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SID 5 Research Project Final Report

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NoteIn line with the Freedom of Information Act 2000, Defra aims to place the results of its completed research projects in the public domain wherever possible. The SID 5 (Research Project Final Report) is designed to capture the information on the results and outputs of Defra-funded research in a format that is easily publishable through the Defra website. A SID 5 must be completed for all projects.

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Project identification

1. Defra Project code VM2203

2. Project title

Interventions to reduce carriage of antimicrobial resistance in food producing animals

3. Contractororganisation(s)

Veterinary Laboratories Agency,Woodham Lane,New Haw,Weybridge,Surrey,KT15 3NB.

54. Total Defra project costs £ 219,092(agreed fixed price)

5. Project: start date................ 01 April 2006

end date................. 01 November 2007

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6. It is Defra’s intention to publish this form. Please confirm your agreement to do so...................................................................................YES NO (a) When preparing SID 5s contractors should bear in mind that Defra intends that they be made public. They

should be written in a clear and concise manner and represent a full account of the research project which someone not closely associated with the project can follow.Defra recognises that in a small minority of cases there may be information, such as intellectual property or commercially confidential data, used in or generated by the research project, which should not be disclosed. In these cases, such information should be detailed in a separate annex (not to be published) so that the SID 5 can be placed in the public domain. Where it is impossible to complete the Final Report without including references to any sensitive or confidential data, the information should be included and section (b) completed. NB: only in exceptional circumstances will Defra expect contractors to give a "No" answer.In all cases, reasons for withholding information must be fully in line with exemptions under the Environmental Information Regulations or the Freedom of Information Act 2000.

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Executive Summary7. The executive summary must not exceed 2 sides in total of A4 and should be understandable to the

intelligent non-scientist. It should cover the main objectives, methods and findings of the research, together with any other significant events and options for new work.

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In simple in vitro pair-wise inhibition studies, all short chain fatty acid producing probiotic strains inhibited the growth of two S. Typhimurium test strains. The extent of inhibition varied between probiotic strains and pH neutralisation studies demonstrated that the most likely inhibitory factors were the short chain fatty acids. Indeed, DHPLC approaches proved that the end product of metabolism of the probiotics were a range of short chain fatty acids. One strain of Lactobacillus plantarum, of pig origin, produced a pronounced pH independent inhibition of S. Typhimurium. Further work to identify the nature of this inhibitor is recommended.

Batch culture fermentations simulating the pig caecum were carried out to evaluate the influence of different prebiotics on resistant Salmonella Typhimurium SL1344. The test prebiotics were xylooligosaccharides (XOS), a mixed fructooligosaccharides/inulin (FIN), fructooligosaccharides (FOS), gentiooligosaccharides (GEO) and lactulose (LAC). Compared with control (CTR, no prebiotic), GEO and FIN increased lactic acid (P<0.05), all the prebiotics except LAC increased the production of SCFA (P<0.0001) and modified SCFA profiles. All the prebiotics increased Bifidobacteria counts as detected by Bif164 probing compared to CTR (P<0.005) and all of them except FOS increased Lactic Acid Bacteria counts as detected by Lab158 probe (P<0.01). Salmonella grew in the presence of all the prebiotics, although less with LAC (P<0.0001). Under our experimental fermentation conditions, prebiotics seemed to not exert any inhibitory effect on Salmonella growth, neither by competitive exclusion nor by inhibition by lactic acid and SCFA generated in their fermentation. However, this does not rule out the inhibitory effect of prebiotics on S. Typhimurium, by mechanisms other than those studied in this in vitro system. Therefore, prebiotics and especially LAC should be further investigated in vivo against S. Typhimurium.

Although the fermentation data may be considered equivocal in terms of suppression of S.

INTRODUCTION TO THE PROJECT

Within the European Union since the 1st of January 2006, the use of antibiotics as growth promoters in livestock is no longer permitted (Regulation 1831/2003/EC). Until then, the inclusion of antibiotics in feeds was common practice and it is considered likely to have contributed to the observed increase in bacterial resistance to antibiotics (Randall and Woodward, 2002; Quinn et al., 2006; Herrero et al., 2007; Spiliopoulou et al., 2007). Antibiotic resistance in Salmonella species found in farm animals can lead to increased morbidity and mortality, due to increased bacterial fitness and reduced efficacy of therapeutic antibiotics. In addition, Salmonella species from animals can enter the human food chain where they can cause infections in humans and may not be effectively treated. Therefore, in both animals and humans, there are economic losses associated with Salmonella infection (Sockett and Roberts, 1991; Roberts et al., 2003; de Jong and Ekdahl, 2006). Dietary strategies with alternative to the inclusion of antibiotics in feed are being pursued actively and among these alternatives, prebiotics and probiotics seem to be valuable candidates. In the gastro-intestinal tract, prebiotics and probitoics have been shown to act against Salmonella both directly and indirectly. The direct mechanisms are considered to include inhibition of adhesion to the epithelium (Baumler et al., 1997; Linquist et al., 1987; Naughton et al., 2001; Tzortzis et al., 2005) and stimulation of the immune system (Milo et al., 2004; Benyacoub et al., 2008). The indirect mechanisms are considered to be the promotion of growth of beneficial bacteria (lactic acid bacteria) (Gibson et al., 2005) which can act against Salmonella by mechanisms of competitive exclusion for nutrients or adhesion sites (Salminen et al.1996; Fukata et al., 1999; Lievin-Le et al. 2002; Casey et al., 2004; Klessen & Blaut, 2005), production of antimicrobial compounds including short chain fatty acids (SCFA), Hydrogen peroxide (H2O2) or bacteriocins (Servin 2004; Reid et al. 2003) or by stimulation of the immune system (Tomioka et al. 1992; Park et al. 2005; Vinderola et al., 2007)

OVERALL AIMS OF THE STUDY

Multi-resistant Salmonella Typhimurium DT104 is prevalent in pig production and is therefore of considerable veterinary public health concern both in terms of risks of transmission of S. Typhimurium to man and entry of antibiotic resistance into the food chain. The overall goal of the project was to identify and study the mechanism of action of pre and probiotics for use as potential control strategies for multi-resistant Salmonella Typhimurium DT104 in pigs. Prebiotics are non-digestible complex carbohydrate compounds that enhance fermentation of health beneficial bacterial commensals in the lower gut. Probiotics are live health beneficial bacteria, usually native strains that are commensals in the target

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animal, that can infer a benefit, which in the study to be reported herein is suppression of Salmonella Typhimurium DT104 in a simulated of the pig gut.

The specific objectives of the project were set out in the proposal forms and for brevity are not repeated in full here. This report follows the project proposal and describes each of the objectives in brief and then gives a ‘materials and methods’ section relevant to that objective, a ‘results’ section and brief ‘discussion’ section. At the conclusion of the objectives (5 in total), there is a broader discussion of the whole study.

Project Report to Defra8. As a guide this report should be no longer than 20 sides of A4. This report is to provide Defra with

details of the outputs of the research project for internal purposes; to meet the terms of the contract; and to allow Defra to publish details of the outputs to meet Environmental Information Regulation or Freedom of Information obligations. This short report to Defra does not preclude contractors from also seeking to publish a full, formal scientific report/paper in an appropriate scientific or other journal/publication. Indeed, Defra actively encourages such publications as part of the contract terms. The report to Defra should include: the scientific objectives as set out in the contract; the extent to which the objectives set out in the contract have been met; details of methods used and the results obtained, including statistical analysis (if appropriate); a discussion of the results and their reliability; the main implications of the findings; possible future work; and any action resulting from the research (e.g. IP, Knowledge Transfer).

Objective 01:

Selection of probiotic strains: Determination of the inhibitory spectrum of probiotics against multi-resistant S. Typhimurium in co-cultureOne of the principal values for probiotic use in agriculture is to reduce the incidence of gastrointestinal infection and thereby improve not only the health of the animal but other factors such as yield, carcass quality, etc. ( Fox, 1988; Veterinary Medicine 9, 806-830). There are many candidate probiotics for use on the market but none make specific claims for the reduction of S. Typhimurium in any farmed animal species as such claims would immediately convert the probiotic into a medicinal product. However, many research studies have indicated that probiotics can inhibit certain bacterial pathogens including S. Typhimurium by a range of mechanisms including:-

Competition for nutrients Formation of lactic and acetic acids that are inhibitory to pathogens The excretion of antimicrobial peptides Inhibitory properties by colonisation of receptor sites for pathogens Immune stimulation (non specific)

In this objective, we screened a wide range of probiotic species from a variety of sources for anti-Salmonella Typhimurium activity.

Materials and Methods

Bacterial Culture Conditions

Probiotics were cultured statically in 20ml Mann, Rogosa, Sharpe Broth (MRS) (Difco, UK) in an anaerobic cabinet (Don Whitley, Yorkshire, UK; 10:10:80, H2:CO2:N2 ) at 37C. Salmonella Typhimurium strains and E. coli DH5-α were cultured aerobically in Luria Bertani broth without glucose (LB-G broth) at 37°C, or with gentle agitation (225 rpm) in LB-G supplemented with 0.5% Bacto agar for plates.

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Table of Strains: Enterobacteriaceae

Species Strain PT Antibiotic Resistance Pattern SourceS.Typhimurium SL1344 104 S, SU Gift from Dr O’Connor

S05461-03 104 S, SU VLA, WeybridgeS06674-02 104 S, SU VLA, WeybridgeS07552-02 104 T, AM, SXT, C, S, SU VLA, WeybridgeS01760-03 104 Sensitive to all 16 antibiotics tested VLA, WeybridgeS02980-03 104 Sensitive to all 16 antibiotics tested VLA, WeybridgeS02981-03 104 Sensitive to all 16 antibiotics tested VLA, WeybridgeS02982-03 104 T, AM, C, S, SU VLA, WeybridgeS02442-05 104 T, AM, C, S, SU VLA, WeybridgeS02486-06 104 T, AM, C, S, SU VLA, Weybridge

E. coli Strain DH5α - InvitrogenPT-Phage type, S-Sulphonamide, S- Streptomycin, T-Tetracycline, A-Ampicillin, C, Chloramphenicol, SXT- sulfamethoxazole and trimethoprim.

Probiotic strains.

Probiotic strains used in the studies were provided by the following:-Probiotics International Ltd, Stoke-sub-Hamdon, Somerset, UK [PIL], Institute of Food Research, Norwich, UK [IFR] Reading University [RU]Veterinary Laboratories Agency [VLA] For commercial in confidence reasons strains from PIL and IFR were offered for the study on the understanding that strain identities were not revealed without prior permission from the suppliers.

Strain Number of strains SourcesLactobacillus acidophilus 3 IFR, VLA, URLactobacillus rhamnosus 3 UR, PIL (x2)Lactobacillus reuteri 1 URLactobacillus casei 3 UR, IFR, PILLactobacillus gasseri 1 IFRLactobacillus plantarum 1 VLALactobacillus johnsonii 1 IFRBifidobacterium bifidum 2 UR, PILBifidobacterium longum 2 UR, PILBifidobacterium breve 1 PILBifidobacterium adolescentis 2 UR, PILBifidobacterium infantis 2 UR, PILBifidobacterium lactis 2 UR, PILEnterococcus faecium 3 PILBacillus subtilis 4 VLA (ex EU project Sporebiotics)

Disc diffusion assay.

Each of the probiotic strains were grown in appropriate broth media (MRSA, HIB, LB) at 37oC under appropriate conditions (e.g. anaerobiosis for all Bifidobacteria) to stationary phase. The bacteria were removed from the broth medium by centrifugation and subsequent filtration through 0.2um filter. Sterility was checked by plating samples (5 x 100ul drops) of the filtrates onto air-dried plates of the relevant broth medium solidified with agar (2% w/v). The plates were then incubated for 96h at 37oC under appropriate conditions with visual inspection for bacterial colonies daily. The sterile spent medium was portioned into two equal volumes. Both samples were measured for pH and one sample neutralised by addition of 1M NaOH drop-wise to achieve pH7.2. Filter discs were soaked in the broths and then laid onto LA plates on which 105 cfu S. Typhimurium (prepared by serial dilution in PBS of a 16h LB culture grown at 37oC with vigorous aeration) had been spread. These plates were incubated at 37oC for 24h with regular observation. As controls, filter discs soaked in fresh un-inoculated broth alone were also applied to the plates. The radius of the zone of inhibition, if observed, was measured in mm from the edge of the disc and recordedFor all these studies, the discs were used immediately upon preparation in case any substance in the probiotic broth was volatile or unstable.

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Spent medium Culture (a).

Spent media were prepared as described for the disc diffusion assay. To confirm the inhibitory capacity against multi-resistant S. Typhimurium, aliquots of dilutions of spent medium (neutralised and un-neutralised) were separately added to LB broth in the following ratios:- 0 + 20, 1 + 19, 2 + 18 and 5 + 15. S. Typhimurium cultures were prepared as described above and 100ul was added to each culture vessel and incubation continued at 37oC with vigorous aeration for at least 12h. OD540 measurements of the S. Typhimurium cultures were taken and recorded as growth curves.

Spent medium Culture (b).

There was some evidence to suggest that the MRS medium that was used to grow Lactic Acid bacteria, whilst able to support the growth of S. Typhimurium, also caused some inhibition. To clarify the contribution of MRS diluted into LB, experiments were set up as above but in which the basal medium was MRS. No other variant was introduced. The spent culture medium of only four Lactic Acid Bacteria were selected for this more detailed study.

Salmonella Inhibition Assay

To test the impact on growth of Salmonella strains mediated by probiotic strains in a faecal slurry, the following system was set up. Sterile 1 lt volumes of basal nutrient medium were prepared. The medium comprised peptone water (2 g L-1), yeast extract (2 g L-1), NaCl (0.1 g L-1), KH2PO4 (0.04 g L-1), MgSO4.7H2O (0.01 g L-1), CaCl2.6H2O (0.01 g L-1), NaHCO3 (2 g L-1), Tween 80 (2 mL L-1), haemin (0.05g L-1), vitamin K (10 µL L-1), L-cysteine hydrochloride (0.5 g L-1) and bile salts (sodium glycocholate and sodium taurocholate) (0.5 g L -1). The medium was adjusted to pH 7.0 and 4 mL of 0.025 % (w/v) resazurin solution were added prior to autoclaving. All media and chemicals were purchased from Oxoid and Sigma, respectively. The sterile medium was sparged with O2-free N2 (15 mL min-1) overnight to establish anaerobic conditions. The following day, a faecal slurry was prepared by collecting and combining fresh faeces from 8 pigs (body weight 25–30 kg, proven free of Salmonella infection by enrichment in selenite broth at 37 °C for 16 h and plating on BGA) and mixing them with pre-reduced sterile phosphate-buffered saline (PBS) (to give a 10 %, w/v, faecal slurry). The faecal matter used was assumed to have a microbial population representative of the large intestinal microflora of pigs (Coates et al., 1988; Williams et al., 1998; Bauer et al., 2004). To a series of sterile 1oz McCartney bottles, 4.5mL of basal medium and 0.5 mL of the freshly prepared faecal slurry were added. Each probiotic was grown o/n at 37oC in MRS medium, diluted in pre-warmed basal medium to give 106 cfu ml-1 of which 100l was added immediately to prepared McCartney bottles. Each test Salmonella and control E. coli strains were grown o/n at 37oC in LB broth medium, diluted in pre-warmed basal medium to give 106 cfu ml-1 of which 100l was added immediately to prepared McCartney bottles. Pair-wise combinations of Salmonella and Probiotic were prepared and control s were inoculated with only the test strain or the probiotic alone.The cultures were grown for 24h at 37oC. The caps were sealed tight to reduce oxygen egress. After incubation, serial dilutions were made in pre-warmed basal medium and samples plated out on MRS and BGA agars. Plates were incubated at 37°C and colonies counted to give a final count per ml -1. Counts were compared between test and controls and percentage value for test and probiotic strains determined.

Results

In this project 31 probiotic strains were collected from four sources. As far as was feasible, probiotics that were originally isolated from the pig gut were used because one selection criterion for use of a strain was that it should have been generated from the same species as the intended host (Fooks et al. 2000. Clinical Nutrition 9, 29-40). The history of the probiotic strains from PIL and IFR were not available for commercial in confidence reasons but those from VLA and Reading had been selected on the basis of being of pig origin with the exception of the B. subitlis strains which were of chicken origin but well characterised from a previous EU funded project.

Characterisation of bacterial isolatesThe panel of Lactobacillus strains were tested for there unique fermentation profiles, to distinguish between different Lactobacilli species. Lactobacillus Plantarum B2028 was able to metabolise the most carbon sources (data not shown). Interestingly, all of the porcine isolates did metabolise raffinose and D Xylose, whereas all the commercial strains did not. All of the test isolates metabolised; glucose, galactose, ribose, maltose, lactose, sucrose and gluconate. In contrast, none of the test isolates could metabolise D+L fucose, 2-keto-glutarate, 5- keto-glutarate, D+L-arabinose, glycerol, erythritol, inositol, dulcitol, starch, xylitol, D+L xylose and glycogen. It was possible to identify all of the test isolates based upon there unique fermentation patterns.

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Disc diffusion studies

The disc diffusion data are presented in Appendix 1 page 1 table 1. All of the probiotic strains, with the exception of one Bacillus subtilis strain, produced spent medium that when un-neutralised inhibited the growth of the two test S. Typhimurium strains. The size of the inhibition zone varied from one probiotic to another but the spent medium from four of the strains were particularly inhibitory inducing inhibition zones of 9mm in radius or more. The spent medium from L. plantarum strain B2028 induced a zone of inhibition measuring 13mm in radius.

In all cases neutralisation of the spent medium reduced the zones of inhibition of S. Typhimurium. For 25 strains, the reduction was absolute. However, the inhibitory effect of neutralised spent medium from the remaining 6 probiotics was not eliminated. Three of those six were B. subtilis strains, two were lactic acid bacteria and one was a Bifidobacterium strain.

Growth curve studies

Four strains (B2206 VLA, B1775 IFR, B2028 VLA and B2207 IFR) induced large zones of inhibition in the disc diffusion studies described above and these were selected for more detailed studies. The first was to quantify the effect of the spent medium on actual growth rate of the Salmonella strains. Thus, broth cultures of two S. Typhimurium strains (Salmonella Typhimurium SL1344 and a penta-resistant Salmonella Typhimurium DT104 strain S01760 which exhibits the R-type ACCsSuT phenotype and is also a field isolate from a pig) were set up in LB broth to which dilutions of spent medium and neutralised spent medium had been added at the ratios described in material and methods. Growth was monitored and two examples of the data arising are shown in Appendix 1 page 2 Figure 1.

In all control experiments, both Salmonella strains grew as anticipated. However, any dilution of un-neutralised spent medium added to the LB caused a very significant reduction in growth rate. However, only the neutralised and un-neutralised spent medium from strain B2028 caused significant reductions in growth. These data are shown in Appendix 1 page 2 Figure 1 and indicate that this one strain had both pH and pH independent mechanisms of inhibition of S. Typhimurium. This was found for both strains of S. Typhimurium tested.

It was possible that the dilution of MRS, the basal medium for the growth of the probiotic Lactic Acid Bacterial strains, into LB had some subtle inhibitory effects. To test this, the experiments were repeated but this time the basal medium for S. Typhimurium growth was MRS therefore nullifying any potential issues arising from diluting MRS into LB. In these studies, the spent medium was prepared and added to MRS to which the S. Typhimurium strains were inoculated as described above. The spent medium from the four strains that produced the most significant inhibitions in disc diffusion assays were tested and the data are shown in Appendix 1, page 3, table 2. For this study, instead of presenting the growth curve as a graph, a more sophisticated statistical approach was used. Here, the ‘area under the growth curve’ was converted to a numerical value and these were then compared in student pair-wise T-tests. Each experiment was repeated three times and the data are presented as mean values with standard deviations. The data confirmed the same findings as before, namely that pH was the primary factor in suppression of Growth of Salmonella and that this effect was real and not an artefact of mixed medium components.

Survival of Salmonella after growth in faecal slurry supplemented with probiotic strains

The table below gives values in percentage terms of the relative growth of both the Salmonella and probiotic strains in faecal slurry together compared with when grown in the faecal slurry alone. The trend shown was that Salmonella strains did not achieve the final bacterial cell density in the presence of the probiotic compared with when grown alone. L. plantarum B2028 appeared to suppress the growth of Salmonella strain consistently more than any other probiotic strain. However, L. acidophilus suppressed one Salmonella (S07552-02) more than in any other pair-wise test.

Strain survival values after mixed culture growth 24h in faecal slurry

Salmonella Probiotic24h exposure% survival Salmonella

24h exposure% survival Probiotic

SL1344

L. plantarumL. fermentum L. rhamnosus L. casei L. acidophilus

78.887.394.395.387.3

102.397.899.5

103.4102.2

S05461-03 L. plantarumL. fermentum L. rhamnosus

88.893.7ND

107.3107.8ND

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L. casei L. acidophilus

95.689.6

104.6106.2

S06674-02


80.897.397.397.497.3

106.3102.899.5

104.497.2

S07552-02


58.8NDND93.747.9

107.3NDND

104.499.8

S01760-03


65.897.394.989.687.3

109.3100.899.5

109.8107.3

S02980-03 L. plantarum 67.8 109.6S02981-03 L. plantarum 54.8 102.2S02982-03 L. plantarum 69.7 100.8S02442-05 L. plantarum 87.9 110.6S02486-06 L. plantarum 67.9 108.7Strain DH5α L. plantarum 0.0 103.8

Discussion There are numerous descriptions in the literature indicating that probiotic strains that produce short chain fatty acids and bacteriocins are capable of inhibiting gram negative bacteria and Salmonella in particular (Servin 2004; Reid et al. 2003). The disc diffusion assays are relatively crude but the data generated gave clear indications that the products of probiotic bacterial metabolism were all inhibitory to the growth of Salmonella. That pH neutralisation nullified these effects indicates conclusively that the inhibitory factor was primarily low pH as might be anticipated. In several cases evidence was gained that the probiotic strain produced an inhibitory effect that persisted after pH neutralisation. One strain in particular, L. plantarum B2028, produced very significant inhibition when the spent medium was un-neutralised and neutralised. Without further investigation, it can only be assumed that the effector of inhibition after neutralisation is a bacteriocin.

The next goal was to test whether probiotics in a simulated gut environment would suppress the growth of Salmonella. Not unsurprisingly given the data from disc diffusion studies, evidence was gained that all the probiotics tested in the faecal slurry culture system did indeed reduce the final density of Salmonella achieved compared with Salmonella grown in the system alone. There was a clear trend of suppression but the individual % suppression values were not remarkable, with the highest level of suppression being in the region of 0.5 log 10. A weakness of these experiments were the lack of true anaerobiosis because the medium was sparged prior top addition to the McCartney bottles which themselves were only sealed tightly. A question arises as to the impact of residual oxygen in the head space upon the probiotic. Compared with the latter fermentation and continuous culture systems (see objectives 02 and 03), this was a crude experiment and in hindsight is far removed from the gut environment. Whilst oxygen may be rapidly reduced in the system through bacterial metabolism, its’ presence may have had a negative impact on the extent of acid production for example.

For subsequent studies, it was not feasible to analyse all the 31 strains investigated by the simple tests described in this section. A pragmatic selection was made based on strains that showed an effect.

Objective 02: Selection of pre/probiotic combinations: In vitro batch experiments using mixed culture inocula containing probiotics and prebiotics

Based on the above information, a number of probiotics were selected for further experimentation in a series of studies. The aims were to (a) test the ability of the probiotic to survive/grow in mixed culture, the model being the luminal contents from pig colon, (b) to determine the capacity of a range of prebiotic carbohydrates to enhance the survival/growth of the probiotic and (c) to test whether either probiotic or pro-prebiotic (symbiotic) suppressed the growth/survival of S. Typhimurium. The probiotic strains selected for further study were B. bifidum aUR1, B. breve aPIL, B. infantis aUR1, B. longum aUR1, E. faecium PIL1, L. acidophilus B2206, L. casei aUR1, L.

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plantarum B2028 and Lactobacillus rhamnosus aUR1. A prebiotic is a dietary ingredient that is resistant to upper gut digestion but exerts a selective fermentation on indigenous populations of hindgut bacteria, in that beneficial organisms such as the bifidobacteria become stimulated (Gibson & Roberfroid 1995. Journal of Nutrition, 125, 1401-1412). The prebiotics selected for inclusion in the study included those that are commercially available and were fructooligosaccharides (FOS, inulin), galactooligosaccharides (GOS), lactulose, xylooligosaccharides (XOS), mannooligosaccharides (MOS) and gentiooligosaccharides (GeOS) as well as novel forms such as pectic oligosaccharides (POS) and alternan oligosaccharides (AOS).

The aim of fermentation studies was to establish what effects were induced by addition of probiotic strains and prebiotics (synbiotics, both pro and pre-biotics) on both ‘good’ bacteria (ie/ Bifidobacteria) and pathogenic bacteria (ie/ Salmonella). A probiotic strain surviving in the fermentation is taken as a measure of success, while the prebiotic approach will define the most efficient forms at stimulating ‘positive’ gut flora genera. In both, effects upon the microbial composition will be determined by the genetic probing strategy FISH (see objective 03).

Prior to incorporation into the batch culture systems (which are set up to reflect colonic events) the probiotics and prebiotics were treated with digesta that simulates the porcine upper gut. This substrate digestion procedure will be required to simulate material entering the colon.

Materials and methods Selected cultures for further study For the pure cultures assays, nine bacterial strains were studied: Lactobacillus acidophilus 72 NCIMB 30179 (PXN23) [cUR], Lactobacillus rhamnosus NCIMB 30188 (PXN54) [aUR1], Lactobacillus casei NCIMB 30185 (PXN37) [aUR1], Lactobacillus plantarum NCIMB 30187 (PXN47) [B2028 VLA], Bifidobacterium bifidum NCIMB 30179 (PXN23) [aUR1], Bifidobacterium longum NCIMB 30182 (PXN30) [aUR1], Bifidobacterium breve NCIMB 30180 (PXN25) [aPIL], Bifidobacterium infantis NCIMB 30181 (PXN27) aUR1] and E. faecium [PIL1]. Strains labelled with PIL were kindly provided by Probiotics International Ltd (Protexin) (Somerset, UK). Strains were maintained at -70 ºC in 15 % (w/w) glycerol. The Salmonella Typhimurium strain used was a fully virulent naladixic-acid resistant derivative of the wild-type strain SL1344, a kind gift from David O’Connor (Southampton University).

Substrates

The following potential prebiotics were evaluated. XOS (95 %; Suntory Limited, Osaka, Japan), a mixture of fructooligosaccharides/inulin (FIN) (8 % glucose, fructose and sucrose; Orafti, Tienen, Belgium), gentiooligosaccharides (GEO) (95 %; Wako Pure Chemicals, Japan), FOS (5 % glucose, fructose and sucrose; Orafti) and LAC (100 %; Solvay Pharmaceuticals, Southampton, UK).

Growth curves

The growth rates of the nine bacterial strains listed above and Salmonella in pure cultures were determined in the presence of individual prebiotics. Plates of Mueller–Rogosa–Sharpe (MRS) agar (Oxoid Ltd, Basingstoke, Hampshire, UK) (for the nine bacterial strains) or brilliant green agar (BGA; Oxoid) containing Nalidixic acid (15 µg mL-1) (for Salmonella) were inoculated from the stock culture collection and incubated for 24 h at 37 ºC under anaerobic conditions in a Don Whitley anaerobic cabinet (10:10:80 %; H2:CO2:N2). Hungate tubes were then inoculated with one colony from each plate. For the Lactobacillus and Bifidobacterium strains, the Hungate tubes contained MRS broth (10 mL) (Oxoid); for Salmonella, the tubes contained 10 mL of Luria–Bertani broth (Difco Laboratories, Detroit, MI, USA). Hungate tubes were incubated overnight in a shaking incubator at 37 °C. After the overnight incubation, 0.1 mL of each bacterial cell suspension (107 CFU mL was inoculated into Hungate tubes containing 9 mL glucose-free MRS medium (Oxoid) and 1 mL of the prebiotic substrates (1 %, w/w). Tubes were then incubated for 24 h at 37 °C in a shaking incubator. The optical density at 660 nm of each culture was determined at hourly intervals for up to 24 h. Subsequently, the exponential-phase growth rate (OD units h-1) of the probiotics in each prebiotic-containing medium was calculated. This experiment was carried out in triplicate.

Batch culture fermentations

Batch culture fermentations were performed in order to characterize the fermentation pattern of the prebiotics selected. Sterile stirred batch culture fermentation vessels (100 mL working volume) were prepared and aseptically filled with 45 mL of sterilized basal nutrient medium. The medium comprised peptone water (2 g L -1), yeast extract (2 g L-1), NaCl (0.1 g L-1), KH2PO4 (0.04 g L-1), MgSO4.7H2O (0.01 g L-1), CaCl2.6H2O (0.01 g L-1),

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NaHCO3 (2 g L-1), Tween 80 (2 mL L-1), haemin (0.05g L-1), vitamin K (10 µL L-1), L-cysteine hydrochloride (0.5 g L-1) and bile salts (sodium glycocholate and sodium taurocholate) (0.5 g L -1). The medium was adjusted to pH 7.0 and 4 mL of 0.025 % (w/v) resazurin solution were added prior to autoclaving. All media and chemicals were purchased from Oxoid and Sigma, respectively. Once in the fermentation vessels, the sterile medium was sparged with O2-free N2 (15 mL min-1) overnight to maintain anaerobic conditions. The following day, a faecal slurry was prepared by collecting and combining fresh faeces from 8 pigs (body weight 25–30 kg, proven free of Salmonella infection by enrichment in selenite broth at 37 °C for 16 h and plating on BGA) and mixing them with pre-reduced sterile phosphate-buffered saline (PBS) (to give a 10 %, w/v, faecal slurry). The faecal matter used was assumed to have a microbial population representative of the large intestinal microflora of pigs (Coates et al., 1988; Williams et al., 1998; Bauer et al., 2004). Each vessel was inoculated with 5 mL of the freshly prepared faecal slurry. Each prebiotic was immediately added at a concentration of 1 % (w/v) to the vessels. Also included as a control was an extra vessel with no added prebiotic (referred to here as CTR).

Batch culture fermentations were run for 24 h after inoculation with Salmonella. In the first experiment, 1 ml of sample was taken at 0, 5, 10 and 24 h for the analyses of lactic acid and SCFA by high-performance liquid chromatography (HLPC). In the second experiment, samples (5 mL) were taken at 0, 5, 10 and 24 h for analysis of bacterial populations by fluorescence in situ hybridisation (FISH) and for analyses of lactic acid and SCFA by HPLC. The first experiment was carried out in triplicate and the second in duplicate.

Lactic acid and SCFA analysis

Samples taken from the batch culture vessels were centrifuged at 13,000 g for 5 min to remove all particulate matter. Supernatants were then filtered using 0.2 µm polycarbonate syringe filters (Whatman, UK) and injected (20 µL) into an HPLC system (MERCK, New Jersey, USA) equipped with RI detection. The column used was an ion-exclusion REZEXROA organic acid column (Phenomenex, Inc.) maintained at 85 °C. Sulphuric acid in HPLC grade H2O (0.0025 mmol L-1) was used as the eluent and the flow rate was maintained at 0.5 mL min -1. Quantification of the samples was obtained through calibration curves of lactic, acetic, propionic, butyric and valeric acids and branched-chain fatty acids (BCFA) in concentrations ranging between 12.5 mM and 100 mM.

Statistical analyses

All data were analyzed by ANOVA with the GLM procedure of SAS (v. 9.1.; SAS Institute Incorporated, Cary, NC, USA). Growth rates from pure cultures were analysed according to the following model:

Yijkh = µ + i+ xi Where Yijkk is the dependent variable, µ is the overall mean, i is the effect of the prebiotic and N (0,2 ) represents the unexplained random error.

Batch culture data were analysed according to the following model: Yijkh = µ + i+ j+ )ij + k+ ij

Where Yijkk is the dependent variable, µ is the overall mean, i is the effect of the prebiotic, j is the effect of the time, )ij the interaction between prebiotic and time and e N(0,2) represents the unexplained random error. Data were analysed initially including the effect of the inoculum and its interactions into the model. However, because no significant effect was found, they were finally excluded.For analysis of SCFA (mM and %) and lactic acid, differences between means were assessed by time point including the effect of the inoculum. Means were assessed with a least significant difference (LSD) test. Statistical significance was accepted at P<0.05 and a trend when P<0.10.

Results

Influence of prebiotics on selected pig bacteria and on Salmonella

Table 3 (appendix 1 page 4) summarises bacterial growth rates following incubation of pure cultures with different prebiotics. The highest growth rates for all the Lactobacillus strains and B. infantis occurred in presence of LAC. B. longum showed a similar high growth rate on FIN as on LAC. The growth of B. breve was highest on FOS, FIN and LAC. Salmonella grew slowest on LAC and FOS.

In vitro batch culture fermentations

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Figure 2 (appendix 1 page 5) illustrates the production of lactic acid in absence (Fig. 2a) and presence (Fig. 2b) of S. Typhimurium. In the absence of Salmonella, as overall mean, fermentation of FOS and especially FIN and GEO generated higher amounts of Lactic acid than the control (CTR) (2.269, 4.514 and 4.489 vs 0.379 mM; SEM=0.629;P<0.0001). These differences started at 10 h, especially with the fermentation of GEO. At 24 hours, a marked decrease in the amount of Lactic acid generated from the fermentation of GEO was observed and only FIN showed higher concentrations of lactic acid compared with CTR. In presence of Salmonella, Lactic acid increased over time for all the treatments compared with CTR (SEM=0.928; P=0.0005). As overall mean only FIN and specially GEO showed a significant increase in Lactic acid compared with CTR (4.046 and 6.180 vs 0.030 mM; SEM=1.136; P<0.05). Differences along the fermentation occurred at 5 and 10 hours, when XOS, FIN and GEO showed higher values than CTR.

Discussion The objective of this part of the project was to investigate in vitro the potential of selected prebiotics to indirectly inhibit Salmonella through the promotion of indigenous beneficial bacteria, which would compete for nutrients and would produce lactic acid and SCFA as inhibitory compounds. The effect on the growth of potentially beneficial bacteria and on the inhibition of S. Typhimurium was assessed in pure cultures. In these studies, Lactulose (LAC) was the most promising prebiotic regarding both effects.

Objective 03:

In vitro demonstration of competitive effects: Continuous culture model systems with synbiotics – salmonellae challenge experiments

In this objective, the model system becomes more complex in that it exploits a development of the batch culture to simulate different transit times throughout the pig hindgut by conversion to a continuous flow system. Following a short stabilisation period after inoculation, the cultures were challenged with multi-resistant S. Typhimurium in the presence and absence of the test synbiotics. The following parameters will be assayed:

SCFA generation (rate, amount, type) FISH analysis of predominant microflora components Survival of S. typhimurium

In addition, studies were undertaken to understand the possible mechanisms of inhibition of S. Typhimurium. These included the effect of organic acid and L. plantarum on adherence and invasion of pig cells by S. Typhimurium.

Materials and Methods

Continuous culture fermentations

A total of 5 lt of the basal medium was prepared as described previously for each vessel. Each culture fermentation vessel (100 mL working volume) was filled aseptically with 45 mL of sterilized basal nutrient medium as described previously. The remainder was stored in a reservoir. Once in the fermentation and reservoir vessels, the sterile medium was sparged with O2-free N2 (15 mL min-1) overnight to maintain anaerobic conditions. The following day, a faecal slurry was prepared as described previously and 5 mL of the freshly prepared faecal slurry was added to the fermenter. Additions of probiotic, prebiotic and Salmonella were as described previously. The temperature of the fermentation vessels was held at 37 °C by using a circulating water bath, pH values were kept between 6.4 and 6.6 (average 6.5) by the addition of 0.5 M NaOH or HCl to the vessels, pH was monitored via pH meter controllers (Electrolab260, UK), anaerobic conditions were maintained by sparging the vessels with O2-free N2 (15 mL min-1) and fresh medium was pumped into the fermenter at a rate of 2.1ml per hour (representing a volume change approximately every 24h). Culture fermentations were run for varying periods up to five days after inoculation with Salmonella. Samples were taken (6 mL: 1ml for acid analysis and 5ml for FISH) at varying times during the run.

Reverse phase fluorescence detection high-pressure liquid chromatography for the detection of organic acids.

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The method of Wang et al., (2001) [Wang Y, Liu Y, Ito Y, Hashiguchi T, Kitajima I, Yamakuchi M, Shimizu H, Matsuo S, Imaizumi H and Maruyama I (2001) Simultaneous measurement of anandamide and 2-arachidonoylglycerol by polymyxin B-selective adsorption and subsequent high-performance liquid chromatography analysis: increase in endogenous cannabinoids in the sera of patients with endotoxic shock. Analytical Biochemistry. 294:73-82.] was employed to detect and quantify acids.

Enumeration of bacterial populations by FISH

FISH was performed essentially as described by Daims et al. (2005). Briefly, aliquots (160 µl) of culture samples were fixed in three volumes of ice-cold 4 % (w/v) paraformaldehyde for 4 h at 4 °C. They were then centrifuged at 13,000 g for 5 min and washed twice in 1 mL of sterile PBS. The cells were pelleted by centrifugation and resuspended in 150 µl of sterile PBS, to which 150 µl of ethanol was added. The samples were then vortexed and stored at -20 °C until used in hybridisations studies. For hybridizations, 20 µl of each sample was pipetted onto Teflon- and poly-L-lysine coated, six-well (10 mm diameter each) slides (Tekdon Inc., Myakka City, FL, USA). The samples were dried onto the slides at 46 °C for 15 min, then dehydrated in an alcohol series (50, 80 and 96%, 3 min each). The ethanol was allowed to evaporate from the slides before the probes were applied to the samples. To permeabilise the cells for use with probes Lab158 and Rfla729/Rbro730, samples were treated with 50 µl of lysozyme (1 mg mL-1 in 100 mM Tris-HCl, pH 8.0) at 37 °C for 15 min prior to being washed briefly (2–3 s) in H2O then dehydrated in the ethanol series. A probe/hybridization buffer mixture (5 µl of a 50 ng µl -1 stock of probe plus 45 µl of hybridization buffer) was applied to the surface of each well. Hybridizations were performed for 4 h in an ISO20 oven (Grant Boekel). For the washing step, slides were placed in 50 mL of wash buffer containing 20 µl of 4,6-diamidino-2-phenylindole dihydrochloride (DAPI; 50 ng µL-1; Sigma) for 15 min. They were then briefly washed (2–3 s) in ice-cold H2O and dried under a stream of compressed air. Five microlitres of antifade reagent (polyvinyl alcohol mounting medium with DABCOTM antifading; Sigma) was added to each well and a coverslip applied. Slides were stored in the dark at 4 °C (for a maximum of 3 days) until cells were counted under a Nikon E400 Eclipse microscope. DAPI slides were visualized with the aid of a DM 400 filter and probe slides with the aid of a DM 575 filter. Numbers of specific bacteria and DAPI-stained entities were determined using the following equation:

DF × ACC × 6732.42 × 50 × DF samplewhere DF is the dilution factor (300/500=0.6), ACC is the average cell count of 15 fields of view and DFsample refers to the dilution of sample used with a particular probe or stain (e.g. 50× for Bif164 counts, depending on inoculum). The figure 6732.42 refers to the area of the well divided by the area of the field of view and the factor 50 takes the cell count back to per millilitre of sample.

All probes were Cy3-labelled and synthesized by Sigma Aldrich.

Short name

Accession no.*

Full name† Target species

Sal303 ND L-S-Sal-1713-a-A-18 Different serovars of Salmonella spp.Bif164 pB-00037 S-G-Bif-0164-a-A-18 Most Bifidobacterium spp. and Parascardovia denticolensLab158 ND S-G-Lab-0158-a-A-20 Most Lactobacillus, Leuconostoc and Weissella spp.;

Lactococcus lactis; all Vagococcus, Enterococcus, Melisococcus, Tetragenococcus, Catellicoccus, Pediococcus and Paralactobacillus spp.

Bac303 pB-00031 S-*-Bacto-0303-a-A-17 Most Bacteroides sensu stricto and Prevotella spp.; all Parabacteroides; Barnesiella viscericola and Odoribacter splanchnicus

Chis150 pB-00962 S-*-Chis-0150-a-A-23 Most members of Clostridium cluster I; all members of Clostridium cluster II; Clostridium tyrobutyricum; Adhaeribacter aquaticus and Flexibacter canadensis (family Flexibacteriaceae); [Eubacterium] combesii (family Propionibacteriaceae)

Rbro730‡ pB-00558 S-*-Rbro-730-a-A-18 Ruminococcus bromii-like; Clostridium sporosphaeroides and Clostridium leptum

Rfla729‡ pB-00557 S-*-Rfla-729-a-A-18 Ruminococcus albus and Ruminococcus flavefaciensAto291 pB-00943 S-*-Ato-0291-a-A-17 Atopobium, Colinsella, Olsenella and Eggerthella spp.;

Cryptobacterium curtum; Mycoplasma equigenitalium and Mycoplasma elephantis

Erec482 pB-00963 S-*-Erec-0482-a-A-19 Most members of Clostridium cluster XIVa; Syntrophococcus sucromutans, [Bacteroides] galacturonicus and [Bacteroides] xylanolyticus, Lachnospira pectinschiza and

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Clostridium saccharolyticumProp853 ND ND Clostridium cluster IX

Short name Temperature Sequence (5´ to 3´) Reference

Hybridisation Washing

Sal303 37 37 AATCACTTCACCTACGTG Nordentoft et al., 1997

Bif164 50 50 CATCCGGCATTACCACCC Langendijk et al., 1995

Lab158 50 50 GGTATTAGCAYCTGTTTCCA Harmsen et al., 1999

Bac303 46 48 CCAATGTGGGGGACCTT Manz et al., 1996

Chis150 50 50 TTATGCGGTATTAATCTYCCTTT Franks et al., 1998

Rbro730‡ 50 50 TAAAGCCCAGYAGGCCGC Harmsen et al., 2002

Rfla729‡ 50 50 AAAGCCCAGTAAGCCGCC Harmsen et al., 2002

Ato291 50 50 GGTCGGTCTCTCAACCC Harmsen et al., 2000

Erec482 50 50 GCTTCTTAGTCARGTACCG Franks et al., 1998

Prop853 50 50 ATTGCGTTAACTCCGGCAC Walker et al., 2005

Bacterial strains and growth conditions for adhesion/invasion assays

All infection assays were performed using wild-type Salmonella Typhimurium SL1344. Bacterial cells were cultured in LB-G broth overnight at 37C, cultures were then diluted 1:200 for infection assays. Probiotics were cultured statically in 20ml Mann, Rogosa, Sharpe Broth (MRS) (Difco, UK) in an anaerobic cabinet (Don Whitley, Yorkshire, UK; 10:10:80, H2:CO2:N2 ) at 37C. For infection assays cultures were diluted 1:200 from an overnight culture. Unless otherwise stated all infection assays were performed using a multiplicity of exposure (MOE) of approximately 1000:1 bacteria to host cells. The MOI was calculated following and was ~ :1.

Adhesion/Invasion assay

Adhesion and Invasion assays were performed according to the methods of Dibb-Fuller et al. (1999). IPEC-J2 or IPI-21 cells were grown to confluency in 24 well tissue culture plates, yielding approximately 5 X 105 cells per ml (BD biosciences. UK). 3D aggregates were removed from the RWV diluted in 120ml of fresh medium and then seeded evenly into 24 well tissue culture plates. Aliquots (1ml) which were representative of a tissue culture well, had the culture medium removed and were subsequently resuspended in 1ml cell dissociation solution for 30 minutes. Cells were passed through a cell strainer (70 mesh, BD Biosciences) and were enumerated in a haemocytometer to determine the approximate number of eukaryotic cells present in each well. Cell counts were between 5 X 10 5

and 9 X 105 cells per ml. Each of the six test conditions were run in quadruplicate on two identical plates (one for Association, one for invasion). Plates were incubated at 37°C in a 5% CO2

atmosphere. For time course studies, the incubation times were 5, 15, 30, 45, 60 and 120 minutes. For standard adhesion and invasion studies the incubation time used was 60 minutes. A sixty minute time point was chosen because after 2 hours there was a marked decrease in cell viability due to the accumulation of Organic acids from the probiotics, monolayers grown on collagen also detached due to the acidic conditions at the 2 hour time point.

After the required incubation period cells were aspirated of the spent culture medium and washed three times in hanks balanced salt solution. Cells were then incubated for two hours in fresh pre warmed culture medium containing 50g/ml gentamicin. After incubation, spent medium was aspirated and cells were subsequently washed with Hanks balanced salt solution three times and then re-suspended in 1% Triton X-100 in PBS. Cells were subsequently disrupted using magnetic stirrers for 10 minutes, after which serial dilutions of each well were

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plated onto LB-G agar. To determine the total number of associated bacteria (adhered and invaded Salmonella) by subtracting the mean of the invaded bacteria from the mean of the associated bacteria.

Development of a porcine 3D cell culture model models

For this study we developed a porcine jejunal and ileal 3D cell culture model based upon the methods of Nickerson et al (2001). The porcine jejunal cell line IPEC-J2 was obtained from Peter Schierach (Schierach et al., 2005). The IPEC-J2 cell line is non-transformed and was originally derived from a neonatal un-suckled piglet and obtained in continuous culture (H.M Berschneider 1989, Abstract of the Annual Meeting of the American Gastroenterological Association). IPEC-J2’s were cultured according to Schierack et al., 2005. The porcine ileal cell line IPI-21 was obtained from Phillippe Velge (Kaeffer et al., 1993). The IPI-21 cell line was derived from the Ileum of an adult histocompatible miniature male pig and were immortalized by transfection with the SV40 plasmid pSV3-neo (Kaeffer et al., 1993).

Both cell lines were initially grown as monolayers in a medium formulated for the growth of porcine cells in 3D cell culture. This medium consists of DMEM/ Ham’s F12 (1:1) medium (Sigma, Poole) supplemented with 5% porcine serum (Sigma, Poole) containing gentamicin (50ug/ml). The medium for these cells is supplemented with 5ug/ml, 5ug/ml, 5ng/ml ITSS (Insulin, Transferrin, Sodium Selenite), 5ng/ml epidermal growth factor and 1% L-Glutamine. Three sugars; glucose, galactose and fructose were added at (1g/l, 0.25g/l, 0.13g/l) which mimic human physiological levels of these sugars in vivo (Goodwin et al., 1993). The levels are not dissimilar to that found in other monogastrics including pig. The monolayers were cultured until they became confluent ~100% upon which they were washed three times with Hanks balanced salt solution (Sigma, Poole) and removed from the flask with 0.25%. trypsin (Sigma, Poole). The IPEC-J2’s or IPI-21’s were resuspended in the medium as indicated above at 2 X 105 cells/ml. Cells were counted using a haemocytometer and assayed for viability using Trypan Blue, before the cell suspensions were mixed with ~5mg/ml porous Cytodex-3 microcarrier beads and allowed 30 minutes to attach to the microcarriers before being introduced into the RWV (Cellon, UK, Belgium) at a ratio of 10 beads per cell. The Cytodex beads are ~175um and coated in Type-I-Collagen from pigs (Sigma, Poole) and are believed to promote cellular differentiation and aid attachment of Fastidious cell lines. Cells were cultured in the RWV at a rotation speed sufficient to keep the aggregates in suspension without the aggregates making contact with the high aspect rotary vessel (HARV) wall. The medium was replenished by removing the luer port and aspirating 90% of the culture medium every 24hrs and cells were cultured in the RWV for 20-24 days at 37°C, 5% CO2.

Adherence and Invasion assays using 3D cells

The method was similar to standard tissue culture adherence and invasion assays. Samples of 3D cells were diluted into fresh pre-warmed medium, counted by haemocytometer and further diluted in tissue culture plates (8 wells per plate) in fresh pre-warmed medium inoculated with S. Typhimurium SL1344 or L. plantarum B2028, or both organisms at 107 cfu per ml. Incubation continued at 37oC. Gentamicin was added after three hours to give a final concentration of 15 m per ml. Samples were taken for analysis at varying times during incubation and analysed as described below.

Microscopy

Antibodies for Confocal Scanning Laser Microscopy

Antibodies used were Alexa Fluor 647 conjugated to phalloidin (Molecular Probes), Salmonella common structural antigens 01-91-99 (CSA-1) (Kirkegard & Perry), Anti-Goat IgG TRITC conjugate (T7028), Anti-Goat IgG FITC conjugate (F7367), cytokeratin -18 (AB668) (Abcam, Cambridge), villin (AB739) and beta catenin (AB6302).

Analysis of cellular markers of differentiation by confocal microscopy.

Aliquots of 3D IPEC-J2’s or IPEC-J2 monolayers (prepared on collagen-1 coated 12mm glass coverslips) were fixed in either 4% paraformaldehyde (for actin staining) or 100% methanol at -20C, for all other stains. Cells were subsequently washed in PBS and antigen de-masking/permeabilisation of cells was performed by incubation with 0.5% Triton X-100 for 15 minutes. Aggregates or monolayers were added to the relevant primary antibodies at dilutions of (1/100). Aggregates or monolayers were subsequently washed in PBS and then incubated with either FITC anti-rabbit (Sigma, Poole) or FITC anti-mouse (Sigma, Poole) for 45 minutes in the dark at room temperature.

Evaluation of S. Typhimurium Association to Actin in 3D aggregates.

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Aliquots of IPEC-J2 cells from the 1 hour time point were taken in quadruplicate from three separate batches of IPEC-J2’s and fixed in 4% Paraformaldehyde prepared in a 0.1M phosphate buffered saline (PBS). The cells were then washed using PBS to remove any non-adherent bacteria and remove any paraformaldehyde. Association of S. Typhimurium (both Adhered and Invaded) were determined by permeabilisation of 3D aggregates by incubation with PBS containing 0.1% Triton X-100 for 15 minutes. Cells were subsequently washed with room temperature PBS and the incubated for 1 hour with Phalloidin (Molecular Probes, UK) and with goat anti-Salmonella antibody (KPL, affinity-purified antibody) and Alexa Fluor 488 rabbit anti-goat immunoglobulin. The 3D aggregates were washed with PBS and mounted in Vectashield containing DAPI (Vector Laboratories). Images of the Alexa Fluor 488 and TRITC labelled actin were obtained by confocal laser scanning microscopy using a Leica TCS SP2 AOBS confocal system attached to a Leica DM IRE2 microscope equipped with ArKr laser excitation (488nm), HeNe laser excitation (594nm) and a diode laser (405nm). Oil-immersion objective lenses (40x and 63x) were used, and imaging parameters were selected to optimise resolution.

Salmonella differential stain to identify adhered and invaded bacteria.

Samples of 3D IPEC-J2’s were taken at various time points after incubation with salmonella were fixed using 4% paraformaldehyde prepared in a 0.1M phosphate buffered saline (PBS). The cells were then washed using PBS to remove any non-adherent bacteria. The extent of bacterial attachment and invasion was then assessed by differential immunocytochemical staining as described previously (Jepson et al. 1996 and 2003). Briefly, the cells were incubated sequentially with goat anti-Salmonella antibodies (KPL, affinity-purified antibody) and Alexa Fluor 488 rabbit anti-goat immunoglobulin to label extracellular bacteria. Cells were then washed using PBS and permeabilised using PBS containing 0.1% Triton X-100. The cells were then incubated again with anti-Salmonella antibodies and Alexa Fluor 594 rabbit anti-goat immunoglobulin to label extracellular and intracellular bacteria. The cells were then washed thoroughly using PBS and mounted in Vectashield containing DAPI (Vector Laboratories). Images of the Alexa Fluor 488 and 594-labelled bacteria were obtained by confocal laser scanning microscopy using a Leica TCS SP2 AOBS confocal system attached to a Leica DM IRE2 microscope equipped with ArKr laser excitation (488nm), HeNe laser excitation (594) and a diode laser (405nm). Oil-immersion objective lenses (40x and 63x) were used, and imaging parameters were selected to optimise resolution.

Scanning Electron Microscopy

Aliquots of 3D IPEC-J2 aggregates were fixed using 3% gluteradehyde prepared in a 0.1M phosphate buffer during a time course infection assay, which included the following time points; 0, 15, 30, 45, 60, and 120 minutes. Infection of the aggregates were carried out according to (infection of 3D aggregates above). Processing of the samples for SEM was carried out as described previously (Walker et al., 1999). Briefly, the cells were washed in 0.1M phosphate buffer, post fixed in 1% osmium tetroxide, dehydrated through a gradual series of ethanol up to 100% and then treated with hexamethyldisilazane for 5 minutes. The air-dried cells were allowed to settle on to poly-l-lysine coated glass coverslips. The coverslips were then attached to aluminium stubs, sputter-coated with gold and examined by SEM using a Stereoscan S250 Mark III SEM at 10-20 KV.

Transmission Electron Microscopy

TEM monolayers had the spent culture medium aspirated and cells were immediately fixed in 3% glutaraldehyde/PBS pH 7.2. Tissue aggregates from the 3D cell culture were processed as above with the exception that the cells are in an 2.5 ml eppendorf. The culture medium was aspirated, after the aggregates were allowed to settle out of solution. The pellet of aggregates was then resuspended in 2mls of 3% glutaraldehyde/PBS pH 7.2. TEM was carried out using standard techniques (Walker et al., 1999).

Fluostar Salmonella Inhibition Assay

For preparation of cell free supernatants (CFS), overnight cultures of L. plantarum were centrifuged (4800rpm) on a Sigma Howie centrifuge for 10 minutes. The supernatants were decanted and then subsequently filter-sterilised using 0.45μm and 0.2μm filters (Millipore, UK). For determination of the inhibitory activity of probiotic CFS on a variety of S. Typhimurium strains, standardised inocula containing the relevant S. Typhimurium strain was prepared by a 1/1000 dilution of an O/N S. Typhimurium culture into fresh LB-G. Subsequently 180l of 106 CFU S. Typhimurium are added to each test well. Finally 20l of the probiotic CFS were added (Total well volume 200l). Plates were incubated at 37°C and growth was monitored at A600nm. as a function of time over 12 hours. All samples were run in triplicate and each experiment was repeated in triplicate on separate days.

Results

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Determination of acid profile of Lactic Acid Bacteria

The reverse phase fluorescence detection high-pressure liquid chromatography method of Wang et al., (2001) was established and used to detect organic acids in spent medium. Spent media were extracted and analysed by RPF-HPLC. One example of the detection of organic acid detected is shown in Figure 3 (Appendix 1 page 6). As anticipated the primary organic acid produced by all strains was lactic acid. However, there were significant traces of butyric, propionic and acetic acids plus small traces of an as yet unidentified volatile.

Production of Lactic acid and SCFA in the fermentation systems

Figure 4 (appendix 1 page 8) illustrates the accumulated generation of total SCFA along the 24 h of fermentation in the cultures in the absence and presence of Salmonella. In its’ absence, SCFA increased over time for all the treatments compared with the control (CTR) (SEM=0.975; P< 0.0001). Taking overall mean results, all prebiotics and especially GEO (10.999mM) and XOS (10.246 mM) generated higher amounts than CTR (from 6.984 to 10.999 vs 3.415 mM; SEM=1.195; P=0.0005). Differences with CTR started at 10 hours, when all prebiotics except LAC showed higher concentrations of SCFA than CTR. However, at 24 h all prebiotics except FOS generated higher concentrations of SCFA than CTR, especially LAC and GEO. In the presence of Salmonella, SCFA increased over time for all the treatments except LAC compared with CTR (SEM=1.168; P<0.0001). In the overall mean, all prebiotics except LAC generated higher concentrations of SCFA than CTR (from 5.790 to 12.896 vs 0.799 mM; SEM=1.430; P<0.0001) specially FIN. Differences were found at 5h, 10 and 24 h, with XOS, FIN and GEO generating higher concentrations of total SCFA than CTR.

Table 4 (appendix 1 page 7) shows the profiles for the SCFA found in the cultures at 24 h of fermentation without and with Salmonella. In the absence of Salmonella, the inclusion of prebiotics modified the percentages of acetic and butyric acids and branched chain fatty acids BCFA compared with CTR. Whereas FIN and especially GEO and LAC generated lower percentages of acetic acid than CTR, they showed higher percentages of butyric acid (250%) than CTR. Fermentation of FOS and XOS increased the percentages of BCFA compared with CTR. In the control culture in the presence of Salmonella, high levels of acetic acid were detected whereas propionic, butyric and other BCFAs were not detected. However, the overall amount of acetic acid was only 2.045mM. In the test cultures, none of the prebiotics generated higher percentages of acetic acid, but all produced varying levels of propionic, butyric or BCF acids than CTR. Importantly, the total concentration of these acids were significantly higher in all test cultures that the control with concentrations up to 33.88mM. The lowest percentages of all acids were found in cultures supplemented LAC.

The intention of setting up a continuous culture system was to study SCFA and bacterial changes over greater periods of time than in batch culture (objective 02) and in a model that reflected the flow through of digesta at a rate perhaps related to the natural gut. We set up the system for a complete medium flow through every 24h. Data beyond about 36h was inherently highly variable whereas the data for the first 24h was similar to that for batch culture.

In a supplementary experiment, the luminal content from three sites of the of the piglet gastro-intestinal tract were collected at necropsy (Materials and methods not described here) and tested by reverse phase fluorescence HPLC. The data are shown in Figure 5 (appendix 1 page 9). The highest concentrations of SCFAs were observed as anticipated in the colon.

FISH identified population changes

Population levels of the dominant members of the pig microbiota in the fermenter system as determined by FISH are shown in Table 5 (appendix 1 page 10). Numbers of total bacteria were not affected by the prebiotic fermented. All prebiotics induced the growth of Salmonella when compared to CTR, although this increase was lower for LAC. Bif164 counts increased with all the prebiotics. With the exception of FOS, all the prebiotics increased Lab158 counts. Counts with Bac303 decreased in the presence of FOS and LAC compared to CTR. The effect of each prebiotic was different and these differences were dependent on time (data not shown, P interaction <0.05). After 5 h, none of the prebiotics generated higher Bac303 counts than CTR, whereas LAC and FOS generated lower counts than CTR (5.85, 5.55 and 6.49 log10 (cells mL-1) for LAC, FOS and CTR, respectively). However, after 24 h, GEO and FIN generated higher counts than CTR (7.20, 7.08 and 6.46 log10 (cells mL-1) for GEO, FIN and CTR, respectively). Chis150 counts increased only with FIN compared to CTR. Counts of Rfla729/Rbro730 increased for all the prebiotics except with LAC when compared to CTR.

Growth in the RWV causes the formation of well differentiated aggregates of IPEC-J2’s.

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Int 407 and IPEC-J2’s grown in the RWV cause the formation of ‘tissue like’ multi-layered aggregates of cells (examples of data for Int 407 and IPEC-J2 cells are shown in Figures 6 onward, Appendix 1 page 12 onward). The 3D Int 407 and IPEC-J2 aggregates were compared to monolayers grown in the same medium (JISM) and on the same ECM scaffold Type-1 collagen, cells were evaluated by SEM for cellular morphology, microvilli and the response to S. Typhimurium infection.

IPEC-J2’s were grown in the RWV exhibited a raised morphology by SEM and form large regular tessellating epithelial like cells (Figure 6c/d Appendix 1 page 12). In contrast monolayers appear to be flat, striated and the cell-cell junctions are not as apparent (Figure 6 a/b). Also, 3D IPEC-J2’s under SEM have a confluent layer of microvilli across the surface of all the epithelial like cells, whereas the monolayer controls exhibit some cell surface processes, however they are irregular and only present on some cells (Figure 8). Microvilli were identified by TEM on the apical surface of 3D IPEC-J2’s but not on monolayers. A major protein involved in the formation of microvilli, villin was used as a marker of brush border formation on IPEC-J2’s, 3D IPEC-J2’s show dense apical staining consistent with the observed microvilli by SEM and TEM. Monolayers in contrast showed irregular and sparse villin staining (Figure 8 Appendix 1 page 14).

Interestingly, IPEC-J2’s grown in monolayers don’t interact in the same way with Salmonella Typhimurium SL1344 as the 3D aggregates (Figure 7 Appendix 1 page 13). The membrane ruffles formed by invading Salmonella are rough and undefined in monolayers, whereas on 3D aggregates the monolayers are large and filamentous, which is consistent with previous findings. Identification of M-Cell like formations in 3D IPEC-J2’s

SEM analysis of 3D aggregates revealed cells with no microvilli and abnormal cell surface processes which exhibited an M-Cell like morphology. Cytokeratin 18 has been used as a marker for M-Cells in porcine tissues and this intermediate filament is present in M-Cells and surrounding enterocytes. Identification of M-Cells is determined by the intensity of the cytokeratin 18 staining. However, all 3D aggregates and monolayers exhibited strong cytokeratin 18 staining (data not shown). These data suggest that this cell line is of a lineage that may be able to form M-Cells under the appropriate conditions.

Organic Acids alone inhibit Salmonella

In separate experiments, the two test Salmonella Typhimurium strains were grown in LB and MRS to which each of the four acids (lactic, acetic, propionic and butyric acid) identified by RPF-HPLC in the spent medium. Concentrations ranged from 0 – 20mM. In each case, the growth of both Salmonella test strains was inhibited in a concentration dependent manner with the degree of inhibition greatest for lactic >butyric >propionic=acetic acids (data not shown).

Organic Acids inhibit Salmonella invasion of host cells

Salmonella enterica spp are invasive, and S. Typhimurium especially so. In previous studies we have developed invasion assays for Salmonella enterica spp and these were exploited to study the impact of organic acids on Salmonella invasion. To do this, the two S. Typhimurium strains were grown in LB broth overnight and then acclimatised separately in the presence of 20mM lactic, butyric, propionic and acetic acids. Acclimatised bacteria were then applied to the tissue adherence and invasion cell culture assay and the data are shown in Figure 12 Appendix 1 page 18. Both Salmonella strains adhered as efficiently in all tests as in control experiments. However, when assayed for invasion, there were significant differences in the extent of invasion. These differences were specific to the acid type. Invasion was reduced most by the presence of propionic acid and then butyric.

Lactobacillus plantarum inhibits invasion of host cells by Salmonella

The data from the preceding experiments indicated that short chain fatty acids (SCFAs) if delivered to Salmonella in vivo, may reduce invasion and therefore limit systemic effects of Salmonellosis. One strain of Lactobacillus plantarum strain B2028 produced high levels of Lactic acid and low levels of other SCFAs (see objective 01) and this strain was used in co-infection studies to assess whether the presence of this strain inhibited the invasion of host cells by S. Typhimurium. To test this hypothesis, assays were established using a 3D cell culture system in which IPEC-J2 cells were used in various combinations with L. plantarum and S. Typhimurium.

A time course study was undertaken using SEM to identify morphological changes to the surface of 3D-IPEC-J2’s and control monolayers after exposure to L. Plantarum, S. Typhimurium or both in response to Competition, Exclusion or displacement assays (as outlined in methods and materials). The 15 minute and 45 minute time points were chosen as good indicators of early and late stages of infection of the aggregates. There

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were clear time dependant effects observed for the Salmonella invasion of 3D IPEC-J2’s (Figure 9 Appendix 1 page 15). Early signs of Salmonella infection of the 3D cells appear as membrane ruffles, whereas late stages of infection result in the death of the enterocytes and formation of apoptotic bodies (Figure 10 Appendix 1 page 16). The competition assay showed no visible signs of infection, and no membrane ruffles were visible at any time point. The exclusion assay showed extremely large membrane ruffles at 15 minutes infection, which were a lot more developed than the membrane ruffles observed for the SL1344 control. However, at 45 minutes there was no sign of damage to the cell, associated bacteria were present but not causing cellular changes.

Discussion

Contrary to expectation, the inclusion of the prebiotics into the culture systems did not decrease Salmonella growth compared with the control (CTR), although among the prebiotics tested, LAC promoted the least growth of Salmonella. In the present study all the prebiotics were fermented by pig microflora, evidenced by the increase in the production of SCFAs compared with the control in the fermentations without Salmonella and all of them except LAC when Salmonella was present.

Fermentation of prebiotics was evidenced as well by FISH that defined the modifications in the bacterial populations analysed; all the prebiotics fermented in the batch cultures increased bifidobacterial counts as detected by the Bif164 probe and all, except FOS, increased lactic Acid Bacteria as detected by the probe Lab158 counts compared to the control (CTR). These data indicate that presumed beneficial bacteria are enhanced in terms of absolute numbers.

Our hypothesis was that the fermentation of the prebiotics would result in the production of lactic acid and SCFAs that could inhibit Salmonella growth. However, besides the increased SCFA generation observed in the fermentation of the prebiotics, there was a lack of inhibition of Salmonella. However when tests were performed in vitro, we gained definitive evidence that organic acids did indeed inhibit their growth. The reasons for this require investigation.

The fermenter system that was used attempted to mimic flow through of digesta but in reality worked like a static culture for the first 24-36h where flow through was 1/25 th of the volume every hour. Thereafter, results were highly variable and not reproducible and are not reported here. Further work is required to establish a genuine pig gut continuous culture system. However, the findings from the system were comparable with that for static culture early in the process (presumably little dilution effect early on) and data for the first 24h were taken as reliable especially as there were striking similarities between data sets for lactic acid and total SCFA (Figure 2 appendix 1 page 5 and Figure 4 appendix 1 page 8).

The exposure of S. Typhimurium to either SCFAs or the high SCFA producing strain of L. plantarum in two component assays resulted in inhibition of growth and prevention of cellular invasion. These findings are very positive in terms of potential mechanisms for the control of Salmonella in vivo. However, the fermentation studies seemed to indicate that although SCFAs were produced, suppression of S. Typhimurium was not observed in all cases except with the use of LAC. These data suggest that, although SCFAs were produced and produced in abundance in the fermentations, growth of S. Typhimurium was not inhibited. The reasons for this disparity between in vitro texts and the simulated GIT environment requires further analysis but it may be assumed that the complexity of the gut fermenter system, possibly more closely modelling the actual gut in vivo, nullifies or at least diminishes the inhibitory effects.

However, there are some promising lines to follow. First, SCFAs do have profound effects on S. Typhimurium in terms of growth and pathobiology. Therefore studies looking into acidification of feeds should be considered. Second, Lactulose was associated with suppression of S. Typhimurium later in the simulated GIT system. Studies are warranted to assess whether this effect can be replicated in vivo. Third, one strain was shown to be highly suppressive of S. Typhimurium in terms of growth and pathobiology. The Lactobacillus plantarum B2028 strain produced SCFAs and also a presumed bacteriocin. Further study on the combined effects in vitro and in vivo of LAC and L. plantarum on S. Typhimurium suppression are recommended.

Objective 04:

Development of analytical tools: Development of antimicrobial resistance gene probes for in vitro and in vivo real time studies

Gut microbiology is usually carried out by plating faecal microorganisms onto selective agars designed to recover numerically predominant groups of micro-organisms. However, the media used are only semi-selective, do not recover non-culturable bacteria and allow operator subjectivity in terms of microbial characterisation - which is

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usually based on limited phenotypic procedures. Additionally, the methods are costly and labour intensive. As such, detection mechanisms, based around gene probes, to more effectively characterise the microflora involved in fermentation studies have been developed already. This culture independent approach allows the processing of stored fermenter and/or faecal specimens.

The original aims of this component of the project were to develop probes for fluorescent in situ hybridisation technology (FISH) that would specifically detect the prevalent bacterial groups in the gut microflora and to detect antibiotic resistance genes in situ. The FISH techniques were worked up and applied to the fermenter studies as described in the previous objective specifically to identify and enumerate principal anaerobic bacteria in these fermentation studies. (see above)

Fish technology was applied in this study using the probes described earlier. This method was translated to the VLA from Reading University so that it is now in use in support of other projects. However, this was a demanding technological development and the original aim to produce FISH probes for each antibiotic resistance genes proved to be overly stretching within the project. The bioinformatic analysis for the design of probes for each resistance gene with identical annealing temperatures as used by the other probes for strain differentiation proved to be very expensive in time and failed to deliver probes that in silico would meet experimental requirements. Therefore to meet the same overall goal of detecting antibiotic resistance genes, it was decided to further develop the AMR array (see VMO2136) for the specific detection of antimicrobial resistance genes in samples. The overall goal has been achieved and is to be described here but the specific minor variation to the project was not agreed with the customer.

Materials and methodsArray platform and applicationThe prototype array platform for the specific detection of antibiotic resistance genes was developed in project VMO2136. The details of the method are therefore not repeated here and the reader is referred to the recently published paper by Batchelor et al., 2008 [Bathelor, M., Hopkins, K.L., Liebana, E., Slickers, P., Ehricht, R., Mafura,. M., Aarestrup, F., Mevius, D., Clifton-Hadley, F.A., Woodward, M.J., Davies, R.H., Threlfall, E.J. and Anjum, M.F. (2008). Development of a miniaturised micro-array based assay for the rapid identification of antimicrobial resistance genes in Gram-negative bacteria. Int. J. Antimicrobial Ag. 31: 440-451] for the specific details of the array and its use.

DNA extraction from samplesSamples (1ml) from fermentation studies were collected and inoculated into 9ml LB broth. The subcultures were incubated with vigorous aeration for six hours at which point the bacteria were concentrated by centrifugation. Genomic DNA was isolated from the bacterial pellet using the DNeasy tissue kit (Qiagen) or Nucleospin tissue kit (Abgene). DNA was assayed (OD260/280). Two micrograms of genomic DNA was linearly amplified using the array primers (see above) and biotin labelled as described previously (see above). Labelled amplified products were hybridised to the array tubes as described previously (see above). The signal intensity value was determined for each spot on the array using Iconoclust software (version 2; CLONDIAG). The mean signal value for three replicate spots per probe was used for analysis. Probes with intensity values of greater than 0.4 were considered positive whilst those less than 0.3 were considered negative. Probes with intensity values between 0.3 and 0.4 were considered ambiguous.

ResultsAntimicrobial resistance genes of Salmonella Typhimurium DT104 strain S01760 which exhibits the R-type ACCsSuT phenotype that was used in one fermentation experiment were detected (Figure 13, Appendix 1 page 19) by the method developed in this study. The raw data shows in figure 13a the number of genes detected without enrichment and figure 13b with enrichment.

Discussion

The array was developed originally for use with pure bacterial cultures, but in this study complex samples were taken from the fermenter and incubated for 6h to enrich facultative anaerobes such as the Salmonella Typhimurium DT104 strain S01760 that exhibits the R-type ACCsSuT phenotype. The DNA from the bacterial

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pellet was extracted and was used successfully to detect the genes of the inoculated penta-resistant S. Typhimurium. This short study indicated that the array may be useful in the detection of not only antibiotic resistance genes but other genes, such as virulence determinants, from complex bacterial populations. However, there is a caveat. First, there was an enrichment stage that selected for facultative anaerobes and second this one study did not assess the sensitivity of detection. It is possible that other genes were present but not detected.

Compared with FISH, the array does not locate the resistance gene(s) to a specific bacterium within the complex microbiota. Also, the FISH approach can be semi-quantitative. No studies were performed to assess whether or not the array can be used to quantify the number of antibiotic resistant bacteria in a sample but, in principle, it should be possible to do so. This is worthy of further effort.

Objective 05:

Dissemination of information

A key objective of this study is the provision of sound science that will underpin advice and possibly policy development on the control of antibiotic resistance in farmed animal species by the use of pre- and probiotics that are simple to administer and potentially cost effective. Here we list the outputs from this 20 month project and the list includes peer reviewed papers (submitted and/or in preparation), poster and oral presentations.

The peer reviewed papers are planned, in preparation or submitted as indicated.

Please see Section 9 below for details of all publications produced from this project.

Discussion.

This short project has been highly productive in terms of new information gained that has been shared by various methods with a wide audience. The research team is still in the process of finalising major papers which will be submitted over the coming months.

OVERALL DISCUSSION

The objective of this project was to investigate in vitro potential of selected probiotics and prebiotics to inhibit Salmonella either directly and/or indirectly through the promotion of indigenous beneficial bacteria, which would compete for nutrients and would produce lactic acid and SCFA as inhibitory compounds. Firstly, clear evidence was gained that probiotic strains appeared to inhibit the growth of Salmonella in various assays. The fact that pH neutralisation eliminated this effect for the vast majority of probiotic strains indicated that acid production was a key factor in growth suppression. A few strains also possessed a suppressive capacity that was independent of acid production. Without further detailed analysis the effector molecule is unknown but may be a bacteriocin or perhaps a small metabolite that controls bacterial cell densities (quorum sensing factor); this is worthy of further analysis. Of interest, Lactobacillus plantarum B2028 which was derived from pig in the first instance, was the most significant probiotic in terms of acid and non-acid suppression of Salmonella and much effort later in the project focused in this probiotic. Secondly, the effect on the growth of potentially beneficial bacteria and on the inhibition of S. Typhimurium was assessed. In these studies, Lactulose (LAC) was the most promising prebiotic regarding both effects. To further investigate these initial results, batch culture fermentations were carried out; such mixed culture work is essential to determine the prebiotic selectivity of a substrate (Gibson & Roberfroid, 1995). In a first set of fermentations the fermentation profile of the prebiotics by the gut microflora was assessed, in terms of lactic acid and SCFA production. In a second set of fermentations oin which a n attempt was made to establish a continuous culture system was made, changes in bacterial populations and fermentation products (lactic acid and SCFA) and also Salmonella survival were investigated. Contrary to the expected, the inclusion of the prebiotics into the culture systems did not decrease Salmonella growth compared with the controls lacking any prebiotic, although among the prebiotics tested, LAC promoted the lowest Salmonella growth. Prebiotics are thought to be utilized preferentially by Lactobacillus and Bifidobacterium species (Kaplan & Hutkins, 2000; Bailey et al., 1991) and may suppress or have no stimulatory effect upon undesirable bacteria such as Salmonella (Gibson & Roberfroid, 1995; Oyarzabal & Conner, 1995). In this way, Lactobacillus and Bifidobacterium species could have a competitive advantage, limiting the nutrient sources for pathogenic bacteria. Thus, Tzortzis et al. (2005) observed how a novel GOS mixture when added to a commercial diet (4%) increased the density of

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bifidobacteria and acetate and decreased the pH compared with the control diet supplemented with inulin. Apanavicius et al. (2007) observed an increase in Lactobacillus in puppies fed 1% inulin together with an increase in acetate and total SCFA compared with those fed with 1% FOS and the control. In the present study all the prebiotics were fermented by pig microflora, evidenced by the increase in the production of SCFA compared with the control in the fermentations without Salmonella and all of them except LAC when Salmonella was present. Fermentation of prebiotics was evidenced as well by the modifications in the bacterial populations analyzed; all the prebiotics fermented in the batch cultures increased the counts of Bifidobacteria as detected by the Bif164 probe, and all of them except FOS increased the counts of Lactic Acid Bacteria as detected by the Lab158 probe compared to CTR. However, Salmonella was able to grow in presence of the prebiotics, especially XOS, FIN, FOS and GEO. Salmonella could have utilized the prebiotics for its growth, generating SCFA from their fermentation. As an example, it has been seen how FOS can stimulate the growth of enterobacteria (Oli et al., 1998; Sakai et al., 2001). Salmonella may not have consumed the prebiotics directly but rather products of their breakdown. It is known that members of the family Enterobacteriaceae are able to take up carbohydrates (mostly C6 carbohydrates such as glucose, mannose, fructose, and hexitols) via the phosphoenolpyruvate-dependent phosphotransferase system (PTS) (Postma et al., 1986). It has also been seen that rapid xylose fermentation is almost universal amongst enteric bacteria. Differences observed in SCFA concentrations and profiles between our in vitro fermentations with and without Salmonella would support this hypothesis.

Apart from the competition for nutrients, our hypothesis was that the production of lactic acid and SCFA could inhibit Salmonella growth, since this effect has been reported by several authors (Prohaszka et al., 1990; McHan, F., Shotts, E.B., 1993; Durant et al., 1999; Van Immerseel et al., 2004 a,b). However, besides the increased SCFA generation observed in the fermentation of the prebiotics, there was a lack of inhibition of Salmonella in the fermenter systems. This could have happened for two reasons. One would be the utilization of the SCFA by Salmonella. The ability of microorganisms to metabolize SCFA may be a primary line of defence against the negative effects of these compounds on cell growth (Cherrington et al., 1991; Lück & Jager, 997). For example, Horswill & Escalante-Semerena (1999) observed how Salmonella enterica serovar Typhimurium LT2 was able to catabolise propionate through the methylcitric acid cycle. This mechanism could have been especially important for its survival in presence of LAC, since Salmonella is not able to ferment this prebiotic (Liao et al., 1994). A second reason would be that SCFA exposure could have contributed to enhanced acid resistance (ATR). The ATR is known to increase significantly following exposure to SCFA, enhanced by acid pH, anaerobiosis and prolonged exposure to these compounds (Kwon & Ricke, 1998). The ATR of S. Typhimurium is able to protect against two types of acid stress: organic (weak acids) and inorganic (low pH) (Baik et al., 1996; Bearson et al., 1998). Regulators of the ATR, which are involved in protection against these two types of acid stress, have been identified in Salmonella Typhimurium, with RpoS and Fur protecting against organic acid stress and PhoP and RpoS protecting against inorganic acid stress (Bearson et al., 1998). Although the pH in our in vitro system was controlled, batch cultures are closed systems and the continuous flow system behaved more like a batch system than desired!, in which the SCFA produced are not absorbed by the epithelium, so high amounts of SCFA can accumulate inside the vessels. Even in vivo, results are also controversial. For example, butyrate has shown antimicrobial effect on E. coli and Salmonella spp (Chrerrington et al., 1991) being very important in the maintenance of the GIT epithelium, in this way preventing foreign bacteria from infiltrating the small bowel and colon. However some authors have demonstrated possible negative effects regarding the integrity of the intestinal epithelial barrier (Bovee-Oudenhoven et al., 2003; Ten Bruggencate et al., 2003, 2005). Furthermore SCFA exposure may contribute to enhanced virulence (Rishi et al., 2005) or invasion of the epithelium (Ten Bruggencate et al., 2003, 2005). Lactic acid bacteria display numerous antimicrobial activities. These are mainly due to their production of organic acids, but also of other compounds, such as bacteriocins (De Vuyst & Leroy, 2007). All the prebiotics fermented in the batch cultures increased Bif164 counts, and all of them except FOS increased Lab158 counts compared to the control. However, the relative amounts of Lab158- and Bif164-dectectable species from the faeces introduced into the batch culture systems were lower than that of Salmonella (107 viable cells, in order to simulate a Salmonella gut infection), so their increase may not have been sufficient to exert any inhibitory effect on Salmonella, although this is only speculative. In order to observe an inhibitory effect, the addition of higher amounts of lactic acid bacteria than that naturally present in the GIT (probiotics) would be needed. Thus, the addition of probiotic species in the feed has demonstrated to be effective against Salmonella Typhimurium (Hudault et al., 1997; Silva et al., 1999; Chiu et al., 2007; Casey et al., 2007).

The afore going discussion leads us to the conclusion that the prebiotics tested in the simulated gut fermenter systems did not have a dramatic impact on the S. Typhimurium that was inoculated into it. The discussion has suggested weaknesses of the system and there is a need to develop better continuous flow models of the pig gut. Whilst Lactulose gave promising data suggesting a suppressive effect on S. Typhimurium, probably by indirect mechanisms, the most promising data for positive intervention came from far simpler analytical systems. The growth and tissue culture systems focused on the direct effects of SCFA and metabolic end products from L. plantarum on the growth and pathobiology of S. Typhimurium. The exciting findings generated proved conclusively that, in isolation from the complex microbiota, SCFAs and L. plantarum inhibit S. Typhimurium. Our observations are not particularly new but they do build upon a body of data indicating these two approaches should be considered as possible interventions. The question is, how can we translate these positive findings into an appropriate on farm intervention? This cannot be done without the in vivo studies. The data we have

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generated strongly suggests that S. Typhimurium can be suppressed in simple analytical systems but the closed fermenter systems do not fully mimic the gut and seem to obviate the effects seen in the simpler test systems. We need to understand why the fermenter system obviates the effects and we need to test whether the in vivo model will show suppressive effects.

Conclusions

All probiotic strains exert a suppressive effect upon Salmonella and this was shown to be acid dependent. Some probiotic strains exert a suppressive effect upon Salmonella and this was shown to be acid

independent Under our experimental gut simulating fermenter conditions, the majority of prebiotics tested seemed not

to exert substantive inhibitory effect on the growth Salmonella. Of the various prebiotics tested, only Lactulose has a noticeable suppressive effect against the growth of

S. Typhimurium but this was after initial growth of the pathogen suggesting a time dependent effect: it is possible that the system that used a large S. Typhimurium inoculum which does not represent the in vivo situation where very low inocula are likey; modifying the inoculum to better reflect real challenge may yield more appropriate data.

All prebiotics tested increased the amounts of short chain fatty acids that are known to have health benefits.

All prebiotics tested increased the amounts of lactic acid and bifidobacteria that are known to have health benefits.

Increased SCFAs especially butyric acid which was detected as an end product of all the prebiotics and it is known that butyric acid is a key nutrient for epithelial cell growth and integrity.

Some SCFAs suppressed the invasion of S. Typhimurium into host cells: that was noted to varying degrees with propionic > butyric > acetic/formic having the greatest suppressive effect. Lactic acid appeared to have little or no effect.

All Lactic Acid Bacteria produced SCFAs that inhibit the growth of S. Typhimurium in simple non-complex environments (e.g. disc diffusion assays).

Lactobacillus plantarum produced high level of SCFAs and another pH independent mechanism of inhibition of S. Typhimurium.

Recommendations for inventions

Prebiotics and Lactulose specifically, may have health benefits in terms of inducing carbohydrate fermentation in the lower gut that will produce nutrients for the host.

Prebiotics may have a suppressive effect on pathogens: it is known that carbohydrate fermentation in the gut, induced by prebiotics, is better in terms of productivity and health status than high Nitrogen clastic anaerobic fermentation that leads to the increase of Clostridial species (e.g. C. Perfringens induced enteritis)

Short chain fatty acids are key factors that should be enhanced in the lower gut and they have inhibitory effects on S. Typhimurium. Methods to enhance organic acids in the gut should be considered and this can be done by acidification of feed and/or application or pre and pro-biotics.

Probiotics can be selected that have very powerful suppressive effects against S. Typhimurium and these could be added to feed.

Further study is required to assess the real impact of such a measure.

Additional figures and tables are to be found in the accompanying appendix 1

Papers cited in this report are to be found in the accompanying appendix 2

Redrafted by Prof M J Woodward

28th October 2008

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References to published material9. This section should be used to record links (hypertext links where possible) or references to other

published material generated by, or relating to this project.

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Papers

Martín-Peláez, S., Costabile, A., Martín–Orúe S. M., Rastall, R. A., Gibson, G. R., La Ragione, R.M., Woodward, M. J. 2008. Determination in vitro of the inhibitory spectrum of different prebiotics against Salmonella Typhimurium in pigs. FEMS Microbial Ecology (accepted for publication 28th March 2008)

Collins, J. W., W. A. Cooley, R. A. Rastall, G. Gibson, M. J. Woodward, R. M. La Ragione. Lactobacillus plantarum reduces S. Typhimurium invasion in a novel 3d porcine jejunum model. In preparation.

Collins, J. W., W. A. Cooley, R. A. Rastall, G. Gibson, M. J. Woodward, R. M. La Ragione. Fluorescence detection HPLC for the quantification of organic acids in biological matrixes. In preparation.

Martín-Peláez, S., Costabile, A., Martín–Orúe S. M., Gibson, G. R., La Ragione, R.M., Woodward, M. J. and Rastall. R. A. In vivo efficacy of prebiotics against Salmonella Typhimurium in a pig model of infection. In preparation*

* note: this paper is not part of the VMO2203 funded project and is work carried out in collaboration with Barcelona University who, at no expense to the project, took the in vitro findings of the project to the pig in vivo model.

Poster Presentations.Martín-Peláez, S.M. Martín-Orúe, R.M. La Ragione, M.J. Woodward, R.A. Rastall and G.R. Gibson. Conference on Gastrointestinal Function 16th-18th April 2007 “Interventions to reduce the carriage of antimicrobial resistance in pigs. Determination of the inhibitory spectrum of pre, pro and synbiotics against Salmonella Typhimurium”. Costabile1*, S. Chigaco.

Martín-Peláez S, Costabile A, Rastall RA, La Ragione RM, Woodward MJ, Martín-Orúe S.M. and Gibson GR ISAPP 2007. Competition submission: “In vitro determination of the inhibitory spectrum of pre-probiotics against Salmonella Typhimurium in pigs”. ISAPP Open Forum, London UK, June 26-28, 2007.

Collins, J. W., N G Coldham, R A Rastall, G R Gibson, M J Woodward, R M La Ragione. 2007. Quantification of Organic Acids Produced by Porcine Microflora & their Effect on S. Typhimurium Invasion & Viability In Vitro James. Med-Vet-Net, Lucca, Italy.

Oral Presentations.Collins, J. W., W. A. Cooley, R. A. Rastall, G. Gibson, M. J. Woodward, R. M. La Ragione. 2007. Lactobacillus plantarum reduces S. Typhimurium invasion in a novel 3d porcine jejunum model. Reading Uni.

Collins, J. W., W. A. Cooley, R. A. Rastall, G. Gibson, M. J. Woodward, R. M. La Ragione. 2007. Lactobacillus plantarum reduces S. Typhimurium invasion in a novel 3d porcine jejunum model. SGM, 161 meeting, Edinburgh Uni.

Collins, J. W., W. A. Cooley, R. A. Rastall, G. Gibson, M. J. Woodward, R. M. La Ragione. 2007. Lactobacillus plantarum reduces S. Typhimurium invasion in a novel 3d porcine jejunum model. Reading Uni. Annual Research day.

Costabile A. et al. Department of Food Biosciences Research Day 2006, Reading University Title: Interventions to reduce the carriage of antimicrobial resistance in pigs. Determination of the inhibitory spectrum of pre, pro and synbiotics against Salmonella Typhimurium in in vitro studies

Costabile A. et al. 30th March 2006 - Food Chain and Health in School of Agriculture, Reading University. Title: Dietary strategies to reduce the Salmonella Typhimurium growth chances in pigs

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