"Human Lactobacilli strains"

Introduction

Clostridium difficile is a Gram positive, cytotoxin-producing anaerobic intestinal pathogen with an asymptomatic carriage rate of up to 30 % of people in long-term care facilities (Ziakas et al, 2015). However, when the intestinal microbiota is altered following broad spectrum antibiotic therapy, C. difficile may flourish and cause illness varying from mild diarrhoea (usually self- limiting) to pseudomembraneous colitis, fulminant colitis, toxic megacolon and even death (Kachrimanidou and Malisiovas, 2011). The incidences of C. difficile infection (CD I) have rapidly increased since the 1990s, and the mortality rate has also grown markedly (Wiegand et al., 2012). Recent studies indicate that the economic burden of C. difficile is mounting as a result of increased number of cases of infection in hospitalised patients. Latest estimates show that the economic health-care costs of CDI are over $ 4.8 billion per annum in the U.S. and over€ 3 billion per annum in Europe (DePestel and Aronoff, 2013b).

The European Society of Clinical Microbiology and Infection (ESCMID) guidelines for treatment of CDI include antibiotics, toxin-binding resins and polymers, immunotherapy, probiotics and faecal or bacterial intestinal transplantation (Debast et ah, 2014). Antibiotic treatment is typically advised, including the use of metronidazole, vancomycin and fidaxomicin (Debast et ah, 2014). However, as standard therapies for CDI frequently have limited efficacy, the search for alternative therapies such as live therapeutics and bacteriocins that may help to reduce incidences and recurring infections are gaining credence (Evans and Johnson, 2015; Goldstein et ah, 2015; Rea et ah, 2013). One of the strategies used to modulate the gut microbiota is the dietary administration of live microorganisms, (Hill et al., 2014). A meta- analysis of the literature, from 1985 to 2013, relating to the ability of probiotics to prevent paediatric antibiotic-associated diarrhoea and CDI found that the use of probiotics significantly prevented CDI and antibiotic-associated diarrhoea in children, but that the effect of probiotics was strain dependant (McFarland and Goh, 2013). The mode of action of live microbes is multi- faceted and includes an improvement of epithelial barrier function, immune-modulation, secretion of antimicrobial substances (e.g. bacteriocins and H2O2 and bioactive metabolites (e.g. CLA), inhibition of the expression of virulence factors, playing a role in competitive exclusion possibly through colonization resistance or through the production of neurotransmitters such as Gamma- aminobutyric acid (GAB A) which may impact on brain function (Dinan et ah, 2015; Dobson et al., 2012; Fernandez et al., 2015; Nebot-Vivinus et al., 2014; O'Shea et al., 2012; Sultana et al., 2013). However, the effect of pure cultures of bacterial strains administered as live therapeutics has been shown in many instances to be strain rather than species dependant indicating that careful strain selection is required (Wall et al 2012).

The effect of bacteria frequently associated with the human gut, such as C. difficle is being increasingly understood within the whole context of the human gut microbiome. The human gut microbiota consists of greater than 1000 bacterial species, with every individual hosting at least 160 different species (Eckburg et al., 2005; Qin et al., 2010; Tap et al., 2009; Walker et al., 2011). The gut microbiota of a healthy adult is, in general, stable with Firmicutes and Bacteroidetes typically accounting for over 90% of bacteria with smaller fractions of Actinobacteria and Proteobacteria (Eckburg et al., 2005; Claesson et al., 2011; Ley et al., 2006; Human Microbiome Project Consortium, 2012). However, considerable inter-individual variability exists at species and strain level (Lozupone et al., 2012). Although it is not yet fully clear what constitutes a 'healthy' gut microbiota diversity and composition are key factors (Backhed et al., 2012; Claesson et al., 2012; Jeffery et al., 2016; Le Chatelier et al., 2013) and a gut microbiota with low diversity may have negative consequences for health (Thomas et al., 2014). This low microbial diversity can arise in several human life stages or due particular therapeutic interventions.

Noncommunicable diseases (NCDs), also known as chronic diseases, are diseases that are not caused by infectious agents and are not passed from person to person. They are generally of long duration and slow progression (WHO, 2016) and include diseases such as autoimmune disease, cardiovascular disease, stroke, cancer, osteoporosis, depression, anxiety, autism, Alzheimer's disease, chronic kidney disease, diabetes and obesity. NCDs are the leading cause of death globally (WHO, 2016).

Reduced microbial diversity is centrally implicated in many NCDs. Studies have demonstrated associations between reduced microbial diversity and eczema (Bisgaard et al., 2011; Abrahamsson et al., 2012; Ismail et al., 2012; van Nimwegen et al., 2011), asthma (Ege et al., 2011; Abrahamsson et al., 2014) autoimmune disease (Kostic et al., 2015; Knip & Siljander, 2016), cardiovascular disease (Kelly et al., 2016) and obesity and type 2 diabetes (Le Chatelier et al., 2013; Remely et al., 2014; Turnbaugh et al., 2009). Reduced diversity has been observed in patients with Crohn's disease (Sokol et al., 2008), type 1 diabetes, coeliac disease, allergy, autism and cystic fibrosis (Spor et al., 2011), all non-communicable diseases. Reduced diversity has also been associated with an increased risk of adiposity, insulin resistance, high blood lipid levels and inflammation (Cotillard et al, 2013; Le Chatelier et al, 2013) - features that can lead to Non- alcoholic liver disease (NAFLD) and its more serious follow-on Non-alcoholic steatohepatitis (NASH) as well as contributing to many of the disease highlighted above.

Obesity as a growing health issue and again it has been highlighted that the diversity of the gut microbiota has relevance. Gut microbiota from obese mice had a lower bacterial diversity than that from lean mice (Turnbaugh et al, 2008). Similarly, 16S rRNA gene surveys revealed a reduced diversity of gut microbiota in human obese twins compared to their lean twin counterparts (Turnbaugh et al, 2009).

Furthermore, gut microbiota diversity may be a contributing factor to disorders of brain function, such as depression, anxiety and autism, through bidirectional signalling via the gut-brain axis (Collins et al, 2012; Cryan and Dinan, 2012; Mayer et al, 2014; Mayer et al, 2015; Stilling et al, 2014).

Finally, compositional diversity also decreases as we age (Claesson et al, 2011), which has been associated with a less diverse diet and correlates with poor health, increased frailty and markers of inflammation (Claesson et al, 2012).

The impact of the microbiota also extends to infectious disease. Individuals with a less diverse gut microbiota are more susceptible to antibiotic-associated dysbiosis (O'Sullivan et al, 2013). A reduction in diversity was observed in patients with Clostridium dijficile (C dijficile) associated diarrhoea while asymptomatic subjects from whom C dijjicile was isolated showed no significant difference in diversity compared to the control group. C dijjicile infection is normally the result of perturbation of the gut microbiota as a result of broad- spectrum antibiotic treatment which results in a decrease in diversity of the gut microbiota (Rea et al, 2012a). Introducing a probiotic strain in a disease state could increase diversity, thus reducing the ability of C. dijjicile to survive and multiply due to competition for nutrients. Older adults (>65 years) have an increased risk of C. difficile infection (Goorhuis et ah, 2008; Vardakas et ah, 2012). Additionally, the risk of hospitalization associated with a . difficile infection increases with age (Lucado et al., 2006). Although C. difficile has historically been considered a nosocomial pathogen associated with antibiotic exposure C. difficile infections have emerged in populations previously considered low risk, such as healthy peripartum women, children, antibiotic-naive patients, and those with minimal or no recent healthcare exposure (DePestel and Aronoff, 2013a; Kim et ah, 2008; Wilcox et ah, 2008). This expansion of disease prevalence and virulence highlights the unmet need for safe, effective treatments for both acute infections and to maintain the health status of at-risk populations.

Recently faecal microbiota transplantation (FMT) has also been used with some success for the treatment of refractory CDI and the interest in this area has risen as evidenced by the large increase in publications relating to FMT over the last 3 years (Bojanova and Bordenstein 2016). To date the mechanism of action of FMT to break the cycle of recurrent CDI remains poorly understood. However, intra colonic bile acid has been suggested to play a role in both spore germination and inhibition of vegetative cells of C. difficile (Weingarden et al 2016). Clostridium scindens, a member of the intestinal microbiota capable of dehyrdoxylating bile acid, was shown to be associated with resistance to C. difficile and enhanced resistance to infection in a murine model of CDI (Buffie et al 2015).

Although, in the short-term, serious adverse effects directly attributable to FMT in patients with normal immune function are uncommon the long-term safety of FMT is unknown (Rao and Safdar, 2016). The interactions between the gut microbiome and the host are complex system and associations with disease processes have been demonstrated. FMT may therefore have unintended consequences in a patient after successful FMT due to alteration of the gut microbiota. FMT in experimental animals has shown that immunologic, behavioural and metabolic phenotypes can be transferred from donor to recipient ((Collins et al, 2013; Di Luccia et al, 2015; Pamer, 2014). This raises questions about the selection of FMT donors. Since FMT in humans has been shown to transfer an improved metabolic phenotype from lean donors to individuals with metabolic syndrome (Vrieze et al, 2012), the reverse could also be true. This implies that donor selection for FMT should not be based solely on exclusion of transmissible infections (Shanahan, 2015). In summary there is an ongoing need to provide therapies for prophylaxis and/or treatment of C. difficile infection, post-antibiotic infection and non-communicable diseases that are caused by or exacerbated by perturbations and reduced diversity in the human gut microbiome Statements of Invention

According to the invention there is provided Lactobacillus gasseri strain APC678 having NCIMB accession number 42658.

In one use the strain is probiotic.

The strain may be in the form of a biologically pure culture.

Also provided is an isolated strain of Lactobacillus gasseri APC678 (NCIMB 42658). The strain may be in the form of viable cells.

The strain may be in the form of non-viable cells. The strain may be isolated from human faeces.

In one case the strain is in the form of a bacterial broth. In another case the strain is in the form of a freeze-dried powder. Also provided is a formulation comprising a strain of the invention. The formulation may comprise an ingestible carrier.

The ingestible carrier may be a pharmaceutically acceptable carrier. The ingestible carrier in some cases is a food product. The food product may, for example, be selected from the group comprising acidified milk, yoghurt, frozen yoghurt, ice cream, milk powder, milk concentrate, cheese spread, dressing and beverage. The formulation may be in the form of a fermented food product. The formulation may be in the form of a fermented milk product. In some cases the carrier does not occur in nature.

The formulation, in some embodiments, is in the form of a capsule, a tablet, a pellet, or a powder.

In some cases the strain is present in the formulation at more than 10 ⁶ cfu per gram of ingestible carrier.

Also provided is a vaccine comprising a strain of the invention.

The invention further provides a strain or a formulation of the invention for use as a probiotic.

The strain or the formulation in some cases is used for the prophylaxis and/or treatment of a Clostridium difficile colonisation or infection.

The strain or the formulation in some cases is used for generating a protective immune response against a pathogenic organism.

Also provided is a method for the prophylaxis and/or treatment of a Clostridium difficile colonisation or infection comprising administering a strain or a formulation of the invention. In some embodiments the invention provides a method of generating a protective immune response in a subject against a pathogenic organism comprising administering to the subject a strain or a formulation of the invention. The invention also provides a method of increasing the bacterial biodiversity in the gastrointestinal tract comprising the step of administering to a subject a strain or a formulation of the invention.

In some cases the strain or formulation is administered in association with antibiotic treatment.

The subject, in some embodiments, has a compromised immune system. In some cases the subject is more than 65 years of age.

The strain may be used in the prophylaxis or treatment of low bacterial biodiversity of the gastrointestinal tract.

The formulation may be used in the prophylaxis or treatment of low bacterial biodiversity of the gastrointestinal tract.

According to the invention there is provided a strain selected from one or more of:-

Lactobacillus gasseri strain APC678 having NCIMB accession number 42658;

Lactobacillus rhamnosus DPC6111 having NCIMB accession number 42661;

Lactobacillus gasseri strain DPC6112 having NCIMB accession number 42659; and

Lactobacillus paracasei strain APC1483 having NCIMB accession number 42660 for use in the prophylaxis and/or treatment of a Clostridium difficile colonisation or infection. invention also provides a formulation comprising one or more strains selected from:

Lactobacillus gasseri strain APC678 having NCIMB accession number 42658;

Lactobacillus rhamnosus DPC6111 having NCIMB accession number 42661;

Lactobacillus gasseri strain DPC6112 having NCIMB accession number 42659; and Lactobacillus paracasei strain APC1483 having NCIMB accession number 42660. The formulation may comprise an ingestible carrier.

The ingestible may be a pharmaceutically acceptable carrier. In some cases the ingestible carrier is a food product.

The food product may be selected from the group comprising acidified milk, yoghurt, frozen yoghurt, milk powder, milk concentrate, cheese spread, dressing and beverage.

In one case the formulation is in the form of a fermented food product.

In one embodiment the formulation is in the form of a fermented milk product. In some embodiments the carrier does not occur in nature.

In some cases the formulation is in the form of a capsule, a tablet, a pellet, or a powder.

In some embodiments the strain is present in the formulation at more than 10 ⁶ cfu per gram of ingestible carrier.

The invention also provides Lactobacillus gasseri strain APC678 having NCIMB accession number 42658 or mutants or variants thereof. The invention also provides Lactobacillus rhamnosus DPC6111 having NCIMB accession number 42661 or mutants or variants thereof.

The invention also provides Lactobacillus gasseri strain DPC6112 having NCIMB accession number 42659 or mutants or variants thereof.

The invention also provides Lactobacillus paracasei strain APC1483 having NCIMB accession number 42660 or mutants or variants thereof.

In some embodiments the mutant is a genetically modified mutant.

In some embodiments the variant is a naturally occurring variant. The strain(s) may be probiotic. The strain may be in the form of a biologically pure culture.

The invention also provides an isolated strain of Lactobacillus gasseri APC678 (NCIMB 42658).

The invention also provides an isolated strain of Lactobacillus rhamnosus DPC6111 (NCIMB 42661).

The invention also provides an isolated strain of Lactobacillus gasseri DPC6112 (NCIMB 42659).

The invention also provides an isolated strain of Lactobacillus paracasei APC1483 (NCIMB 42660). The strain may be in the form of viable cells.

The strain may be in the form of non-viable cells.

In some cases the strain is isolated from human faeces.

In some embodiments the strain is in the form of a bacterial broth. In some cases the strain is in the form of a freeze-dried powder. The invention also provides a formulation comprising at least one isolated strain of the invention. The formulation may comprise an ingestible carrier. The ingestible may be a pharmaceutically acceptable carrier.

In some cases the ingestible carrier is a food product. The food product may be selected from the group comprising acidified milk, yoghurt, frozen yoghurt, milk powder, milk concentrate, cheese spread, dressing and beverage.

In one case the formulation is in the form of a fermented food product.

In one embodiment the formulation is in the form of a fermented milk product.

In some embodiments the carrier does not occur in nature. In some cases the formulation is in the form of a capsule, a tablet, a pellet, or a powder.

In some embodiments the strain is present in the formulation at more than 10 ⁶ cfu per gram of ingestible carrier. The invention also provides a vaccine comprising one or more of the strains of the invention The strain or a formulation may be for use as a probiotic.

The strain or a formulation may be for the prophylaxis and/or treatment of a Clostridium difficile colonisation or infection.

The invention also provides a method for the prophylaxis and/or treatment of a Clostridium difficile colonisation or infection comprising administering a strain or a formulation of one or more of :

Lactobacillus gasseri strain APC678 having NCIMB accession number 42658;

Lactobacillus rhamnosus DPC6111 having NCIMB accession number 42661;

Lactobacillus gasseri strain DPC6112 having NCIMB accession number 42659;

Lactobacillus paracasei strain APC1483 having NCIMB accession number 42660; and Lactobacillus gasseri strain having NTCC accession number 33323;

The invention further provides a method for screening a bacterial strain for activity against C difficile comprising: co-culturing a strain of interest with C. difficile in a co-culture medium comprising sugars (such as glucose) in a concentration of from 0.01 g/1 to 2 g/1, the co-culture medium having a pH of from 6.7 to 6.9. The invention also provides a method for screening a bacterial strain for activity against C. difficile comprising:

co-culturing a strain of interest with C. difficile in a co-culture medium comprising sugars (such as glucose) in a concentration of from 0.01 g/1 to 01.0 g/1, the co-culture medium having a pH of 6.8

In some cases the co-culture medium comprises sugars (such as glucose) in a concentration of from 0.05 g/1 to 0.5 g/1.

The co-culture medium in some cases comprises sugars (such as glucose) in a concentration of from 0.09 g/1 to 0.11 g/1.

The strain of interest may, for example, be a Lactobacillus.

The invention also provides a co-culture medium for use in screening a bacterial strain for activity against C. difficile wherein the medium comprises sugars (such as glucose) in a concentration of from 0.01 g/1 to 2.0 g/1 and wherein the co-culture medium has a pH of from 6.7 to 6.9.

Also provided is a co-culture medium for use in screening a bacterial strain for activity against C. difficile wherein the medium comprises sugars (such as glucose) in a concentration of from 0.01 g/1 to 1.0 g/1 and wherein the co-culture medium has a pH of 6.8.

In some cases the co-culture medium comprises sugars (such as glucose) in a concentration of from 0.05 g/1 to 0.5 g/1.

The co-culture medium in some cases comprises sugars (such as glucose) in a concentration of from 0.09 g/1 to 0.1 lg/1. The invention also provides a method of increasing the bacterial biodiversity in the gastrointestinal tract comprising the step of administering to a subject a strain or a formulation of the invention. In some embodiments the strain or formulation is administered in association with antibiotic treatment.

In some embodiments the subject has a compromised immune system. The subject may be an elderly patient who may be more than 65 years of age.

The strain or formulation may be for use in the prophylaxis or treatment of low bacterial biodiversity of the gastrointestinal tract. The isolated strains may be in the form of viable cells.

The isolated strains may be in the form of non- viable cells.

Formulations comprising one or more of the strains may also comprise an ingestible carrier. The ingestible carrier may be a pharmaceutically acceptable carrier such as a capsule, tablet or powder. The ingestible carrier may be a food product such as acidified milk, yoghurt, frozen yoghurt, milk powder, milk concentrate, cheese spreads, dressings or beverages.

The strains of the invention may be administered to animals (including humans) in an orally ingestible form in a conventional preparation such as capsules, microcapsules, tablets, granules, powder, troches, pills, suppositories, suspensions and syrups. Suitable formulations may be prepared by methods commonly employed using conventional organic and inorganic additives. The amount of active ingredient in the medical composition may be at a level that will exercise the desired therapeutic effect.

A formulation comprising one or more of the strains may also include a bacterial component, a drug entity or a biological compound. In addition a vaccine comprising at least one strain of the invention may be prepared using any suitable known method and may include a pharmaceutically acceptable carrier or adjuvant.

The invention also includes mutants and variants of the deposited strains. Throughout the specification the terms mutant, variant and genetically modified mutant include a strain whose genetic and/or phenotypic properties are altered compared to the parent strain. Naturally occurring variant includes the spontaneous alterations of targeted properties selectively isolated. Deliberate alteration of parent strain properties is accomplished by conventional (in vitro) genetic manipulation technologies, such as gene disruption, conjugative transfer, etc. Genetic modification includes introduction of exogenous and/or endogenous DNA sequences into the genome of a strain, for example by insertion into the genome of the bacterial strain by vectors, including plasmid DNA, or bacteriophages.

Natural or induced mutations include at least single base alterations such as deletion, insertion, transversion or other DNA modifications which may result in alteration of the amino acid sequence encoded by the DNA sequence.

The terms mutant, variant and genetically modified mutant also include a strain that has undergone genetic alterations that accumulate in a genome at a rate which is consistent in nature for all micro-organisms and/or genetic alterations which occur through spontaneous mutation and/or acquisition of genes and/or loss of genes which is not achieved by deliberate (in vitro) manipulation of the genome but is achieved through the natural selection of variants and/or mutants that provide a selective advantage to support the survival of the bacterium when exposed to environmental pressures such as antibiotics. A mutant can be created by the deliberate (in vitro) insertion of specific genes into the genome which do not fundamentally alter the biochemical functionality of the organism but whose products can be used for identification or selection of the bacterium, for example antibiotic resistance.

A person skilled in the art would appreciate that mutant or variant strains can be identified by DNA sequence homology analysis with the parent strain. Strains having a close sequence identity with the parent strain without demonstrable phenotypic or measurable functional differences are considered to be mutant or variant strains. A strain with a sequence identity (homology) of 99.5% or more with the parent DNA sequence may be considered to be a mutant or variant. Sequence homology may be determined using on-line homology algorithm "BLAST" program, publicly available at http://www.ncbi.nlm.nih,gov/BLAST/.

Mutants of the parent strain also include derived strains having at least 95.5% sequence homology to the 16S rRNA polynucleotide sequence of the parent strain. These mutants may further comprise DNA mutations in other DNA sequences in the bacterial genome.

Brief Description of the Drawings

The invention will be more clearly understood from the following description thereof, given by way of example only, in which :-

Fig. 1 is Effect of lactobacilli on the survival of Clostridium difficile in co-culture. Black bars (■) show the cell numbers of C difficile at TO and T 24 in the absence of Lactobacillus strains. Bars with dashed lines (Ea) show the cell numbers of C difficile following 24 h co-culture with: L. gasseri APC 678, L. rhamnosus DPC 6111, L. gasseri

DPC 6112, L. paracasei APC 1483 or L. gasseri ATCC 33323. The horizontal dashed line (— ) indicates the starting inoculum of C difficile at 0 h; and

Fig. 2 is Clostridium difficile detected during faecal shedding. C difficile detected in mouse faeces (CFU g ^"1 faeces) following (a) 24 h, (b) 4 days, (c) 7 days administration of lactobacillus strains and (d) C. difficile levels in mouse colon (CFU colon ^"1 ) following 7 days injestion of the lactobacillus strains (or control). Control: 10 % RSM only; Lactobacillus gasseri APC 678; Lactobacillus rhamnosus DPC 6111 and Lactobacillus gasseri ATCC 33323.

Detailed Description

A deposit of Lactobacillus gasseri strain APC678 was made at the National Collections of Industrial and Marine Bacteria Limited (NCIMB) Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB21 9YA, Scotland, UK on September 20, 2016 and accorded the accession number NCIMB42658.

A deposit of Lactobacillus rhamnosus strain DPC6111 was made at the National Collections of Industrial and Marine Bacteria Limited (NCIMB) Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB21 9YA, Scotland, UK on September 20, 2016 and accorded the accession number NCIMB 42661.

A deposit of Lactobacillus gasseri strain DPC6112 was made at the National Collections of Industrial and Marine Bacteria Limited (NCIMB) Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB21 9YA, Scotland, UK on September 20, 2016 and accorded the accession number NCIMB 42659.

A deposit of Lactobacillus paracasei strain APC1483 was made at the National Collections of Industrial and Marine Bacteria Limited (NCIMB) Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB21 9YA, Scotland, UK on September 20, 2016 and accorded the accession number NCIMB 42660.

Screening method for anti-C. difficile probiotics.

Lactobacilli when they grow in the normal growth medium used- MRS (Man Rogasa Sharpe) produce significant amounts of lactic acid from the metabolism of glucose and a fully grown lactobacillus culture can drop the pH over night to -4.4-4.2.

Clostridium difficile strains are quite sensitive to acidic conditions and these conditions would not be encountered in the colon (normal pH -6.8) which is where C. difficile would normally be found.

Also the concentration of simple sugars like glucose would be negligible in the colon as monosaccharaides are more likely to be metabolised further up the GI tract.

We have developed a screening method to test lactobacilli strains and C. difficile when grown together in co-culture but without the drop in pH that would occur in the presence of high concentrations of sugar. We have eliminated the killing effect resulting from acid production by the lactobacilli therefore providing a method to isolate strains based on their ability to selectively target C. difficle through specific and unique mechanisms, not just generalised acid effects.

A novel co-culture medium containing minimized nutritional composition was developed. The new medium support limited growth of both lactobacilli and C. difficile cells when grown separately. The medium was rationally designed to simulate the lower gut environment in its composition so that cell growth was limited mainly by the amount of energy supply available. Furthermore, the medium was designed to have high buffering capacity to prevent a drop in pH during cell growth.

Table 1: Comparison of de Man, Rogosa and Sharpe (MRS), Reinforced Clostridium Medium (RCM) and developed co-culture medium.

Ingredient MRS (g/1) RCM (g/1) Co-culture medium (g/1)

Peptone 10 10 2

Meat Extract 8 10 2

Yeast extract 4 3 1

Glucose 20 5 0.1

Sodium acetate 5 3 0.5

Starch 1

Sodium chloride - 5 5

Triammonium 2

citrate

Magnesium sulphate 0.2 - -

Manganese sulphate 0.05 - -

Dipotassium 2

phosphate

Tween 80 1 - -

L-cysteine HC1 - 0.5 0.5

Dihydrogen Sodium 3.7

Phosphate

DiSodium Hydrogen 6.2

Phospahte

pH 6.2 6.8 6.8 Example 1 Screening of Lactobacillus strains for anti-bacterial activity against Clostridium difficile

Bacterial strains and growth conditions

Lactobacillus strains were maintained at -80°C in 40 % (v/v) glycerol and routinely cultured anaerobically at 37°C on de Man Rogosa Sharpe (MRS) agar (Difco, Beckton Dickinson & Co., New Jersey, USA) for 48 h or overnight in MRS broth. C difficile strains EM304 (ribotype 027) and VPI 10463 (see Table 2 for details) were maintained at -80°C on micro-bank beads (Pro-Lab Diagnostics, Merseyside, UK) and cultured on Fastidious Anaerobic Agar (Lab M, Heywood, Lancashire, UK) supplemented with 7 % defibrinated horse blood (Cruinn Diagnostics, Dublin, Ireland) at 37 °C for 3 days. Fresh cultures were grown overnight at 37 °C in Reinforced Clostridium Medium (RCM) (Merck, Darmstadt, Germany) pre -boiled and cooled under anaerobic conditions. The lactobacilli and Clostridia were grown in an anaerobic chamber (Don Whitley, West Yorkshire, UK) under an anoxic atmosphere (10 % H ₂ , 0 % 0 ₂ , 0 % N ₂ ), unless otherwise stated.

Table 2: Source and origin of critical bacterial strains either used or isolated in this study

Bacterial strain Source Origin

Clostridium difficile EM304 ELDERMET Culture elderly adult faeces

(ribotype 027) Collection ¹

Clostridium difficile VPI American Type Culture human abdominal wound

10463 Collection

(ATCC® 43255FZ™)

L. paracasei strain APC1483 APC Culture Collection ¹ Human faeces

L. gasseri APC 678 APC Culture Collection ¹ human faeces

L. rhamnosus DPC 6111 DPC Culture Collection ¹ human faeces

L. gasseri DPC 6112 DPC Culture Collection ¹ healthy adult faeces

L. gasseri ATCC 33323 American Type Culture human faeces

Collection

2"

Teagasc, Moorepark, Fermoy, Co. Cork, Ireland; Manassas, VA 20110, USA Agar diffusion assay

Initially one thousand five hundred Lactobacillus isolates of food, human and animal origin were assessed for anti-bacterial activity against C. difficile EM304 using agar diffusion assays. Briefly, RCM agar was seeded with C. difficile and overnight cultures of lactobacilli, grown in MRS, were stabbed into the C. difficile seeded agar. Agar plates were incubated anaerobically at 37°C and assessed at 72 h for zones of inhibition against C. difficile. In addition L. gasseri APC 678 was assessed for bacteriocin activity against a range of target organisms as previously described (Rea et ah, 2010). The full list of target strains, together with their growth conditions are outlined in Table 3. Target organisms were chosen to test the effect of the novel lactobacillus strains on both potential gut pathogens and also gut commensals.

Table 3: Target strains, growth medium and incubation conditions for well diffusion assays to detect bacteriocin production by strains of interest.

Target strain Culture Incubation Growth

Collection No. Conditions medium

Lactobacillus delbrueckii subsp. bulgaricus LMG ¹ 6901 37 °C, anaerobic MRS

Lactobacillus delbrueckii subsp. lactis LMG 7942 37 °C, anaerobic MRS

Lactobacillus amylovorus LMG 9496 37 °C, anaerobic MRS

Enterococcus saccharolyticus LMG 11427 37 °C, anaerobic MRS

Enterococcus mundtii LMG 10758 37 °C, anaerobic MRS

Listeria innocua DPC ² 3572 37 °C, aerobic BHI*

Enterococcus faecium LMG 11423 37 °C, anaerobic MRS

Lactobacillus plantarum LMG 6907 37 °C, anaerobic MRS

Micrococcus luteus DPC 6275 30°C, aerobic BHI

Lactobacillus acidophilus LMG 9433 37 °C, anaerobic MRS

Staphylococcus aureus DPC 5246 37 °C, aerobic BHI

Salmonella typhimurium DPC 6048 37 °C, aerobic BHI

Lactobacillus agilis LMG 9186 37 °C, anaerobic MRS

Lactobacillus rhamnosus GG ATCC ³ 53103 37 °C, anaerobic MRS

Lactobacillus casei LMG 6904 37 °C, anaerobic MRS

Lactobacillus crispatus LMG 9479 37 °C, anaerobic MRS

Lactobacillus fermentum LMG 6902 37 °C, anaerobic MRS ^MG Culture Collection, B-9000 Gent, Belgium; ² DPC Culture Collection, Teagasc, Moorepark, Fermoy, Co. Cork, Ireland; American Type Culture Collection, Manassas, VA 20110, USA. *BHI: Brain heart infusion medium, Merck, Darmstadt, Germany. Initial screening for the production of antibacterial compounds by lactobacilli against C. difficile using the antagonistic agar assay failed to reveal zones of inhibition. Therefore, a low nutrient media (MGM) - the novel co-culture medium - was developed which represented more closely the low concentration of simple carbohydrates in the human gut. Co-culture broth

This media allowed growth of both Lactobacillus and C. difficile strains by ~1 to 2 logs over a 24h period. Due to the buffering capacity of the medium, the pH was maintained at near neutral (~pH 6.5) following growth for 24 h, eliminating concerns relating to the reduction of C. difficile merely as a result of acid production.

Fifty eight Lactobacillus strains (Table 4) from human and animal origin were selected for further screening using a co-culture method, with C. difficile EM 304 (ribotype 027) (Rea et ah, 2012b) as the target strain. A minimal growth medium (MGM) - the co-culture medium - was developed to represent the nutrient limited content of the human gut and to allow co-culturing of Lactobacillus and Clostridium strains. The media composition (g L ^"1 ) was: meat extract, 2 g; peptone, 2 g; yeast extract, 1 g; NaCl, 5 g; sodium acetate, 0.5 g; L-cysteine hydrochloride, 0.5 g; glucose, 0.1 g; NaH ₂ PO ₄ .H ₂ 0, 3.7 g; Na ₂ HP0 ₄ .7H ₂ 0, 6.2 g; pH 6.8 (+ 0.2). Following overnight growth, 1 ml of C. difficile EM304 and each Lactobacillus strain were centrifuged at 14,000 x g. Pelleted cells were washed once in phosphate buffered saline (PBS) under anaerobic conditions and resuspended in fresh PBS. The MGM was inoculated with a test strain of Lactobacillus and C. difficile EM304 at 10 ⁶ CFU ml ^"1 . The tubes were incubated at 37°C for 24 h. Survival of C. difficile was determined by plating onto Brazier's cefoxitin cycloserine and egg yolk agar (CCEY) (Lab M), and Lactobacillus counts were determined by plating onto MRS agar. Plates were incubated anaerobically at 37°C for 2-3 days and the anti-bacterial ability was determined as a reduction in C. difficile counts compared to the control in the absence of Lactobacillus. Table 4: Lactobacilli screened for anti-Clostridium difficile activity.

Lactobacillus spp. No. of Origin

strains

tested

Lactobacillus gasseri 10 human faeces

Lactobacillus casei 7 human faeces

Lactobacillus paracasei 4 human faeces

Lactobacillus salivarius 5 4 human faeces; 1 pig faeces

Lactobacillus rhamnosus 6 human faeces

Lactobacillus brevis 4 2 human faeces; 1 cow faeces; 1 silage

Lactobacillus mucosae 3 cow faeces

Lactobacillus plantarum 4 cow faeces; hand wash water; milking yard water

Lactobacillus 2 1 pig caecum; 1 milk

parabuchneri/kefir

Lactobacillus ruminis 2 human faeces

Lactobacillus reuteri 2 1 human faeces; 1 pig faeces

Lactobacillus acidophilus 1 human faeces

Lactobacillus murinus 1 pig caecum

Uncharacterised lactobacilli 7 human faeces

The co-culture assay showed that 4/58 lactobacilli tested (L. gasseri APC 678, L. rhamnosus DPC 6111, L. gasseri DPC 6112 and L. paracasei APC 1483) had the ability to reduce the growth/survival of C. difficile in vitro (Fig. 1). Of these, the best performing L. gasseri strain in numerically reducing C. dijficle shedding was L. gasseri APC 678. Further replicates of this experiment supported this finding. In contrast the well characterised L. gasseri ATCC 33323 strain was not as effective. Example 2 - Identity of L. gasseri APC678 was confirmed by BLAST analysis of the

16S rRNA gene region.

Method 16s rRNA gene sequencing (16S) was performed to identify L. gasseri APC678. Briefly, total DNA was isolated from the strains using 100 μΐ of Extraction Solution and 25 μΐ of Tissue Preparation solution (Sigma-Aldrich, XNAT2 Kit). The samples were incubated for 5 minutes at room temperature followed by 2 h at 95 °C. 100 μΐ of Neutralization Solution (Sigma-Aldrich, XNAT2 kit) was then added. DNA solution was quantified using a Nanodrop spectrophotometer and stored at 4°C. PCR was performed using the 16S primers. The primer pairs used for identification of the both strain were 16S Forward 5'- CTG ATC TCG AGG GCG GTG TGT ACA AGG -3' and 16S Reverse 5'- CTG ATG AAT TCG AGA CAC GGT CCA GAC TCC-3'. The cycling conditions were 94°C for 4 min (1 cycle), 94°C for 45 sec, 56°C for 45 sec, 72°C for 45 sec (30 cycles). The PCR reaction contained 2 μΐ (100 ng) of DNA, PCR mix (Sigma-Aldrich, Red Taq), 0.025 nM 16S L and R primer (MWG Biotech, Germany). The PCR reactions were performed on an Eppendorf thermocycler. The PCR products were run alongside a molecular weight marker (100 bp Ladder, Roche) on a 2 % agarose EtBr stained gel in TAE, to determine the 16S profile. PCR products were purified using the Promega Wizard PCR purification kit. The purified PCR products were sequenced at Beckman Coulter Genomics (UK) using the primer sequences (above) for the 16S rRNA gene region. Sequence data was then searched against the NCBI nucleotide database to determine the identity of the strain by nucleotide homology. The resultant DNA sequence data was subjected to the NCBI standard nucleotide-to-nucleotide homology BLAST search engine (http://www.ncbi.nlm.nih.gov/BLAST/) to identify the nearest match to the sequence.

Results

Identity of L. gasseri APC678 was confirmed by BLAST analysis of the 16S rRNA gene region. Table 5: Blast results of the 16S rRNA gene region of L. gasseri APC678.

Sample ID Accession # Closest Match on NCBI BLAST Identities % match bp Query Coverage E value

L. gasseri APC678 KU710510.1 Lactobacillus gasseri strain LG202 1024/1028 99% 1026 100% 0% Table 6: Sequence of the 16S rRNA gene region of L. gasseri APC678.

16s rRNA sequence of L. gasseri APC678 (1026 nt):

CGATACTAGCGATTCCGCTTCGTGTAGGCGAGTTGCAGCCTACAGTCCGAACTGAGA

ACGGCTTTCAGAGATCCGCTTGCCTTCGCAGGTTCGCTTCTCGTTGTACCGTCCATT G

TAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGACTTGACGTCATCCCCACCT

TCCTCCGGTTTGTCACCGGCAGTCTCATTAGAGTGCCCAACTTAATGATGGCAACTA

ATGACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTG

ACGACAGCCATGCACCACCTGTCTCAGCGTCCCCGAAGGGAACACCTAATCTCTTAG

GTTTGCACTGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCA

CATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGT C

GTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTGAGAGGCGGAAACC

TCCCAACACTTAGCACTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGT

TCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTGCAGACCAGAGAGCCGCCTTCGC

CACTGGTGTTCTTCCATATATCTACGCATTCCACCGCTACACATGGAGTTCCACTCT C

CTCTTCTGCACTCAAGTTCAACAGTTTCTGATGCAATTCTCCGGTTGAGCCGAAGGC

TTTCACATCAGACTTATTGAACCGCCTGCACTCGCTTTACGCCCAATAAATCCGGAC

AACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGACTTTC

TAAGTAATTACCGTCAAATAAAGGCCAGTTACTACCTCTATCTTTCTTCACTACCAA

CAGAGCTTTACGAGCCGAAACCCTTCTTCACTCACGCGGCGTTGCTCCATCAGACTT

GCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGACCGTGTC

Example 3 - Screening for survival through gastrointestinal transit

Among the important traits required for live microbes intended for use in the gastrointestinal tract (GIT) is the ability to survive the acidic conditions of the stomach and the presence of bile in the upper small intestine. The ability of the selected bacterial strains to survive a simulated gastric environment was assessed. Briefly, MRS broth was inoculated at 1 % with the Lactobacillus strains and incubated anaerobically at 37°C for 16 h. One ml of cells was centrifuged at 14,000 x g, washed in PBS and re-centrifuged. The cells were then resuspended in

PBS or 10 % reconstituted skim milk (RSM). A suspension of 10 8 CFU ml - ^" 1 Lactobacillus was suspended in artificial gastric juice with the following composition: NaCl, 125 mmol L ^"1 ; KC1, 7 mmol L ^"1 ; NaHC0 ₃ , 45 mmol L ^"1 and pepsin, 3 g L ^"1 . The final pH was adjusted with HC1 to pH 2 and pH 3 and with NaOH to pH 7. The bacterial suspensions were incubated at 37 °C with agitation (200 rev min ^" ) to stimulate peristalsis. Aliquots were taken for enumeration of viability after 0, 90 and 180 min.

Following 180 min suspension in simulated gastric juice, the cells were suspended in simulated intestinal fluid, which was prepared with 0.1 % (w/v) pancreatin (Sigma) and 0.15 % oxgall bile salts (Difco), in water adjusted to pH 8.0 with NaOH, for a further 180 min. The suspensions were incubated at 37°C and samples taken for total viability after 90 and 180 min. Viability was assessed by plating serial dilutions on MRS agar and incubating at 37°C for 48 h. Survival was expressed as log reduction from 0 h.

We have demonstrated the survival of L. gasseri APC 678 and L. rhamnosus DPC 6111 in a simulated GIT environment (Table 7). Both strains, when suspended in PBS before being added to simulated gastric juice at pH 3 were very stable. However, L. gasseri APC 678 was the more stable of the two at pH 2, showing a 1.5 log reduction in total viable counts compared to 3.5 log reduction for L. rhamnosus DPC 6111. Viable counts indicated that neither strain survived the subsequent 3 h incubation in simulated ileal juice. However, if the strains were suspended in 10 % RSM prior to treatment with gastric/ileal juice, their survival was markedly improved both in simulated gastric juice at both pH 2 and 3 and also after a further 3 h incubation in ileal juice. In 10 % RSM L. rhamnosus DPC 6111 was more sensitive to the conditions of the stomach and ileum than L. gasseri APC 678, showing -4.5 log reduction at the end of the incubation period compared with a reduction of 2.8 logs for L. gasseri APC 678.

Table 7: Survival of lactobacilli during simulated gastrointestinal tract transit. The effect of initial suspension medium (PBS or RSM) on the subsequent survival of L. gasseri APC 678 and L. rhamnosus DPC 6111 in simulated gastric juice followed by simulated ileal juice.

Log reduction (CFU/ml) after 3 h Log reduction (CFU/ml) after a in simulated gastric juice further 3 h in simulated ileal juice

Suspension pH L. gasseri L. rhamnosus L. gasseri L. rhamnosus medium APC 678 DPC 6111 APC 678 DPC 6111

PBS o θ Ϊ6 02Ϊ 052 043

3.0 0.96 1.01 2.19 1.78

2.0 1.52 3.53 8.00 8.00 RSM 7.0 0.84 0.49 0.52 0.13

3.0 1.92 0.91 1.89 2.47

2.0 1.91 1.49 2.82 4.39

*Cells were transferred to simulated ileal juice following 3 h incubation in gastric juice and incubated for an additional 3 h.

These results demonstrated the suitability of these strains for consumption due to their inherent ability to survive gastric transit in an in vitro model system.

Example 4 - In vivo assessment of strains in C. difficile carriage mouse model

The ability of L. gasseri APC 678 and L. rhamnosus DPC 6111 to reduce C. difficile in vivo in a murine model was investigated. This model serves two purposes. It acts as a model of diversity reduction post-antibiotic use in addition to being a model of c. difficile colonisation and disease. In addition, the well characterised strain L. gasseri ATCC 33323 (Azcarate-Peril et ah, 2008), which did not show any inhibition of C. difficile using the afore-mentioned in vitro assays, was selected as a negative control for the animal study. Mouse model

Materials and Methods

All procedures involving animals were approved by the UCC Animal Experimentation Ethics Committee (#2011/17). For the C. difficile model, 40 female C57BL/6 mice (7 weeks old) were obtained from Harlan Laboratories UK (Bicester, Oxfordshire, UK). All mice used in the experiment were housed in groups of 5 animals per cage under the same conditions. Food, water, bedding and cages were autoclaved before use.

Antibiotic administration

All mice were made susceptible to C. difficile infection by altering the gut microbiota using a previously described protocol (Chen et al, 2008). Briefly, an antibiotic mixture comprising of kanamycin (0.4 mg mL ^"1 ), gentamicin (0.035 mg mL ^"1 ), colistin (850 U mL ^"1 ), metronidazole (0.215 mg L ^"1 ) and vancomycin (0.045 mg mL ^"1 ) was prepared in water (all antibiotics were purchased from Sigma Aldrich, Ireland). This corresponds to the approximate daily dose for each antibiotic as follows kanamycin (40 mg kg ^"1 ), gentamicin (3.5 mg kg ^"1 ), colistin (4.2 mg kg ^"1 ), metronidazole (21.5 mg kg ^"1 ) and vancomycin (4.5 mg kg ^"1 ). The concentrations of antibiotics in the water were calculated based on the average weight of the animals and expected daily water consumption of the mice. All mice received the antibiotic cocktail in water for 3 days, followed by 2 days of water without antibiotics. All mice received a single dose of clindamycin 10 mg kg ^{" 1} intra-peritoneally 1 day before C. difficile challenge.

Preparation of bacterial cultures

Adhering to strict anaerobic conditions, C. difficile strain VPI 10463 was grown overnight in RCM. Bacterial cells were collected by centrifugation at 4,050 x g for 5 minutes, washed once in PBS and resuspended in PBS to achieve a preparation of 5 x 10 ⁵ CFU per mouse. Lactobacillus strains for each group - L. gasseri APC 678, L. rhamnosus DPC 6111 and L. gasseri ATCC 33323 (see Table 4 for details) were prepared by growing the strains overnight in MRS broth under anaerobic conditions. Cells were collected by centrifugation at 4,050 x g for 5 minutes, washed once in saline solution and resuspended in 10 % (w/v) reconstituted skim milk (RSM) to achieve 1 x 10 CFU per mouse in 100 μΐ RSM. The control group were fed 10 % RSM only. At the start of the experiment all mice (10/group) received an individual inoculum of C. difficile (5 x 10 5 CFU/mouse). Five hours later 100 μΐ of the target strain (equivalent to 1 x 108 CFU) or RSM (control group) was administered by oral gavage daily for 7 days.

Sample collection and C. difficile counts

Prior to commencement of the trial and before antibiotic treatment, faecal samples were collected from all animals and plated on CCEY agar to confirm that the mice were C. difficile- free. Subsequently, faecal pellets were collected at 24 h, 4 days and 7 days post-infection with C. difficile and stored anaerobically before being assessed for viable C. difficile (CFU g ^"1 faeces). At the end of the trial the mice were sacrificed and total numbers of C. difficile per colon were counted (CFU colon ^"1 ). C. difficile survival was determined by culturing anaerobically at 37°C on CCEY agar for 48 h. C. difficile colonies were confirmed using the Oxoid C. difficile test kit (Oxoid, Basingstoke, UK). At the time of culling caecal contents were collected for compositional sequencing from each individual mouse, snap frozen and stored at -80°C until required.

Microbial DNA extraction, 16s rRNA amplification and Illumina MiSeq sequencing

Total metagenomic DNA was extracted for each mouse caecum, following thawing at 4°C, with the QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany) with an additional bead beating step (Murphy et aL , 2010). DNA was quantified using the Nanodrop 1000 spectrophotometer (Thermo Scientific, Ireland). Initially the template DNA was amplified using primers specific to the V3-V4 region of the 16s rRNA gene which also allowed for the Illumina overhang adaptor, where the forward

(5 ' TCGTCGGC AGCGTC AG ATGTGTAT A AG AG AC AGCCT ACGGGNGGCWGC AG) and reverse primers

(5 ' GTCTCGTGGGCTCGG AG ATGTGT ATA AG AG AC AGG ACT ACH VGGGTATCT A ATCC

)

were used. Each PCR reaction contained 2.5 μΐ DNA template (5 ng), 5 μΐ forward primer (1 μΜ), 5 μΐ reverse primer (1 μΜ) (Sigma) and 12.5 μΐ Kapa HiFi Hotstart Readymix (2X) (Anachem, Dublin, Ireland). The template DNA was amplified under the following PCR conditions for a total of 25 cycles: 95°C for 3 minutes and 30 seconds respectively (initialization and denaturation), 55°C for 30 seconds (annealing) and 72°C for 30 seconds (elongation), followed by a final elongation step of 5 minutes. PCR products were visualised using gel electrophoresis (IX TAE buffer, 1.5 % agarose gel, 100 V) post PCR reaction. Successful amplicons were cleaned using the AMpure XP purification system (Labplan, Dublin, Ireland). A second PCR reaction was completed using the previously amplified and purified DNA as the template. Two indexing primers (Illumina Nextera XT indexing primers, Illumina, Sweden) were used per sample to allow all samples to be pooled, sequenced on one flow cell and subsequently identified bioinformatically. Each reaction contained 25 μΐ Kapa HiFi HotStart ReadyMix (2X), 5 μΐ template DNA, 5 μΐ index primer 1 (N7xx), 5 μΐ index primer 2 (S5xx) and 10 μΐ PCR grade water. PCR conditions were the same as previously described with the samples undergoing just 8 cycles instead of 25. PCR products then underwent the same electrophoresis and cleaning protocols as described above. Samples were quantified using the Qubit 2.0 fluorometer (Invitrogen, Carlsbad, CA, USA) in conjunction with the broad range DNA quantification assay kit (Biosciences, Dublin, Ireland). All samples were pooled to an eqimolar concentration. Quality of the pool was determined by running on the Agilent Bioanalyser prior to sequencing. The sample pool was then denatured with 0.2 M NaOH, diluted to 4 pM and combined with 10 % (v/v) denatured 4 pM PhiX. Samples were sequenced on the MiSeq sequencing platform (Teagasc Sequencing Centre, Moorepark, Fermoy, Co. Cork, Ireland) using a 2300 cycle V3 Kit following protocols outlined by Illumina. Bioinformatic analysis

Raw niumina 300 base pair paired-end sequence reads were merged using Flash (Magoc and Salzberg, 2011) and quality checked using the split libraries script from the Qiime package (Caporaso et ah, 2010). Reads were then clustered into operational taxonomical units (OTUs) and chimeras removed with the 64-bit version of USEARCH (Edgar, 2010). Subsequently OTUs were aligned and a phylogenetic tree generated within Qiime. Taxonomical assignments were reached using the SILVA 16S specific database (version 111) (Quast et al, 2013). PICRUSt (phylogenetic investigation of communities by reconstruction of unobserved states) analysis was performed on the OTU tables to infer function (Langille et ah, 2013). Alpha and beta diversity analysis was also implemented within Qiime. Principal coordinate analysis (PCoA) plots were then visualised using EMPeror v0.9.3-dev (Vazquez-Baeza et ah, 2013).

Statistical analysis

Non-parametric Mann- Whitney statistical analysis was applied on MiniTab (Version 15; faecal and colon culture data) and SPSS (PASW Statistics version 18; caecal microbiota compositional data) statistical packages, to assess whether differences in C. difficile shedding, microbiota composition and diversity between the control and probiotic-fed groups were significant. Statistical significance was accepted at p<0.05, adjusted for ties, where the null hypothesis was rejected.

Results

The reduction of C. difficile shedding in the faeces, the reduction of total viable C. difficile in the mouse colon and changes in the microbiota composition of the mouse caecum were assessed. Mice were infected with ~5 x 10 ⁵ CFU of C. difficile and at day 1 the mean C. difficile counts were >10 ⁷ /g of faeces in all groups, which compares well with C. difficile counts in murine studies where the animals received clindamycin or metronidazole prior to infection with C. difficile (Schubert et al, 2015b). There was no significant reduction in the numbers of C. difficile shed in the faeces between the control group (fed RSM only) and the lactobacillus-fed mice after 24 h (Fig. 2a). However, after 4 and 7 days, L. gasseri APC 678 significantly reduced C. difficile faecal shedding (p<0.05) compared to the control mice, while, surprisingly, given the results of exemplar 1 there was no significant reduction in C. difficile in the faeces of those mice receiving either L. rhamnosus DPC 6111 or L. gasseri ATCC 33323 compared to the control mice (Fig. 2b and 2c). It was also noted that, by day 7, both L. gasseri APC 678 and L. gasseri ATCC 33323 significantly reduced the numbers of C. difficile that had adhered to the colon (p=0.003 and p= 0,014 respectively); Fig. 2d). The difference between L. gasseri APC 678 and L. gasseri ATCC 33323 whereby L. gasseri APC 678 reduces fecal shedding much more quickly than L. gasseri ATCC 33323 is important as C. dijficle is an infectious agent that is spread through contact.

Effects of the chosen bacteria on the gut microbiota

Significant effects of the chosen bacteria on the gut microbiota were seen. These effects were strain specific and surprising.

Diversity Indices higher in APC687 fed mice compared to controls and other strains tested.

Following total metagenomic DNA extraction of the caecal contents, V3-V4 16S rRNA gene amplicons were generated and sequenced using the Illumina MiSeq. Following quality filtering, 4,985,283 sequence reads remained. Diversity, richness and coverage estimations were calculated for each data set (Table 6), all of which indicated good sample richness throughout and the presence of a diverse microbiota. Interestingly, the Simpson and Shannon diversity metrics were significantly higher in the L. gasseri APC 678-fed mice compared to the control mice, and all alpha diversity indices tested were significantly higher in the L. gasseri APC 678- fed mice compared to the those fed L. gasseri ATCC33323 or L. rhamnosus DPC 6111, indicating that L. gasseri APC 678 had a greater impact on diversity than the other probiotics tested. Beta-diversity was estimated using distance matrices built from unweighted Unifrac distances and, subsequently, principal co-ordinate analysis (PCoA) was performed on the distance matrices. The different groups cluster on the basis of strain administrated.

Differences in effect on abundance of specific phyla seen between strains Sequence analysis revealed that the microbiota was comprised of 7 main phyla (Table 7, and Table 8), with Firmicutes and Bacteroidetes dominating, and relative abundance corresponding to 28-55 % and 43-71%, respectively. Unlike the group fed L. gasseri APC 678, where no significant change in the abundance of Firmicutes or Bacteroidetes relative to controls was observed, the groups fed L. rhamnosus DPC 6111 or L. gasseri ATCC 33323 showed a significant decrease in the relative abundance of Firmicutes (p=0.002 and 0.019, respectively) and a significant increase in Bacteroidetes (p=0.002 and 0.023, respectively) relative to the control. The relative abundance of the Phylum Proteobacteria significantly decreased in the mice fed APC 678 or DPC 6111 relative to the control mice or the animals fed ATCC 33323 which in fact showed an increase in Proteobacteria relative to the control. This change was mirrored in the significant reduction of the relative abundance of the genera Escherichia/Shigella in the groups fed L. gasseri APC 678 and L. rhamnosus DPC6111. Elevated Proteobacteria are normally associated with antibiotic use and are elevated in patients with CDI (Cotter et al and Milani et al 2016) indicating that the abundance profiles seen post feeding APC678 or with L. rhamnosus DPC6111 are more consistent with a healthy diversity profile at the phylum level. Differences in effect on abundance of specific bacterial families and genera within phyla seen on administration of different bacterial strains.

A diverse range of microbial families (Table 9) and genera (Table 10) were also detected across the 4 feeding groups. A number of statistical differences were found in OTUs at family and genus level (Table 9).

The relative abundance of the genus Alistipes significantly increased in mice fed all Lactobacillus strains. The increase was highest in animals L. gasseri ATCC 33323-fed group. A study by Schubert et al (2015) showed that in a murine model Alistipes protected against C. difficile colonisation and the relative abundance of this genus has been shown to be decreased in CDI patients compared to non CDI patients not in receipt of antibiotics (Milani et al., 2016; Schubert et al., 2015a). The L. gasseri ATCC 33323-fed group did not work in the in vivo mouse model however. Further interrogation of the diversity indices did indicate that L. gasseri APC 678 and to a lesser extent L. rhamnosus DPC 6111 had multiple effects on different bacterial families all of which have been linked to effects in CDI.

The relative abundance of the genera Escherichia/Shigella was significantly reduced in the groups fed L. gasseri APC 678 and L. rhamnosus DPC 6111. The decrease in the L. gasseri ATCC 33233-fed group was not significant. The reduction in the genera Escherichia/Shigella would be considered a positive attribute and the relative abundance of these genera was shown to increase in CDI patients in a study where the faecal microbiota of 25 CDI positive patients was compared to a control group (n=30) who were CDI negative and where not exposed to antibiotics (Milani et al., 2016). The relative abundance of Rikenellaceae RC9 gut group significantly increased in all lactobacillus-fed groups compared to the control group with the largest increase associated with L. gasseri APC 678-fed group. The relative abundance of RC9 gut group was shown to be decreased in a cohort of CDI patients relative to that group who were CDI negative and had not been exposed to antibiotics (Milani et al, 2016). The relative abundance of Peptostreptococcaceae Incertae Sedis were significantly reduced in all Lactobacillus-fed groups with both L. gasseri strains showing the largest decrease. This family encompasses C. difficile which has now been reclassified as Peptoclostridium difficile (Yutin and Galperin, 2013)(6).

Strain specific effects were seen for L. gasseri APC 678 and L. rhamnosus DPC 6111 which are associated with the presence or absence of non-communicable disease.

The relative abundance of Roseburia significantly increased in all Lactobacillus-fed groups but the largest increase was seen in the L. gasseri APC 678-fed group. Roseburia are associated with the production of short chain fatty acid production in the gut. SCFA are known to have antiinflammatory, anti-tumorigenic, and antimicrobial activity and can be metabolised by host epithelial cells in the colon (Rios-Covian et ah, 2016). The relative abundance of Oscillibacter significantly increased in the groups fed both L. gasseri APC 678 or L. rhamnosus DPC 6111 but not in the L. gasseri ATCC 33323-fed group. Some species of Oscillibacter are associated with the production of SCFA producing predominantly valerate (lino et ah, 2007). Table 8: Alpha diversity indices for sequencing coverage and diversity of microbiota of caecum samples at Day 7 from control and the mice fed the test strains.

L. gasseri L. rhamnosus L. gasseri

Alpha diversity index Control

APC 678 DPC 6111 ATCC 33323

Chaol richness estimate 210 272 ^a 194 168

Simpson diversity index 0.92 0.94*' ^a 0.92 0.89

Shannon diversity index 4.77 5.45*' ^a 4.69 4.41

PD whole tree 11.69 14.06 ^a 11.06 9.73

Number of observed species 197 259 ^a 182 153 * significantly different compared to the control; Significantly different compared to L. rhamnosus DPC 6111 and L. gasseri ATCC 33323; non-parametric Mann- Whitney test was used to estimate the relationship between the groups; statistical significance was accepted at p<0.05.

Table 9: Relative abundance (%) of bacterial phyla in the caecum at Day 7 of the control and probiotic-fed mice (Lactobacillus gasseri APC 678, Lactobacillus rhamnosus DPC 6111 and Lactobacillus gasseri ATCC 33323).

Group Control APC 678 DPC 6111 ATCC 33323

relative abundance (%)

Firmicutes 54.51 37.22 28.44* 31.29*

Bacteroidetes 43.17 62.24 70.62* 66.25*

Verrucomicrobia 1.53 0.08 0.35 0.93

Proteobacteria 0.47 0.19* 0.09* 1.29

Actinobacteria 0.26 0.18 0.19 0.13*

Tenericutes 0.01 0.03 0.26 0.10

Cyanobacteria 0.00 0.00 0.00 0.00

Other 0.04 0.05 0.05 0.02

* significantly different compared to the control; significance was determined by p<0.05.

Table 10: Relative abundance (%) at bacterial phylum, family and genus level in the caecum at Day 7 of the control and test mice (Lactobacillus gasseri APC 678, Lactobacillus rhamnosus DPC 6111 and Lactobacillus gasseri ATCC 33323). Only phyla, families and genera with significant differences compared to the control mice are represented.

Group Control APC 678 DPC 6111 ATCC 33323

Relative abundance (%)

Phylum:

Firmicutes 54.51 37.22 28.44* 31.29*

Bacteroidetes 43.17 62.24 70.62* 66.25*

Proteobacteria 0.47 0.19* 0.09* 1.29

Actinobacteria 0.26 0.18 0.19 0.13*

Family:

Lachno spiraceae 40.35 27.46 20.14* 23.60* S24-7 24.10 31.18 40.72* 23.21

Bacteroidaceae 9.29 21.60 18.61 28.21 uncultured Clostridiales 2.01 0.64 0.21* 0.58*

Rikenellaceae 1.86 7.31* 6.52* 8.11*

Erysipelotrichaceae 1.80 0.62* 0.83* 0.95*

Peptostreptococcaceae 0.60 0.08* 0.02* 0.05*

Alcaligenaceae 0.29 0.09* 0.069* 1.24

Enterobacteriaceae 0.18 0.01* 0.02* 0.06

Bifidobacteriaceae 0.13 0.04* 0.02* 0.07*

Enterococcaceae 0.03 0.02 0.01* 0.01

Peptococcaceae 0.00 0.04* 0.02 0.07

Prevotellaceae 0.00 0.53 0.56* 0.00

Xanthomonadaceae 0.00 0.06* 0.00 0.00

Genus:

uncultured Lachnospiraceae 29.99 20.93 14.78* 18.00 uncultured S24-7 24.10 31.18 40.72* 23.21

Bacteroides 9.29 21.60 18.61 28.21

Ruminococcaceae Incertae Sedis 5.36 2.19* 1.44* 2.01* uncultured Clostridiales 2.01 0.64 0.21* 0.58*

Alistipes 1.41 3.40* 4.95* 6.03* uncultured Ruminococcaceae 1.08 2.03* 0.77 1.31

Peptostreptococcaceae Incertae 0.60 0.08* 0.02* 0.05*

Sedis

Anaerotruncus 0.60 1.09 1.85* 0.76

Rikenellaceae RC9 gut group 0.45 3.90* 1.57* 2.08

Oscillibacter 0.36 1.15* 1.81* 0.79

Parasutterella 0.29 0.09* 0.07* 1.24

Flavonifractor 0.25 0.01* 0.01* 0.09

Roseburia 0.21 0.90* 0.21 0.29

Escherichia-Shigella 0.18 0.01* 0.02* 0.06 uncultured Erysipelotrichaceae 0.14 0.04* 0.05* 0.04*

Bifidobacterium 0.13 0.04* 0.02* 0.07*

Enterococcus 0.03 0.02 0.007* 0.01 uncultured Peptococcaceae 0.00 0.03* 0.01 0.07

Prevotella 0.00 0.53 0.56* 0.00

Hydrogenoanaerobacterium 0.00 0.004* 0.00 0.00

Stenotrophomonas 0.00 0.06* 0.00 0.00

* significantly different compared to the control; significance was determined by p<0.05, where the null hypothesis was rejected.

Table 11: Relative abundance (%) of bacterial families in the caecum at Day 7 of the control and test mice (fed Lactobacillus gasseri APC 678, Lactobacillus rhamnosus DPC 6111 and

Lactobacillus gasseri ATCC 33323).

Group Control APC 678 DPC 6111 ATCC 33323

Lachno spiraceae 40.346 27.461 20.141* 23.600*

S24-7 24.104 31.182 40.720* 23.214

Bacteroidaceae 9.289 21.603 18.614 28.214*

Porphyromonadaceae 7.918 1.611 4.213 6.710

Ruminococcaceae 7.743 6.576 5.874 4.957 uncultured Clostridiales 2.008 0.639 0.213* 0.582*

Rikenellaceae 1.859 7.314* 6.515* 8.112*

Lactobacillaceae 1.835 1.078 1.259 1.036

Erysipelotrichaceae 1.804 0.616* 0.829* 0.948*

Verrucomicrobiaceae 1.531 0.084 0.354 0.928

Peptostreptococcaceae 0.603 0.078* 0.019* 0.050*

Alcaligenaceae 0.286 0.085* 0.069* 1.235

Enterobacteriaceae 0.177 0.010* 0.017* 0.055

Coriobacteriaceae 0.130 0.140 0.167 0.063

Bifidobacteriaceae 0.126 0.039* 0.024* 0.065*

Christensenellaceae 0.103 0.375 0.000 0.012

Enterococcaceae 0.030 0.023 0.007* 0.013

Clostridiales Family XIII Incertae 0.019 0.028 0.029 0.019

Sedis

Clostridiaceae 0.016 0.000 0.002 0.001

Anaeroplasmataceae 0.013 0.033 0.258 0.097

Oxalobacteraceae 0.005 0.000 0.000 0.000 Peptococcaceae 0.004 0.041* 0.020 0.066

Rhodo spirillaceae 0.002 0.008 0.000 0.000

Staphylococcaceae 0.002 0.288 0.045 0.000

Streptococcaceae 0.001 0.001 0.000 0.000

Rickettsiales mitochondria 0.001 0.000 0.000 0.000

Cyanobacteria 4C0d-2 uncultured 0.001 0.000 0.000 0.000

Moraxellaceae 0.001 0.005 0.000 0.000

Planococcaceae 0.000 0.012 0.000 0.000

Prevotellaceae 0.000 0.533 0.561* 0.001

Bacteroidales ratAN060301C 0.000 0.000 0.000 0.000

Bacillaceae 0.000 0.001 0.000 0.000

Methylobacteriaceae 0.000 0.000 0.000 0.001

Comamonadaceae 0.000 0.002 0.000 0.000

Desulfovibrionaceae 0.000 0.024 0.000 0.000

Xanthomonadaceae 0.000 0.057* 0.001 0.000

Others 0.045 0.051 0.047 0.018 significantly different compared to the control; significance was determined by p<0.05.

Table 12: Relative abundance (%) of bacterial genera in the caecum at Day 7 of the control and test mice (fed Lactobacillus gasseri APC 678, Lactobacillus rhamnosus DPC 6111 and Lactobacillus gasseri ATCC 33323).

Genus Control APC 678 DPC 6111 ATCC 33323 uncultured Lachnospiraceae A 29.985 20.933 14.777* 18.000* uncultured S24-7 24.104 31.182 40.720* 23.214

Lachnospiraceae Incertae Sedis 9.665 5.321 4.994 4.906

Bacteroides 9.289 21.603 18.614 28.214*

Parabacteroides 7.917 1.611 4.213 6.710

Ruminococcaceae Incertae Sedis 5.364 2.185* 1.441* 2.014* uncultured Clostridiales 2.008 0.639 0.213* 0.582*

Lactobacillus 1.835 1.078 1.259 1.036

Akkermansia 1.531 0.084 0.354 0.928

Alistipes 1.406 3.395* 4.949* 6.034* uncultured Ruminococcaceae 1.081 2.031* 0.771 1.308

Erysipelotrichaceae Incertae Sedis 0.953 0.171 0.290 0.268

Allobaculum 0.691 0.401 0.449 0.294

Peptostreptococcaceae Incertae Sedis 0.603 0.078* 0.019* 0.050 ^:

Anaerotruncus 0.601 1.090 1.846* 0.755

Rikenellaceae RC9 gut group 0.453 3.895* 1.566* 2.078

Oscillibacter 0.363 1.151* 1.805* 0.787

Parasutterella 0.286 0.085* 0.069* 1.235

Blautia 0.267 0.076 0.030 0.132

Flavonifractor 0.251 0.012* 0.007* 0.087

Roseburia 0.207 0.903* 0.212 0.288

Escherichia/Shigella 0.177 0.010* 0.017* 0.055 uncultured Erysipelotrichaceae A 0.138 0.036* 0.054* 0.039 ^:

Anaerostipes 0.129 0.064 0.064 0.065

Enterorhabdus 0.128 0.135 0.159 0.059

Bifidobacterium 0.126 0.039* 0.024* 0.065 ^: uncultured Christensenellaceae 0.102 0.375 0.000 0.008

Ruminococcus 0.082 0.102 0.001 0.005

Coprococcus 0.053 0.063 0.017 0.020

Marvinbryantia 0.038 0.099 0.047 0.188

Enterococcus 0.030 0.023 0.007* 0.013

Clostridiales Family XIII Incertae 0.019 0.025 0.029 0.012

Sedis

uncultured Erysipelotrichaceae B 0.017 0.009 0.026 0.011

Clostridium 0.016 0.000 0.002 0.001

Anaeroplasma 0.013 0.033 0.258 0.097

Oxalobacter 0.005 0.000 0.000 0.000

Coprobacillus 0.004 0.000 0.010 0.337 uncultured Peptococcaceae 0.004 0.025* 0.006 0.066

Acetitomaculum 0.002 0.000 0.000 0.000

Thalassospira 0.002 0.008 0.000 0.000

Staphylococcus 0.002 0.287 0.045 0.000

Streptococcus 0.001 0.001 0.000 0.000 Christensenella 0.001 0.000 0.000 0.004 uncultured Coriobacteriaceae 0.001 0.005 0.007 0.004

Idiospermum 0.001 0.000 0.000 0.000 uncultured Cyanobacteria 4C0d-2 0.001 0.000 0.000 0.000

Acinetobacter 0.001 0.005 0.000 0.000

Sporosarcina 0.000 0.012 0.000 0.000

Barnesiella 0.000 0.000 0.000 0.000

Prevotella 0.000 0.533 0.561* 0.000 uncultured Prevotellaceae 0.000 0.000 0.000 0.001

Rikenella 0.000 0.025 0.000 0.000 uncultured Bacteroidales 0.000 0.000 0.000 0.000 ratAN060301C

Bacillus 0.000 0.001 0.000 0.000

Salinicoccus 0.000 0.001 0.000 0.000

Anaerovorax 0.000 0.000 0.000 0.001 uncultured Clostridiales Family XIII 0.000 0.003 0.000 0.007

Incertae Sedis

uncultured Lachnospiraceae B 0.000 0.002 0.000 0.000

Peptococcus 0.000 0.016 0.015 0.000

Hydrogenoanaerobacterium 0.000 0.004* 0.000 0.000

Subdoligranulum 0.000 0.000 0.003 0.000

Methylobacterium 0.000 0.000 0.000 0.001

Variovorax 0.000 0.002 0.000 0.000

Bilophila 0.000 0.024 0.000 0.000

Stenotrophomonas 0.000 0.057* 0.001 0.000

Other 0.045 0.051 0.047 0.018 significantly different compared to the control; significance was determined by p<0.05.

Discussion

Because of the economic burden of the frequency of CDI in hospitals, facilities for the elderly and, more recently, community settings (Chitnis et ah, 2013), alternative treatments for the prevention of CDI are being investigated. The use of microbial therapy as a preventive measure is one such avenue under investigation. Surveys of the literature have shown that for paediatric use, lactobacillus strains significantly prevented antibiotic associated diarrhoea and CDI in children (Goldenberg et ah, 2013). However, while there was some evidence that microbial therapy was effective in preventing primary CDI in adults, there was insufficient evidence to confirm the efficacy of these strains to prevent recurrent CDI (Evans and Johnson, 2015; Goldstein et ah, 2015).

The concept of strain specific benefits of probiotics is not new and has also already been shown to be true of probiotics for other applications (Wall et ah, 2012). In this context our study pre- screened a range of Lactobacillus species for their ability to inhibit C. difficile with a view to identifying a potential strains to target CDI in humans. To that end we developed a culture medium which allowed the growth of both the Lactobacillus and the C. difficile strains in co- culture, without the drop in pH normally associated with growth of lactobacilli in other media such as MRS. We identified 4/58 Lactobacillus strains (2 L. gasseri strains APC 678 and DPC 6112, 1 L. rhamnosus strain DPC 6111 and 1 L. paracasei strain APC1483), all of human origin, which inhibited the growth of C. difficile in vitro. Among the strains screened but which did not show efficacy was L. gasseri ATCC 33323, which has been shown to have a number of traits encoded on its genome which are important for its survival and retention in the gastrointestinal tract (Azcarate-Peril et ah, 2008). L. gasseri APC 678 was the lead candidate from this screening and, interestingly while 10 strains of L. gasseri were screened in vitro only 2 L. gasseri strains, L. gasseri APC 678 and L. gasseri DPC 6112, inhibited C. difficile lending credence to the theory that not all strains of the same species have the same effect.

The two lead candidates from the in vitro work, namely L. gasseri APC 678 and L. rhamnosus DPC 6111, were selected for in vivo analysis. These strains had the added advantage of being capable of survival in significant numbers during simulated gastric transit, at low pH and in the presence of bile and the digestive enzymes encountered in the stomach and upper GIT. The increased survival rate in these environments in the presence of milk is a further bonus as fermented milk products such as yoghurts and cheese are often used as vehicles for oral delivery of live bacteria (Gardiner et ah, 1998; Hickson et ah, 2007).

As a first step to determine the efficacy of these strains with respect to decreasing CDI in vivo, the strains (L. gasseri APC 678, L. rhamnosus DPC 6111 and the aforementioned well- characterised strain L. gasseri ATCC 33323) were tested for their ability to reduce faecal shedding of C. difficile in a murine model of CDI over 7 days. The ability to reduce faecal shedding was significant, when compared to the control group fed RSM, in those animals fed L. gasseri APC 678 four days post infection and this reduction was maintained up to 7 days at which time the animals were euthanized. No significant effect was seen in terms of faecal shedding of C. difficile in those mice fed either L. gasseri 33323 or L. rhamnosus DPC 6111. Interestingly, when the level of viable C. difficile in the colon was assessed the numbers were significantly reduced in those mice which were fed either of the L. gasseri strains, which is possibly a result of competitive exclusion of C. difficile by the L. gasseri strains. The absence of evidence of bacteriocin activity in vitro would suggest that inhibition by bacteriocins is not the reason.

CDI is normally the result of perturbation of the gut microbiota as a result of broad-spectrum antibiotic treatment which results in a decrease in microbial diversity (Rea et ah, 2012b). One function of a live therapeutic in a disease state would be to increase diversity, thus reducing the ability of C. difficile to survive and multiply due to competition for nutrients. Compositional sequencing showed that in the control fed mice and the test groups there was a diverse microbiota despite the prior administration of antibiotics to make the animals more susceptible to infection. L. gasseri APC 678 increased diversity for all the indices tested, including the number of observed species compared to the other strains studied. It has been recognised that a decrease in diversity has been linked to CDI (Gu et ah, 2016; Rea et ah, 2012b). However, unlike the mice fed L. gasseri APC 678, where no change in the relative abundance of the main phyla was observed when compared to the control, there was a significant change in the Firmicutes and Bacteroides levels in the groups fed either L. rhamnosus DPC 6111 or L. gasseri ATCC 33323 when compared to the control with a significant increase in Bacteroidetes. In a recent paper by Schubert et al. (2015) it was shown that, when the gut microbiota was changed as a result of antibiotics in a murine model, populations of Porphyromonadaceae, Lachnospiraceae, Lactobacillus and Alistipes protected against C. difficile colonisation (Schubert et al., 2015). While our study showed that Lachnospiraceae were significantly reduced in the groups fed L. rhamnosus DPC 6111 and L. gasseri ATCC 33323, it was notable that Alistipes were significantly increased in all probiotic-fed animals relative to the control group.

In the fight against CDI it is likely that live therapeutics, either as well characterised single/multiple strains of lactobacilli and bifidobacteria as described here or the more complex less defined microbiota in FMT will play a role in addressing the reduction in microbial diversity in the GIT that results from broad spectrum antibiotic treatment leading to CDI. There are advantages of using well characterised strains with QPS status which have proven efficacy against C. difficile in vivo and that translate into positive changes in the gut microbiota profile. For instance, the interactions between the gut microbiome and the host are complex and FMT may therefore have unintended consequences in a patient after successful FMT due to alteration of the gut microbiota. FMT in experimental animals has shown that immunologic, behavioural and metabolic phenotypes can be transferred from donor to recipient which may not always be beneficial to the recipient in the long term. (Collins et al., 2013; Di Luccia et al., 2015; Pamer, 2014).

Protective immunity against C difficile

L. gasseri APC678 was used successfully to control (clear/reduce significantly) C. difficile infection in an in vivo mouse model. L. gasseri APC678 by reducing the C difficile infection will reduce the immune pathogenesis of Clostridium difficile- associated disease through a reduction of the neutrophil-mediated inflammatory response such as bactericidal reactive oxygen intermediates, defensins and pro-inflammatory cytokines and chemokines thereby stopping the tissue damage and persistent clinical disease. In certain embodiments, the compositions described herein exhibit immunological properties (Protective immunity) following appropriate administration to a subject. The presence of Protective immunity may be demonstrated as described above and/or by showing that infection by a pathogen (e.g., C. difficile) is affected (e.g., decreased) in individuals (e.g., human being or other animal) to whom the materials described herein have been administered as compared to individuals to whom the materials have not been administered. For instance, one or more test subjects (e.g., human or non-human) may be administered by any suitable route and schedule a composition described herein, and then after a suitable amount of time (such as 1 week in the model) challenged by a pathogenic organism. Protective immunity may be one that is detrimental to the infectious organism corresponding to the C. difficile and beneficial to the host (e.g., by reducing or preventing infection). As used herein, protective cellular responses may be reactive with the C. difficile strain, especially when administered in an effective amount and/or schedule. Those cellular responses may reduce or inhibit the severity, time, and/or lethality of C. difficile infection when tested in animals. As shown in the examples, the compositions described herein may be used to induce an immune response against C. difficile. An immunological composition that, upon administration to a host, results in a therapeutic (e.g., typically administered during an active infection) and/or protective (e.g., typically administered before or after an active infection).

The present invention generally relates to compositions and methods for the prevention or treatment of bacterial infection by the Gram-positive organism, Clostridium difficile, in a vertebrate subject. The methods provide administering an agent to the vertebrate subject in need thereof in an amount effective to reduce, eliminate, or prevent Clostridium difficile bacterial infection or bacterial carriage.

As used herein, the term "immunity" refers to the response of immune system cells to external or internal stimuli {e.g., antigen, cell surface receptors, cytokines, chemokines, and other cells) producing biochemical changes in the immune cells that result in immune cell migration, killing of target cells, phagocytosis, production of antibodies, other soluble effectors of the immune response, and the like.

"Protective immunity" means that the subject mounts an active immune response to a composition, such that upon subsequent exposure to Clostridium difficile bacteria, the subject is able to combat the infection. Thus, a protective immune response will generally decrease the incidence of morbidity and mortality from subsequent exposure to Clostridium difficile bacteria among subjects. Protective immunity will also generally decrease colonization by Clostridium difficile bacteria in the subjects.

Pathogenesis of Clostridium difficile-associated disease

C. difficile release toxins A and B in the colon, which are the key virulence determinants of C. difficile- associated disease (CDAD). The toxins translocate to cytosol of target cells including IECs, mast cells, fibroblasts, smooth muscle cells, and monocytes (Na et al 2008), eventually resulting in a disruption of the actin cytoskeleton (Voth et al 2005), epithelial cell rounding and eventually cell death and necrosis. This cellular stress response triggers secretion of inflammatory cytokines and expression of leukocyte adhesion molecules (integrins and selectins (Savidge et al 2003) which in turn facilitate neutrophil infiltration into the colon. The infiltrated neutrophils are activated at the site of infection and produce bactericidal reactive oxygen intermediates (He et al 2000), defensins and pro-inflammatory cytokines and chemokines (Kelly et al 2011), leading to an intense neutrophil-mediated inflammatory response, which is believed to be one of the key determinants of disease severity an over exuberant neutrophil response is clearly associated with tissue damage and persistent clinical disease (Bulusu et al 2000).

Formulations

One or more of the strains of the invention may be administered to animals (including humans) in an orally ingestible form in a conventional preparation such as capsules, microcapsules, tablets, granules, powder, troches, pills, suppositories, suspensions and syrups. Suitable formulations may be prepared by methods commonly employed using conventional organic and inorganic additives. The amount of active ingredient in the medical composition may be at a level that will exercise the desired therapeutic effect. The formulation may also include a bacterial component, a drug entity or a biological compound.

In addition a vaccine comprising one or more of the strains of the invention may be prepared using any suitable known method and may include a pharmaceutically acceptable carrier or adjuvant.

The introduction of probiotic organisms is accomplished by the ingestion of the micro-organism in a suitable carrier. It would be advantageous to provide a medium that would promote the growth of these probiotic strains in the large bowel. The addition of one or more oligosaccharides, polysaccharides, or other prebiotics enhances the growth of lactic acid bacteria in the gastrointestinal tract. Prebiotics refers to any non-viable food component that is specifically fermented in the colon by indigenous bacteria thought to be of positive value, e.g. bifidobacteria, lactobacilli. Types of prebiotics may include those that contain fructose, xylose, soya, galactose, glucose and mannose. The combined administration of a probiotic strain with one or more prebiotic compounds may enhance the growth of the administered probiotic in vivo resulting in a more pronounced health benefit, and is termed synbiotic.

It will be appreciated that the probiotic strains may be administered prophylactically or as a method of treatment either on its own or with other probiotic and/or prebiotic materials as described above. In addition, the bacteria may be used as part of a prophylactic or treatment regime using other active materials such as those used for treating inflammation or other disorders especially those with an immunological involvement. Such combinations may be administered in a single formulation or as separate formulations administered at the same or different times and using the same or different routes of administration.

The strains of the invention may be formulated to facilitate controlled release such as a delayed release of the strain. For example, the formulation may be adapted to release the strain at a particular location in the gastrointestinal tract such as the small intestine or in the colon. To achieve such a controlled release the strain may be formulated in a capsule which has a coating which is adapted to release the strain at a particular location. A range of coatings are available to facilitate such controlled release. One such family of coatings are those available under the Trade Mark Eudragit. The invention is not limited to the embodiments hereinbefore described, which may be varied in detail.

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