WO2025224309A1 - Live lactic acid bacteria for use in the treatment or prevention of an infection with a multi-drug resistant (mdr) microorganism - Google Patents

Abstract

The present invention relates to lactic acid bacteria (LAB) for use alone or in combination with antimicrobial agents in the treatment or prevention of an infection with a multi-drug resistant (MDR) microorganism.

Description

LIVE LACTIC ACID BACTERIA FOR USE IN THE TREATMENT OR PREVENTION OF AN

INFECTION WITH A MULTI-DRUG RESISTANT (MDR) MICROORGANISM

Field

The invention and disclosure relate generally to the treatment or prevention

5 of infections caused by multi-drug resistant (MDR) microorganisms and in particular, to a new medical use of lactic acid bacteria in treating or preventing MDR infections. The lactic acid bacteria may be administered alone or in combination with an antibiotic, and may be engineered recombinantly to express a therapeutic protein.

Background

Antimicrobial resistance (AMR) of pathogenic bacteria to available antibiotics is rapidly increasing and has become one of the main public health concerns of the 21^st century. In 2019, 1.27 million deaths were directly attributed to,

15 and 4.9 million deaths were associated with, resistant bacteria globally. It is becoming increasingly challenging for healthcare professionals to effectively treat and prevent the ever-increasing range of infections caused by microorganisms, particularly, bacteria and fungi, which are no longer susceptible to the common medicines which have been used to treat them previously. AMR is particularly

20 problematic in bacteria, which has resulted in an ever-dwindling number of antibiotics being effective in their treatment. Most concerningly, it has become clear that bacteria responsible for causing common or severe infections are developing AMR to each new antibiotic which comes to the market. A similar growing health concern is the increasing number of AMR fungi which are no longer susceptible to

25 common antifungal (also known as antimycotic) medications. This is a particularly pressing issue due to the limited number of antifungal medications available on the market.

Further compounding the problems surrounding the treatment of increasing numbers of AMR microorganisms is that the current antimicrobial pipeline is struggling, particularly the late-stage pipeline. In the course of 2019-2022, only two drugs were approved for the treatment of AMR pathogens. These drugs are modified fluoroquinolones and thus are not even a truly new class of antimicrobials, but only a chemically modified version of the original. This is true of the vast majority of new compounds in advanced stages of clinical development. For 10

35 years (2013-2023), no new first-in-class antimicrobials have been approved. Clearly, there is an increasing need for new classes of antimicrobials or new, non- traditional approaches to infection and wound care.

A particular cause for concern is multi-drug resistant (MDR) microorganisms, that is AMR microorganisms which have become resistant to at least three antimicrobial drugs. The multiple drug resistance further reduces the treatment options that are available (and effective). At present, there are currently very few treatment options for MDR microorganisms and combination regimens are often required to increase the chance of successfully treating the infection.

AMR and MDR microorganisms may cause infections in almost any part of the body including the bloodstream, the lungs, the urinary tract, genital areas, wounds (e.g. cutaneous and mucosal etc), skin and at surgical sites. Generally, young children, the elderly or people who have an existing severe illness or a health condition such as chronic lung, heart, or kidney disease are most at risk of developing an infection with an MDR microorganism and recently MDR infections have been reported to be most likely to develop in immune-metabolically compromised patients such as patients with type 2 diabetes and for infection in the lungs, urinary tract and skin wounds. However, any individual may develop an infection with an MDR microorganism.

AMR and MDR is particularly problematic in health-care settings, especially in hospitals and one of the major risk factors associated with developing an infection with an MDR microorganism is a prolonged stay in a healthcare facility (e.g. a hospital). This is due to the fact that patients in these facilities are commonly exposed to antibiotics repeatedly and receive hands-on care, thus increasing the risk of cross-contamination.

The influx of new, severely wounded people, due to geopolitical conflicts (i.e. as a result of the conflict in Ukraine), has exacerbated this already troublesome issue. AMR and MDR is particularly problematic in military hospitals as patients are often in close proximity to one another and such conditions allow (albeit unintentionally) for the spread of multiple MDR microorganisms, particularly MDR bacteria. The more widespread the distribution becomes, the more risk that these new MDR strains will become dominant in the bacterial population. A recent report, highlights this issue and documents that this is already happening. This report isolated and characterized, with EUCAST standard methods, many isolates from the soft tissue of intensive care war wounds that were resistant to multiple, relevant antibiotic treatments. Alarmingly, 6% of the isolates demonstrated resistance to all antibiotics examined. Furthermore, in a case report from earlier this year, a number of isolates from one service member being treated for burn wounds were determined to be non-susceptible to most antibiotics and carried multiple antibiotic resistant genes. Especially relevant for skin wounds is that three of the isolated strains were distinct Pseudomonas aeruginosa strains.

Until recently, it was unclear in the art whether MDR microorganisms retained their virulence and pathogenicity following the acquisition of antimicrobial resistance. It had been suggested that carrying plasmids with AMR genes impaired bacterial fitness, virulence and reduced essential cellular functions. However, it has recently been found that a pandrug-resistant (PDR) strain (i.e. resistant to all antimicrobial drugs in all antimicrobial categories) of Klebsiella pneumoniae isolated from war victims treated in Ukraine was hypervirulent and retained its pathogenicity (Ljungquist et al. (2024), J Infect. 89(6)). This finding further highlights the urgent need for effective treatment of MDR microorganisms to prevent life threatening infections and lethal sepsis events.

Certain probiotic bacteria have been shown to exert an antimicrobial effect through the release of various antimicrobial mediators such as hydrogen peroxide and bacteriocins. This is one of the reasons that probiotics including lactic acid bacteria (Lactobacillales) are used in the food industry where they are used to prevent the growth of undesirable microorganisms in or on food products.

It should be noted that whilst certain antimicrobial effects of lactic acid bacteria have been described, particularly in relation to the food industry, their efficacy against MDR microorganisms has not been reported. Indeed, a person skilled in the art would recognise that an antimicrobial effect against a single, non- MDR microorganism could not be used to predict a beneficial effect against an MDR microorganism, especially not as products in a clinically relevant situation.

Summary

It has surprisingly been found that lactic acid bacteria (LAB) can be used to overcome AMR and render microorganisms that are MDR (resistant to multiple antimicrobials) susceptible to antibiotics (more specifically susceptible to antibiotic(s) to which they are resistant). In particular, we have observed that, surprisingly, LAB, which may be wild-type (i.e. unmodified) or which have been modified, e.g. transformed to express a heterologous protein, are effective in inhibiting the growth of multi-drug resistant microorganisms, and in particular that such effects are truly microbicidal rather than microbiostatic. Based on this, we propose that live LAB are useful in treating or preventing infections with such MDR microorganisms.

Furthermore, the combination of lactic acid bacteria with antibiotics constitutes a highly effective approach to the combat of infections caused by MDR microorganisms.

Accordingly, in a first aspect, we provide herein live lactic acid bacteria (LAB) for use in the treatment or prevention of an infection with a multi-drug resistant (MDR) microorganism.

Alternatively defined, this aspect provides a pharmaceutical composition comprising live LAB for use in the treatment or prevention of an infection with a MDR microorganism.

The LAB may be any genus, species or strain of LAB, but as discussed further below, the LAB is particularly a Lactobacillus, Limosilacobacillus or Lactococcus. In an embodiment the LAB are Limosilactobacillus reuteri, formerly known as Lactobacillus reuteri, Lactobacillus rhamnosus or Lactococcus lactis.

The LAB may be transformed or untransformed. Put another way, the LAB may be wild-type or non-wild-type.

In an embodiment, the LAB have been transformed, or in other words recombinantly engineered to express a heterologous protein, particularly a mammalian protein, or to over-express an endogenous protein. More particularly, the protein is a therapeutic protein. In an embodiment the protein is antimicrobial, or promotes resolution of inflammation and/or wound healing. More particularly, the protein may be selected from the group consisting of CXCL12, CXCL17 and Ym1.

The infection with an MDR microorganism (or “MDR infection”) may be in any soft tissue or mucosal surface or in blood.

The live LAB may be used fresh or lyophilised.

In advantageous embodiments, the LAB are formulated in a composition comprising a sugar, notably sucrose.

In a particular embodiment, the LAB are for use in combination with an antimicrobial agent, and particularly an antibiotic. The antibiotic may be any antibiotic, preferably selected from: the beta-lactams, the sulphonamides, the quinolones, the macrolides, the tetracyclines, the aminoglycosides, the lincosamides, the polypeptide antibiotics, the streptogramins or the oxazolidinones. We have observed evidence of synergy between the LAB and antibiotics. Accordingly, in particular embodiments, the LAB and antibiotic act synergistically to treat or prevent the infection.

In another aspect, we provide a kit comprising live LAB and an antimicrobial agent, e.g. an antibiotic.

In particular, the kit, which may be viewed a pharmaceutical product, notably a combined pharmaceutical product or a combined preparation, is for use in treating or preventing an infection with a MDR microorganism.

Accordingly, further provided according to this aspect is a product comprising live LAB and an antimicrobial agent (e.g. an antibiotic) as a combined preparation for separate, simultaneous or sequential use in the treatment or prevention of an infection with a MDR microorganism.

Other aspects provide a wound dressing or medical device comprising said product.

In another aspect, provided herein is a method of treating or preventing an infection with an MDR microorganism in a subject, said method comprising administering live lactic acid bacteria to said subject.

In a further aspect, said method may further comprise administering an antimicrobial agent, e.g. an antibiotic.

Still another aspect provides the use of live lactic acid bacteria in the manufacture of a medicament for treating or preventing an infection with an MDR microorganism in a subject.

In a further aspect, said LAB are for use in combination with an antimicrobial agent (e.g. an antibiotic).

The medicament may accordingly be seen to include pharmaceutical compositions comprising LAB, as well as kits, and products (combination products and combined preparations etc.) as discussed above.

Further provided is the use of live LAB and an antimicrobial agent (e.g. an antibiotic) in the manufacture of a medicament for treating or preventing an infection with an MDR microorganism in a subject.

Also provided is an antibiotic for use in combination with live LAB in the treatment or prevention an infection with an MDR microorganism, wherein the antibiotic potentiates the anti-microbial effect of the live LAB. Still further provided is live LAB for use in combination with an antibiotic in the treatment or prevention an infection with an MDR microorganism, wherein the live lactic acid bacteria potentiate the anti-microbial effect of the antibiotic.

Also provided herein is a method of potentiating the effect of live LAB in the treatment or prevention an infection with an MDR microorganism, comprising administering the live LAB in combination with an antibiotic.

Yet still provided herein is a method of potentiating the effect of an antibiotic in the treatment or prevention an infection with an MDR microorganism, comprising administering the antibiotic in combination with live LAB.

Put in other words, these aspects can be seen to provide: live LAB for use together with an antimicrobial agent (e.g. an antibiotic) in treating a subject infected, suspected to be infected, or at risk of infection with an MDR microorganism; use of live LAB for the manufacture of a medicament for use together with an antimicrobial agent (e.g. an antibiotic) to treat in treating a subject infected, suspected to be infected, or at risk of infection with an MDR microorganism; a product comprising live LAB and an antimicrobial agent (e.g. an antibiotic) as a combined preparation for separate, simultaneous or sequential use in treating a subject infected, suspected to be infected, or at risk of infection with an MDR microorganism; a method of treating a subject infected, suspected to be infected, or at risk of infection with an MDR microorganism, said method comprising administering to said subject live LAB together with an antimicrobial agent.

In such aspects, the LAB may be used to overcome or reduce resistance to the antimicrobial agent in said microorganism. In certain embodiments, as noted above, there may be a synergistic effect.

Detailed description

The medical uses, products and methods herein are directed to the use of live lactic acid bacteria (LAB) for the treatment or prevention of an infection with a multi-drug resistant (MDR) microorganism.

LAB are a group of Gram-positive, low-GC, acid-tolerant, generally nonsporulating, non-respiring, either rod-shaped (bacilli), or spherical (cocci) bacteria which share common metabolic and physiological characteristics. These bacteria produce lactic acid as the major metabolic end-product of carbohydrate fermentation and are characterized by an increased tolerance to acidity (low pH range). These characteristics of LAB allow them to outcompete other bacteria in a natural fermentation because LAB can withstand the increased acidity from organic acid production (e.g., lactic acid). Thus, LAB play an important role in food fermentations, as acidification inhibits the growth of spoilage microorganisms. Several LAB strains also produce proteinaceous bacteriocins which further inhibit spoilage and growth of pathogenic microorganisms. LAB often have a generally recognized safe (GRAS) status and are amongst the most important groups of microorganisms used in the food industry.

The core genera that make up the lactic acid bacteria group are Lactobacillus*, Limosilacobacillus, Leuconostoc, Pediococcus, Lactococcus, Enterococcus, Weissella, and Streptococcus, as well as the more peripheral Aerococcus, Carnobacterium, Enterococcus, Oenococcus, Sporolactobacillus, Tetragenococcus, and Vagococcus. The term “lactic acid bacteria” as used herein in synonymous with and interchangeable with the terms “lactic acid bacteria group” and “Lactobacillales”. Any lactic acid bacterium from these genera is included within the scope of the present invention, but particularly bacteria from the genera Lactobacillus, Limosilacobacillus or Lactococcus, and more particularly from Limosilacobacillus. (*Lactobacillus, Pediococcus, Weissella and Leuconostoc have recently been unified but together also been divided into 25 genera. Reference: Zheng, J., Wittouck, S., Salvetti, E., Franz, C. M. A. P., Harris, H. M. B., Mattarelli, P., et al. (2020). A taxonomic note on the genus Lactobacillus: Description of 23 novel genera, amended description of the genus Lactobacillus Beijerinck 1901 , and union of Lactobacillaceae and Leuconostocaceae. International Journal of Systematic and Evolutionary Microbiology, 70(4), 2782-2858. http://doi.Org/10.1099/iisem.0.004107).

Thus, the LAB for use herein are not limited and may be any bacteria which are classified as LAB. In other words, the LAB of the present invention may be any bacteria which falls under the order of Lactobacillales.

More particularly, the LAB is a Lactobacillus, a Limosilacobacillus or a Lactococcus. This may include any species of these genera, or any strain of the species, many of which are known and have been deposited, and are accordingly available from culture collections. More particularly, the LAB are Limosilacobacillus Reuteri, Lactobacillus Rhamnosus or Lactococcus Lactis. Limosilactobacillus reuteri is a recent reclassification of bacteria formerly identified by the taxonomic name Lactobacillus reuteri.. Thus, the terms “Limosilactobacillus reuteri" and “Lactobacillus reuteri” are synonymous and may be used interchangeably herein. Furthermore, references to bacterial species may be abbreviated throughout the specification, i.e. Limosilactobacillus reuteri may be referred to as “L. reuteri’’ and Lactobacillus Rhamnosus may be referred to as “L. Rhamnosus" etc.

In one embodiment, the LAB are Limosilactobacillus reuteri, and may be any strain of this species, a number of which are known and reported in the art. Many strains of Limosilactobacillus reuteri are publicly available from culture collections, including under the name Lactobacillus reuteri for example Lactobacillus reuteri DSM20016 or Lactobacillus reuteri ATCC PTA 6475.

Particular mention may be made of the strain L. reuteri R2LC. In other words, the LAB may be Limosilactobacillus reuteri R2LC (also identified as Lactobacillus reuteri R2LC). As noted above, these names may be used interchangeably whilst referring to the same bacteria.

Limosilactobacillus reuteri R2LC, and indeed Limosilactobacillus reuteri more generally, and Lactococcus lactis are not found on human skin as determined by phylogenetic analysis of the forearm skin biota of six subjects.

Limosilactobacillus reuteri R2LC has been reported in the literature and is available on request from Prof. Siv Ahnre, Lund University, Sweden (see Ahnre et al., Nutrients 2011 , 3, 104-117). Limosilactobacillus reuteri R2LC has been deposited under the name Lactobacillus reuteri strain R2LC at the Culture Collection of the University of Gothenburg (CCUG) in December 2021 with the preliminary deposit number R2LC20211221. Further, under the name Lactobacillus reuteri R2LC, Limosilactobacillus reuteri R2LC has been deposited under the terms of the Budapest Treaty at the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (InhoffenstraBe 7 B, D-38124 Braunschweig, Germany) on 26 August 2022 with the accession number DSM 34372.

In an embodiment, the LAB is of a single population or type, i.e. of a particular strain, rather than a mixed culture or mixed population.

As the problems of drug resistance in bacteria has continued to grow, efforts have been made to introduce a more harmonised definition system to describe and classify bacteria that are resistant to multiple antimicrobial agents. The Clinical Laboratory Standards Institute (CLSI), the European Committee on Antimicrobial Susceptibility Testing (EUCAST) and the United States Food and Drug Administration (FDA) have developed several categories of drug-resistant microorganisms which are classified based on the extent of their antimicrobial resistance (Magiorakos et al. (2011) Clin Microbiol Infect., 18(3), 268-281). These categories are multidrug-resistant bacteria (MDR), extensively drug-resistant (XDR) and pandrug-resistant (PDR).

At its broadest, the term MDR in microorganisms describes the situation where a microorganism has acquired non-susceptibility to at least one antimicrobial drug in three or more antimicrobial categories. Thus, the term “MDR microorganism” refers to a microorganism which is non-susceptible, or in other words, resistant, to at least one antimicrobial drug in three or more antimicrobial categories.

At its broadest, the term XDR in microorganisms describes the situation where a microorganism has acquired non-susceptibility to at least one antimicrobial drug in all but two or fewer antimicrobial categories. Thus, the term “XDR microorganism” refers to a microorganism which is non-susceptible, or in other words, resistant, to at least one antimicrobial drug in all but two or fewer antimicrobial categories.

At its broadest, the term PDR in microorganisms describes the situation where a microorganism has acquired non-susceptibility to all antimicrobial drugs in all antimicrobial categories. Thus, the term “PDR microorganism” refers to a microorganism which is non-susceptible, or in other words, resistant, to all antimicrobial drugs in all antimicrobial categories.

It will be appreciated that XDR and PDR may be seen to be a more extreme cases of MDR. Accordingly, the term “MDR microorganism” as used herein may also encompass PDR and XDR microorganisms, unless stated otherwise.

There is as yet no clearly defined or accepted definition of an antimicrobial category, but for the purposes herein this can be taken as including all categories of antibacterial or antifungal agents. Put another way, an MDR microorganism may be defined as non-susceptible, or resistant, to at least one antimicrobial agent in three or more classes of antimicrobial agent. Similarly, an XDR microorganism may be defined as non-susceptible, or resistant, to a least one antimicrobial agent in all but two or fewer classes of antimicrobial agent. Furthermore, a PDR microorganism may be defined as non-susceptible, or resistant, to all antimicrobial agents in all classes of antimicrobial agents. Even if an XDR bacteria is susceptible to one class of antibiotics, it is not necessarily so that the antibiotic it is susceptible to can be used to treat the patient. Infections are in the majority of cases are polymicrobial. A class of agent may be defined in functional or structural terms. An antimicrobial agent in one class may be functionally unrelated, structurally unrelated, or both, to antimicrobial agents in a different class. The terms “antimicrobial agent” and “antimicrobial drug” are used interchangeably herein.

The term “microorganism” as used herein broadly includes all types of microbial organisms which are recognised under this term in the art, including single cell and multicellular organisms. Particularly, however, the microorganisms are bacteria or fungi. In certain embodiments, the microorganisms may be pathogenic, and may according be referred to as MDR pathogens.

The term "infection with an MDR microorganism” is used broadly herein to indicate that a subject, which may be any human or non-human animal subject, but typically a mammalian subject, is infected with, or may comprise, or contain, or carry, the MDR microorganism (e.g. bacteria or fungi) question, i.e. that the MDR microorganism may simply be present in or on the subject, and this may include any site or location in or on the body of the subject. It is not necessary that the infection of the subject be manifest as a clinical disease (i.e. that the infection result in clinical symptoms in the subject), although this is of course encompassed. A subject who is suspected to be infected or who is at risk of infection may be a subject who has been exposed to the MDR microorganism or to an infected subject, or a subject presenting with clinical signs or symptoms of infection (in the case of a suspected infection), or a subject who is susceptible to infection, whether generally e.g. due to the clinical status of the subject) or particularly to the MDR microorganism in question. The term “infection with an MDR microorganism” as used herein is used synonymously and interchangeably with the term “MDR infection”.

The uses herein include the treatment of an infection with an MDR microorganism. The terms “treating" or “treatment” as used herein refer broadly to any effect or step (or intervention) beneficial in the management of a clinical condition or disorder. It includes any therapeutic effect. Treatment therefore may refer to reducing, alleviating, ameliorating, slowing the development of, or eliminating one or more symptoms of the MDR infection that is being treated, relative to the symptoms prior to treatment, or in any way improving the clinical status of the subject. A treatment may include any clinical step or intervention which contributes to, or is a part of, a treatment programme or regimen. Thus, not only included is eradication or elimination of the infection, or cure of the subject or infection, but also an improvement in the infection or condition of the subject. Thus, included for example, is an improvement in any symptom or sign of the infection or condition, or in any clinically accepted indicator of the infection/condition (for example a decrease in wound size or an acceleration of healing time). Treatment thus includes both curative and palliative therapy, e.g. of a pre-existing or diagnosed infection/condition

A therapeutic affect may be achieved by any means, e.g. by inhibiting the growth of the microorganism, reducing its viability or killing it. Thus, in some embodiments, the LAB may inhibit the growth of the MDR microorganism without killing it. In other embodiments, treatment with the LAB of the present invention may result in the death of the MDR microorganism. In other words, the LAB may kill the MDR microorganism. Thus, the LAB may be bactericidal or fungicidal.

By "inhibiting the growth of an MDR microorganism”, it is meant that measurable growth (e.g. replication) of an MDR microorganism, or the rate thereof, is reduced. This may include bacteriostatic or fungistatic effects. Preferably measurable growth (e.g. replication) of an MDR microorganism, or the rate thereof, is reduced by at least 50%, more preferably at least 60%, 70%, 80% or 90%, e.g. at least 95%. Preferably, measurable growth (e.g. replication) is ceased. Growth in terms of microbial size increase or expansion etc. may be inhibited independently of replication and vice versa.

By “killing an MDR microorganism”, it is meant that the microorganism is no longer viable (e.g. able to reproduce or grow). That is to say, the microorganism is damaged to the point that the extent of injury is beyond the ability of a cell to resume growth. Alternatively viewed, the LAB may reduce the survival of the MDR microorganism.

In some embodiments, the LAB may result in the death of at least 10% of the MDR microorganisms. In other embodiments, the LAB may result in the death of at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the MDR microorganisms. In particular embodiments, the LAB may result in the death of at least 96%, at least 97%, at least 98% or at least 99% or 100% of the MDR microorganisms. The viability or survival of the MDR microorganism may be measured by any techniques known in the art. In some embodiments, the LAB may be used to treat an established infection, e.g. wherein the LAB is administered after the infection with an MDR microorganism has been identified or diagnosed in a subject. This represents a particularly advantageous aspect of the therapies disclosed herein. In another embodiment, the LAB may be administered to a subject who is suspected to have an infection with an MDR microorganism. In other words, the subject may be treated with the LAB before the MDR infection has been diagnosed. In some embodiments, the subject may therefore be treated before the MDR infection has been diagnosed as they have begun to exhibit the symptoms of an infection. In other words, the MDR infection may not have been diagnosed by testing. An MDR infection may be suspected if other treatments, e.g. with antibiotics, have been unsuccessful (i.e. the infection has persisted or not resolved). Alternatively, the uses or therapies described herein may involve the treatment of an MDR infection once the symptoms of said infection have begun to manifest.

The MDR infection may be diagnosed or identified by any accepted means in the art. Such diagnosis methods may include culture-based or molecular analysis. In other words, such diagnostic methods may involve phenotypic or genotypic testing. An example of phenotypic tests are those which investigate the direct activity of antibiotics on bacteria, i.e. disk diffusion or spot-drop methods. Such methods are well known and widely described in the literature. Genotypic tests include those tests which search for target genes in the bacteria to see whether they are expected to be susceptible or resistant to an antibiotic, e.g. PCR, real-time PCR (qPCR) or whole genome sequencing. In other words, genotypic tests may be used to identify the presence of antibiotic resistance genes (ARGs) in a pathogen. Generally, it is found that phenotypic testing methods are more accurate and relevant to clinical situations than genotypic testing methods.

Alternatively viewed, a treatment may include delaying, limiting, reducing or preventing the onset of one or more symptoms of the MDR infection, for example relative to the symptom prior to the treatment. Thus, treatment explicitly includes both absolute prevention of occurrence or development of symptoms of the MDR infection, and any delay in the development of the MDR infection or symptom thereof, or reduction or limitation on the development or progression of the MDR infection or symptom thereof.

In other words, the LAB may be administered before the infection has begun or when an infection is not present. In other words, the LAB may be used prophylactically to prevent or stop the MDR infection from developing. Alternatively viewed, the LAB may prevent colonisation by the MDR microorganism.

The term “prophylactic” as used herein means that the LAB prevents or protects against an MDR infection. Thus, the terms “prophylaxis” and “prevention” as used herein are synonymous. Thus, in one embodiment, the LAB may be seen to be preventative or protective against an MDR infection.

The LAB which are used may be native or wild-type, i.e. as they occur in nature, of they may be modified in some way, for example, adapted, mutated or genetically engineered etc. Thus, non-wildtype or non-native LAB may be used. In some embodiments, the LAB may be transformed to express a protein, in particular a heterologous protein. In accordance with its usual meaning, “heterologous” means that the protein is not expressed by the native LAB species or strain in question, or indeed by LAB of that genus, or any LAB. In other embodiments, the LAB may be non-transformed (not genetically engineered by introduction of foreign or heterologous nucleic acid) but may be modified in some other way, e.g. adapted, evolved or mutated. In one embodiment, the LAB may be engineered (e.g. transformed) to overexpress an endogenous protein. Thus, non-transformed bacteria may or may not be native or wild-type.

Thus, the term “wild-type” as used herein means that the LAB are native LAB, or LAB which occur in nature, and which have not been transformed, engineered or modified in any way. In particular, the LAB have not been modified by human intervention.

Conversely non-wild-type or non-native LAB have been modified or altered by human intervention. Accordingly, the term “non-wild-type” means that the LAB are non-native, or do not occur in nature. In other words, non-wild-type LAB are LAB which have been subject to some sort of modification, notably a modification which alters a property of the bacteria. For example, they have been subjected to mutation, or genetic engineering, which includes all forms of genetic modification or gene editing, recombinant gene expression etc. Thus, non-wild type LAB may be modified to introduce a heterologous nucleic acid sequence or molecule into their genome, for example to express a protein, particularly a heterologous protein, or they may be subject to gene knock-out, or gene inactivation, or gene duplication etc. In a particular embodiment, the LAB are modified or engineered to express a heterologous protein, or in other words they are transformed. The term “transformed” as used herein means the introduction of genetic material (nucleic acid) into the microorganism, in particular for the purpose of expressing a protein. The term is used herein synonymously with “engineered” and in particular “genetically engineered”.

An example of such a heterologous protein may be a mammalian protein. In some embodiments, the mammalian protein may be a therapeutic protein, such as one that is antimicrobial, promotes resolution of inflammation and/or wound healing, as discussed further below. One or more heterologous proteins may be expressed. Whilst the above embodiments are preferred, the LAB may be genetically modified in any way. Non-wild type LAB also include those that have not undergone gene editing/genetic engineering and have been subjected to adaptation or evolution by culture methods. Such modifications may be made for example to introduce or improve a function or property of the bacteria, for example to improve growth, or viability, or nutrient usage or assimilation etc., or to introduce or modify a biological, e.g. biosynthetic or degradative, pathway etc.

The term “nucleotide sequence” is used herein synonymously and interchangeably with “gene” or “gene sequence” to refer to a sequence encoding the protein in question. In particular, the use of the term “gene” herein does not imply or require the presence with the coding sequence of any promoter sequence or other expression control sequence. Thus, the term “gene” does not imply or require that the native promoter or other control sequence of the native gene is present, merely a coding sequence encoding the stated protein.

The nucleic acid molecule encoding the desired heterologous protein may be introduced into the LAB in, or as part of an autonomously replicating element, e.g. a plasmid, or another vector, or it may be integrated into the chromosome of the recipient, or host, LAB. Thus, the nucleotide sequence encoding the protein may be present in the engineered LAB integrated in the host genome, or independent of the host genome, in a vector that is present in the engineered LAB. This may be done by any accepted means in the art.

The protein which the LAB are transformed to express may be any protein which is beneficial or useful in the context of treating or preventing a microbial infection. It may thus have a therapeutic effect which is beneficial to or contributes to treating or preventing an infection. It may be directly or indirectly anti-microbial (i.e. a direct or indirect antimicrobial effect), or it may be any protein which has an effect of promoting healing of a wound or in promoting or aiding the resolution of inflammation. In other words, a protein which promotes or aids the resolution inflammation may be referred to as an “anti-inflammatory” protein. The protein may advantageously be an immunomodulatory protein, that is a protein which has an effect in modulating the activity of immune cells. Thus, in an embodiment, the protein may be defined as an immune-active protein, in particular an immune-active protein which is active locally on immune cells present in the vicinity of the MDR infection.

In one embodiment, the protein is an antimicrobial protein or a protein with antimicrobial activity. The term “antimicrobial protein” as used herein is used interchangeably with the terms “antimicrobial peptide” and “host defense peptide”. Antimicrobial proteins/peptides (AMPs) are small proteins that provide defense against microbial infections and form one of the key components to an effective host defense system. AMPs have been found in all classes of life, including mammals. Interestingly, the development of resistance by microorganisms to AMPs is slower (or delayed) in comparison to conventional antibiotics. The mechanism of action by which AMPs kill microorganisms are varied and not limited to a single mechanism of action. For example, AMPs may disrupt the cytoplasmic membrane of microorganisms, thus causing cell death. Other mechanisms of action include, but are not limited to, interference with DNA and protein synthesis, protein folding, and cell wall synthesis. Generally, AMPs have broad spectrum antimicrobial activity. That is to say, they are effective against an extensive range of microorganisms, including bacterial, viral, and fungal pathogens.

In another particular embodiment, the protein is an anti-inflammatory protein, or a protein with anti-inflammatory activity. In particular, the protein may modulate the activity of immune cells. In some embodiments, the protein may therefore act to stimulate the growth and/or activity of immune cells. In preferred embodiments, the protein may act to promote or increase the anti-inflammatory effect of the immune cells (e.g. macrophages). The protein may stimulate the proliferation of local macrophages and/or other immune cells and may induce a phenotypic shift to an anti-inflammatory phenotype. Such anti-inflammatory activity may be particularly beneficial in the healing of wounds. Thus, in some embodiments, the anti-inflammatory protein may also be a wound healing protein.

In another embodiment, the protein is a wound healing protein. In other words, the protein may promote or accelerate wound healing. Preferably, the said protein is an interleukin, a chitinase-like protein, a cytokine, or a chemokine, more preferably a CXC protein. In an embodiment the protein is selected from one or more of CXCL12, CXCL17, Ym1, TGF-p, IL-22, IL- 27 IL-4, IL-10, IL-12, IL-8, or SP1. In a more particular embodiment the protein is selected from one or more of CXCL12, CXCL17, Ym1 or TGF-p. In some embodiments, the protein is selected from the group consisting of CXCL12, CXCL17 and Ym1.

CXCL12 (also known as SDF-1; SEQ ID NO: 3 and 6) is constitutively expressed in tissues and acts through the receptor CXCR4 found on leukocytes and endothelial cells inducing multiple cellular actions. CXCL12 is found in high levels in macrophages specialized in tissue remodeling. CXCL12 has also been reported to have anti-fibrotic effects.

CXCL17 (SEQ ID NO: 9 and 12), originally classified as a chemokine, has similar effects on the phenotype of tissue macrophages as CXCL12. In similarity with CXCL12, CXCL17 is co-regulated with VEGF-A measured in cell culture. CXCL17 is found mainly in mucosal tissues and have been reported to be directly antimicrobial to pathogenic bacteria that are also found on skin whilst showing no effect on survival of Lactobacillus casei. As well as anti-microbial effects, it has been reported to have microbial and anti-fibrotic effects and chemotactic properties. More recently, the classification of CXCL17 as a chemokine has been questioned, but this is not relevant to its proposed use herein.

A further protein of interest is Ym1 (SEQ ID NO: 15 and 18), which is a chitinase-like protein. Chitin is a common polysaccharide in bacterial biofilm. Ym1 both counteracts biofilm production and induces macrophage functions important for tissue remodeling and wound healing and is specific to macrophages since it is not taken up by either vascular cells or epithelial cells.

Another protein of interest is TGF-p. TGF-p occurs in three different isoforms, TGF-p 1 , 2 and 3, all of which are included herein. TGF-p is a multifunctional cytokine, and is secreted by many cell types, including macrophages, and plays a role in the regulation of inflammatory processes, including in the gut.

The said protein may be of any species, e.g. murine or human. More specifically, the protein may be murine CXCL12, in particular murine CXCL12-1a (SEQ ID NO: 3); human CXCL12, in particular human CXCL12-1a (SEQ ID NO: 6); murine CXCL17 (SEQ ID NO: 9); human CXCL17 (SEQ ID NO: 12); murine Ym1 (SEQ ID NO: 15) and human Ym1 (SEQ ID NO: 18). In certain embodiments, human proteins are preferred.

In one embodiment, the protein is selected from murine CXCL12-1a having an amino acid sequence as shown in SEQ ID NO: 3 or 2, or an amino acid sequence with at least 80% sequence identity thereto; human CXCL12-1a having an amino acid sequence as shown in SEQ ID NO: 6 or 5, or an amino acid sequence with at least 80% sequence identity thereto; murine CXCL17 having an amino acid sequence as shown in SEQ ID NO: 9 or 8, or an amino acid sequence with at least 80% sequence identity thereto; human CXCL17 having an amino acid sequence as shown in SEQ I D NO: 12 or 11 , or an amino acid sequence with at least 80% sequence identity thereto; murine Ym1 having an amino acid sequence as shown in SEQ ID NO: 15 or 14, or an amino acid sequence with at least 80% sequence identity thereto; and human Ym1 as shown in SEQ ID NO: 18 or 17 or an amino acid sequence with at least 80% sequence identity thereto.

In other embodiments the protein(s) may have an amino acid sequence which has at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% 90%, 91% 92% 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with any aforesaid amino acid sequence.

Sequence identity may readily be determined by methods and software known and readily available in the art. Thus, sequence identity may be assessed by any convenient method. However, for determining the degree of sequence identity between sequences, computer programs that make multiple alignments of sequences are useful, for instance Clustal W (Thompson et al., (1994) Nucleic Acids Res., 22: 4673-4680). Programs that compare and align pairs of sequences, like ALIGN (Myers et al., (1988) CABIOS, 4: 11-17), FASTA (Pearson et al., (1988) PNAS, 85:2444-2448; Pearson (1990), Methods Enzymol., 183: 63-98), BLAST and gapped BLAST (Altschul et al., (1997) Nucleic Acids Res., 25: 3389-3402) are also useful for this purpose, and may be used using default settings. Furthermore, the Dali server at the European Bioinformatics institute offers structure-based alignments of protein sequences (Holm (1993) J. Mol. Biol., 233: 123-38; Holm (1995) Trends Biochem. Sci., 20: 478-480; Holm (1998) Nucleic Acid Res., 26: 316- 9). Multiple sequence alignments and percent identity calculations may be determined using the standard BLAST parameters, (e.g., using sequences from all organisms available, matrix Blosum 62, gap costs: existence 11, extension 1). Alternatively, the following program and parameters may be used: Program: Align Plus 4, version 4.10 (Sci Ed Central Clone Manager Professional Suite). DNA comparison: Global comparison, Standard Linear Scoring matrix, Mismatch penalty = 2, Open gap penalty = 4, Extend gap penalty = 1. Amino acid comparison: Global comparison, BLOSLIM 62 Scoring matrix.

Variants of the naturally occurring polypeptide sequences as defined herein can be generated synthetically, e.g., by using standard molecular biology techniques that are known in the art, for example standard mutagenesis techniques such as site-directed or random mutagenesis (e.g., using gene shuffling or error prone PCR).

Derivatives of the proteins as defined herein are also encompassed. By derivative is meant a protein as described above or a variant thereof in which the amino acid is chemically modified, e.g., by glycosylation and the like, etc.

Where a protein comprises an amino acid substitution relative to the sequence of the native protein, the substitution may preferably be a conservative substitution. The term “a conservative amino acid substitution” refers to any amino acid substitution in which an amino acid is replaced (substituted) with an amino acid having similar physicochemical properties, i.e., an amino acid of the same class/group. For instance, small residues Glycine (G), Alanine (A) Serine (S) or Threonine (T); hydrophobic or aliphatic residues Leucine (L), Isoleucine (I); Valine (V) or Methionine (M); hydrophilic residues Asparagine (N) and Glutamine (Q); acidic residues Aspartic acid (D) and Glutamic acid (E); positively-charged (basic) residues Arginine (R), Lysine (K) or Histidine (H); or aromatic residues Phenylalanine (F), Tyrosine (Y) and Tryptophan (W), may be substituted interchangeably without substantially altering the function or activity of the protein.

As is well known in the art, doses and dosage regimens for any one subject depend upon many factors, including the subject's size, body surface area, surface of area to be treated, age, gender, time and route of administration, general health, stage of the disease, underlying health conditions and other drugs being administered concurrently.

Thus, the LAB may be provided, or administered to the subject in a therapeutically effective amount. A “therapeutically effective amount” or a “effective amount” refers to at least an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result, i.e. at least the minimum concentration required to affect a measurable improvement of the MDR infection. In some embodiments, an effective amount or therapeutically effective amount can be provided in one or more administrations. A therapeutically effective amount may also be an amount in which any detrimental effects of the agents or pharmaceutical compositions are outweighed by the therapeutically beneficial effects. In other words, the LAB may be provided or administered at any dose which treats or prevents an infection with an MDR microorganism.

The dose may be determined based on the size of the area of the subject to be treated, for example at the site of the infection.

In one embodiment, the LAB may be applied in a dose of 10² CFU/cm² to 10¹³ CFU/cm². That is to say, the LAB may be applied at a dose selected from 10² CFU/cm², 10³ CFU/cm², 10⁴ CFU/cm², 10⁵ CFU/cm², 10⁶ CFU/cm², 10⁷ CFU/cm², 10⁸ CFU/cm², 10⁹ CFU/cm², 1O¹⁰ CFU/cm², 10¹¹ CFU/cm², 10¹² CFU/cm² and 10¹³ CFU/cm², or in range between any two of these values. In one embodiment, the LAB may be applied at a dose of 10³ CFU/cm² to 10¹² CFU/cm², 10⁴ CFU/cm² to 10¹¹ CFU/cm², 10⁵ CFU/cm² to 1O¹⁰ CFU/cm², 10⁶ CFU/cm² to 10⁹ CFU/cm² and 10⁷ CFU/cm² to 10⁸ CFU/cm². In a preferred embodiment, the LAB may be applied at a dose of 10⁴ CFU/cm² to 10¹¹ CFU/cm². In other embodiments, the LAB may be applied at a dose of 10⁴ CFU/cm² to 10⁹ CFU/cm² or 10³ CFU/cm² to 10⁹ CFU/cm² .

As noted above, the dose to be applied will depend on the surface area to be treated and/or the location of the infection or potential infection, or nature of the infection or body site etc., or the purpose for which the treatment is applied. Thus, in some embodiments, the LAB may be applied at a dose of 2.5 x 10⁴ to 2.5 x 10⁶ per cm. Alternatively viewed, the LAB may be applied at a dose of 2.5 x 10⁴ per cm, 2.5 x 10⁵ per cm or 2.5 x 10⁶ per cm. In a preferred embodiment, the LAB may be applied at a dose of 2.5x10⁵ per cm.

For surgical wounds, a dose of about 2.5 x 10⁵ per cm may be appropriate.

In certain embodiments, the LAB may be used, or applied, at a dose in the range of 100:1 to 1000; 1 of LAB relative to the amount (e.g. number or CFU) of the single strain pathogenic bacteria or polymicrobial composition of pathogenic bacteria. Representative doses include 100:1 , 200: 1 , 500; 1 and 700:1 or indeed any integer between 100 and 1000:1.

The LAB may be used to treat or prevent an MDR infection located at any site in or on the body of the subject. This may be in or on any soft tissue, mucosal surface, or in blood. In a preferred embodiment, the MDR infection is located or expected or suspected, or at risk of occurring, in a wound, in the respiratory tract, e.g. in the lungs, or the urinary tract. Preferably, the wound is a cutaneous wound, a mucosal wound or a surgical wound. Also included are fibrotic lesions, or sites of fibrosis. These may occur in or at different sites in the body, e.g. in the lungs.

The term “soft tissue” refers to any tissue in the body that is not bone and includes, but is not limited to, muscle, fat, fibrous tissue and blood vessels.

The term “mucosal surface” as used herein is interchangeable with the terms “mucosa”, “mucosal membrane” and “mucosal tissue” and refers to a surface composed of epithelial cells which form a barrier between the host organism and the environment. Particularly, the mucosal membrane lines the body's canals and organs in the digestive, respiratory and reproductive systems. Some mucous membranes secrete a thick protective fluid called mucus.

The term “wound” is used broadly herein to include any breach of the integrity of a tissue, namely any damage, trauma or injury to tissue or any lesion, howsoever caused (e.g., due to accidental injury or trauma, surgical or other intended or purposeful injury or disease). The trauma may include any physical or mechanical injury or any damage caused by an external agent including pathogens or biological or chemical agents. Wounds may include any type of burn. The wound may be acute or chronic. A chronic wound may be described as any wound stalled in a healing stage, e.g., in the inflammatory phase, or any wound that has not healed in 30, 40, 50 or 60 days or more. The wound may be present in or on an internal or external surface or tissue of the body. The wound may exhibit a delayed healing response, e.g. the wound has failed to progress through the normal stages of healing in a timely manner. In other words, the wound may be persistent.

For some conditions, the term “ulcer” is used alternative to the term “wound”. Ulcers falling within the same type of description as wounds are included herein.

In a particular embodiment the wound is on an external surface or tissue of the body, e.g., it is a skin (i.e. , cutaneous) wound or a mucosal wound, in particular a wound in an external mucosal tissue or surface of the body (e.g., in the eye, ear or nose, etc.).

Thus, the wound to be healed can include any injury, trauma or damage to any portion of the body of a subject wherein the wound has been infected or is at risk of infection with an MDR microorganism. Examples of wounds that can be treated include acute conditions or wounds; such as thermal burns (hot or cold), chemical burns, radiation burns, electrical burns, burns caused by excess exposure to ultraviolet radiation (e.g., sunburn); damage to bodily tissues, such as the perineum as a result of labour and childbirth; injuries sustained during medical procedures, such as episiotomies, trauma-induced injuries including cuts, incisions, excoriations; injuries sustained from accidents; post-surgical injuries, as well as chronic conditions; such as pressure sores, bedsores, ulcers, conditions related to diabetes and poor circulation, and all types of acne, as well as wounds caused by genetic defects. In addition, the wound can include dermatitis, wounds following dental surgery; periodontal disease; wounds following trauma; and tumour associated wounds. Further examples are gastrointestinal wounds occurring during for instance gastritis or inflammatory bowel disease.

MDR microorganism have particularly been found to cause problems in the context of war wounds and war injuries (that is wounds and injuries sustained in the course of combat, or caused by military equipment), and these represent a particular group of MDR infections of interest herein. In other words, the subject to be treated in accordance with the medical uses and methods herein may be injured service or military personnel or patients contaminated with MDR strains from war victims.

Biofilms may be present in some wounds, and this may contribute to intractability of wound healing. The term “biofilm” as used herein may also refer to a bacterial or fungal aggregate which has formed on the surface of a wound. In some instances, a biofilm may be described as a microbial colony encased in a polysaccharide matrix which can become attached to the surface of a wound. The presence of a biofilm can delay the healing of a wound due to the production of various substances such as inflammatory mediators and proteases which can promote a chronic inflammatory state within the wound. It is believed that the presence of biofilms is one of the factors which results in the delayed healing, particularly in chronic wounds. Biofilm formation has been found to be a hallmark feature of MDR microorganisms, particularly in healthcare settings. Notably, biofilms allow for the exchange of plasmid-mediated antimicrobial resistance genes among bacteria. Thus, the effective treatment of biofilms is imperative to allow for wound healing to progress normally.

Thus, in some embodiments, the MDR microorganism will be in a biofilm. In other embodiments, the MDR microorganism will not be in a biofilm (e.g. will be growing planktonically). In another embodiment, the MDR microorganism to be treated may be a biofilm which is located in a wound, preferably a skin wound. By "in a biofilm", it is meant that the MDR microorganism targeted by the LAB is within (completely or in part), on or associated with the polysaccharide matrix of a biofilm. Viewed differently, MDR microorganisms that are "not in a biofilm" are organisms that are either in isolation, e.g. planktonic, or if in an aggregation of a plurality of organisms, that aggregation is unorganised and/or is devoid of the matrix characteristic of a biofilm. In each case, the individual microorganisms do not exhibit an altered phenotype that is observed in their biofilm dwelling counterparts.

Broadly speaking, the LAB may be used to treat or prevent an MDR infection location at any site in the respiratory tract. That is, the MDR infection may be located in any part of the body associated with breathing. In some embodiments, the LAB may be used to treat or prevent an MDR infection in the lungs. Thus, the LAB may be seen to treat a lung infection.

A lung infection may be defined as the presence of an MDR microorganism within one or both of the lungs. The term “respiratory tract infection” may comprise an upper respiratory tract infection or a lower respiratory tract infection. A respiratory tract infection may be present in any part of the body involved in breathing, including in the sinuses, throat, airways or lungs. Upper respiratory tract infections comprise infections located in the nose, sinuses, pharynx or larynx. Lower respiratory tract infections comprise infections located below the larynx including the trachea and within the lungs, the bronchi, bronchioles, and alveoli. Examples of lung infections which may be treated with a LAB of the invention include, but are not limited to, pneumonia, or infections occurring in the context of cystic fibrosis, chronic obstructive pulmonary disease (COPD), chronic obstructive airway disease (COAD), community- or hospital acquired pneumonia and other respiratory diseases.

A urinary tract infection is an infection in any part of the urinary system, including the kidneys, ureters, bladder and urethra. Examples of urinary tract infections which may be treated with a LAB of the invention include, but are not limited to, cystitis, urethritis and pyelonephritis. Another condition of particular interest is STING-associated vasculopathy with onset in infancy (SAVI). This is a very rare condition involving abnormal inflammation throughout the body, especially in the skin, blood vessels and lungs. It is characterised by severe skin lesions, resulting in ulceration, eschar formation and necrosis. Many patients have interstitial lung disease. The various wounds which result from this disease are susceptible to infection, including by MDR microorganisms. The treatment of wounds in SAVI or in any subjects with genetic defects resulting in skin or other wounds is included herein.

As discussed above, it is contemplated that the LAB may not only eradicate or eliminate the infection, but that the LAB it may also improve the condition of the subject. Improving the condition of the subject may be achieved by treating other symptoms of the disease in addition to the infection. Thus, it may be viewed that the LAB has the additional benefit of treating other symptoms of the disease. By way of example, the LAB may treat or reduce fibrosis, treat or reduce inflammation or may promote or accelerate resolution of inflammation and regain of function of the tissue and healing (e.g. wound healing).

As discussed above, the LAB may have been transformed to express a heterologous protein or to over-express an endogenous protein. Such proteins may be selected for their therapeutic effects (e.g. CXCL12, CXCL17 or Ym1). In particular, it is contemplated that the LAB may additionally treat or reduce fibrosis, resolution of inflammation and regain of tissue function within the lungs and/or that the LAB may be transformed to express a therapeutic protein (e.g. CXCL12 or CXCL17) to treat or reduce fibrosis within the lungs. In view of the known anti- fibrotic effects of CXCL12 and CXCL17, a LAB transformed to express CXCL12 and/or CXCL17 may be particularly beneficial in the treatment of lung diseases including cystic fibrosis, COPD, COAD, community- or hospital acquired pneumonia, and other respiratory diseases, particularly cystic fibrosis (CF). In particular, LAB delivered CXCL17 may be of benefit in the treatment of lung diseases, such as CF in view of its potential direct potent anti-microbial effects on a range of pathogenic bacteria. It is also known that during homeostasis CXCL17 is present on the mucosal side of the epithelial surface in the lungs, where is it believed to function as part of the immune system. There is thus a rationale to using LAB modified to express CXCL17 to treat or prevent MDR infections in the lung, particularly in the context of CF and other diseases of the lung, as noted above. It is believed that CXCL17 will act on the innate local resident immune cells in the tissue, e.g. macrophages and neutrophils in the mucosa, to promote resolution of inflammation and restoration of tissue function. The efficiency of bacterial killing by neutrophils and macrophages in the mucosa may be potentiated by CXCL17 (or indeed CXCL12). In this regard, it has been shown that modified LAB may be present up to 1-2 days following administration. The modified bacteria may be used alongside antibiotics, as discussed herein. This may help to clear infections faster than using antibiotics alone. The dose of antibiotic may be reduced, and thereby the possible side-effects of the antibiotics. This may be of particular relevance to CF patients with limited tolerability for repeated use of available indicated antibiotics. As described above, an MDR microorganism is a microorganism which is non- susceptible to at least one antimicrobial drug in three or more antimicrobial categories or classes. In bacteria, MDR is often termed multiple anti-bacterial drug resistance or multiple antibiotic resistance (MAR) - these terms are used interchangeably in the art and herein. Bacteria displaying multidrug resistance phenotypes (or multiple antibacterial/antibiotic drug resistance phenotypes) are referred to as MDR bacteria (or sometimes MAR bacteria). Again, these terms are used interchangeably in the art and herein.

In terms of antimicrobial resistance, the term “susceptible” may refer to the microorganism’s ability to grow if the antimicrobial is present. Thus, if a microorganism is susceptible to an antimicrobial, they are unable to grow in the presence of the antimicrobial. Conversely, if a microorganism is non-susceptible to an antimicrobial, they are able to grow in its presence. The susceptibility of a microorganism to an antimicrobial may be determined by analysing the minimum concentration of an antimicrobial (usually expressed in pg/ml or mg/L) that inhibits the growth of a specific microorganism. In terms of bacteria, this may involve determining the minimum concentration of an antibiotic that inhibits the growth of a specific bacterial strain. A minimum inhibitory concentration (MIC) is one of the factors used to determine if a microorganism is susceptible or not to an antimicrobial. Depending on the MIC value, a microorganism could be assigned to three different clinical categories: susceptible, intermediate or resistant. Susceptible may mean that the growth of the microorganism is inhibited in vitro by a concentration of an antimicrobial agent that is associated with a high probability of therapeutic success. Intermediate may mean that the growth of the microorganism is inhibited in vitro by a concentration of an antimicrobial agent that is associated with an uncertain therapeutic effect. Resistant may mean that the growth of the microorganism is inhibited in vitro by a concentration of an antimicrobial agent that is associated with a high probability of therapeutic failure. In the art, the “Intermediate” classification is often interpreted to be a form of antimicrobial resistance. Thus, in the context of the present invention, the microorganism may be classed as intermediate or resistant using this classification system. By "resistant to an antimicrobial" it is meant that the microorganism displays a substantially greater tolerance (reduced susceptibility) to an antimicrobial as compared to a reference microorganism sensitive to the antimicrobial or a typical, or a wild type, version of the microorganism. Such a substantially greater tolerance may be a statistically significant decrease in susceptibility to the antimicrobial, as measured for example in standard assays, such as MIC assays. In some cases, a microorganism can be completely unaffected by exposure to an antimicrobial. In this instance the microorganism can be considered fully resistant to that antimicrobial.

A suitable reference bacterium is Oxford Staphylococcus aureus (NCTC 6571) although many others are known in the art and are readily available. Typical, or wild type, versions of a bacterium can be obtained easily from laboratories and culture collections throughout the world.

A suitable reference fungus is Candida Albicans, although many others are known in the art and are readily available. Typical, or wild type, versions of a fungi can be obtained easily from laboratories and culture collections throughout the world.

Susceptibility (and conversely resistance and tolerance) to antimicrobial can be measured in any convenient way, e.g. with dilution susceptibility tests and/or disk diffusion tests. The skilled person would appreciate that the extent of the difference in tolerance/susceptibility sufficient to constitute resistance will vary depending on the antibiotic and organism under test and the test used However, a resistant microorganism will preferably be at least twice, e.g. at least 3, 4, 5, 6, 10, 20, or 50 times as tolerant to the antimicrobial as the reference microorganism sensitive to the antimicrobial or a typical or a wild type version of the microorganism. Preferably resistance of a particular microorganism to an antimicrobial is determined using microorganisms which are not in a biofilm or which do not have a biofilm phenotype.

In the context of an MDR microorganism infection, a microorganism may be considered resistant to an antimicrobial if the microorganism has a MIC value for the antimicrobial that is greater than then maximum safe circulating concentration of the antimicrobial in the subject (which may be determined easily by the skilled person). More functionally, a microorganism is resistant to an antimicrobial if an infection associated with that microorganism is unresponsive (i.e. there is no change in the clinical indicia of the infection) to the maximum safe dose of the antimicrobial.

As noted above, in some embodiments, the LAB may be seen as having an effect which overcomes resistance. "Overcoming resistance" should be construed accordingly as a measurable reduction in the above-described indicators of the resistance (or measurable increase in susceptibility or measurable decrease in tolerance) to the antimicrobial displayed by the microorganism. Therefore "overcoming resistance" can alternatively be expressed as "reducing resistance". It is a reference to the observed phenotype of the target microorganism and should not necessarily be considered to equate to a reversal, to any extent, at the mechanistic level of any particular resistance mechanism.

The mechanisms by which a microorganism may be resistant to an antimicrobial are numerous. For instance, resistance may arise from permeability mechanisms which physically prevent the antimicrobial from reaching its site of action in or on the microorganism; efflux mechanisms which prevent effective amounts of the antimicrobial reaching its site of action in or on the microorganism by rapidly removing the antimicrobial from the microorganism; metabolic mechanisms which breakdown the antimicrobial or convert the antimicrobial into a harmless (or less harmful) compound, or a compound more easily excreted; bypass mechanisms in which the microorganism uses alternative pathways to those inhibited by the antimicrobial; or through the microorganism having a form of the antimicrobial target (e.g. enzyme) that is less sensitive to the antimicrobial or not having the target at all.

Resistance to a particular antimicrobial or class of antimicrobials may be intrinsic to the microorganism, but it can also be developed or acquired, e.g. through mutation or genetic transfer between microorganisms. Generally intrinsic resistance may be seen to a particular type or class of antimicrobial, but the number of different antimicrobial classes to which resistance is seen is usually restricted. Resistance to numerous classes of antimicrobials (including to multiple classes of antimicrobials, which is defined herein as at least three classes of antimicrobials) may be an acquired (or developed) phenomenon, but this is not exclusively the case. In the case of MDR bacteria, the bacteria may acquire or develop resistance to particular antimicrobial, e.g. antibiotic, classes (e.g. to one or more or two or more classes, for example additional classes, or to 3 or more classes), or in certain cases the bacteria may be intrinsically resistant to multiple classes. In the case of MDR fungi, the fungi may acquire or develop resistance to particular antifungal classes (e.g., to one or more or two or more classes, for example additional classes, or to 3 or more classes), or in certain cases the fungi may be intrinsically resistant to multiple classes.

The microorganism targeted by the LAB can be any microorganism that is MDR, which according to the present invention means that the microorganism is resistant to at least 3, or at least 4, 5, 6, 7, 8, 9 or 10 antimicrobial classes. As noted above antimicrobials in different classes are structurally and/or functionally different. In other embodiments the MDR microorganism can be any microorganism that has extreme drug resistance, which means that the microorganism is resistant to the majority of, or all, antimicrobials. Alternatively viewed, the MDR microorganism may be an XDR or PDR microorganism, as defined above. That is to say, the microorganism targeted by the LAB may be non- susceptible (or in other words resistant) to at least one antimicrobial drugs in all but two or fewer antimicrobial categories (i.e. an XDR microorganism) or all antimicrobial drugs in all antimicrobial categories (i.e. a PDR microorganism). In the case of antibiotics, extreme drug resistant bacteria are resistant to at least one antibiotic of last resort (e.g. vancomycin, linezolid, etc.). In the case of antifungals, extreme drug resistant fungi are resistant to at least one antifungal of last resort (e.g. Amphotericin B etc.).As a consequence of the inherent selective pressure antimicrobials exert on a microorganism population, the use of antimicrobials selects for resistant members of that population. The sequential use of different antimicrobials, particularly antibiotics, in a treatment regime can therefore give rise to MDR microorganisms, particularly bacteria. Many MDR bacterial and fungal strains and species exist today. Bacterial families from which MDR species and strains pose significant problems for human and animal health include, but are not limited to Pseudomonadaceae, Enterococcaceae, Streptococcaceae, Moraxellaceae, Enterobacteriaceae, Staphylococcaceae, Helicobacteraceae, Campylobacteraceae, Neisseriaceae, Pasteurellaceae, Corynebacteriaceae, Lachnospiraceae, Bacillaceae and Mycobacteriaceae.

Particular bacterial genera from which MDR species and strains pose significant problems for human and animal health include, but are not limited to Pseudomonas, Acinetobacter, Enterobacter, Enterococcus, Proteus, Klebsiella, Staphylococcus, Clostridium and Bacillus. Thus, the MDR bacteria may be a strain or species from any of these bacterial families or genera. Pseudomonas is a genus of strictly aerobic, gram-negative bacteria of relatively low virulence. Nevertheless, Pseudomonas species can act as opportunistic pathogens and infections have been reported with Pseudomonas aeruginosa, Pseudomonas oryzihabitans, Pseudomonas luteola, Pseudomonas anguilliseptica and Pseudomonas plecoglossicida. In a particular embodiment, the MDR bacteria may be Pseudomonas aeruginosa.

P. plecoglossicida and P. anguilliseptica are fish pathogens. P. oryzihabitans can be a human pathogen causing peritonitis, endophthalmitis, septicemia and bacteriaemia. Similar infections can be caused by P. luteola. The majority of Pseudomonas infections in humans are, however, caused by P. aeruginosa. However, other species such as P. putida, P. fluorescens, P. mendocina, P. fulva and P. monteilii have also been shown to cause human clinical infections.

P. aeruginosa is a widespread and extremely versatile bacteria that can be considered a part of the natural flora of a healthy subject and is capable of colonising most man-made environments. This ubiquity and versatility has seen colonisation of healthcare environments by P. aeruginosa. Problematically, the same versatility enables P. aeruginosa to act as an opportunistic human pathogen in impaired subjects, most commonly immunocompromised patients (e.g. those with cystic fibrosis or AIDS) and patients with a compromised barrier to infections (e.g. those with chronic wounds and burns and those with in-dwelling medical devices such as intravenous catheters, urinary catheters, dialysis catheters, endotracheal tubes).

P. aeruginosa infection can affect many different parts of the body, but infections typically target the respiratory tract, the Gl tract, the urinary tract and cutaneous wounds and burns and in-dwelling medical devices. This problem is compounded by the presence of intrinsic resistance to many of the p-lactam antibiotics. Acquired resistance of certain strains to further antibiotics is also being reported. The ability of certain strains of P. aeruginosa to form biofilms adds further to these problems because biofilm-dwelling bacteria are often more resistant to anti-microbials than their non-biofilm counterparts. As such, there is an urgent need for safe and effective treatments for MDR Pseudomonas infections.

Acinetobacter is a genus of bacteria that are strictly aerobic non- fermentative gram-negative bacilli. Acinetobacter species are generally considered to be non-pathogenic to healthy subjects, but it is becoming increasingly apparent that Acinetobacter species persist in hospital environments for a long period of time and can be responsible for nosocomial infections in compromised patients. Acinetobacter baumannii is a frequent cause of nosocomial pneumonia, especially of late-onset ventilator associated pneumonia and it can cause various other infections including skin and wound infections, bacteraemia, and meningitis. It is frequently found in infections of war wounds Acinetobacter Iwoffii has also been associated with meningitis. Other species including Acinetobacter haemolyticus, Acinetobacter johnsonii, Acinetobacter junii, Acinetobacter radioresistens, Acinetobacter tandoii, Acinetobacter tjernbergiae, Acinetobacter towneri, or Acinetobacter ursingii have also been linked to infection. Of concern is the fact that many Acinetobacter strains appear to be multidrug resistant, thus making the combat of Acinetobacter infections and contamination difficult. As such, there is an urgent need for safe and effective treatments for Acinetobacter infections.

Enterobacter is a genus of gram-negative, facultatively anaerobic, rodshaped, non-spore-forming bacteria of the family. Several strains of Enterobacter are pathogenic and act as an opportunistic human pathogen in impaired subjects, most commonly immunocompromised patients (especially hospitalised patients) and those who are on mechanical ventilation. Particularly, infection most commonly occurs in the urinary tract and respiratory tract.

Enterobacter cloacae is a particular species of Enterobacter which has been associated with pathogenic infections. Common sites of infection include the urinary tract, the respiratory tract, the blood and soft tissue. E. cloacae has been found to contribute to nosocomial infections. The presence of intravascular devices (e.g. venous catheters) increases the likelihood of nosocomial infection with E. cloacae. As antibiotic resistance is a growing problem in Enterobacter infections, safe and effective treatments for Enterobacter infections are needed urgently.

Enterococcus is a genus of gram-positive cocci that often occur in pairs (diplococci) or short chains. Enterococci are capable of respiration in both oxygenrich and oxygen-poor environments and are very tolerant in a wide range of environmental conditions including extreme temperature (10-45°C), pH (4.6-9.9), and high sodium chloride concentrations. As this genus are so robust under a wide range of conditions, it allows them to act as an opportunistic human pathogen at a variety of sites including in the urinary tract, the endocardium, soft tissues, the skin and the abdomen. Enterococcus species include Enterococcus faecalis, Enterococcus faecium Enterococcus avium, Enterococcus casseliflavus, Enterococcus durans and Enterococcus gallinarum. Whilst these species have been found to cause human infection, E. faecalis and E. faecium infections are the most common in humans. Common sites of infection include the urinary tract, intra-abdominally, the pelvis, soft tissue and the endocardium. Infection with Enterococci can also lead to bacteraemia which can progress to sepsis if left untreated. Less common infections associated with Enterococci include meningitis, hematogenous osteomyelitis, septic arthritis, and pneumonia. Enterococci is a frequent cause of nosocomial infections, particularly in patients who have in-dwelling medical devices such as central or peripheral intravenous catheters, urinary catheters, dialysis catheters, endotracheal tubes.

Problematically, Enterococci species are often intrinsically resistant to several antibiotics including cephalosporins, clindamycin, aminoglycosides, and trimethoprim-sulfamethoxazole. Thus, there is a vital need for safe and effective treatments for Enterococci infections, particularly treatments which are able to treat resistant species.

Klebsiella is a genus of non-motile, gram-negative, rod shaped bacteria. Klebsiella species are ubiquitous in nature. In humans, they may colonize the skin, pharynx, and gastrointestinal tract and may be regarded as normal flora in many parts of the colon, the intestinal tract and in the biliary tract.

Klebsiella species include, Klebsiella pneumoniae, Klebsiella granulomatis, Klebsiella oxytoca, Klebsiella singaporensis, and Klebsiella variicola, although K. pneumoniae and K. oxytoca are the members of this genus responsible for most human infections. Such infections include pneumonia, bacteraemia, thrombophlebitis, urinary tract infection, cholecystitis, diarrhoea, upper respiratory tract infection, wound infection, osteomyelitis, and meningitis. Rhinoscleroma and ozena are two other infections caused by Klebsiella species. Rhinoscleroma is a chronic inflammatory process involving the nasopharynx, whereas ozena is a chronic atrophic rhinitis characterized by necrosis of nasal mucosa and mucopurulent nasal discharge.

Klebsiellae often contribute to nosocomial infections. Common sites include the urinary tract, lower respiratory tract, biliary tract, and wounds. The presence of invasive devices, in particular respiratory support equipment and urinary catheters, increase the likelihood of nosocomial infection with Klebsiella species. Sepsis and septic shock may follow entry of organisms into the blood from these sources.

K. pneumoniae is an important cause of community-acquired pneumonia in elderly persons and subjects with impaired respiratory host defences. Untreated, infection with K. pneumoniae may lead to severe pneumonia and sepsis. In addition to being associated with pneumonia, K. pneumoniae is an emerging cause of bacterial meningitis and peritonitis. K. oxytoca has been implicated in neonatal bacteraemia, especially among premature infants and in neonatal intensive care units. Increasingly, the organism is being isolated from patients with neonatal septicaemia.

Problematically, resistance of Klebsiella species to antibiotics is increasing. As such, there is an urgent need for safe and effective treatments for Klebsiella infections and contamination and, in particular, treatments that overcome antibiotic resistance in Klebsiella species.

Staphylococcus is a genus of gram-positive, sphere shaped bacteria which form in grape-like clusters. In humans, they may harmlessly colonise the skin, particularly on the armpits, on the buttocks or in the nose. Staphylococcus is one of the leading infections in hospital settings and several strains of this genera have become antibiotic resistant. Staphylococcus species include S. aureus, S. intermedius, Staphylococcus hyicus, Staphylococcus pseudintermedius and Staphylococcus schleiferi.

In particular, S. aureus is one of the key species responsible for causing infections and is often MDR. Such infections include bacteraemia, endocarditis, skin and soft tissue infections (e.g. impetigo, folliculitis, furuncles, carbuncles, cellulitis, scalded skin syndrome, and others), osteomyelitis, septic arthritis, prosthetic device infections, respiratory tract infections (e.g. pneumonia), gastroenteritis, meningitis, toxic shock syndrome and urinary tract infections. Particularly, S. aureus is one of the leading causes of sepsis. S. aureus has been found to be resistant to a wide range of antibiotics, primarily due to intrinsic resistance.

Two particularly problematic antibiotic resistant S. aureus strains are methicillin-resistant S. aureus (MRSA) and vancomycin-resistant S. aureus (VRSA). While MRSA is resistant to methicillin in particular, more broadly it is characterised by its resistance to p-lactam antibiotics which act by inhibiting cell wall biosynthesis. Several genes and factors have been demonstrated to confer antibiotic resistance on MRSA including Staphylococcal cassette chromosome mec (SCCmec), mecA and the arginine catabolic mobile element (ACME). As indicated by its name, VRSA strains are resistant to the glycopeptide antibiotic vancomycin. Vancomycin- resistance is understood to be mediated by the vanA gene cluster which was transferred from vancomycin-resistant enterococcus, particularly E. faecium or E. faecalis.

In terms of other species which may exhibit MDR, particular mention may be made of the gangrene-causing Clostridium pefringens, Clostridium difficile and Proteus mirabilis.

Bacillus is a genus of Gram-positive, rod-shaped bacteria. Bacillus can reduce themselves into oval endospores and can remain dormant in said state for many years. This is particularly problematic as these spores are resistant to heat, cold, radiation, desiccation, disinfectants and antibiotics which makes them very challenging to successfully eliminate. This resistance allows Bacillus species to survive for many years and especially in a controlled environment. Anthrax is a particularly concerning infection which is caused by the spores of Bacillus anthracis or Bacillus cereus biovar anthracis. Early antibiotic treatment of individuals who have been exposed to anthrax is essential to survival. The fatality rate for inhalation Anthrax is approximately 45%. Concerningly, Bacillus anthracis and Bacillus cereus biovar anthracis are increasingly developing MDR which can delay the commencement of an effective treatment, thus jeopardising patient survival.

Whilst representative, the foregoing examples of MDR bacteria are not exclusive, and they may be found in other families and genera. These include for example, Corynebacteriaceae, for example in the genus Corynebacterium.

Fungal families from which MDR species and strains pose significant problems for human and animal health include, but are not limited to Mucoraceae, Trichocomaceae, Aspergillaceae, Saccharomycetaceae (particularly genus Candida), Cryptococcaceae and Arthrodermataceae. Particular fungal species include Candida albicans, and Trichophyton rubrum, which are involved in infection in skin, soft tissues and mucosal surfaces.

As noted above, MDR microorganisms are classified as microorganisms with non-susceptibility to at least one antimicrobial in three or more antimicrobial classes or categories. In particular, in the case of MDR bacteria, MDR bacteria are classified as bacteria with acquired non-susceptibility to at least one antibiotic in three or more antibiotic classes or categories. In the case of MDR fungi, MDR fungi are classified as fungi with acquired non-susceptibility to at least one antifungal in three or more antifungal classes or categories, which may be anti-fungal antibiotics.

Classes of antibiotics and representative constituents thereof include, but are not limited to the aminoglycosides (e.g. amikacin, gentamicin, kanamycin, neomycin, netilmicin, streptomycin, tobramycin); the carbapenems (e.g. loracarbef); the 1st generation cephalosporins (e.g. cefadroxil, cefazolin, cephalexin); 2nd generation cephalosporins (e.g. cefaclor, cefamandole, cephalexin, cefoxitin, cefprozil, cefuroxime); 3rd generation cephalosporins (e.g. cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone); 4th generation cephalosporins (e.g. cefepime); the macrolides (e.g. azithromycin, clarithromycin, dirithromycin, erythromycin, troleandomycin); the monobactams (e.g. aztreonam); the penicillins (e.g. amoxicillin, ampicillin, carbenicillin, cioxacillin, dicloxacillin, nafcillin, oxacillin, penicillin G, penicillin V, piperacillin, ticarcillin); the polypeptide antibiotics (e.g. bacitracin, colistin, polymyxin B); the quinolones (e.g. ciprofloxacin, enoxacin, gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, norfloxacin, ofloxacin, trovafloxacin); the sulfonamides (e.g. mafenide, sulfacetamide, sulfamethizole, sulfasalazine, sulfisoxazole, trimethoprim- sulfamethoxazole); the tetracyclines (e.g. demeclocycline, doxycycline, minocycline, oxytetracycline, tetracycline); the glycylcyclines (e.g. tigecycline); the carbapenems (e.g. imipenem, meropenem, ertapenem, doripenem, panipenem/betamipron, biapenem, PZ-601); the streptogramins (e.g. quinupristin/dalfopristin, pristinamycin, virginiamycin); the oxazolidinones (e.g. linezolid, posizolid, tedizolid, radezolid); the lincosamides (e.g. lincomycin, clindamycin, pirlimycin); other antibiotics include chloramphenicol, ethambutol, Fosfomycin, isoniazid, metronidazole, nitrofurantoin, pyrazinamide, rifampin, spectinomycin, and vancomycin.

Classes of antifungals and representative constituents thereof include, but are not limited to the allylamines (e.g. naftifine, terbinafine), the azoles (e.g. clotrimazole, miconazole, ketoconazole), the polyenes (e.g. nystatin, amphotericin B, pimaricin) and the echinocandins (e.g. rezafungin, micafungin, anidulafungin, caspofungin).

In particular embodiments, the MDR microorganism is resistant to at least one class of antimicrobial drug selected from the beta-lactams, the sulphonamides, the quinolones, the macrolides, the tetracyclines, the aminoglycosides, the lincosamides, the polypeptide antibiotics, the streptogramins, the oxazolidinones, the allylamines, the azoles, the polyenes and the echinocandins.

It will be noted that the LAB may result in the overcoming of resistance to one or more classes to which the MDR microorganism is resistant, but it is not necessarily implied that resistance is overcome to all of the classes of antimicrobials to which an MDR microorganism may be resistant. Thus, for example, resistance to a macrolide and/or a p-lactam and/or a quinolone may be overcome in an MDR bacteria which is also resistant to other antibiotics e.g. aminoglycosides. Similarly, resistance to an allylamine and/or an azole may be overcome in an MDR fungus which is also resistant to other antifungals e.g. polyenes.

In one embodiment, the MDR bacterium is resistant to at least one an antimicrobial selected from piperacillin-tazobactam, ceftazidime-avibactam, meropenem-vaborbactam, ceftolozane-tazobactam, imipenem/avibactam, cefiderocol, ciprofloxacin, gentamycin, tobramycin, trimethoprim-sulfamethoxazole, meropenem, imipenem, vancomycin and erythromycin.

Individuals at a higher risk of infection with an MDR microorganism include individuals who have an underlying disease or condition, invasive procedures, the use of medical devices, previous prolonged use of antibiotics, repeated contact with healthcare systems (i.e. hospitals) or prolonged periods of hospitalisation, previous MDR infections, age and immune suppressing medications.

As noted above, individuals who have an underlying disease or condition are at higher risk of developing an MDR infection. Examples of such diseases or conditions include individuals with diabetes, chronic kidney disease and wounds (e.g. chronic wounds), including e.g. subjects with SAVI. It has been found that individuals with diabetes, particularly type 2 diabetes which is characterised by high blood sugar, insulin resistance and lack of insulin, are at greater risk of MDR infections in the urinary tract and MDR respiratory infections. Individuals with chronic kidney disease are also at increased risk of developing an MDR infection, particularly if they are undergoing dialysis. Such examples of individuals with underlying diseases or conditions should not be seen as limiting.

Thus, individuals who are at increased risk of developing an MDR infection as discussed above may particularly benefit from the prophylactic or preventative treatment with a LAB to prevent the development of an MDR infection. As mentioned above, individuals may be at higher risk of developing an MDR infection if they are exposed to medical devices. This may be due directly or indirectly to the medical device. The medical device may directly lead to the development of an MDR if, for example, the device is contaminated with an MDR organism. The medical device may indirectly lead to the development of an MDR if, for example, it requires the assistance of a healthcare professional to use who may be transiently contaminated (i.e. they have encountered a previous patient or unsterile item which was contaminated with an MDR microorganism.). Thus, it may be beneficial for the LAB of the invention to be used on patients who use medical devices.

The medical device may include any medical or surgical equipment used in or on the body of a subject. This may include any kind of line, including catheters (e.g. central venous and urinary catheters), prosthetic devices e.g., heart valves, artificial joints, false teeth, dental crowns, dental caps and soft tissue implants (e.g. breast, buttock and lip implants). Any kind of implantable (or "in-dwelling") medical device is included (e.g. stents, intrauterine devices, pacemakers, intubation tubes (e.g. endotracheal or tracheostomy tubes), prostheses or prosthetic devices, lines or catheters). An "in-dwelling" medical device may include a device in which any part of it is contained within the body, i.e. the device may be wholly or partly indwelling.

The aspect of the invention concerning the treatment or prevention of infection by an MDR microorganism with a LAB is of particular utility in the care of hospitalised patients as the risk of contracting a nosocomial infection (commonly known as hospital related/acquired infection or healthcare-associated infection) by an MDR bacterium can be minimised with a prophylactic regime of the LAB defined herein. This aspect of the invention is also of particular utility in the care of subjects suffering from trauma, subjects with a burn and subjects with wounds, all of which, as discussed above, are more susceptible to infection by MDR microorganism than a subject that is not affected similarly.

Prevention or prophylaxis may be maintained at least about 36 hours. In other embodiments, prevention or prophylaxis may be maintained at least about 48 hours, at least about 24 hours, at least about 12 hours, at least about 6 hours, at least about 3 hours or at least about 3 hours. Thus, prevention may be maintained for the duration of a surgical procedure, a dental procedure or an invasive medical procedure. In the case of medical uses, the LAB may be administered in any convenient or desired way, e.g., orally, or topically, or by direct administration to a wound site e.g., by direct infusion or application or introduction of the LAB. In other embodiments it may be administered to the oral cavity, or intranasally or by inhalation, rectally or vaginally. The LAB may thus be administered to, or via, any orifice of the body.

For topical administration, the LAB may be formulated as a liquid e.g., a suspension, freeze-dried cake, or a spray or aerosol (powder or liquid), gel, cream, lotion, paste, ointment or salve, etc. or as any form of dressing, e.g., bandage, plaster, pad, strip, swab, sponge, mat, etc., with or without a solid support or substrate. Further the LAB may be provided on (e.g., coated on) the surface of a medical device such as an implant (e.g., a prosthetic implant), tube, line or catheter, etc.

Oral administration forms include powders, tablets, capsules and liquids etc. Further the LAB may be provided on (e.g. coated on) the surface of a medical device such as an implant (e.g. a prosthetic implant), tube, line or catheter etc.

The LAB can be formulated for topical or oral administration to treat any soft tissue or mucosal surface. For example, for topical application, the LAB may be provided as a lotion or a lotion-soaked wound dressing.

Where LAB are provided in lyophilized or freeze-dried form, it may be desirable to reconstitute, or resuspend, them prior to administration e.g., prior to or during use. This may depend on the format in which the LAB is used. For example, in the case of some wounds there may be sufficient liquid present to allow for the LAB to be reconstituted/resuspended and become active. However, in other embodiments it may be desirable to provide a liquid for reconstitution (or alternatively expressed, for suspension or resuspension) of the LAB. This may be provided in a separate vessel or container (e.g., as part of a kit or combination product or in a separate compartment of a container, or vessel or device). The liquid may be any suitable liquid for reconstitution or suspension of freeze-dried bacteria, e.g., water, or an aqueous solution, or buffer or growth or culture medium.

Viable LAB may also be comprised in a hydrocolloid, for example a natural gelatin. The LAB can be incorporated by crosslinking into hydrocolloid e.g., gelatin films, plasticized and dried, retaining viability during storage until hydration. Viable LAB may also be encapsulated within cross-linked electrospun hydrogel fibers. In this format the LAB need not be freeze-dried. For wounds in the mouth (e.g., on the gums), the LAB can be administered in a high viscous paste.

Specifically, formulations for topical administration to the skin can include ointments, creams, gels, and pastes to be administered in a pharmaceutically acceptable carrier. Topical formulations can be prepared using oleaginous or water- soluble ointment bases, as is well known to those in the art. For example, these formulations may include vegetable oils, animal fats, and more preferably semisolid hydrocarbons obtained from petroleum. Particular components used may include white ointment, yellow ointment, acetyl esters wax, oleic acid, olive oil, paraffin, petrolatum, white petrolatum, spermaceti, starch glycerite, white wax, yellow wax, lanolin, anhydrous lanolin, and glyceryl monostearate. Various water-soluble ointment bases may also be used including, for example, glycol ethers and derivatives, polyethylene glycols, polyoxyl 40 stearate, and polysorbates.

The LAB can be provided in and/or on a substrate, solid support, and/or wound dressing for delivery of active substances to the wound. The solid support or substrate may be a medical device or a part thereof. As used herein, the term “substrate” or “solid support” and “wound dressing” refer broadly to any substrate when prepared for, and applied to, a wound for protection, absorbance, drainage, etc. An embodiment provides a wound healing material or dressing comprising the LAB for use in the treatment of an infection MDR microorganism. Alternatively, the vehicle may be a plaster or bandage. Also included are any of the numerous types of substrates and/or backings that are commercially available, the choice of wound healing material will depend on the nature of the wound to be treated.

The most commonly used wound dressings include: transparent film dressings (e.g., synthetic films made of polyurethane, polyamide, or gelatin, which are permeable to water vapor oxygen and other gases but impermeable to water and bacteria); hydrocolloids (e.g. hydrophilic colloidal particles bound to polyurethane foam); hydrogels (cross-linked polymers containing about at least 60% water); foams (hydrophilic or hydrophobic e.g., polymeric foam dressings produced through the modification of polyurethane foam); calcium alginates (e.g. non-woven composites of fibres from calcium alginate from the phycocolloid group), and cellophane (cellulose with a plasticizer).

The shape and size of a wound may be determined and the wound dressing customized for the exact site based on the measurements provided for the wound. As wound sites can vary in terms of mechanical strength, thickness, sensitivity, etc., the substrate can be molded to specifically address the mechanical and/or other needs of the site.

As noted above, the LAB may be provided for administration in any convenient or desired form, e.g. fresh, for example, as an active or growing culture or in lyophilized or freeze-dried form. The LAB may be lyophilised or freeze-dried by any technique known in the art.

Lyophilised preparations of LAB are convenient for use, and will typically include a cryoprotectant, or more particularly a lyoprotectant. Many such protectants suitable for use with microorganisms are known in the art, and any of these may be used. Typically, they include sugars, such as sucrose, and sugar alcohols, e.g. sorbitol.

Our experiments have shown that LAB used directly from lyophilised products are as effective, in terms of antimicrobial effect, as freshly grown optimally vital LAB. More particularly, we have observed that lyophilised transformed LAB are as effective as the corresponding non-transformed or wild-type species, and that is comparable to the effect of freshly grown bacteria.

Further, our data appear to indicate that reconstituted lyophilised bacteria may perform better than their freshly cultured counterparts. Without wishing to be bound by theory, we believe that this effect may be contributed to, at least in part, by the components present in lyophilised preparations, and particularly the protectant components.

In the course of testing lyophilised preparations we have found that the LAB may exhibit a tolerance to high levels of sucrose. In other words, the LAB may retain its biological activity when exposed to high sucrose concentration and also rapid and immediate changes in sucrose levels. Thus, the provision of the LAB in a composition comprising sucrose may impart several benefits. In particular, the sucrose may provide an energy source for the LABs once they are reconstituted. Alternatively, or additionally, a high concentration of sucrose is hypothesised to “stun” or temporarily inhibit the MDR microorganisms due to the high osmolarity of sucrose solutions. This may place the MDR microorganism into a state of hyperosmotic stress which may inhibit their growth.

Accordingly, the LAB may advantageously be provided in a composition (particularly a pharmaceutical composition) comprising a sugar or sugar alcohol. In another embodiment the composition may be a lyophilised, or a reconstituted lyophilised, composition comprising a cryoprotectant or lyoprotectant. The sugar may for example be sucrose, glucose, dextrose, lactose, trehalose, maltose, raffinose, fructose and galactose, and in particular sucrose. The sugar alcohol may be sorbitol.

In a further embodiment, the composition may further comprise polyols, polymers, salts, proteins, acids and surfactants.

The polyols may for example be mannitol, sorbitol, xylitol, and erythritol.

The polymers may for example be maltodextrin, polyethylene glycol (PEG) and dextran.

The salts may for example be sodium chloride and potassium phosphate.

The acid may for example be ascorbic acid.

The surfactants may for example be polysorbate 80.

In particular embodiments, the LAB may be used or provided in combination with a second therapeutic agent. This may be any agent which may be effective or of benefit in the treatment or prevention of the MDR infection. Typically, this will be an anti-microbial agent, and particularly an antibiotic, which includes antifungal antibiotics. Alternatively speaking, it may be seen that an antibiotic may be used in combination with the LAB.

The antibiotic may be selected from any antibiotic disclosed above. Preferred antibiotics may be selected from the aminoglycosides (e.g. amikacin, gentamicin, kanamycin, neomycin, netilmicin, streptomycin, tobramycin); the carbapenems (e.g. loracarbef); the 1st generation cephalosporins (e.g. cefadroxil, cefazolin, cephalexin); 2nd generation cephalosporins (e.g. cefaclor, cefamandole, cephalexin, cefoxitin, cefprozil, cefuroxime); 3rd generation cephalosporins (e.g. cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone); 4th generation cephalosporins (e.g. cefepime); the macrolides (e.g. azithromycin, clarithromycin, dirithromycin, erythromycin, troleandomycin); the monobactams (e.g. aztreonam); the penicillins (e.g. amoxicillin, ampicillin, carbenicillin, cioxacillin, dicloxacillin, nafcillin, oxacillin, penicillin G, penicillin V, piperacillin, ticarcillin); the polypeptide antibiotics (e.g. bacitracin, colistin, polymyxin B); the quinolones (e.g. ciprofloxacin, enoxacin, gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, norfloxacin, ofloxacin, trovafloxacin); the sulfonamides (e.g. mafenide, sulfacetamide, sulfamethizole, sulfasalazine, sulfisoxazole, trimethoprim- sulfamethoxazole); the tetracyclines (e.g. demeclocycline, doxycycline, minocycline, oxytetracycline, tetracycline); the glycylcyclines (e.g. tigecycline); the carbapenems (e.g. imipenem, meropenem, ertapenem, doripenem, panipenem/betamipron, biapenem, PZ-601); the streptogramins (e.g. quinupristin/dalfopristin, pristinamycin, virginiamycin); the oxazolidinones (e.g. linezolid, posizolid, tedizolid, radezolid); the lincosamides (e.g. lincomycin, clindamycin, pirlimycin); other antibiotics include chloramphenicol, ethambutol, Fosfomycin, isoniazid, metronidazole, nitrofurantoin, pyrazinamide, rifampin, spectinomycin, and vancomycin.

More preferably the antibiotic is selected from piperacillin-tazobactam, ceftazidime-avibactam, meropenem-vaborbactam, ceftolozane-tazobactam, imipenem/avibactam, cefiderocol, ciprofloxacin, gentamycin, tobramycin, trimethoprim-sulfamethoxazole, meropenem, imipenem, vancomycin and erythromycin.

The LAB may be used in combination with a single antibiotic or a mixture (multiplicity/plurality) of different antibiotics. Thus, for example, a combination of different antibiotics (e.g. two or more) may be used. The MDR microorganism may be sensitive to the further antibiotic(s) used or may be resistant to the further antibiotic(s) used.

The antimicrobial effects of the LAB and antibiotic may be additive or more than additive. Further the use of the LAB may allow an antibiotic which is not effective when used on its own to exhibit an antimicrobial effect. Thus, the LAB may enhance, or potentiate the effect of antibiotic, or as noted above it may allow resistance to an antibiotic to be overcome at least to a degree.

In certain embodiments a synergistic effect may be seen.

As can be seen from the Examples, the LAB and antibiotic may have a combinatorial, e.g. synergistic, effect that makes microorganisms (e.g. bacteria) with a phenotype that is resistant to an antibiotic more susceptible to that antibiotic. In particular, a synergistic effect between the LAB and an antibiotic may be observed in embodiments wherein the LAB is modified (e.g. transformed) to express a heterologous protein or to over-express an endogenous protein (including CXCL12, CXCL17 and/or Ym1 , or any other protein), as described above. In one embodiment, the LAB which acts in synergy with an antibiotic may be Limosilactobacillus reuteri, particularly Limosilactobacillus reuteri R2LC. The Limosilactobacillus reuteri may be modified, and in particular may be modified to express a heterologous protein, or more particularly a therapeutic protein, e.g. a wound-healing, anti-microbial, anti-inflammatory, anti-fibrotic, or immunomodulatory protein (or more generally any of the proteins discussed above). In one embodiment the Limosilactobacillus reuteri may be transformed to express CXCL12. In a further embodiment, the LAB may be ILP100 (Limosilactobacillus reuteri R2LC _pSIP_CXCL12). In a further embodiment, the LAB which acts in synergy with an antibiotic may be Umosilactobacillus reuteri, particularly Limosilactobacillus reuteri R2LC, transformed to express CXCL17. The LAB as described herein, including particularly Limosilactobacillus reuteri, may act synergistically with piperacillin/tazobactam, ceftazidime/avibactam and/or meropenem/vaborbactam.

The above-described synergistic effect may be more pronounced in specific bacterial genera and/or specific bacterial species, including those discussed herein. Indeed, the inventors have found that combining Limosilactobacillus reuteri modified to express CXCL12 with various different antibiotics (e.g. piperacillin/tazobactam (PZT), ceftolozane/tazobactam (CT), ceftazidime/avibactam (CZA), meropenem/vaborbactm (MEV), imipenem/avibactam (IMR), cefiderocol (FDC)) has a synergistic effect against MDR pathogens. That is to say, the combination of Limosilactobacillus reuteri modified to express CXCL12 with antibiotics resulted in a synergistically increased pathogen clearance. The synergistic effect (i.e. of the LAB and antibiotics) was particularly notable in the bacterial genus Pseudomonas. In other words, the combined effect of the LAB (particularly Limosilactobacillus reuteri expressing CXCL12) and an antibiotic may be at least 2-fold as effective against a pathogen when compared to the efficacy of the LAB or antibiotic alone. Such comparisons can be conducted using any appropriate means in the art (e.g. spot dropping and disk diffusion as discussed herein). It will be appreciated that the synergistic effect of the LAB and antibiotic may be greater than 2-fold, e.g. that it may be 3-, 4-, 5-, 6- or 7-fold more effective when compared to the efficacy of the LAB or antibiotic alone. In a particular embodiment the Limosilactobacillus reuteri in the above-noted synergistic combinations is Limosilactobacillus reuteri R2LC. This synergistic effect may be particularly beneficial in a range of diseases, particularly cystic fibrosis.

In one embodiment the LAB will measurably reduce the MIC value of the resistant microorganism to the antibiotic, e.g. the MIC value will be at least 50%, 25%, 20%, 15%, 10%, 5%, 2% or 1% of the MIC value of the microorganism for the antibiotic without the LAB.

Furthermore, the LAB may potentiate the effect of an antibiotic (or increase or improve its efficacy). It may render usable (or effective) an antibiotic previously thought not to be usable/effective against a particular organism, or an antibiotic which is not normally effective against a given organism (e.g. bacterium or bacterial species in question). It may also enable an antibiotic to be used at a reduced dose. Alternatively, the antibiotic may potentiate the effect of the LAB.

The microorganism may be contacted with more than one antibiotic. The additional antibiotic(s) can be any antibiotic, e.g. those listed above. The additional antibiotic(s) may be an antibiotic to which the microorganism is susceptible. The additional antibiotic(s) may be an antibiotic to which the microorganism is resistant. The additional antibiotic(s) may be used together with (in conjunction or combination with) the first or other antibiotics and/or the LAB. More particularly, the step of using may comprise contacting the microorganism with the LAB at the same or substantially the same time or prior to contacting the microorganism with some or all of the antibiotics.

Thus, the antibiotic(s) may conveniently be applied or administered simultaneously with the LAB, or immediately or almost immediately before or after the LAB. However, the antibiotic(s) may be applied or administered at a different time point e.g. least 1 hour, at least 3 hours, at least 6 hours after the LAB. It is within the skill of the medical practitioner to develop dosage regimes which optimise the effect of the LAB and antibiotic. In these embodiments the antibiotic(s) can be applied or administered with or without a further application of the LAB. The LAB can be applied or administered in a plurality of applications prior to or with the antibiotic(s). In other embodiments the antibiotic(s) may conveniently be applied or administered before the LAB, e.g. at least 1 hour, at least 3 hours, at least 6 hours before the LAB. In these embodiments the LAB can be applied or administered with or without a further application of the antibiotic(s). The antibiotic(s) can be applied or administered in a plurality of applications prior to or with the LAB. The skilled persons can easily determine what would be an appropriate dosing regime for the LAB and antibiotic(s) he intends to use.

In long term treatments the LAB and the antibiotic can also be used repeatedly. The LAB can be applied as frequently as the antibiotic but will typically be less or more frequent depending on the dose of the antibiotics and route of administration, e.g. oral or intravenous. The frequency required will depend on the location of the MDR bacteria, colony composition and the anti-microbial used and the skilled person is able to optimise the dosage or usage patterns to optimise results. In the case of some infections, e.g. in soft tissues or wounds, it may not be possible to determine the exact tissue concentration of the administered antibiotic or determine dose precisely at the site of infection where it is needed. However, it is within the routine skill of a clinician to manage such conditions and determine appropriate dosages to the best of practice. Tissue levels are rarely measured.

For example, the antibiotic may be applied or administered prior to, during or after surgery and the LAB may be applied to the incision wound. It is hypothesised that this may prevent an MDR infection from forming at the incision site with greater efficacy than if the LAB was to be used alone.

In an advantageous embodiment the LAB and/or the antibiotic may be used or applied after physical removal or reduction (e.g. debridement) of the colony/population comprising the MDR microorganism causing the infection at the location undergoing treatment.

Following removal of, or an attempt to remove, the colony/population comprising the MDR microorganism, the location may be contacted with the LAB for between 0 and 24 hours, particularly 2 and 12 hours, more particularly 4 and 8 hours, most particularly 5 and 7 hours, e.g. 6 hours. Following this, the antibiotic may be applied. Such a scenario may be desirable or particularly applicable in a clinical setting. In the case of wounds infected by an MDR microorganism, the duration of incubation can be conveniently be designed to correspond to scheduled changes of the wound dressing.

Physical removal of the colony/population comprising the MDR microorganism can be carried out with any suitable surgical, mechanical or chemical means. Conveniently this can be the use of a liquid, gel, gel-sol, semisolid compositions or gas applied at pressure to the colony/population, sonication, laser, or by abrasive implement. A composition used in the removal itself or as a wash solution before, during or afterwards may conveniently contain the LAB and/or the antibiotic.

Accordingly, in one specific embodiment there is provided a debridement or wash composition e.g. solution for wounds containing the LAB as herein defined, and/or an antibiotic, particularly any antibiotic as herein defined for use as described herein. Such a debridement composition will typically be a sterile solution, particularly an aqueous sterile solution or an oil-based sterile solution, and may additionally contain proteolysis enzymes (e.g. collagenase, trypsin, pepsin, elastase), an abrasive solid phase (e.g. colloidal silica, ground pumice, ground plant or animal shell).

A further supporting but non-limiting disclosure is provided in the Examples below with reference to the figures.

Description of Figures

Figure 1. Representative images of agar plates showing the dose-relevant growth inhibition data from isolate KR6027 where ILP100 was precoated in low dose (5 x 10⁷ CFU) or high dose (5 x 10⁸ CFU) for 3 or 24 hours before addition of the KR6027.

Figure 2. Representative images of agar plates showing spot drop assays in P. aerigunosa (KR6004, KR6006, KR6008 and KR6165), E. cloacae (KR6041), P. mirabilis (KR6091) and A. baumannii (KR6082, KR6132 and KR614plated at 1 x 10⁶ CFU/ml treated with ILP100 (IP; left) or Limosilactobacillus reuteri R2LC (WT; right).

Figure 3. Illustration of spot-drop method showing the steps used to measure the antimicrobial zone inhibition of pathogens by the LABs. The LAB species were measured alone in the triangular configuration shown above.

Figure 4. Illustration of spot-drop method showing the steps used measure the antimicrobial zone inhibition of pathogens by ILP100. This illustrates the “halfmoon” configuration adopted for the direct comparison with antibiotic (Abs) discs. A small addition to step 3 allowed for the placement of Abs discs immediately before adding the pathogen inoculated overlay.

Figure 5. (a) Representative images of agar plates showing disk diffusion and spot dropping measurement comparing the antibiotic effect of ILP100^|OW (left) versus PTZ 30/6, CZA 30/20 or MEV 20/10 (right, top to bottom) in MDR Acinetobacter baumannii (KR6038) isolated from war victims in Ukraine in three concentrations of 1 x 10⁶ CFU (left) 1x10⁵ CFU (middle) and 1x10⁴ CFU (right); (b) A graph showing the results of the disk diffusion and spot dropping experiment described in (a). Figure 6. A graph showing the results from the co-culture experiments. The solid black line represents the highest treatment ratio I LP100: Pathogen in each case, 1000:1. The solid gray line represents the lowest treatment ratio, 100:1. The dashed-gray line shows the normal growth of the pathogen in the same media if not inhibited by any treatment. Note: The solid lines are derived from the dashed line data; therefore, dashed gray line cannot be directly comparable to the solid lines. This graph displays one Enterobacter cloacae MDR isolate, and a similar trend was observed for all isolates tested; all co-culture results show that the MDR isolates were killed over time to a level not detected for the 1000:1 ratio, meaning a CFU log reduction of >5 (greater than 99.999%).

Figure 7. A representative graph showing the mean cleared area (MAC) in P. aeruginosa plated at 1 x 10⁶ CFU with piperacillin/tazobactam (PZT), ceftazidime/avibactam (CZA), meropenem/vaborbactm (MEV), ceftolozane/tazobactam (CT), imipenem/avibactam (IMR), cefiderocol (FDC) or ILP100. Some synergistic influence of the same antibiotics tested on the same plate as ILP100 can be seen when certain antibiotics are present (ILP100 + Group 1 abs + MDR and ILP100 + Group 2 abs + MDR).

Figure 8. Bar graph showing the percentage survival at 5 hours of L. reuteri, S. aureus, S. Pyrogenes and S. Gordonii treated with 0.00%, 1.18%, 2.25%, 4.50%, 9.50% and 19.00%.

Figure 9. A graph showing the mean area cleared zone (MAC, mm²) of MDR and non-MDR isolates treated with ILP100^mid (5 x 10⁷ CFU/drop). Results are mean ± SD, and differences were evaluated using Welch’s t test, where p < 0.05 (*) was significant [p < 0.005 (**), p < 0.0005 (***), p < 0.0001 (****)].

EXAMPLES

Example 1 - Pre-treatment with LAB limits and prevents bacterial growth Dose-relative growth inhibition experiments were conducted to investigate the bactericidal effects of ILP100 (Limosilactobacillus reuteri R2LC transformed with plasmid pSIP to express CXCL12 as described in WO2016/102660) in a clinically relevant situation. The entire surface area of an LSM agar plate which had been inoculated with 10⁶ CFU of the pathogen and incubated for 24 hours was coated with ILP100 (5x10⁷ CFU) or ILP1OO (5x10⁸ CFU) using clinically relevant doseconcentration. This experiment may reflect the situation of, for example, a diabetic foot ulcer or surgical wound that is cleaned and treated with ILP100 to accelerate the wound healing and limit complications including infections.

For the plates pre-incubated with ILP100 (ratio of 100:1) for 3 hours, growth of the pathogen was seen in all 17 samples. For the plates pre-incubated with ILP100 at 5 x 10⁷ for 24 hours, growth of the pathogen was completely inhibited in all 17 samples (see Table 1).

For the plates pre-incubated with ILP100 (ratio of 1000:1) for 3 hours, complete clearance was detected for 7 pathogens and partial growth inhibition of the pathogen was seen in 10 samples. For the plates pre-incubated with ILP100 at 5x10⁸ CFU for 24 hours, growth of the pathogen was completely inhibited in all 17 samples (see Table 2).

A representative image of the inoculated and ILP100 pre-treated agar plates can be seen in Figure 1.

Table. 1 Prevention of infections by ILP100 precoating in a dose of 5x10⁷ CFU.

* Partially cleared area of the plate was measured in percentage of area cleared in relation to the entire plate area. (- )=No; (X)=Yes (or designated result). Table 2. Prevention of infection by pathogens by ILP100 precoating in a dose of 5x10⁸CFU

* Partially cleared area of the plate was measured in percentage of area cleared in relation to the entire plate area. (- )=No (X)=Yes (or designated result)

Example 2 - Standardized Spot-Drop Zone of Inhibition demonstrates the antibiotic effect of WT LABs

The spot-drop technique was used to mimic standardized antibiotic disc diffusion methods which are typically employed in susceptibility studies with standard antibiotics.

To evaluate the antimicrobial effect of different LABs (L. Lactis, L. Rhamnosus, L. Reuteri and ILP100) on the pathogens, different strengths of both the LABs and the pathogen were used and the Mean Area Cleared was measured. The size of the LAB spot drops is larger than the antibiotic discs and an average of 50 measurements resulted in average size of a spot to 71.58 ± 10.45 SD mm². In general, the low dose spread out slightly easier (due to less viscosity) and therefore normally resulted in a slightly larger spot. The high/mid dose spot sizes were more similar to each other.

The inhibition zone induced by mid dose (5 x 10⁷ CFU) of L. Lactis and L. Rhamnosus resulted in clearance for all pathogen isolates in the range of 27- 379mm², 92-541mm² and 158-540mm² (1x10⁶ and 1x10⁴ CFU, respectively), whereas low dose (5 x 10⁶ CFU) L. Lactis and L. Rhamnosus resulted in 29-356 mm², 105-436 mm² and 154-669mm² (1x10⁶ and 1x10⁴ CFU, respectively). See Tables 3 and 4. Table 3. Mean area cleared by L. lactis in two different doses at 18-20 hours

NA= not available

Table 4. Mean area cleared by L rhamnosus in two different doses at 18-20 hours

NA= not available Example 3 - The antibiotic effect of WT L. reuteri is comparable to ILP100.

The aforementioned spot-drop technique used in Example 2 was conducted to compare the efficacy of WT L. reuteri and ILP100. The inhibition zones between the WT L. reuteri and ILP100 were evaluated in a small pilot study. As no major differences were observed between them, ILP100 was used for all further experiments (See Table 5).

Table 5. Mean area cleared by L. reuteri vs ILP100 at 18-20 hours

NA= not available

Example 3 - Standardized Spot-Drop Zone of Inhibition demonstrates the antibiotic effect of I LP 100 The antibiotic effects of I LP100^high (1 x 10⁸ CFU/drop), ILP100^mid (5 x 10⁷ CFU/drop) and ILP100^|OW (5 x 10⁶ CFU/drop) dose was demonstrated, where higher ILP100 doses generally resulted in larger clearance zones. The inhibition zone induced by ILP100^high resulted in clearance for all pathogen isolates in the range of 35-258mm² and 37-281 mm² and 40-426 mm² at 1 x 10⁶, 1 x 10⁵ and 1x10⁴ CFU, respectively. ILP100^mid resulted in clearance for all pathogen isolates in the range of 56-273 mm², 62-308 mm² and 77-457 mm² whereas ILP100^|OW resulted in 37-205mm2 and 59-288mm² and 50-361 mm², respectively (Table 6).

Pseudomonas aeruginosa and one Klebsiella pneumoniae isolate displayed larger clear zones in general. The rest of the species/isolates displayed smaller cleared zones in comparison. Also, larger cleared zones were measured with lower levels of pathogen inoculation for both Pseudomonas aeruginosa and Acinetobacter baumannii isolates. No difference was observed between the levels of pathogen inoculation for Enterobacter cloacae, Klebsiella pneumoniae, Proteus mirabilis and Staphylococcus aureus, except for the two Klebsiella pneumoniae isolates, where a difference was observed between isolates, no significant difference was observed between isolates from the other species. For all pathogens, even ILP100^|OW demonstrated clearing effects, for most pathogens it was similar to ILP100^high.

Table 6. Mean area cleared by ILP100 in three different doses at 18-20 hours.

NA= not available Example 4 - ILP100 exerts an antibiotic effect on multidruq resistant bacteria alone and in combination with antibiotics

The spot-drop technique was used to measure antibiotic effect of ILP100^high (1 x 10⁸ CFU), ILP100^mid (5 x 10⁷ CFU) and ILP100^|OW (5 x 10⁶ CFU) against MDR isolates.

The antibiotic effect of ILP100 was also compared to the effect of disc antibiotics that the MDR pathogens were susceptible to. When evaluating the antimicrobial effect of ILP100 on MDR bacteria, the initial approach was to directly compare the antimicrobial effect of ILP100 to six antibiotics on the same plate (previously verified) using the modified disc diffusion method (see Figure 3).

The method included dividing the six antibiotics into 2 arbitrary groups, with 3 in each group: group 1 (PZT, CZA, MEV) and group 2 (CT, FDC, IMR). Unexpectedly, the antimicrobial effects of ILP100 were increased for most isolates when analyzing both groups of antibiotics with a consistently larger effect using group 2 antibiotic (CT, FDC, IMR).

The inhibition zone induced by ILP100^high resulted in clearance for all MDR isolates, while screening group 1 antibiotics (PZT, CZA, MEV) in the range of 125-470mm² and 185-479mm² and 214-509mm² at 1 x 10⁶, 1 x 10⁵ and 1x10⁴ CFU, respectively. ILP100^mid resulted in clearance for all pathogen isolates in the range of IOS-

311 mm², 158-475mm² and 185-511 mm² whereas ILP100^|OW resulted in 56-320mm² and 110-465mm² and 178-484mm² respectively (Table 7). This resulted in a raised lower clearance level by an average -60% compared to non-MDR pathogens.

Table 7. Mean area of MDR pathogens cleared by ILP100 in three different doses at 18-20 hours together with antibiotic discs (PTZ, CZA, MEV).

5 The inhibition zone induced by ILP100^high resulted in clearance for all MDR isolates, while screening group 2 antibiotics (CT, FDC, IMR) in the range of 216-464mm² and 286-508mm² and 353-527mm² at 1 x 10⁶, 1 x 10⁵ and 1 x 10⁴ CFU, respectively. ILP100^mid resulted in clearance for all pathogen isolates in the range of 310-486mm², 355-507mm² and 403-590mm² whereas ILP100^|OW resulted in 66-320mm² and 122- 10 452mm² and 202-469mm² respectively (Table 8). This resulted in a raised lower clearance level by an average -60% compared to non-MDR pathogens, but an - 40% raised lower clearance level than with the group 1 antibiotics. Table 8. Mean area of MDR pathogens cleared by ILP100 in three different doses at 18-20 hours together with antibiotic discs (CT, FDC, IMR).

Overall, ILP100^mid gave consistently larger clear zones than ILP100^|OW but was also occasionally better or similar to ILP100^high, indicating a maximum effect was reached already at ILP100^mid.

To confirm that the antibiotic discs were providing for an enhanced antimicrobial effect overall, ILP100^mid dose was trialed alone on plates inoculated with a subset of the MDR isolates. ILP100^mid resulted in clearance for all pathogen isolates in the range of 138-286mm², 245-358mm² and 286-450mm² respectively (Table 9). This resulted in a comparable level to group 1 antibiotics (60 vs 70% increase), but the group 2 antibiotics sustained a 30% raised lower clearance level than the MDR isolates alone.

Table 9. Mean area of MDR pathogens cleared by ILP100 in three different doses at 18-20 hours without antibiotic discs on the plate.

The above-described experiment to determine the mean area cleared by ILP100 in three different doses at 18-20 hours without antibiotic discs on the plate was also performed on non-MDR isolates. Interestingly, when comparing the efficacy of ILP100^mid (5 x 10⁷ CFU) against both non-MDR and MDR isolates, it was found that the MDR pathogens were significantly more sensitive to ILP100 than the respective non-MDR isolates were (Figure 9).

5 - Co-qrowth with ILP100 results in 99.999% killing of the over

12 hours

Growth of each pathogen over 12 hours in LSM broth resulted in an average increase of 2-4 logs which represents a 100-10000x increase in number of pathogens, depending on the pathogen isolate, reflecting a situation of an infection of optimal growth if not inhibited (Figure 6). ILP100 was co-cultured with pathogenic isolates in a ratio of 100:1 or 1000:1 (ILP100/pathogen) in LSM broth to determine if ILP100 was able to inhibit the growth of non-MDR and MDR pathogen isolates. The results of these experiments can be seen in Table 10 and 11.

The results demonstrated that co-culturing ILP100 with a subset of isolates inhibited the pathogenic species in all co-habitation trials by 5-7 log (log 7 is limit of method) when the ratio of treatment to pathogen was at least 1000:1 (or higher) and reduced growth by up to 4.3 log when ratio was approximately 100:1 after a treatment for 12 hours. Specifically, the 1000:1 concentration ratio of ILP100 inhibited the growth of MDR pathogens by between log 5.7 to 6.4 CFU/mL, while the 100:1 ratio resulted in an inhibition of up to log 4.3 CFU/mL. Additionally, most pathogens exhibited a detectable reduction in growth at 3 and 6 hours (Figure 6; full data set not shown). Thus, the inhibition effect on growth occurred in a dose/concentration dependent manner. A similar dramatic effect was observed with all non-MDR pathogen isolates and MDR pathogen isolates.

This effect was seen not only in the LSM media, but also in the simulated body fluid substrate as well. All isolates in the subset used gave similar results.

Table 10. Growth rates (delta log™) of non-MDR pathogens following co-culture with

ILP100 after 12 hours

* control of isolate growth without the presence of ILP 100 used to generate total log reduction values for treatment scenarios 1000:1 and 100:1. Table 11. Growth rates (delta log™) of MDR pathogens following co-culture with

ILP100 after 12 hours

* control of isolate growth without the presence of ILP 100 used to generate total log reduction values for treatment scenarios 1000:1 and 100:1.

Example 6 - Combined administration of ILP100 and antibiotics exhibits a synergistic effect on pathogenic bacteria and MDR bacteria

It was determined that resistance of two isolate results in little to no clear zone for several antibiotics, while ILP100 has a measurably better effect. Cefiderocol (FDC) gives better than expected results in our lab, otherwise results are as expected with the other antibiotic discs tested for these isolates. FDC is included in the group 2 antibiotics.

Some synergistic influence of the same antibiotics tested on the same plate may be seen in the additively larger clear zones when certain antibiotics are present (Table 8 and 9). This data has been guantified and is presented in Figure 7. The ILP100 clear zone increases against MDR pathogens compared to less resistant pathogenic isolates of the same species.

Example 7 - LAB exhibit tolerance to high levels of sucrose

To study the effect of a local change of osmolality, reflecting the situation in the microenvironment as a result of treatment with the formulated lyophilized ILP100, the growth media with isolates of three known wound pathogens, S. aureus, S. pyogenes and S. gordonii cultures was changed to media containing 0, 1.18%, 2.25%, 4.5%, 9.5% or 19% sucrose and then incubated in 37°C for 5 hours. The growth inhibition due to change of osmolality was compared to the condition with 0% sucrose serving as the base-case of no change in osmolality.

L. reuteri was the least affected by change in osmolality and was only partially inhibited by the higher concentrations, whilst the three wound pathogens, S. aureus, S. pyogenes and S. gordonii were almost completely inhibited by a change to 4.5%, 9,5% and 19% sucrose for 5 hours (Figure 8).

The tolerance to high levels of sucrose of LABs is critical for two reasons, 1) sucrose is commonly used as protectant during lyophilization processes and energy source for the LABs once resuscitated, and 2) provides a “stunning” effect to pathogenic bacteria due to the high osmolarity of sucrose solutions. These two factors together result in a head-start for the LABs, thus providing an environment that favours LAB and disfavours pathogens.

MATERIALS AND METHODS

Bacterial strains

The soft-tissue isolates (KR6000-6200; shown in Table 12) originated from war victims isolated in Ukraine and were kindly donated by Professor Kristian Risbeck, Lund University Sweden. The S. aureus JE2, DA28823, (Fey et al., 2013) is a kind donation from Professor Dan Andersson, Uppsala University, Sweden. The S. aureus strain EV101 is Zen-29 purchased commercially from Perkin Elmer. Two human wound isolates were obtained from Culture Collection of University of Gothenburg (CCUG) EV102 which is S. Pyogenes and EV103 which is S. gordonni. Lactic acid bacteria (LABs) used: The LAB isolates (SR1001 and SR1002, Lactococcus lactis MG1363 and Lactobacillus rhamnosus Kx151A1, respectively) were kindly donated by Professor Stefan Roos, Swedish University of Agricultural Sciences, Sweden. ILP100 (Limosilactobacillus reuteri R2LC _pSIP_CXCL12) and ILP101 (Limosilactobacillus reuteri R2LC _pSIP_CXCL17) are drug candidates developed by Ilya Pharma AB, Sweden (as prepared in WO2016/102660).

Table 12 - Bacteria strains, species and MDR classification used

Verification of multi drug resistance

The strains detailed in Table 12 were tested functionally to identify multidrug resistance using common antibiotics including piperacillin/tazobactam (PZT), ceftolozane/tazobactam (CT), ceftazidime/avibactam (CZA), meropenem/vaborbactam (MEV), imipenem/avibactam (IMR), cefiderocol (FDC), ciprofloxacin (CIP), gentamycin (GEN), tobramycin TOB), trimethoprim/sulfamethoxazole(TSU), colistin (COL), meropenem (MER), imipenem (IMI), vancomycin (VAN) and erythromycin (ERM). The results of this experiment can be seen in Table 13. It should be noted that the bacterial strains that have not been identified as MDR may be resistant to three or more antimicrobials that have not been tested and still may therefore be MDR in nature. It will be understood that extensive testing with every antimicrobial is not feasible. ELICAST (European Committee on Antimicrobial Susceptibility Testing) were followed when performing disc diffusion assays and broth dilution was performed according to ISO (International Organization for Standardization). For the currently reported results with LABs, the EUCAST disc diffusion assay (using 6 mm discs) was adapted to utilize a soft-agar overlay with media suggested by ISO for use with antimicrobial testing for lactobacilli. The reason for the adaptation was to be able to include both antibiotic disc diffusion testing and LAB antimicrobial properties within the same agar plate. The method is otherwise similar to the EUCAST methods.

Table 13. Measured mean area cleared by the six antibiotic discs

Bold = corresponds to a resistant interpretation according to EUCAST v. 14 Breakpoint Tables

Media

LSM media and agar defined as 90% Iso-sensitest (Oxoid; Thermofisher) and 10% MRS (Oxoid; Thermofisher) was used as it allows the growth of the pathogens investigated and is suited for lactic acid bacteria susceptibility testing and recommended in the ISO 10932/1 DF233 standard for antimicrobial testing of lactobacilli specifically. For some experiments, MRS agar, prepared according to manufacturer's instruction (Merck) was used for recovery of LABs and ILP100.

The spread, or plating, of bacterial samples was performed using glass beads (3 mm). Approximately 10 beads were used per plating and the beads were randomly moved around the plate until no liquid could be detected. The plates used were Sarstedt 92 x 16 mm.

Dose-Relative Growth Inhibition

ILP100 at two doses of 5x10⁸ and 5x10⁷ (CFU/plate) of was spread evenly across the entire surface area of standard petri-dish agar plate (LSM). The dose-inoculate was incubated for either 3 or 24 hours at 37°C under anaerobic conditions. The soft- agar overlay (5 mL) was inoculated with ~1 x 10⁶ CFU MDR pathogens using a 1000X dilution of overnight culture and incubated for another 24 hours. Plates were visually inspected and imaged. Assessment of plates were done by classifications into either “Totally Cleared”, “Partially Cleared” and “Totally Overgrown (by the MDR pathogen)”, see examples in Figure 1. The data analysis was performed either by visual inspection in the “Totally Cleared” and the “Totally Overgrown” cases, or using standardized imaging and image analysis (Imaged) with a similar method as for the zone inhibition method. The partially overgrown area was measured in mm² and was subtracted from the total plate surface area to obtain the “Partially Cleared” area.

Zone of Inhibition measured with spot-dropping

This method was employed to visualize and allow measurements from standardized images of the killing effect of LABs, ILP100 and ILP101 against the pathogenic isolates. The method was developed and standardized for spots of LABs to be able to demonstrate visually their antimicrobial activity and to limit the cumulative killing effect (i.e. clear-zone merging) as observed when spot-drops were located too close to each other or when the effect was very potent, see Figure 3.

By spot-dropping the LABs, ILP100 and ILP101 onto LSM agar and by utilizing an overlay technique with 5m L LSM soft agar (0.7%) inoculated with pathogenic bacteria, visualization of the killing effect in the form of a cleared zone of growth inhibition could be observed and measured.

Triplicate, spot-drops (1 x 10⁸, 5 x 10⁷ or 5 x 10⁶ CFU of ILP100; 5 x 10⁷ or 5 x 10⁶ CFU of LABs) were evenly distributed on the agar surface. For the triplicate, measurements varied generally less than 10%. The spot-drops were incubated at 37°C, anaerobically for 22-24 hrs. Following incubation, soft-agar was inoculated with pathogenic bacteria in three different concentrations for each isolate and was immediately poured onto the agar surface containing the pre-incubated drops of ILP100. This was followed by another 18-20 hours of aerobic incubation at 37°C. Each plate was visually inspected and then imaged.

The method was optimized only in terms of where the drops were placed on the plate to measure the killing effect of LABs, ILP100 and ILP101 against known pathogenic isolates that were verified MDR allowing simultaneous testing of sensitivity to six different clinically relevant antibiotics. The antibiotics used are shown in Table 14.

Table 14. Antibiotic discs used

*EUCAST v.14 Breakpoint Tables, "Calculated - Diameter (6 mm) converted to area- less area for the disc (30.7 mm²).

Osmolality

To study the effects by a local change of osmolality, reflecting the situation in the microenvironment as a result of treatment with the formulated lyophilized ILP100 the growth media with isolates of three known wound pathogens, S. aureus, S. pyogenes and S. gordonii cultures was changed to media containing 0, 1.18%, 2.25%, 4.5%, 9.5% or 19% sucrose and then incubated in 37°C for 5 hours in FBS- free cell media. The growth inhibition due to change of osmolality was related to the condition with 0% sucrose serving as the base-case of no change in osmolality. Co-Culture Growth Inhibition in optimal conditions for bacteria

The bactericidal, effects were also tested in vitro using co-culturing of the LAB strains together with the pathogens, which would more closely reflect the clinically relevant condition in a mucosal surface or in a wound bed, albeit in a controlled setting wherein potential outcomes can be monitored. Overnight cultures of pathogen isolates were started in LSM broth from fresh, single colony streaks. The cultures were incubated at 37°C, shaking for ~20 hours. A 1000X dilution was inoculated (aim was 5x10⁵ - 1x10⁶) into fresh LSM and LAB or ILP100 bacteria were added at a ratio of 100:1 (ILP100^|OW) or 1000:1 (ILP100^high) (depending on pathogen overnight growth density) and samples were taken and serially diluted and plated for surviving CFU at 0, 3, 6 and 12 hours. For each experiment the pathogen was also allowed to grow over 12 hours without any addition of a LAB or ILP100 reflecting a situation of optimal growth if not inhibited. Pathogenic isolate strains were recovered on LSM plates and incubated aerobically at 37°C and LABs or ILP100 were recovered on MRS plates with anaerobic incubation at 37°C. Notably, in these experiments, it was found that ILP100 had a lower growth rate than the pathogens and showed a slower increase of colony forming units/mL (CFU/mL). This demonstrates that the experimental conditions did not favor the growth of ILP100 over the pathogens, thus lending credence to the results of these experiments.

Co-Culture Growth Inhibition in simulated body fluid

In order to evaluate the bactericidal effects to the pathogenic MDR isolates in an even more clinically relevant situation, the same co-culture experiment was repeated using RPMI cell medium mimicking body fluid, like wound fluid. To allow LABs and ILP100 to grow the RPMI was supplemented with trace amounts of manganese, yeast extract (protein substitute for FBS) and 0-19% of sucrose which is approximately equivalent to the concentration of sugars upon wound treatment with the ILP100-Topical drug candidate. Except for the media, the method described above for LSM co-culture experiment above was used.

Image Analysis

All plates were then inspected visually, and images was acquired by utilizing a Nikon D5600 camera set in automatic mode and devoid of flash, images were acquired with a resolution of 24.2 megapixels. To ensure optimal contrast and clarity, a black background was incorporated during the imaging process. Moreover, a controlled light source was deployed to facilitate image acquisition.

The area of the zone of inhibition were measured using Imaged (version 1.53/4) and analysed blinded for the initial scope of describing the difference in potency between the drug candidate (ILP100) and the other LABs used. For experiments when only ILP100 was used in triplicates, blinding procedures were not employed. The diameter of the plate (90 mm) was used as a reference to set the scale and define the size of a pixel. The area of the cleared zone (including the drop/antibiotic disc region) and the drop/antibiotic disc were then measured. The area of the drops/discs were then subtracted from the cleared zone area. For each condition, the average cleared area (MAC) of the occasional replicates but in most cases triplicates was used.

Measurements varied generally less than 10%. All raw image measurements have been marked and saved together with the images. The antibiotic disc itself was measured for every MAC calculated for the disc and 100 measurements were used to calculate the accuracy and precision. The theoretical 6 mm area of a disc is 28.27 mm². The mean of 100 measurements was 30.72 ± 0.73 mm².

SEQUENCES

Summary of Sequence Listing

Claims

Claims

1. Live lactic acid bacteria for use in the treatment or prevention of an infection with a multi-drug resistant (MDR) microorganism.

2. The lactic acid bacteria for use according to claim 1 , wherein the lactic acid bacteria are from the genus Lactobacillus, Limosilacobacillus or Lactococcus.

3. The lactic acid bacteria for use according to claim 2, wherein the lactic acid bacteria are Limosilacobacillus reuteri, Lactobacillus rhamnosus or Lactococcus lactis.

4. The lactic acid bacteria for use according to claim 3, wherein the lactic acid bacteria are Limosilacobacillus reuteri R2LC.

5. The lactic acid bacteria for use according to any one of claims 1 to 4, wherein the lactic acid bacteria are transformed to express a heterologous protein or to over-express an endogenous protein.

6. The lactic acid bacteria for use according to any one of claims 1 to 5, wherein the lactic acid bacteria are transformed to express a mammalian protein which is antimicrobial, is an immune-modulating protein which modulates the activity of immune cells, or which promotes resolution of inflammation and/or wound healing.

7. The lactic acid bacteria for use according to claim 5 or 6, wherein the protein is selected from an interleukin, a chitinase-like protein, a cytokine, or a chemokine, preferably a CXC protein.

8. The lactic acid bacteria for use according to any one of claims 5 to 7, wherein the protein is selected from the group consisting of CXCL12, CXCL17 and Ym1.

9. The lactic acid bacteria for use according to any one of claims 5 to 8, wherein the protein is selected from: i) murine CXCL12-1a having an amino acid sequence as shown in SEQ ID NO: 3 or 2, or an amino acid sequence with at least 80% sequence identity thereto; ii) human CXCL12-1a having an amino acid sequence as shown in SEQ ID NO: 6 or 5, or an amino acid sequence with at least 80% sequence identity thereto; iii) murine CXCL17 having an amino acid sequence as shown in SEQ ID NO: 9 or 8, or an amino acid sequence with at least 80% sequence identity thereto; iv) human CXCL17 having an amino acid sequence as shown in SEQ ID NO: 12 or 11 , or an amino acid sequence with at least 80% sequence identity thereto; v) murine Ym1 having an amino acid sequence as shown in SEQ ID NO: 15 or 14, or an amino acid sequence with at least 80% sequence identity thereto; and vi) human Ym1 as shown in SEQ ID NO: 18 or 17 or an amino acid sequence with at least 80% sequence identity thereto.

10. The lactic acid bacteria for use according to any one of claims 1 to 9, wherein the lactic acid bacteria are applied in a dose of: i) 10² CFU/cm² to 10¹³ CFU/cm², preferably 10⁴ CFU/cm² to 10¹¹ CFU/cm²; or ii) 2.5 x 10⁴ to 2.5 x 10⁶ per cm, preferably 2.5x10⁵ per cm.

11. The lactic acid bacteria for use according to any one of claims 1 to 10, wherein the lactic acid bacteria are lyophilized.

12. The lactic acid bacteria for use according to any one of claims 1 to 11 , wherein the lactic acid bacteria are provided in a pharmaceutical composition further comprising a sugar, preferably wherein said sugar is sucrose.

13. The lactic acid bacteria for use according to any one of claims 1 to 12, wherein the infection is located in any soft tissue, mucosal surface, or in blood, optionally in a wound, the respiratory tract or the urinary tract.

14. The lactic acid bacteria for use according to claim 13, wherein the wound is a cutaneous wound, a mucosal wound or a surgical wound.

15 The lactic acid bacteria for use according to any one of claims 1 to 14, wherein the MDR microorganisms are bacteria or fungi, preferably bacteria.

16. The lactic acid bacteria for use according to any one of claims 1 to 15, wherein the MDR microorganism is selected from the following families: Pseudomonadaceae, Enterococcaceae, Streptococcaceae, Moraxellaceae, Enterobacteriaceae, Staphylococcaceae, Helicobacteraceae, Campylobacteraceae, Neisseriaceae, Pasteurellaceae, Corynebacteriaceae, Lachnospiraceae, Bacillaceae, Mycobacteriaceae, Mucoraceae, Trichocomaceae, Candidaceae, Aspergillaceae, Saccharomycetaceae, Cryptococcaceae and Arthrodermataceae.

17. The lactic acid bacteria for use according to any one of claims 1 to 16, wherein the MDR microorganism is selected from Pseudomonas aeruginosa, Acinetobacter baumannii, Enterobacter cloacae, Proteus mirabilis, Klebsiella pneumoniae, Staphylococcus Aureus, Candida albicans, Trichophyton rubrum, Clostridium perfringens Bacillus anthracis and Bacillus cereus biovar anthracis..

18. The lactic acid bacteria according to any one of claims 1 to 17, wherein the MDR microorganism is resistant to at least one class of antimicrobial drug selected from the beta-lactams, the sulphonamides, the quinolones, the macrolides, the tetracyclines, the aminoglycosides, the lincosamides, the polypeptide antibiotics, the streptogramins, the oxazolidinones, the allylamines, the azoles, the polyenes and the echinocandins.

19. The lactic acid bacteria according to any one of claims 1 to 18, wherein the MDR microorganism is resistant to at least one an antimicrobial drug selected from piperacillin-tazobactam, ceftazidime-avibactam, meropenem-vaborbactam, ceftolozane-tazobactam, imipenem/avibactam, cefiderocol, ciprofloxacin, gentamycin, tobramycin, trimethoprim-sulfamethoxazole, meropenem, imipenem, vancomycin and erythromycin.

20. The lactic acid bacteria according to any one of claims 1 to 19, wherein the MDR microorganism is resistant to: i) at least two, three, four, five, six, seven, eight, nine or ten classes of antimicrobial agents; ii) at least one antimicrobial drug in three or more classes of antimicrobial agents; iii) at least one antimicrobial drug in all but two or fewer classes of antimicrobial agent; or iv) all antimicrobial drugs in all classes of antimicrobial agents.

21. The lactic acid bacteria according to any one of claims 1 to 20, wherein the MDR microorganism is more sensitive to the lactic acid bacteria in comparison to a non-MDR microorganism.

22. The lactic acid bacteria for use according to any one of claims 1 to 21 , wherein the lactic acid bacteria are for use in combination with an antimicrobial agent, preferably an antibiotic.

23. The lactic acid for use according to claim 22, wherein the antibiotic is selected from: the beta-lactams, the sulphonamides, the quinolones, the macrolides, the tetracyclines, the aminoglycosides, the lincosamides, the polypeptide antibiotics, the streptogramins or the oxazolidinones.

24. The lactic acid bacteria for use according to claim 22 or 23, wherein the lactic acid bacteria and antibiotic act synergistically to treat or prevent the infection.

25. A kit comprising live lactic acid bacteria and antimicrobial agent, preferably an antibiotic.

26. A product comprising:

(i) live lactic acid bacteria; and

(ii) antimicrobial agent, preferably an antibiotic; for separate, simultaneous or sequential use in the treatment or prevention of an infection with a multi-drug resistant (MDR) microorganism.

27. A wound dressing comprising the product according to claim 26.

28. A medical device comprising the product according to claim 26.

29. The kit according to claim 25 or the product according to claim 26, wherein:

(i) the lactic acid bacteria is as defined in any one of claims 2 to 11; (ii) the infection is as defined in claim 13 or 14;

(iii) the MDR microorganism is as defined in 15 to 21 ; and/or

(iv) the antibiotic is as defined in claim 23.

30. An antibiotic for use in combination with live lactic acid bacteria in the treatment or prevention an infection with a multi-drug resistant (MDR) microorganism, wherein the antibiotic potentiates the anti-microbial effect of the live lactic acid bacteria.

31. Live lactic acid bacteria for use in combination with an antibiotic in the treatment or prevention an infection with a multi-drug resistant (MDR) microorganism, wherein the live lactic acid bacteria potentiate the anti-microbial effect of the antibiotic.