Data in Support of Final Report for VM02203

Appendix 1

Data in support of Final Report for VM02203

Table 1.

Zones of inhibition Strain Designation S. Tm S. Tm SL1344 DT104 pH* pH7.2 pH* pH7.2

Lactobacillus acidophilus a IFR 5 0 6 0 B2206 VLA 10 0 9 0 c UR 8 0 8 0 Lactobacillus rhamnosus a UR 3 0 4 0 b PIL 4 0 3 0 c PIL 4 0 5 0 Lactobacillus reuteri a UR 5 1 5 1 Lactobacillus casei a UR 6 0 6 0 b IFR 6 0 6 0 c PIL 7 0 8 0 Lactobacillus gasseri B1775 IFR 9 0 9 0 Lactobacillus plantarum B2028 VLA 13 4 12 2 Lactobacillus johnsonii B2207 IFR 10 0 9 0 Bifidobacterium bifidum a UR 3 0 3 0 b PIL 4 0 3 0 Bifidobacterium longum a UR 4 0 5 0 b PIL 4 0 5 0 Bifidobacterium breve a PIL 4 0 4 0 Bifidobacterium adolescentis a UR 7 1 7 1 b PIL 4 0 4 0 Bifidobacterium infantis a UR 3 0 3 0 b PIL 3 0 3 0 Bifidobacterium lactis a UR 7 0 7 0 b PIL 8 0 8 0 Enterococcus faecium a PIL 6 0 6 0 b PIL 6 0 6 0 c PIL 6 0 6 0 Bacillus subtilis a VLA 2 2 2 2 b VLA 3 3 3 3 c VLA 0 0 0 0 d VLA 3 3 3 3

Table 1 lists the zones of inhibition for two S. Typhimurium stains, SL1344 and S. Typhimurium DT104 strain S01760 (a pentaresistant pig field isolate which exhibits the R-type ACCsSuT phenotype) against spent culture medium and spent culture medium neutralised to pH 7.2 from probiotic strains. The figures are the measurements in mm from the edge of the disc to the edge of the zone.

1 Figure 1.

Effect of Lactobacillus Plantarum (B2028) cell free supernatant on Salmonella Typhimurium (SL1344)

0.90

A 0.85

0.80

0.75

0.70

0.65

0.60

0.55 MRS SL1344

0.50 m

n MRS SL1344 pH 0

0 0.45 6

D 0.40 SL1344 + B2028 sp O

0.35 SL1344 + B2028 sp pH 0.30

0.25

0.20

0.15

0.10

0.05

0.00 0 1 2 3 4 5 6 7 8 9 10 11 12 Time Effect of Lactobacillus Plantarum (B2028) cell free supernatant on Salmonella Typhimurium (S01760) 0.9 B 0.8

0.7

0.6

m 0.5 n 0 0 6

D 0.4 O

0.3

MRS S01760 0.2 MRS S01760 pH

0.1 S0760 + B2028 sp S0760 + B2028 sp pH 0.0 0 1 2 3 4 5 6 7 8 9 10 11 12 Time

Figures 1A and 1B demonstrate the inhibitory effect of Lactobacillus plantarum (B2028) cell free supernatant on Salmonella Typhimurium (SL1344) (A), or a pentaresistant Salmonella Typhimurium DT104 pig field isolate (strain S01760) which exhibits the R-type ACCsSuT phenotype (B). Both strains show similar responses to the L. Plantarum cell free supernatant (CFS0 which exhibits a strong inhibitory response before pH inactivation (lower graph lines) demonstrating a pH dependant effect P=0.00001. Figures A and B both illustrate that adjusting the pH to 7.2 which is the pH of a 24 hour LB S. Typhimurium culture (middle graph lines) still induces a statistically significant antimicrobial effect P=0.00001.

2 Table 2.

Condition Probiotic Strain Salmonella strain Mean AUC SD P Value MRS B2026 SL1344 9.3236833 0.024179582 MRS pH ajusted B2026 SL1344 9.2948 0.036900166 0.548394752 MRS + CFS B2026 SL1344 6.2218958 0.151540804 3.53675E-05 MRS + CFS pH ajusted B2026 SL1344 7.9682125 0.086783676 0.00011371 MRS B2026 S01760 9.3196542 0.003592109 MRS pH ajusted B2026 S01760 9.146925 0.140023302 0.285027665 MRS + CFS B2026 S01760 5.8729792 0.016914584 3.80048E-09 MRS + CFS pH ajusted B2026 S01760 8.0775708 0.040488682 6.8328E-06 MRS B1775 SL1344 9.3236833 0.024179582 MRS pH ajusted B1775 SL1344 9.2948 0.036900166 0.54839475 MRS + CFS B1775 SL1344 4.1179167 0.034456937 0.00000003 MRS + CFS pH ajusted B1775 SL1344 8.3006375 0.074693041 0.00020017 MRS B1775 S01760 9.3196542 0.003592109 MRS pH ajusted B1775 S01760 9.146925 0.140023302 0.28502766 MRS + CFS B1775 S01760 3.9510042 0.150044376 0.00000365 MRS + CFS pH ajusted B1775 S01760 8.5335375 0.063857733 0.00025170 MRS B2027 SL1344 9.3236833 0.024179582 MRS pH ajusted B2027 SL1344 9.2948 0.036900166 0.548394752 MRS + CFS B2027 SL1344 7.1695833 0.033634056 8.1847E-07 MRS + CFS pH ajusted B2027 SL1344 8.5968458 0.093395955 0.00166227 MRS B2027 S01760 9.3196542 0.003592109 MRS pH ajusted B2027 S01760 9.146925 0.140023302 0.285027665 MRS + CFS B2027 S01760 7.0613667 0.086756277 1.29855E-05 MRS + CFS pH ajusted B2027 S01760 8.6040167 0.076623163 0.000734806 MRS B2028 SL1344 9.3236833 0.024179582 MRS pH ajusted B2028 SL1344 9.2948 0.036900166 0.548394752 MRS + CFS B2028 SL1344 4.0019292 0.070930716 2.35605E-07 MRS + CFS pH ajusted B2028 SL1344 7.6097542 0.17414262 0.000620181 MRS B2028 S01760 9.3196542 0.003592109 MRS pH ajusted B2028 S01760 9.146925 0.140023302 0.285027665 MRS + CFS B2028 S01760 5.199275 0.813609465 0.007145232 MRS + CFS pH ajusted B2028 S01760 7.6535167 0.043794171 4.8006E-06

Table 2 iIllustrates the area under the curve (AUC) for the probiotic strains L. acidophilus strain B2026, L. johnsonii strain B2027, L. plantarum strain B2028 and L. gasseri B1775. The P Values are also shown and are calculated as the test condition compared to the control in a two-paired students T-Test with a variance of two. Each probiotic was tested for antimicrobial activity against S. Typhimurium strains SL1344 and S01760. Significant P Values are highlighted in green, high P values are highlighted in red. CFS (Cell Free Supernatant).

3 Table 3. Growth in the exponential phase (ΔOD units/hour) of selected bacterial strains in pure cultures

Bacteria Prebiotic B. bifidum B. breve B. infantis B. longum E. faecium L.acidophilus L. casei L. plantarum L.rhammosus Salmonella spp. XOS 0.059ab 0.053c 0.059b 0.046bc 0.137c 0.050c 0.068d 0.107c 0.036c 0.043a FIN 0.022bc 0.168a 0.067b 0.030a 0.140c 0.064c 0.101cd 0.118c 0.028c 0.038ab GEO 0.080ª 0.104b 0.053b 0.057b 0.176b 0.155b 0.132bc 0.183cb 0.093b 0.044a

FOS 0.019bc 0.196a 0.053b 0.035c 0.140c 0.059c 0.163b 0.083d 0.028c 0.034bc

LAC 0.002c 0.181a 0.086ª 0.081a 0.263ª 0.221ª 0.214ª 0.211a 0.161a 0.029c RSD 0.0293 0.0159 0.0081 0.0106 0.0097 0.0114 0.0096 0.0071 0.0048 0.0033 P 0.048 <0.0001 0.003 0.0012 <0.0001 <0.0001 0.0001 <0.0001 <0.0001 0.0014

XOS, xylooligosaccharides; FIN, mixture of fructooligosaccharides/inulin; FOS, fructooligosaccharides; GEO, gentiooligosaccharides; LAC, lactulose Values are means and residual standard deviations (RSD) (n =3). a,b,c Different superscripts within a column indicate significant differences within the values (P<0.05). Strains for cross referencing to text and table 1 are:- B. bifidum aUR1, B. breve aPIL, B. infantis aUR1, B. longum aUR1, E. faecium aPIL, L.acidophilus cUR1, L. casei aUR1, L. plantarum B2028 and L.rhammosus aUR1.

4 Figure 2.

Figure 2 shows the production of Lactic acid during the 24 h fermentation of the different prebiotics in batch cultures in the absence (a) or the presence (b) of Salmonella. Open circle, XOS; open square, FIN; open triangle, FOS; solid triangle, GEO; solid square, LAC; solid circle, CTR. Values are represented as means. The standard errors of the means (SEM) for the time points where differences among treatments were significant (P<0.10) were (a) SEM10h = 0.908, SEM24h=2.041 (b) SEM5h=0.293, SEM10h=0.815

5 Figure 3.

Figure 3 shows the detection of organic acids by reverse phase fluorescence detection high- pressure liquid chromatography. This example shows a chromatogram of the VFA’s present in the gastrointestinal tract of a six week old male pig. The organic acids acetic (A), butyric (B), propionic (P), valeric (V), heptanoic (H) and the internal standard 2-ethyl-butyric (EB) acid (10mM) with U an unknown species.

6 Table 4. Proportions of acetic, propionic and butyric acids and BCFA, and concentrations of total SCFA at 24 h of fermentation of different carbohydrates without and with inoculation of Salmonella Typhimurium into continuous cultures

SCFA at 24 h without Salmonella SCFA at 24 h with Salmonella Carbohydrate % mM % mM Acetic Propionic Butyric BCFA Total* Acetic Propionic Butyric BCFA Total* XOS 74.76 a 6.26 b 8.14c 10.95 20.44ab 81.91 5.51 4.62 7.76ab 19.63abc FIN 61.60 a b 4.54 b 28.15b 5.67 23.59ab 64.00 14.09 14.56 7.09abc 33.88a GEO 41.07c 9.33 b 47.37a 2.23 28.78a 62.22 12.34 18.96 6.23abc 24.45ab FOS 79.20 a 7.60 b 5.04c 8.16 14.48bc 80.31 1.39 5.56 12.47a 14.10 abc LAC 46.90bc 5.33 b 48.60a 3.18 28.58a 89.96 5.70 0 4.23bc 3.39c CTR 54.60 bc 28.87 a 13.57c 2.960 4.257c 100 0 0 0 c 2.045c RSD 10.13 6.622 7.266 4.292 7.539 12.901 6.444 9.274 2.917 8.029 P-treatment 0.005 0.009 <0.0001 0.184 0.019 0.229 0.301 0.338 0.061 0.076 *Sum of acetic, propionic and butyric acids and BCFA. a,b,c Different superscripts within a column indicate either significant differences within the values (P<0.05) or a tendency to significance (P<0.10) Values are given as Lsmeans and residual standard errors (RSD) (batch without Salmonella, n=3; batch with Salmonella, n=2). BCFA = branched chain fatty acids’ SCFA = short chain fatty acids.

7 Figure 4.

Figure 5 shows the total SCFA (sum of acetic, propionic and butyric acids, and BCFA) produced during the first 24 h period of continuous culture fermentation of the different prebiotics in batch cultures in absence (a) or presence (b) of Salmonella. Please refer to the text (see objective 03) giving an explanation of why only the first 24 h data are presented. Open circle, XOS; open square, FIN; open triangle, FOS; solid triangle, GEO; solid square, LAC; solid circle, CTR. Values are represented as means. The standard errors of the means (SEM) for the time points where differences among treatments were significant (P<0.10) were (a) SEM10h = 1.307, SEM24h=4.242 (b) SEM5h=1.366, SEM10h=1.999, SEM24h=5.677

8 Figure 5.

Acetic Acid 175

150

125

100 A F V

M 75 m

0 Caecum Colon Rectum

Propionic Acid 45

25 A F V 20 M m 15

0 Caecum Colon Rectum

Butyric Acid 30

20 A F V M m

0 Caecum Colon Rectum

Figure 5 illustrates the levels of the volatile fatty acid, acetic, butyric and propionic acid which were detected by reverse phase fluorescence detection HPLC of the porcine caecum, colon and rectum of a six week old pig.

9 Table 5. Bacterial populations analyzed by FISH along 24 h of fermentation of different carbohydrates in continuous culture systems inoculated with Salmonella Typhimurium

Mean (log cells ml-1) Carbohydrate 10 DAPI Sal303 Bif164 Lab158 Bac303* Chis150 Rfla729/Rbro730 Ato291 Erec482 Prop853 XOS 8.69 7.61a 5.60a 6.32a 6.51ab 6.11abc 6.77a 6 6.57 6.69 FIN 8.69 7.57a 6.11a 6.39a 6.62a 6.50a 6.84a 5.92 6.60 6.74 GEO 8.87 7.65a 5.98a 6.39a 6.60a 6.24ab 6.85a 6.12 6.59 6.71 FOS 8.74 7.48a 5.83a 6.13ab 6.25c 6.33ab 6.83a 5.76 6.64 6.70 LAC 8.51 7.06b 5.75a 6.41a 6.31bc 5.76c 6.65ab 5.79 6.68 6.63 CTR 8.46 6.22c 4.92b 5.88b 6.53a 5.91bc 6.32b 5.93 6.60 6.46 RSD 0.426 0.293 0.526 0.285 0.211 0.428 0.364 0.296 0.142 0.226 P-treatment 0.439 <0.0001 0.003 0.007 0.006 0.022 0.060 0.199 0.680 0.189 P-time 0.183 <0.0001 0.296 <0.0001 <0.0001 0.0001 0.047 0.002 <0.0001 0.280

* Effect of interaction (P interaction <0.05) a,b,c Different superscripts within a column indicate either significant differences within the values (P<0.05) or a tendency to significance (P<0.10) Values are given as Lsmeans and residual standard deviation (RSD) (n-treatment=8, n-time=12, n-interaction=2)

10 11