substrates, especially based on animal milks, such as cow milk, goat milk, sheep milk and the like or plant-based milk alternatives based on soy, almond, wheat, barley etc., as culture media and fermenting the medium with lactic acid bacteria.

[0007] During the production of a fermented milk product, phages may attack the bacteria, leading to a reduction in the viable cell count of the lactic acid bacteria.

[0008] In particular fermented milk products which are consumed as health-promoting foods having beneficial physiological effects, such as intestinal function controlling effect and immunopotentiating effect, are dependent on a high number of viable bacteria. It is however difficult to replace a strain susceptible to phage attack by another strain of the same species, as the replacement strain seldom has a similar functionality, such as acidification activity, flavor profile and / or the same probiotic character.

[0009] Diverse treatments have been applied to minimize phage infections in the dairy environment. Predominant approaches include chemical and physical methods for equipment sanitation (Guglielmotti et al. 2012), as well as a culture replacement and strain rotation programs (Derkx et al. 2014). The latter require strains with identical technological performance, but different phage sensitivities (Binetti, Bailo, and Reinheimer 2007).

[0010] Thus, isolation and characterization of bacteriophage insensitive mutants (BIMs) or phage hardened variants (PHV's) of strains used in dairy starter cultures or as probiotics has been widely explored.

[0011] Several methods for generating BIMs or PHV's of S. thermophilus have been proposed. The strategies include insertional mutagenesis (Lucchini, Sidoti, and Brussow 2000), the secondary culture method (Binetti, Bailo, and Reinheimer 2007), serial passaging in the presence of high phage titers (Mills et al. 2007), chemical mutagenesis (Rodnguez et al. 2008) or transformation with an antisense mRNA-generating plasmid (McDonnell et al. 2018). The acquired resistance may be due to the activation of intracellular resistance mechanisms, mainly clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems or restriction-modification (R-M) systems (Lucchini, Sidoti, and Brussow 2000; Binetti et al. 2007; Mills et al. 2010). Additional phage resistance systems, such as abortive infection (Abi) (Larbi et al. 1992) or superinfection exclusion (Sie) (Ali et al. 2014). However, those mechanisms probably are not very widespread and therefore, they do not commonly mediate resistance in BIMs of dairy strains.

[0012] The mode of action of CRISPR-Cas and R-M systems is similar in the way that they both target specific genetic sequences of the invading phage (Dupuis et al. 2013). CRISPR and cas genes provide adaptive immunity that uses sequence memory to target incoming DNA (Barrangou and Horvath 2017). Restriction enzymes of R-M systems recognize and cleave foreign DNA at defined sites within the recognition sequence, while the host DNA is resistant to cleavage due to modifications at these sites (Guimont, Henry, and Linden 1993; Pingoud et al. 2005).

[0013] Spontaneous phage hardened variants (PHVs) are frequently generated to substitute industrial strains infected by phages and to obtain robust phage-resistant LAB strains, strategies have been proposed to select for BIMs whose phage resistance was mediated by mechanisms independent of R-M or CRISPR-Cas systems.

[0014] Common ways to measure the inactivity of the phage receptor is simply to analyze the bacterium for increased resistance to a suitable representative panel of different bacteriophages based on the viability of the bacteria and or the acidification speed of in particular starter cultures used in the production of fermented dairy product. However, alternative methods include immunobased methods where for example, BIMs with inhibited phage adherence to the bacterial cell walls which were selected by immunoprecipitation in flow cytometry (Viscardi et al. 2003).

[0015] To efficiently generate and screen for BIMs or PHV's with inhibited phage adherence, new simple and reliable and high-throughput methods have been explored.

[0016] Over time, several methods for studying phage-bacteria interactions have been proposed.

[0017] Classical adsorption assays, such as a double-layer plaque titration method, were successfully used to study impact of various environmental factors on phage attachment to the host cells (Binetti et aL, 2002; Quiberoni et aL, 2000).

[0018] However, those methods are laborious and time-consuming and thus not suitable for high- throughput screening of large bacterial libraries. More recent approaches rely on fluorescently labeled phages for a visual assessment of a phage infection. Phage proteins can be tagged by FITC, while phage DNA can be directly labeled by fluorophores, such as SYBR. Gold, DAPI, YOYO-1, Syto9, and Sytol3 (Jaye et aL, 2004; Low et aL, 2020; Ohno et aL, 2012; Szymczak et aL, 2018). The limitation of those methods is an unspecific labeling of bacterial cells that are not infected by the tested phages.

[0019] Although different protocols for removing excess dyes have been proposed, none of them have been shown sufficiently effective when tested in phage preparations (Low et aL, 2020; Ohno et aL, 2012).

[0020] A fluorescent phage tagging, alternative to the direct phage labelling, requires genetic engineering of bacteriophages. Fluorescent phage particles or fluorescent phage anti-receptor proteins were successfully generated and used for binding studies before (Lavelle et aL, 2022; Szymczak et aL, 2019; Wu et aL, 2016). However, those methods are very laborious and not applicable for a large consortium of phages.

[0021] To track phage-infected cells, approaches based on a flow cytometry were proposed. In those assays, cells infected by phages give a fluorescent signal due to the phage DNA injection and replication inside the host cells, while phage insensitive cells remain untagged (Melo et aL, 2022; Ohno et aL, 2012).

[0022] Nevertheless, those assays are not suitable for the work with PHVs, which have activated phage-resistance mechanisms that prevent from phage DNA replication.

[0023] Hence despite several developments in this area, new simple and reliable and high-throughput methods are needed for efficient analysis and optionally sorting of PHV's or BIMs of lactic acid bacteria as e.g. from the genera Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, Streptococcus, Enterococcus.

[0024] SUMMARY OF INVENTION

[0025] The present invention relates to the use of fluorescently labeled phages as biomarkers to facilitate an analysis of phage-bacteria interactions.

[0026] The role of different cell surface components in phage adsorption to the dairy bacterium Streptococcus thermophilus is largely unknown. To study phage receptors, a reliable and high- throughput method to sort for phage-hardened variants (PHVs) with receptor modifications is required. In summary, the invention as claimed herein relates to a method for measuring the phage susceptibility of a lactic acid bacterium, said method comprises: a) adding a fluorescent dye to a first composition comprising bacteriophages b) obtaining a second composition comprising lactic acid bacteria c) mixing the first composition of a) and the second composition of b) to obtain a third composition comprising phages, bacteria and dye. d) using the distribution of the fluorescent dye as a measure of phage susceptibility wherein three types of a fluorescent signals indicate different strain-phage interactions: outer fluorescence - representing phage binding, inner fluorescence - specific to dead cells or cells with ongoing phage infection no fluorescence - representing cells without phage adsorption.

[0027] To arrive at the invention, the inventors explored options and performed several experiments as summarized below:

[0028] Methods to generate phage hardened variants (PHV's)

[0029] Several methods are available for the person skilled in the art to make a PHV. One approach is to generate mutants where the phage receptor is essentially inactive, e.g. by introducing a mutation in a phage receptor gene. One may randomly mutagenize (e.g. by UV radiation or chemical mutagenesis) and select for mutations wherein the relevant genes are essentially inactive. Further one could select for relevant spontaneous mutations, wherein the relevant genes are altered, e.g. are essentially inactive. Alternatively, one may use protein engineering (PE) techniques to introduce mutations to render the genes responsible for phage susceptibility dysfunctional and hence inhibit synthesis of the relevant genes.

[0030] Labeled phages as biomarkers of phage-bacteria interactions.

[0031] Eight fluorophores were tested for labeling streptococcal phages.

[0032] Labeling with propidium iodide (PI) is proven as a universal method for tagging S. thermophilus phages as the dye penetration through the phage capsid was independent from the phage species.

[0033] Additionally, four methods were validated for the removal of an excess dye from phage preparations. Labeling phage DNA with propidium iodide (PI) was identified as the most optimal method for our application.

[0034] A significant advantage of the technique is that the excess dye does not enter bacterial cells with an intact membrane, hence it diminishes the chances for a false-positive labeling.

[0035] The established method can be applied to sort for PHVs generated from different backgrounds. In this regard, the event of a phage adsorption and phage DNA injection gives a fluorescent signal. A phage hardened variant with an inactive phage receptor does not display any fluorescence, due to the lack of a phage adsorption.

[0036] Rapid selection for PHVs with a desired phenotype for industrial applications.

[0037] Flow cytometry was applied to sort for cells with and without fluorescence, mediated by the presence or absence of a phage adsorption, respectively.

[0038] Results indicated that flow cytometry can be used and validated to sort for cells with and without fluorescence, mediated by the presence or absence of a phage adsorption, respectively.

[0039] For the proof-of-concept study, we mixed propidium iodide (Pl)-labeled phage with a WT strain and its PHV holding a confirmed modification in a phage receptor. Gates designed in the experiment enabled sorting for the WT and the PHV with 100% accuracy.

[0040] Further, the cells survived the sorting and grew in the presence of labeled phages and the excess fluorophore.

[0041] The experiments are further described in the Examples section herein.

[0042] The use of PI in methods of the present invention is not disclosed nor suggested in the prior art and surprisingly provided a robust phage labeling enabling high-throughput screening and sorting of bacterial strains for bacteriophage resistance status. While PI has been used in viability assays and occasionally in phage-host studies as a marker of dead cells, it has not been systematically validated as a simple, stand-alone method to fluorescently tag bacteriophage DNA. In Low et al, (2020) for example, fluorescent labelling of bacteriophage DNA is performed using SYTO 13, and a separate live / dead assay using SYTO13 in combination with propidium iodide (PI) is used to determine bacterial viability after phage infection. PI is thus only used therein as a viability dye for host cells and not as a tag for phage DNA.

[0043] In contrast, the Examples of the present invention surprisingly show that PI can be used for bacteriophage labelling and has advantageous properties compared to other dyes (e.g. SYTO 13, SYBR Gold) in particular with respect to specificity and reduction of unspecific bacterial labelling. The methods of the present invention have been validated experimentally as disclosed in the Examples herein, such as, but not limited to, identifying and optionally sorting of phage-hardened variants (PHVs).

[0044] By applying the herein disclosed knowledge on an assay to study bacterial phage receptors and phage-host interactions, methods to prevent phage infections in dairy plants are enabled. In summary, the invention relates to the following interrelated aspects:

[0045] Aspect 1 : A method for measuring the phage susceptibility of a lactic acid bacterium, said method comprises: a) adding a fluorescent dye to a first composition comprising bacteriophages b) obtaining a second composition comprising lactic acid bacteria c) mixing the first composition of a) and the second composition of b) to obtain a third composition comprising phages, bacteria and dye. d) using the distribution of the fluorescent dye as a measure of phage susceptibility.

[0046] Aspect 2: A method for sorting lactic acid bacterial cells according to phage susceptibility, said method comprises: a) adding a fluorescent dye to a first composition comprising bacteriophages b) obtaining a second composition comprising lactic acid bacteria c) mixing the first composition of a) and the second composition of b) to obtain a third composition comprising phages, bacteria and dye. d) using the fluorescent dye signal to sort cells based.

[0047] Aspect 3: A method according to aspect 1 or 2, wherein the fluorescent dye is propidium iodide (PI).

[0048] Aspect 4: A method according to aspect 3 wherein propidium iodide (PI) is added in a concentration of lmg / l-25 mg / l, preferably 2-10 mg / L, even more preferably 2-5mg / L, yet even more preferably 2.5 mg / L.

[0049] Aspect 5: A method according to any of the preceding aspects, wherein PI is prepared in dimethyl sulfoxide (DMSO) buffer.

[0050] Aspect 6: A method according to any of the preceding aspects, wherein the lactic acid bacterium is of the genus Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, Streptococcus or Enterococcus.

[0051] Aspect 7: A method according to any of the preceding aspects, wherein the lactic acid bacterium is of the genus Streptococcus spp. such as e.g. the species Streptococcus thermophilus.

[0052] Aspect 8: A method according to any of the preceding aspects, wherein the phage susceptibility is measured by microscopy or microfluidics. Aspect 9: A method according to any of the preceding aspects, wherein the distribution of the fluorescent dye is measured by FACS and optionally sorted according to phage susceptibility.

[0053] Aspect 10: A method according to any of the preceding aspects, wherein the lactic acid bacteria with low phage susceptibility are isolated and optionally cultured.

[0054] Aspect 11 : A method according to any of the preceding aspects, wherein the bacteriophages are present at a multiplicity of infection (MOI) of at least 20, such as at least 60, preferably at least 100, such as at least 150, such as at least 200, such as at least 250, such as at least 300, such as at least 500, such as at least 750, such as at least 1000, such as at least 2000, such as at least 3000, such as at least 5000 bacteriophages per live bacterial cell in the third composition.

[0055] Aspect 12: A method according to any of the preceding aspects, wherein the bacteriophages titer in the first composition is at least 109plaque forming unit per ml (pfu / mL), such as at least 1010pfu / mL, such as at least 1011pfu / ml.

[0056] Aspect 13: A method according to any one of the preceding aspects, wherein the bacteriophages are DNA-containing phages, preferably cos-type bacteriophages or pac-type bacteriophages.

[0057] Aspect 14: A method according to any of the preceding aspects, wherein the bacteriophages are phages of the Moineauvirus (cos), Brussowvirus (pac), or 987 species.

[0058] Aspect 15: A method according to any of the preceding aspects, wherein the bacteriophage are selected from the group consisting of: CHPC661, CHPC933, CHPC951, CHPC1005, CHPC952, CHPC1014, CHPC1152, CHPC1042, CHPC1046, and CHPC1057.

[0059] Aspect 16: A method according to any of the preceding aspects wherein the method is carried out in the absence of dyes permeant to living cells.

[0060] Aspect 17: A method according to any of the preceding aspects, wherein propidium iodide is the only fluorescent nucleic-acid stain present in the first composition and / or the third composition.

[0061] Aspect 18: A method according to any of aspects 1 and 3 to 17, wherein said method comprises the steps of: a) adding propidium iodide to a first composition comprising bacteriophages, preferably wherein: i. propidium iodide (PI) is added in a concentration of lmg / L-25 mg / L, preferably 2-10 mg / L, even more preferably 2-5mg / L, yet even more preferably 2.5 mg / L;

[0062] II. PI is prepared in dimethyl sulfoxide (DMSO) buffer; ill. the bacteriophages titer is at least 109pfu / mL, such as at least IO10pfu / mL, such as at least 1011pfu / ml;and / or iv. the bacteriophages are DNA-containing bacteriophages, preferably cos-type bacteriophages or pac-type bacteriophages; b) obtaining a second composition comprising lactic acid bacteria preferably Streptococcus thermophilus strains. c) mixing the first composition of a) and the second composition of b) to obtain a third composition comprising phages, bacteria and dye, preferably wherein the bacteriophages are present at a multiplicity of infection (MOI) of at least 20, such as at least 60, preferably at least 100, such as at least 150, such as at least 200, such as at least 250, such as at least 300, such as at least 500, such as at least 750, such as at least 1000, such as at least 2000, such as at least 3000, such as at least 5000 bacteriophages per live bacterial cell in the third composition; and d) using the distribution of the fluorescent dye as a measure of phage susceptibility.

[0063] Aspect 19: A method according to any of aspects 2 to 17 wherein said method comprises the steps of: a) adding propidium iodide to a first composition comprising bacteriophages, preferably wherein: i. propidium iodide (PI) is added in a concentration of lmg / L-25 mg / L, preferably 2-10 mg / L, even more preferably 2-5mg / L, yet even more preferably 2.5 mg / L;

[0064] II. PI is prepared in dimethyl sulfoxide (DMSO) buffer; ill. the bacteriophages titer is at least 109pfu / mL, such as at least 1010pfu / mL, such as at least 1011pfu / ml; and / or iv. the bacteriophages are DNA-containing bacteriophages, preferably cos-type bacteriophages or pac-type bacteriophages; b) obtaining a second composition comprising lactic acid bacteria preferably Streptococcus thermophilus strains. c) mixing the first composition of a) and the second composition of b) to obtain a third composition comprising phages, bacteria and dye, preferably wherein the bacteriophages are present at a multiplicity of infection (MOI) of at least 20, such as at least 60, preferably at least 100, such as at least 150, such as at least 200, such as at least 250, such as at least 300, such as at least 500, such as at least 750, such as at least 1000, such as at least 2000, such as at least 3000, such as at least 5000 bacteriophages per live bacterial cell in the third composition; and d) using the fluorescent dye signal to sort cells. Aspect 20: A method according to any of the preceding aspects, wherein step d) comprises the steps of :

[0065] - measuring, at the single-cell level, the distribution of propidium iodide (PI) fluorescence within the lactic acid bacterial population in the third composition; and

[0066] - identifying and optionally sorting lactic acid bacterial cells that remain substantially non-fluorescent for propidium iodide (PI) as phage-hardened variants, i.e. variants exhibiting reduced phage susceptibility under the conditions of step c).

[0067] Aspect 21 : Use of propidium iodide (PI) for labelling bacteriophages.

[0068] Aspect 22: Use according to aspect 21, for labelling bacteriophages in a method according to any of aspects 1 to 20.

[0069] Aspect 23: Use of a lactic acid bacterium obtained or isolated by the method of any of aspects 1 to 20.

[0070] Aspect 24: Use according to aspect 23, wherein the lactic acid bacterium is of the genus Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, Streptococcus or Enterococcus.

[0071] Aspect 25: Use according to aspect 24 wherein the lactic acid bacterium is of the genus Streptococcus spp. such as e.g. the species Streptococcus thermophilus.

[0072] Aspect 26: Use according to any of aspects 23 to 25 for manufacturing a fermented milk product.

[0073] Aspect 27: Use according to any of aspects 23 to 26 for manufacturing a fermented plant based product.

[0074] Aspect 28: Use according to any of aspects 23 to 27 wherein the fermented milk product is selected for a list consisting of: yoghurt, mesophilic fermented milk products, e.g. sour cream and buttermilk, as well as fermented whey and cheese products.

[0075] DEFINITIONS

[0076] The term "cell count stability" should be understood as a measure of the amount of colony forming units (CFU) per gram product. The higher CFU / g over time, the more cell count stable is the tested strain. The cell count stability may be measured using the pour plate method or other cell viability assays. The term "phage receptor" denotes a molecule which by one way or the other is exposed to the exterior of the cell and is synthesized by the collective action of biosynthetic proteins.

[0077] The term "PHV" or "Phage Hardened Variant" or "BIM" or "Bacteriophage Insensitive Mutant" is intended to denote a variant of a microorganism which, relative to the parent from which it is derived, is less susceptible to infection by one or more phages.

[0078] In the present context, the term "resistant to phage" refers to the lactic acid bacterium strain is able to propagate (at optimal growth temperature) in milk which contains 1000 phages per ml, i.e. the bacterium is able to reach a cell density above 10E8 cfu / ml after 48 hours when inoculated at a concentration of 10E5 cfu / ml in a suitable medium, such as e.g. milk or whey, under conditions, such as e.g. neutral pH and / or around 40dC, commonly used for preparing a fermented dairy product. Cfu is "cell forming units".

[0079] The term "improved resistance to a bacteriophage" or "reduced phage sensitivity" denotes that the bacterial strain such as e.g. the PHV or BIM when tested in e.g. a plaque assay, such as the assay described herein or as "Determination of phage resistance by the agar overlay method" or the "Heap Lawrence assay" have an improved phage resistance to at least one phage e.g. expressed as the difference in pfu / ml (plaque forming unit per ml) obtainable with said at least one bacteriophage on the given strain, compared to the pfu / ml obtainable with the same bacteriophage on the parent strain. A strain with improved resistance to a bacteriophage preferably show a reduction of pfu / ml of a factor at least 50, such as at least 100, e.g. 500, preferably at least 1000, more preferably at least a factor 10000 or more.

[0080] As used herein, the term "lactic acid bacterium" or "LAB" designates a gram-positive, microaerophilic or anaerobic bacterium, which ferments sugars with the production of acids including acetic acid, propionic acid and lactic acid as the predominantly produced acid. The industrially most useful lactic acid bacteria are bacteria of the species Lactobacillus and Streptococcus, and are normally supplied to the dairy industry either as frozen or freeze-dried cultures for bulk starter propagation or as so-called "Direct Vat Set" (DVS) cultures, intended for direct inoculation into a fermentation vessel or vat for the production of a dairy product, such as a fermented milk product. Such cultures are in general referred to as "starter cultures" or "starters".

[0081] In the present context, the term "milk substrate" may be any raw and / or processed milk material that can be subjected to fermentation according to the method of the invention. Thus, useful milk substrates include, but are not limited to, solutions / suspensions of any milk or milk like products comprising protein, such as whole or low fat milk, skim milk, buttermilk, reconstituted milk powder, condensed milk, dried milk, whey, whey permeate, lactose, mother liquid from crystallization of lactose, whey protein concentrate, or cream. Obviously, the milk substrate may originate from any mammal, e.g. being substantially pure mammalian milk, or reconstituted milk powder. Preferably, at least part of the protein in the milk substrate is proteins naturally occurring in milk, such as casein or whey protein. However, part of the protein may be proteins which are not naturally occurring in milk. Prior to fermentation, the milk substrate may be homogenized and pasteurized according to methods known in the art.

[0082] The term "milk" is to be understood as the lacteal secretion obtained by milking any mammal, such as cows, sheep, goats, buffaloes or camels. In a preferred embodiment, the milk is cow's milk. The term milk also comprises milks derived from plant material, such as soy milk. Optionally the milk is acidified, e.g. by addition of an acid (such as citric, acetic or lactic acid), or mixed, e.g. with water. The milk may be raw or processed, e.g. by filtering, sterilizing, pasteurizing, homogenizing etc., or it may be reconstituted dried milk. An important example of "bovine milk" according to the present invention is pasteurized cow's milk. It is understood that the milk may be acidified, mixed or processed before, during and / or after the inoculation with bacteria.

[0083] The expression "fermented milk product" means a food or feed product wherein the preparation of the food or feed product involves fermentation of a milk base with a lactic acid bacteria. "Fermented milk product" as used herein includes but is not limited to products such as thermophilic fermented milk products, e.g. yoghurt, mesophilic fermented milk products, e.g. sour cream and buttermilk, as well as fermented whey and cheese products.

[0084] The term "fermented milk drink" is a drinkable product obtained by fermentation of a milk substrate with lactic acid bacteria, such as bacteria of the species S. thermophilus. The product may be drinkable from a cup or a bottle, or via a straw. The product may be homogenized, e.g. after fermentation.

[0085] "Homogenizing" as used herein means intensive mixing to obtain a soluble suspension or emulsion. If homogenization is performed prior to fermentation, it may be performed to break up the milk fat into smaller sizes so that it no longer separates from the milk. This may be accomplished by forcing the milk at high pressure through small orifices.

[0086] "Fermentation" in the methods of the present invention means the conversion of carbohydrates into alcohols or acids through the action of a microorganism. Preferably, fermentation in the methods of the invention comprises conversion of lactose to lactic acid. Fermentation processes to be used in production of fermented milk products are well known and the person of skill in the art will know how to select suitable process conditions, such as temperature, oxygen, amount and characteristics of microorganism(s) and process time. Obviously, fermentation conditions are selected to support the achievement of the present invention, i.e. to obtain a fermented milk product. In the present context, the term "mutant" or "variant" should be understood as a strain derived from another strain (mother strain) by means of e.g. mutagenesis, radiation and / or chemical treatment, and / or selection, adaptation, screening, etc. The term also includes mutants with improved or altered phage resistance, e.g. phage hardened mutants or mutants showing improved cell count stability. It is preferred that the mutant is a functionally equivalent mutant, e.g. a mutant that has substantially the same, or improved, properties (e.g. regarding yield, viscosity, gel stiffness, mouth coating, flavor, post acidification, acidification speed, and / or phage robustness) as the mother strain. Such a mutant or variant is a part of the present invention. Especially, the term "mutant" or "variant" refers to a strain obtained by subjecting a strain of the invention to any conventionally used mutagenization treatment including treatment with a chemical mutagen such as ethane methane sulphonate (EMS) or N-methyl-N'-nitro-N-nitroguanidine (NTG), UV light or to a spontaneously occurring mutant. A mutant may have been subjected to several mutagenization treatments (a single treatment should be understood one mutagenization step followed by a screening / selection step), but it is presently preferred that no more than 1000, no more than 100, no more than 20, no more than 10, or no more than 5, treatments are carried out. In a presently preferred mutant, less than 5%, or less than 1% or even less than 0.1% of the nucleotides in the bacterial genome have been changed (such as by replacement, insertion, deletion or a combination thereof) compared to the mother strain.

[0087] In the present context, the term "variant" should be understood as a strain which is functionally equivalent to a strain of the invention, e.g. having substantially the same, or improved, properties e.g. regarding viscosity, gel stiffness, mouth coating, flavor, post acidification, acidification speed, sedimentation, probiotic activity, and / or phage robustness). Such variants, which may be identified using appropriate screening techniques, are a part of the present invention.

[0088] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising", "having", "including" and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0089] Thus in one aspect, the present invention relates to a method for measuring the phage susceptibility of a lactic acid bacterium, said method comprises: a) adding a fluorescent dye to a first composition comprising bacteriophages b) obtaining a second composition comprising lactic acid bacteria c) mixing the first composition of a) and the second composition of b) to obtain a third composition comprising phages, bacteria and dye. d) using the distribution of the fluorescent dye as a measure of phage susceptibility.

[0090] In a further aspect, the present invention relates to a method for sorting lactic acid bacterial cells according to phage susceptibility, said method comprises: a) adding a fluorescent dye to a first composition comprising bacteriophages b) obtaining a second composition comprising lactic acid bacteria c) mixing the first composition of a) and the second composition of b) to obtain a third composition comprising phages, bacteria and dye. d) using the fluorescent dye signal to sort cells.

[0091] In a preferred embodiment, method for measuring the phage susceptibility of a lactic acid bacterium of the present invention comprises: a) adding propidium iodide to a first composition comprising bacteriophages, preferably wherein : i. propidium iodide (PI) is added in a concentration of lmg / L-25 mg / L, preferably 2-10 mg / L, even more preferably 2-5mg / L, yet even more preferably 2.5 mg / L;

[0092] II. PI is prepared in dimethyl sulfoxide (DMSO) buffer; ill. the bacteriophages titer is at least 109pfu / mL, such as at least IO10pfu / mL, such as at least 1011pfu / ml;and / or iv. the bacteriophages are DNA-containing bacteriophages, preferably cos-type bacteriophages or pac-type bacteriophages; b) obtaining a second composition comprising lactic acid bacteria preferably Streptococcus thermophilus strains. c) mixing the first composition of a) and the second composition of b) to obtain a third composition comprising phages, bacteria and dye, preferably wherein the bacteriophages are present at a multiplicity of infection (MOI) of at least 20, such as at least 60, preferably at least 100, such as at least 150, such as at least 200, such as at least 250, such as at least 300, such as at least 500, such as at least 750, such as at least 1000, such as at least 2000, such as at least 3000, such as at least 5000 bacteriophages per live bacterial cell in the third composition. d) using the distribution of the fluorescent dye as a measure of phage susceptibility.

[0093] In another preferred embodiment, the method for sorting lactic acid bacterial cells according to phage susceptibility of the present invention comprises: a) adding propidium iodide to a first composition comprising bacteriophages, preferably wherein: v. propidium iodide (PI) is added in a concentration of lmg / L-25 mg / L, preferably 2-10 mg / L, even more preferably 2-5mg / L, yet even more preferably 2.5 mg / L; vi. PI is prepared in dimethyl sulfoxide (DMSO) buffer; vii. the bacteriophages titer is at least 109pfu / mL, such as at least IO10pfu / mL, such as at least 1011pfu / ml; and / or viii. the bacteriophages are DNA-containing bacteriophages, preferably cos-type bacteriophages or pac-type bacteriophages; b) obtaining a second composition comprising lactic acid bacteria preferably Streptococcus thermophilus strains. c) mixing the first composition of a) and the second composition of b) to obtain a third composition comprising phages, bacteria and dye, preferably wherein the bacteriophages are present at a multiplicity of infection (MOI) of at least 20, such as at least 60, preferably at least 100, such as at least 150, such as at least 200, such as at least 250, such as at least 300, such as at least 500, such as at least 750, such as at least 1000, such as at least 2000, such as at least 3000, such as at least 5000 bacteriophages per live bacterial cell in the third composition. d) using the fluorescent dye signal to sort cells.

[0094] In preferred embodiments of the methods of the present invention step d) comprises the steps of :

[0095] - measuring, at the single-cell level, the distribution of propidium iodide (PI) fluorescence within the lactic acid bacterial population in the third composition; and

[0096] - identifying and optionally sorting lactic acid bacterial cells that remain substantially non-fluorescent for propidium iodide (PI) as phage-hardened variants, i.e. variants exhibiting reduced phage susceptibility under the conditions of step c).

[0097] The skilled person will know general methods for identifying and / or sorting fluorescent vs non fluorescent events, for example using flow cytometry or FACS, for example but using gating approaches, preferably as described herein.

[0098] In some embodiments of the methods of the present invention, PI is used for labelling phage DNA and enables the measurement of bacterial phage susceptibility upon phage adsorption and infection of the bacterial cell as described herein, with the possibility to differentiate these processes by inner and outer cell fluorescence. Thus, in preferred embodiments of the methods of the present invention, no other intact phage-DNA labelling dye than PI is used, such as any of the other phage-DNA labelling dyes known in the art, for example but not limited to SYBR. Gold, DAPI, YOYO-1, SYTO9, or SYTO13. In further embodiments the methods is thus carried out in the absence of dyes permeant to living cells, such as but not limited to, SYTO 13, SYTO 9, or other intrinsically cell permeable fluorescent nucleic-acid dyes.

[0099] In other preferred embodiments, propidium iodide is the only fluorescent nucleic-acid stain present in the first composition and / or the third composition.

[0100] It is preferable that the bacteriophage titer is determined as close as possible to the performance of the methods of the present invention, in particular for phages susceptible of high degradation during storage at 4°C, for example showing phage titer decrease of 2-fold or more, such as 5-fold or more, such as 10-fold or more over 30 days of storage at 4°C.

[0101] Thus, in preferred embodiments, the method further comprises a step of determining the bacteriophage titer, for example to be able to determine the MOI, preferably no more than 30 days prior to performing step a) and / or c), such as no more than 15 days, even more preferably no more than 7 days, such as no more than 3 days prior to performing step a) and / or c).

[0102] Identification of fluorescently labelled and unlabeled events, such as cells by flow cytometry and FACS, is routinely performed for example by identifying the location of the major cell population in forward scatter (FSC) and back scatter (BSC) (or side scatter SSC) plots, and by setting thresholds in the signal obtained from fluorescence channels to discriminate between labelled and un-labelled events and thereby identify events of interest, which can be analyzed, counted, and sorted, for example when FACS is used. The skilled person will know how to select filters and parameters based on the fluorophore used in the experiment in this process known in the art as "gating". Specific gates for outer fluorescence (cell surface signal) and inner fluorescence (internal cell signal) can also be determined routinely.

[0103] In some embodiments of the methods of the present invention, the fluorescent dye may be used to determine phage susceptibility and / or sort cells based on inner fluorescence, outer fluorescence, preferably both inner fluorescence and outer fluorescence, thereby encompassing cells having adsorbed the phages and cells having internalized the phages, respectively.

[0104] In preferred embodiments of the methods of the present invention , the fluorescent dye may be used to determine phage susceptibility and / or sort cells wherein the gates are configured so that as few as possible of non-fluorescent events are identified in the host strain (e.g. the wild-type or baseline cell population from which PHVs are to be identified), preferably below 1%, ensuring precise detection and sorting. This may be the case for example, but not limited to, to ensure a low baseline of non-fluorescent events (i.e. bacterial cells not susceptible to bacteriophages) in order to be able to identify rare Phage-Hardened Variants (PHVs) in a bacterial cell population, either spontaneous PHVs or from mutagenized libraries of bacterial cells, such as obtained using mutagenesis approaches known in the art.

[0105] EXAMPLES

[0106] Example 1: Applying fluorescently labeled phages as biomarkers to facilitate an analysis of phage-bacteria interactions.

[0107] Bacteria, phages, and growth conditions

[0108] Streptococcus thermophilus strains and phages used for this study are listed in Table 1.

[0109] TABLE 1 List of S. thermophilus strains and phages from the Chr. Hansen A / S collection used in this study

[0110] Strains were stored at -40 °C in growth medium supplemented with 15% (wt / vol) glycerol and cultured overnight at 37 °C in LM17 broth (M17 broth [Oxoid, Denmark] with 2% [wt / vol] lactose) or anaerobically at 37 °C on LM17 agar plates (M17 agar [Oxoid] with 2% [wt / vol] lactose). If the bacterial cells were used for tests with phages, the growth medium was additionally supplemented with 10 mM CaCL and 10 mM MgCL (LM17-Ca / Mg).

[0111] Phages were propagated as previously described in (Szymczak et al. 2017) and stored at 4 °C. Phage particles were purified and concentrated using PEG-NaCI precipitation method and a high-speed centrifugation. Depending on the experimental conditions, the harvested phages were resuspended in one of the following buffers: SM buffer, SMGT buffer (SM buffer supplemented with 0.1% gelatin and 1% Tween20), Ringer's solution, conjugation buffer A (1 M Na2CO3 / l M NaHCO3, pH 9.0), conjugation buffer B (100 mM HEPES, pH 7.0-7.5).

[0112] Phage titers as well as the host ranges of investigated phages with bacterial strains were determined by using the double agar overlay spot test, as described before (Kropinski et al. 2009). Following overnight incubation under the appropriate growth conditions, the PFU per milliliter were calculated.

[0113] Fluorescent labeling techniques

[0114] Dyes tested for labeling phages were listed in Table 2. For conjugating proteins with FITC, samples were incubated at 4°C o / n. Labeling with the other fluorophores was performed at room temperature for 5-15 min with shaking, unless indicated differently. Fluorescently labelled samples were protected from light to limit dye bleaching.

[0115] TABLE 2 List of fluorophores used

[0116] To remove the unbound fluorophore from preparations, the following methods were validated : - Dialysis on Millipore membrane

[0117] A sample was mixed with a fluorescent dye and transferred on Millipore membrane floating on MilliQ water. The sample was left at 4°C o / n to enable the spontaneous diffusion of the excess dye thought the membrane. Afterwards, the sample was collected directly from the membrane.

[0118] - Centrifugation with Millipore membrane

[0119] A sample was incubated with a fluorescent dye and transferred on 100 kDa Millipore membrane. The sample was centrifuged at 7.500 rpm for 10 min at 10°C. The sample was washed twice with Ringer's solution. It was recovered from the Millipore membrane by adding an adequate volume of Ringer's solu-tion and centrifuging the inverted Millipore filter at 2.500 rpm for 2 min at 10°C.

[0120] - PEG precipitation

[0121] Tagged phages were captured by PEG-NaCI solution and pelleted by a high-speed centrifugation as de-scribed previously (Jaye et aL, 2004).

[0122] - Passing through PES membrane

[0123] A sample was incubated with a fluorescent dye for 15 min and slowly filtered through 0.22 pm polyeth-ersulfone (PES) membrane on a syringe filter (Low et aL, 2020).

[0124] Microscopy techniques

[0125] For the fluorescence microscopy, bacterial cells were pelleted at 6.000 rpm for 3 min and resuspended in the prepared buffers or phage lysates. Three microliters of the mix were spotted on 1.5 % agarose in saline. Samples were visualized under Nikon Eclipse Ti2-E microscope equipped with CFI Plan Apochromat DM Lambda objective (100X, 1.45 Oil, Ph3). Images were captured by 01-PRIME-BSI-R-M-16-C camera and processed using Nis-Elements software.

[0126] Flow cytometry

[0127] Flow cytometry and cell sorting was performed on FACS instrument SONY MA900 with 100 pm chip. For establishing sorting gates, Cell Sorter software was used. As a default, 50.000 cells were sorted for each event.

[0128] PCR screening

[0129] RedTaq 2x Master Mix (VWR) was used for PCR screening of the sorted cells. Oligonucleotides CHCC2138_epsE_F (TTCTTAGGGGTCGTCCTT) and CHCC2138_epsE_R (TGTCTTGTTGGAACTCTG) were purchased from Eurofins Genomics. The PCR conditions were set as follows: 95°C for 2 min, followed by 30 cycles of 95°C for 30 s, 45°C for 30 s, and 72°C for 100 s, with a final extension of 72°C for 5 min. The PCR products were visualized on 1% E- gel 96 (Invitrogen).

[0130] Results and Discussion

[0131] Identifying fluorophores for phage labeling

[0132] Eight fluorophores were tested for labeling phage CHPC926 (Table 2). DAPI, SYBR Gold, and PI solution in DMSO (2.5 mg / ml) successfully penetrated capsids and labeled phage DNA. The fluorescent signal was visualized under the fluorescence microscope as glowing spots surrounding the host cells (Figure 1).

[0133] A fluorescent signal from the phage particles was not detected with the alternative DNA- specific dyes: SYTO 9, SYTO 13, Cyto 13, and PI solution in water (1 mg / ml). According to the literature, SYTO 9 and SYTO 13 could be used to visualize phages (Low et aL, 2020).

[0134] However, no labeling of the phage particles with the tested stocks of those dyes was observed, even when the incubation time was prolonged to 2 hours. The negative results obtained in our study can be explained by the partial dye degradation e.g., due to an inappropriate storage or a repetitive thawing and freezing of the same stock solution.

[0135] Notably, all tested fluorophores allowed labeling of the bacterial DNA. These results can be explained by the fact that the bacterial DNA is roughly 50-times bigger compared to the phage DNA (estimated 1.9 Mb for S. thermophilus strains and 35.000 bp for streptococcal phages). Although the fluorescent activity of some dyes was decreased, it was sufficient to give a fluorescent signal with the bacteria but not with the phages.

[0136] This notion was confirmed over several experiments performed with different stocks of PI. This fluorophore was proven applicable for labeling phages. However, using fresh stocks with high concentration of PI (above 2 mg / ml) was obligatory to obtain a fluorescent signal from phage particles.

[0137] Labeling with FITC, obtained by the conjugation of the fluorophore into the proteins, was tested as an alternative to the DNA-specific dyes. Tagging phage proteins with FITC was not achieved, despite using various conjugation buffers: growth medium, Ringer's solution, conjugation buffer A, and conjugation buffer B. In all preparations, unspecific labeling of the bacterial cells and some background fluorescence were observed.

[0138] Conjugation buffer A was the recommend buffer for labeling with FITC due to its high pH. However, it significantly impacted the phage activity. The phage titer measured for CHPC926 in conjugation buffer A was 2-log lower compared to preparations in the alternative buffers. Low number of phage particles in the suspension could result in the untraceable adsorption of the FITC-tagged phages under the fluorescence micro-scope.

[0139] Removing the excess dyes from phage preparations Except for PI, the DNA-specific dyes used in this study are permeant to living cells. Thus, they migrate though the bacterial membrane and label bacterial DNA. To limit the unspecific labeling of the bacteria, four proto-cols were validated for removing the unbound fluorophores from the phage preparations.

[0140] Dialysis on Millipore membrane was highly effective in removing the excess SYBR Gold from the solution without phages. The labeled phage particles after dialysis gave a strong fluorescent signal with the host cells. Samples during dialysis could be efficiently protected from the light, which prevented from the dye bleaching (Figure 2).

[0141] Nevertheless, traces of the unbound dye were still detected in the preparation with phages. It was confirmed by adding SYBR Gold-labeled phage CHPC926 to CHCC28628. This strain carries truncation in epsE gene which prevents from the phage adsorption (Szymczak et aL, 2018). Although the phage binding signal was not detected with CHCC28628, the unspecific labeling of the bacterial cells was observed (Figure 2).

[0142] Centrifugation with Millipore membrane and PEG precipitation were equally effective in removing the excess dye. However, during the sample preparation, SYBR Gold lost its fluorescent properties. It can be ex-plained by the fact that the samples were exposed to the daylight during multiple washing steps. In both methods, the signal from phage particles was only detected with a prolonged exposure time (200 ms instead of 10 ms used for the other preparations).

[0143] Filtration though PES membrane was proven useful in removing SYTO 9, SYTO 13, Cyto 13 from the buffer without phages. PES membrane did not retain SYBR Gold, DAPI or PI. Our results confirmed that this method is specific to some cyanine dyes (Hui Zhi Low, personal communication). Filtration through a PES membrane had some advantages over the other tested methods. As opposed to the dialysis or centrifugation with Millipore membrane, it allowed for a rapid processing of large sample volumes. Moreover, it was the only tested method that did not result in any phage loss during the sample preparation. To truly verify its applicability for our study, the experiment should be repeated with fresh stocks of the cyanine dyes.

[0144] None of the tested methods gave the anticipated outcome, i.e., labeling phages without the unspecific labeling of bacterial cells. In the light of those results, labeling phage DNA with PI was identified as the most optimal method for our application. The excess dye did not enter bacterial cells with an intact membrane. Hence, it diminished the chances for a false-positive labeling that was noted with SYBR Gold and DAPI (Figure 1).

[0145] Sorting for PHVs with modifications in cell surface receptors on FACS We applied flow cytometry to sort for cells with and without fluorescence, mediated by the presence or absence of a phage adsorption, respectively. For the proof-of-concept study we used Pl-labeled phage CHPC926 together with its WT strain (CHCC2138) and its PHV (CHCC28628) with a confirmed modification in a phage receptor. A schematic representation of the anticipated cell separation, based on their fluorescence, was presented in Figure 3. For the control trials on FACS, the WT and the PHV were sorted separately. Difference in the forward scatter (FSC) and the back scatter (BSC), understood as the difference in the cell morphology, was noted between the two strains (Figure 4). It can be explained by the fact that CHCC28628 does not biosynthesize capsular polysaccharide on its cells surface.

[0146] A similar proportion of cells with inner fluorescence, corresponding to dead cells, was identified for the WT and the PHV mixed with PI. Close to 99% of the cells in the exponential growth did not display any fluorescence (Figure 4). The results confirmed that PI did not enter bacterial cells with an intact membrane.

[0147] A major difference in the fluorescent signal was observed when the WT and the PHV were mixed with the Pl-labeled phage. More than 70% of the WT cells were sorted into the gates established for the outer fluorescence and the inner fluorescence. Those signals corresponded to the phage adsorption and the phage DNA injection, respectively. Below 1% of the PHV cells was sorted into those gates. Instead, more than 50% of the PHV cells were sorted into the PI- negative gate, reflecting the lack of the phage adsorption (Figure 4). Since the results of the control trials on FACS reached the expectations, the final sort was performed.

[0148] The WT and the PHV cultures in the exponential growth were mixed in proportion 10: 1 (v / v ratio). Single cells identified for each gate were sorted into 96-well plates, rows A to G. Row H included the control samples: dead cells, the living WT, and the living PHV. The survival after the sort was comparable for the PHV and the WT (82-86%). As anticipated, the sort for the dead cells had a lower survival (45%) (Figure 5).

[0149] To verify the accuracy of sorting, the sorted cells were subjected to a PCR screening. Based on the results, the gates established on FACS enabled cell sorting with 100% accuracy. Sort for no fluorescence resulted in sorting the PHV, sort for the outer fluorescence resulted in sorting the WT, and sort for the inner fluorescence resulted in sorting either the WT, used in this experiment in excess, or the dead cells. A few tested colonies did not give any PCR product, possibly due to a pipetting error (Figure 5).

[0150] Perspectives for applying FACS to sort for PHVs generated from different backgrounds.

[0151] We aimed at establishing a method to sort for PHVs generated from different backgrounds. We hypothesized that permeability of a phage capsid could be dependent on the phage species. Thus, we expended the portfolio of phages tested for Pl-labeling.

[0152] Adsorption of nine S. thermophilus phages to their host strains was examined (Table 1). Among the tested strains, the most visible phage adsorption was noted for CHCC2138, CHCC6008, CHCC6592, and CHCC4323 ( Figure 6). The remaining strains, CHCC9204, CHCC7018, and CHCC4327, had either very weak, or no phage adsorption signal detected (Figure 7). The nature of this difference between the stains is yet to be investigated. It could depend on the characteristic of bacteria e.g., cells surface composition, or the specificity of phages e.g., the number of phage particles in the suspension. Nevertheless, phages from different species were successfully labeled with PI.

[0153] The phage adsorption signal was partially dependent on the bacterial stage of growth. An improved binding was detected for cultures in the stationary phase compared to the cells in the exponential phase. The mature cell wall promoted the host recognition by phages.

[0154] Two strains were selected to visualize their interactions with phages on FACS. CHCC6008 and its phage CHPC1152 represented strains with a strong fluorescent signal, while CHCC9204 and its phage CHPC1057 represented strains with a weak fluorescent signal. The phage-bacteria interactions were measured in 15-20 min intervals over the period of 30 min to 1 hour. The fluorescence measured on FACS significantly differed between the two strains.

[0155] For CHCC6008, nearly 80% of the cells in a stationary phase displayed the outer fluorescence immediately after mixing with Pl-labeled phage CHPC1152. The values for this gate gradually dropped, while the values for the inner fluorescence gradually grew, after 15 and 30 min of incubation. It can be explained by the progression of a phage infection when the phage injects its DNA inside the host cells. The values for no fluorescence were close to 0% and remained at this level over the measured time (Figure 8).

[0156] For CHCC9204 mixed with Pl-labeled phage CHPC1057, the initial number of cells without fluorescence was high (above 50% of the cells). The values for no fluorescence gradually dropped, while the values for the inner fluorescence gradually grew, within the tested incubation time. Below 1% of cells were sorted with the outer fluorescence (Figure 8).

[0157] Those results confirmed that sorting for PHVs on FACS requires sufficient phage binding to the WT cells. If a strong phage absorption cannot be achieved immediately after mixing bacteria with phages, a prolonged incubation is recommended to diminish the chance for sorting falsenegative cells.

[0158] Conclusion

[0159] We established a method to sort for PHVs with modifications in cell surface receptors on FACS. Sorting gates designed in this study enabled sorting for the WT and the PHV with 100% accuracy. The cells survived the sort and grew in the presence of labeled phages and the excess fluorophore.

[0160] In our method, phages were labeled with PI. This fluorescent dye is commonly used for live / dead assays with bacterial cells (Crowley, Marfell, et aL, 2016; Crowley, Scott, et aL, 2016). To our knowledge, it has not been tested for labeling phage particles. The fact that it can only be up taken by the cells with a damaged membrane, limits the unspecific bacteria labeling noted with the other DNA-specific dyes.

[0161] In a similar method published by Low et al. 2020, phages were labeled with SYTO 13. Although the excess dye was removed by a filtration method, some free dye remained in the phage preparation causing a false-positive labeling. The unspecific labeling was tested with various bacterial species, and it was especially visible for L. lactis. In our hands, similar observations were made for S. thermophilus. None of the tested protocols was sufficiently removing the excess fluorophore from the phage preparations.

[0162] Labeling phage proteins by conjugation is an appealing alternative to the DNA-labeling. A reliable protocol for labeling phages with FITC was not established in this study. Other probes that can be conjugated with proteins and peptides, such as NHS esters or maleimides, could be tested.

[0163] Our method aimed at separating cells with phage adsorption from the cells without phage adsorption. Previously, we noted that phages display different adsorption patterns (Szymczak et aL, 2018). Similar observations were made in this study. To better understand phage kinetics and their adsorption types, testing more strains and phages on FACS is recommended. The established method has a potential to generate important benefits at Chr. Hansen. By identifying variants with modifications in cell surface receptors, we will be able to study the identity of phage receptors in various strains. Subsequently, we could validate the stability of this resistance mechanism in comparison to other resistance systems. Finally, the described method would enable reliable and rapid selection for PHVs with a desired phenotype for industrial applications.

[0164] Example 2

[0165] Aim

[0166] In Example 1, the inventors established a method to sort for phage-hardened variants (PHVs) with modifications in cell surface receptors on FACS. In the method, phages were labeled with propidium iodide (PI). PI provides an advantage as compared to other DNA-labelling dyes, because it does not penetrate the bacterial cells easily. Thus, it limits the unspecific bacteria labeling noted with the other DNA-specific dyes. On the other hand, it surprisingly allowed efficient phage labelling as shown herein.

[0167] According to the literature, SYTO 9 and SYTO 13 can be used to label phage DNA and visualize phage adsorption under a fluorescence microscope (Low et aL, 2020). Labeling with these dyes was tested in Example 1. However, no fluorescent signal corresponding to the phage particles was observed. The inventors hypothesized that the negative results can be explained by the partial dye degradation, e.g. due to inappropriate storage or repetitive thawing and freezing of the same stock solution. Here, phage DNA labeling with SYTO 13 was repeated using a fresh stock solution of fluorescent dye and the labeling efficiency was compared to the signal obtained from phage particles labeled with PI.

[0168] In Example 1, the inventors aimed at establishing a universal method to isolate PHVs generated from different backgrounds. Phage-bacteria interactions of S. thermophilus phages from two different species were examined under a fluorescence microscope and on FACS. Among the tested strains, representatives with strong and weak fluorescent signal, mediated by the phage adsorption, were noted. The inventors hypothesized that the observed difference can be related to the number of phage particles in the suspension. Here, it was tested whether the increased multiplicity of infection (MOI), corresponding to the ratio of phage particles to bacterial cells in a suspension, would result in increasing the fluorescent signal detected on FACS.

[0169] The overall aim of the study was to further establish the optimal conditions for using phages as biomarkers to analyze phage-bacteria interactions.

[0170] Materials and Methods

[0171] Material and methods were as in Example 1 as outlined hereafter, unless otherwise indicated.

[0172] Bacteria, phages, and growth conditions

[0173] Streptococcus thermophilus strains and phages used in this study were listed in Table 3. The strains were stored at -40°C in growth medium supplemented with 15% glycerol. They were cultured overnight at 40°C in M17 broth supplemented with 2% lactose (LM17),10 mM CaCI2 and 10 mM MgCI2. The phages were propagated on their corresponding host and enumerated following the published procedure (Szymczak et aL, 2017). Phage particles were purified and concentrated using PEG-NaCI precipitation method and a high-speed centrifugation. The harvested phages were resuspended in SMGT buffer (SM phage buffer (Tris / NaCI / MgSO4), as commonly known in the art, supplemented with 0.1% gelatin and 1% Tween20). Phage preparations were stored at 4°C.

[0174] Fluorescent labeling technioues

[0175] Fresh stock solutions of SYTO 13 Green (Invitrogen) and PI in DMSO (stock solution 2.5 mg / ml) were used in the experiment. 1 pl SYTO 13 and 10 pl of PI stock solution was added to 1 ml phage preparations, thereby obtaining a final Pi concentration in the phage preparation of 25pg / mL, or 25mg / L. Samples were incubated in dark for 15 min. Afterwards, phage preparations with SYTO 13 were slowly filtered through 0.22 pm polyethersulfone (PES) membrane on a syringe filter. Microscopy technique

[0176] For the fluorescence microscopy, bacterial cells were mixed with labeled phage lysates in 1 : 1 (vol / vol) ratio. One microliter of the mix was spotted on 1.5 % agarose in saline. Samples were visualized under Nikon Eclipse Ti2-E microscope equipped with CFI Plan Apochromat DM Lambda objective (100X, 1.45 Oil, Ph3). Images were captured by 01-PR.IME-BSI-R-M-16-C camera and processed using Nis-Elements software.

[0177] Flow cytometry

[0178] For the flow cytometry, bacterial cells were mixed with labeled phage lysates at different concentrations, incubated for 10 min at room temperature, and diluted in SM buffer to obtain the final volume of 1 ml. Control was included in the assay. It contained the mix of bacteria in SM buffer with the addition of PI. The analysis was performed on FACS instrument SONY MA900 with 100 pm chip. Data analysis was done using Cell Sorter software. As a default, 50.000 events were analyzed for each measurement.

[0179] Results and Discussion

[0180] PI solution in DMSO (stock solution 2.5 mg / ml) successfully penetrated capsids and labeled phage DNA. The fluorescent signal was visualized under the fluorescence microscope as glowing spots surrounding the host cells (Figure 9A). The same phage lysates labeled with SYTO 13 and filtered on 0.22 pm PES filter could not be detected under the microscope. Instead, some unspecific labeling of bacterial DNA was noted (Figure 9B). The results supported the previous observations (Example 1). Despite using the fresh solution of SYTO 13, the sufficient labeling of phage DNA could not be achieved with this dye.

[0181] For the microscopy assay, phages and bacteria were mixed in 1 : 1 (vol / vol) ratio. Phage adsorption was more visible for strain CHCC2136 mixed with phage CHPC661, as compared to the signal obtained for phage CHPC933 mixed with strain CHCC3021. Phage CHPC661 used in the experiment had a higher titer, i.e., number of phage particles in one milliliter of the lysate (pfu / ml), as compared to phage CHGPPC933. Thus, the MOI for strain CHCC2136 was around 300, while for strain CHCC3021 it was around 20. The fluorescent signal intensity measured for the two phages corresponded to the number of phage particles per bacterial cells.

[0182] This notion was further verified on FACS. Two gates were established to distinguish fluorescent events (FL cells) from non-fluorescent events (no FL - PHV). Cells were mixed with phages to obtain the MOI ranging from 20 to 5000 (Figure 10). Control mix of bacterial cells in SM buffer with the addition of PI was included.

[0183] The higher the MOI, the more fluorescent events were detected during the analysis. At MOI 20, the percentage of fluorescent events detected for strain CHCC3021 mixed with phage CHPC933 was around 16%. It was comparable to the fluorescent events detected for the control mix (bacterial cells in SM buffer with the addition of PI). At MOI 60, 100, 500, and 5000, the percentage of fluorescent events gradually increased to 42%, 58%, 79%, and 97%, respectively. At the same time, the percentage of non-fluorescent events gradually decreased from 11% at MOI 20 to 0.05% at MOI 5000 (Figure 10A). Similar observations were made for strain CHCC2136 incubated with phage CHPC661. At MOI 300, the percentage of fluorescent events was 91%. It increased to 99% at MOI 3000. The percentage of non- fluorescent events decreased from 2% at MOI 300 to 0.05% at MOI 3000 (Figure 10B).

[0184] A drop of phage titer was noted for phage CHPC933 during the experiment. Phage concentration decreased from 2xl0e9 pfu / ml to 2xl0e8 pfu / ml after 30 days of storage at 4°C. For phage CHPC661, a titer of 3xl0el0 pfu / ml was measured throughout the study period. Thus, the decrease of phage concentration was phage specific. This observation was crucial to calculate the optimal MOI for the assay.

[0185] Conclusions

[0186] Labeling with PI was successfully used to visualize phage adsorption to the bacterial host. Phages belonging to two different species were successfully labeled with this dye. Despite using a fresh stock of SYTO 13, labeling of phage particles could not be achieved with this dye. Although the excess dye was removed by a filtration method, some free dye remained in the phage preparation resulting in an unspecific labeling of bacterial cells. The results supported our previous observations (Example 1).

[0187] Based on the results, the optimal conditions for the assay were established. To avoid falsenegative results when sorting for PHVs without phage adsorption, the percentage of non- fluorescent events measured with a host strain should be as low as possible, preferably below 1%. To achieve this, the minimal MOI for the analysis is 100, and higher MOI is recommended, when possible. The MOI can be optimized per strain by increasing the volume of phage lysate added to the cell suspension. Phage titer should be measured prior to the analysis to account for the drop of phage titer during storage.

[0188] TABLE 3 : List of strains and phages used in this study. DRAWINGS

[0189] Figure 1 : Adsorption of phage CHCC926 to strains CHCC2138 (WT) and CHCC28628 (PHV). The phage was labeled with SYBR Gold (top row), DAPI (middle row), and PI (bottom row), and visualized under the fluorescence microscope.

[0190] Figure 2: Removal of the excess SYBR Gold from the preparation with and without phages by the dialysis on Millipore membrane. Phage CHPC926 with strains CHCC2138 (WT) and CHCC28628 (PHV) were used in the experiment.

[0191] Figure 3 : A schematic representation of the anticipated cell separation on FACS

[0192] Figure 4: Results of the cell sorting on FACS. Top row: Bacterial cells mixed with PI. Bottom row: Bacterial cells mixed with Pl-labeled phages.

[0193] Figure 5: The growth after the sort and the accuracy of FACS sorting obtained for the tested gates.

[0194] Figure 6: Adsorption of Pl-labeled phages to their host strains. Group of strains with a strong fluorescent signal.

[0195] Figure 7: Adsorption of Pl-labeled phages to their host strains. Group of strains with a weak fluorescent signal.

[0196] Figure 8: Progression of a phage infection measured on FACS. Cells were mixed with Pl-labeled phages and sorted based on the fluorescence in 15-20 min intervals. CHCC6008 with CHPC1152 (left) and CHCC9204 with CHPC1057 (right) were used in the experiment.

[0197] Figure 9 : Microscopy images of phage adsorption to the host strains. Phage CHPC661 with strain CHCC2136 (left), phage CHPC933 with strain CHCC3021

[0198] (right). Phages were labeled with PI (A) and with SYTO 13 followed PES filtration (B).

[0199] Figure 10: Adsorption of Pl-labeled phages to their host strains analyzed on FACS. Top row (panel A) : Strain CHCC3021 with phage CHPC933. Bottom row (panel B) : Strain CHCC2136 with phage CHPC661. Control: bacterial cells in SM buffer with the addition of PI. MOI: Multiplicity of infection. FL=fluorescent / fluorescence.

[0200] REFERENCES

[0201] All references cited in this patent document are hereby incorporated herein in their entirety by reference.

Claims

28CLAIMS1. A method for measuring the phage susceptibility of a lactic acid bacterium, said method comprises: a) adding a fluorescent dye to a first composition comprising bacteriophages b) obtaining a second composition comprising lactic acid bacteria c) mixing the first composition of a) and the second composition of b) to obtain a third composition comprising phages, bacteria and dye. d) using the distribution of the fluorescent dye as a measure of phage susceptibility.

2. A method for sorting lactic acid bacterial cells according to phage susceptibility, said method comprises: a) adding a fluorescent dye to a first composition comprising bacteriophages b) obtaining a second composition comprising lactic acid bacteria c) mixing the first composition of a) and the second composition of b) to obtain a third composition comprising phages, bacteria and dye. d) using the fluorescent dye signal to sort cells based.

3. The method according to any one of claim 1 or 2, wherein the fluorescent dye is propidium iodide (PI).

4. The method according to claim 3 wherein propidium iodide (PI) is added in a concentration of lmg / l-25 mg / l, preferably 2-10 mg / L, even more preferably 2-5mg / L, yet even more preferably 2.5 mg / L.

5. The method according to any one of the preceding claims, wherein PI is prepared in dimethyl sulfoxide (DMSO) buffer.

6. The method according to any one of the preceding claims, wherein the lactic acid bacterium is of the genus Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, Streptococcus or Enterococcus.

7. The method according to any one of the preceding claims, wherein the lactic acid bacterium is of the genus Streptococcus spp. such as e.g. the species Streptococcus thermophilus.

8. The method according to any one of the preceding claims, wherein the phage susceptibility is measured by microscopy or microfluidics.

9. The method according to any one of the preceding claims, wherein the distribution of the fluorescent dye is measured by FACS and optionally sorted according to phage susceptibility.

10. The method according to any one of the preceding claims, wherein the lactic acid bacteria with low phage susceptibility are isolated and optionally cultured.

11. The method according to any one of the preceding claims, wherein the bacteriophages are present at a multiplicity of infection (MOI) of at least 20, such as at least 60, preferably at least 100, such as at least 150, such as at least 200, such as at least 250, such as at least 300, such as at least 500, such as at least 750, such as at least 1000, such as at least 2000, such as at least 3000, such as at least 5000 bacteriophages per live bacterial cell in the third composition.

12. The method according to any one of the preceding claims, wherein the bacteriophages titer in the first composition is at least 109plaque forming unit per ml (pfu / mL), such as at least 1010pfu / mL, such as at least 1011pfu / ml.

13. The method according to any one of the preceding claims, wherein the bacteriophages are DNA containing phages, preferably cos-type bacteriophages or pac-type bacteriophages.

14. The method according to any one of the preceding claims, wherein the bacteriophages are phages of the Moineauvirus (cos), Brussowvirus (pac), or 987 species.

15. The method according to any one of the preceding claims, wherein the bacteriophage are selected from the group consisting of: CHPC661, CHPC933, CHPC951, CHPC1005, CHPC952 CHPC1014, CHPC1152, CHPC1042, CHPC1046, and CHPC1057.

16. The method according to any one of the preceding claims wherein the method is carried out in the absence of dyes permeant to living cells.

17. The method according to any one of the preceding claims, wherein propidium iodide is the only fluorescent nucleic-acid stain present in the first composition and / or the third composition.

18. The method according to any one of claims 1 and 3 to 17, wherein said method comprises the steps of: a) adding propidium iodide to a first composition comprising bacteriophages, preferably wherein: i. propidium iodide (PI) is added in a concentration of lmg / L-25 mg / L, preferably 2-10 mg / L, even more preferably 2-5mg / L, yet even more preferably 2.5 mg / L;ii. PI is prepared in dimethyl sulfoxide (DMSO) buffer; ill. the bacteriophages titer is at least 109pfu / mL, such as at least IO10pfu / mL, such as at least 1011pfu / ml;and / or iv. the bacteriophages are DNA-containing bacteriophages, preferably cos-type bacteriophages or pac-type bacteriophages; b) obtaining a second composition comprising lactic acid bacteria preferably Streptococcus thermophilus strains. c) mixing the first composition of a) and the second composition of b) to obtain a third composition comprising phages, bacteria and dye, preferably wherein the bacteriophages are present at a multiplicity of infection (MOI) of at least 20, such as at least 60, preferably at least 100, such as at least 150, such as at least 200, such as at least 250, such as at least 300, such as at least 500, such as at least 750, such as at least 1000, such as at least 2000, such as at least 3000, such as at least 5000 bacteriophages per live bacterial cell in the third composition; and d) using the distribution of the fluorescent dye as a measure of phage susceptibility.

19. The method according to any one of claims 2 to 17 wherein said method comprises the steps of: a) adding propidium iodide to a first composition comprising bacteriophages, preferably wherein: i. propidium iodide (PI) is added in a concentration of lmg / L-25 mg / L, preferably 2-10 mg / L, even more preferably 2-5mg / L, yet even more preferably 2.5 mg / L;II. PI is prepared in dimethyl sulfoxide (DMSO) buffer; ill. the bacteriophages titer is at least 109pfu / mL, such as at least 1010pfu / mL, such as at least 1011pfu / ml; and / or iv. the bacteriophages are DNA-containing bacteriophages, preferably cos-type bacteriophages or pac-type bacteriophages; b) obtaining a second composition comprising lactic acid bacteria preferably Streptococcus thermophilus strains. c) mixing the first composition of a) and the second composition of b) to obtain a third composition comprising phages, bacteria and dye, preferably wherein the bacteriophages are present at a multiplicity of infection (MOI) of at least 20, such as at least 60, preferably at least 100, such as at least 150, such as at least 200, such as at least 250, such as at least 300, such as at least 500, such as at least 750, such as at least 1000, such as at least 2000, such as at least 3000, such as at least 5000 bacteriophages per live bacterial cell in the third composition; and d) using the fluorescent dye signal to sort cells.

20. The method according to any one of the preceding claims, wherein step d) comprises the steps of :- measuring, at the single-cell level, the distribution of propidium iodide (PI) fluorescence within the lactic acid bacterial population in the third composition; and- identifying and optionally sorting lactic acid bacterial cells that remain substantially non-fluorescent for propidium iodide (PI) as phage-hardened variants, i.e. variants exhibiting reduced phage susceptibility under the conditions of step c).

21. Use of propidium iodide (PI) for labelling bacteriophages.

22. The use according to claim 21, for labelling bacteriophages in a method according to any one of claims 1 to 20.

23. Use of a lactic acid bacterium obtained or isolated by the method of any of claims 1 to 20.

24. The use according to claim 23, wherein the lactic acid bacterium is of the genus Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, Streptococcus or Enterococcus.

25. The use according to claim 23 wherein the lactic acid bacterium is of the genus Streptococcus spp. such as e.g. the species Streptococcus thermophilus.

26. The use according to any one of claims 23 to 25 for manufacturing a fermented milk product.

27. The use according to any one of claims 23 to 26 for manufacturing a fermented plant based product.

28. The use according to any one of claims 23 to 27 wherein the fermented milk product is selected for a list consisting of: yoghurt, mesophilic fermented milk products, e.g. sour cream and buttermilk, as well as fermented whey and cheese products.

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