Colonisation of poultry by Salmonella Enteritidis S1400 is reduced by combined

administration of Lactobacillus salivarius 59 and Enterococcus faecium PXN-33

Alun Carter1,2*, Martin Adams2, and Roberto M. La Ragione3 Martin J. Woodward4

1Department of Bacteriology, Animal and Plant Health Agency, Addlestone, Surrey, KT15

3NB, UK.

2Faculty of Health and Medical Sciences, AX Building, University of Surrey, Guildford,

Surrey, GU2 7XH, UK.

3Department of Pathology and Infectious Disease, School of Veterinary Medicine, Faculty of

Health and Medical Sciences, Vet School Main Building, Daphne Jackson Road, University

of Surrey, Guildford, GU2 7AL, UK.

4Department of Food and Nutrition, The University of Reading, Whiteknights Park, Reading,

RG6 6AP, UK.

*Corresponding author.

Current address: Department of Medicine, Division of Oncology, Washington University, St.

Louis, Missouri, USA.

Tel: (314) 757 0427

Email: acarter@dom.wustl.edu

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Funding: The studies presented here were supported by a commercial grant from Probiotics

International Ltd (Protexin).

Key words: Probiotic, Salmonella Enteritidis, Lactobacillus salivarius, Enterococcus

faecium, poultry, competitive exclusion

Title: Colonisation of poultry by Salmonella Enteritidis S1400 is reduced by combined

administration of Lactobacillus salivarius 59 and Enterococcus faecium PXN-33

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Abstract: Salmonella Enteritidis remains a significant issue within the poultry

industry and one potential solution is to use probiotic bacteria to prevent Salmonella

colonisation through competitive exclusion (CE). We demonstrate that combined

administration of Lactobacillus salivarius 59 and Enterococcus faecium PXN33 were

effective competitive excluders of Salmonella Enteritidis S1400 in poultry. Two models were

developed to evaluate the efficacy of probiotic where birds received Salmonella Enteritidis

S1400 by a) oral gavage and b) sentinel bird to bird transmission. A statistically significant

(p<0.001) 2 log reduction of Salmonella Enteritidis S1400 colonisation was observed in the

ileum, caecum and colon at day 43 using combined administration of the two probiotic

bacteria. However, no Salmonella Enteritidis S1400 colonisation reduction was observed

when either probiotic was administered individually. In the sentinel bird model the combined

probiotic administered at days 12 and 20 was more effective than one-off or double

administrations at age 1 and 12 days. In vitro cell free culture supernatant studies suggest the

mechanism of Salmonella Enteritidis S1400 inhibition was due to a reduction in pH by the

probiotic bacteria. Our current study provides further evidence that probiotics can

significantly reduce pathogenic bacterial colonisation in poultry and that mixed preparation

of probiotics provide superior performance when compared to individual bacterial

preparations.

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Introduction

Salmonella is a major cause of food poisoning that accounted for an estimated 88,715

confirmed cases in the EU in 2014 (Osimani et al., 2016). Public health concerns over

Salmonellosis remain due to several prominent out-breaks, including a reported 250,000

cases in Minnesota in 1994 and a recent hospital outbreak of 287 cases in the UK (Hennessy

et al., 1996; Inns et al., 2014). Contaminated eggs and poultry meat are a major source of

food poisoning with 46.1% and 6.4% of Salmonellosis being attributed to eggs and broiler

meat respectively (Osimani et al., 2016). Since the introduction of EU legislation, member

states have targeted the reduction of Salmonella in poultry (O'Brien, 2013). However, there

was a considerable increase (15.3%) in reported Salmonella cases in the EU between 2013

and 2014 (EFSA and ECDC, 2015) despite regular use of vaccines in the layer sector and

improved barrier security in the broiler meat sector.

Growth promoting antibiotics have been used previously to increase bird weight gain

and led to a passive control strategy for Salmonella species (de Oliveira et al., 2004).

However, with the increasing emergence of antimicrobial resistance, withdrawal of

antibiotics in animal feed came into force in 2006 (European-Commission, 1998). Probiotics

and prebiotics remain an appealing alternative control measure due to the potential

competitive exclusion (CE) of pathogens, improved feed conversion rates and relatively low

additional cost to production (Carter et al., 2009). Performance of probiotic preparations

varies and there is a continuing need for product development and safety evaluation. Since

1972 several successful undefined Salmonella Entertitdis CE products have been developed

including Aviguard and BROILACT (Nurmi and Rantala, 1973; Nuotio et al., 1992;

Nakamura, et al., 2002). Concerns with safety and the spread of antibiotic resistance has led

to the development of defined preparations such as the FM-B11 (Higgins et al., 2008;

Vicente et al., 2008; Prado-Rebolledo et al., 2016). Multi-species and single strain probiotic

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cultures have been shown to reduce Salmonella in poultry although complex cultures are

significantly more effective (La Ragione and Woodward, 2003; Timmerman et al., 2004;

Chapman et al., 2011). Our work aimed to evaluate the efficacy of novel probiotic

preparations to reduce Salmonella Enteritidis S1400 colonisation in chickens.

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Materials and Methods

Bacterial strains, culture and enumeration

Enterococcus faecium PXN-33 and Lactobacillus salivarius 59 were kindly supplied

by Probiotics International Ltd. Salmonella Enteritidis (S1400 Nalr) was used for in vivo

model challenges and has been described previously (Clifton-Hadley et al., 2002; La Ragione

and Woodward, 2003). Salmonella Braenderup H9812 was used as the PFGE standard and

was obtained from the Animal and Plant Health Agency (APHA) culture collection.

Escherichia coli O111 and NM B171, E. coli O127:H6 EC2348/69 from the APHA culture

collection was used as the controls for adhesion assays. Lactobacilli were grown for 48hrs

micro-aerophilically using BBL® GasPaks® (Becton and Dickinson™ Oxford, U.K.) on de

Man, Rogosa, Sharpe agar (MRS). Enterococci were grown micro-aerophilically on Slanetz

and Bartley (SB) agar at 37oC for 16hrs. S. Enteritidis was grown for 16hrs aerobically on

brilliant green agar (BGA). Broth cultures for enterococci, lactobacilli and Salmonella were

cultured in Heart Infusion Broth (HIB), MRS and Luria-Bertani without glucose (LB-G),

respectively with agitation for 16hrs at 37oC, unless stated otherwise in the methods. Prior to

experimental dosing of birds, broth cultures were centrifuged at 1700g for 10mins at room

temperature and adjusted to the appropriate bacterial counts in 0.1M phosphate-buffered

saline (PBS) (pH 7.2).

For culture of probiotic and S. Enteritidis S1400 isolates from in vivo studies circa 1g

of tissue was added to 9mls 0.1M PBS (pH 7.2), homogenized using a CAT S620® (SLS)

tissue macerator, serially diluted, plated and enumerated after incubation in a 5% CO2

atmosphere at 37oC for 24hrs: MRS, Slanetz and Bartley (SB) agar and BGA plates

(supplemented with 15µg of nalidixic acid for selection of S1400) were used to culture

lactobacilli, Enterococci and S. Enteritidis S1400, respectively.

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Probiotic adherence to avian in vitro organ culture (IVOC) gut tissue

IVOC assays were performed as previously described with some modifications

(Allen-Vercoe & Woodward, 1999; La Ragione et al., 2000). Day old SPF White Leghorn

chicks (SPAFAS) were killed by cervical dislocation. Approximately 2cm sections of tissue

from the crop, duodenum, jejunum, ileum, caeca and colon, were removed aseptically and

placed in pre-warmed (42oC) sterile Ringer’s solution for immediate use. The tissue loops

were sliced down the longitudinal axis to expose the epithelial surface. Tissue loops were

washed in sterile Ringer’s solution twice, placed in new 10ml of sterile pre-warmed Ringer’s

solution, inoculated with 100µl of 5x108 cfu/ml of L. salivarius 59 or E. faecium PXN33

bacteria and incubated aerobically at 42oC with shaking for 2 hrs. The tissues were

subsequently rinsed in Ringer’s solution three times and homogenized and bacterial counts

were determined. Assays were performed using three chicks from which two duplicate

intestinal sections were aseptically removed and used in the association assays (adhesion and

invasion). Experiments were repeated on two separate occasions. The bacteriological

procedures were as described above.

Adherence of probiotic to human cell monolayers

Tissue culture assays were performed essentially as described previously with minor

modifications (Dibb-Fuller et al., 1999). Briefly, HEp-2 and CaCo-2 cells were reconstituted

in Dulbecco’s Modified Eagles Medium D5671 (DMEM) (Sigma) supplemented with foetal

calf serum (10% v/v, Autogenbioclear), non-essential amino-acids (1% v/v, Sigma) and

gentamicin (50g/ml, Sigma) and grown to confluency in 24 well micro-titre plates. HEp-2

and CaCo-2 mono-layers were washed twice in HBSS and inoculated with 5 x 107 CFU/ml L.

salivarius 59 and E. faecium PXN-33 Mono-layers were then incubated at 37C

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supplemented with 5% CO2 in air for 3hrs. The supernatant was removed and the mono-

layers were washed (x3) to remove non-adherent bacteria. Mono-layers were disrupted with

1% Triton X-100 (Sigma) and adherent bacteria numbers were determined by plating serial

dilutions.

Scanning electron microscopy of HEp-2 cells

Mono-layers were grown on in 24 well plates and prepared for bacterial adherence as

described above. Supernatant was removed from the mono-layers and fixed for 16 hours in

3% (v/v) glutaraldehyde in 0.1M PBS (pH 7.2). Samples were washed in 0.1M PBS (pH 7.2)

and post fixed in 1% (w/v) osmium tetroxide, washed in PBS, dehydrated in ethanol and

placed in hexamethyldisizane. Samples were subjected to critical point drying with liquid

carbon dioxide. Air dried specimens were fixed to aluminium stubs with silver conductive

paint, sputter coated with gold and examined using a Stereo-scan S250 MarkIII SEM at 10-

20KV.

General in vivo poultry methods

Mixed sex SPF white leghorn chicks were used in all in vivo studies. All chicks were

hatched and transferred to sterile Wey-isolators maintained under negative pressure (source;

APHA, Weybridge). A commercial antibiotic free feed (complete mash diet-chick crumbs;

Zootechnical Products) and water were sterilized and made available to the chicks ad libitum.

All licensed procedures were approved by the local ethics committee and performed under

the jurisdiction of project licenses 70/6435 and 70/5282 at the APHA.

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Probiotic feeding trial after S. Enteritidis S1400 oral challenge

The probiotic feeding trial used in this study was performed as described previously

with minor modifications (Pascual et al., 1999). One hundred day old chicks were divided

randomly into 4 groups of 25 birds housed in Wey-isolators. Probiotic and S. Enteritidis

S1400 administration was performed by oral gavage in a final volume of 0.1ml PBS. Four

randomized groups of birds were dosed by oral gavage with 1x109 cfu of probiotic in 100µl

0.1M PBS (pH 7.2) or 100µl of PBS only for control birds; Group 1) PBS by oral gavage,

Group 2) E. faecium PXN-33, Group 3) L. salivarius 59 or Group 4) a 50:50 preparation of

both probiotic strains (combined group). At 2 days old all the birds in the 4 groups were

dosed with 5x104 cfu of S. Enteritidis (S1400 Nalr). To confirm colonisation of the chicks by

S. Enteritidis S1400 10 birds from each group selected at random were cloacally swabbed at

3, 6, and 8 days of age and plated on BGA (supplemented with nalidixic acid). At 2 days of

age and 3 days of age 3 birds were sacrificed to determine probiotic and S. Enteritidis S1400

colonisation, respectively. At 3, 6, 24 and 43 days of age 3 birds were killed by cervical

dislocation and subjected to post-mortem examination. Circa one gram of the ileum, caecum

and colon were aseptically sampled for bacteriology. Probiotic colonisation was determined

at days 24 and 43 days of age by growth on MRS agar or SB agar for lactobacilli and

enterococci, respectively. Recovered isolates were screened by PFGE (see below) to confirm

identity as the challenge probiotic strain.

At 6, 24 and 43 days of age the ileum, caecum and colon samples were plated onto

BGA supplemented with nalidixic acid for the recovery of S. Enteritidis S1400. Where no

Salmonella was recovered by direct plating, homogenates were enriched in Selenite broth at

42oC, aerobically. Samples were re-plated onto selective media after 1 and 7 days.

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Approximately 10% of putative Salmonella isolates recovered from all studies were tested by

O9 slide agglutination.

Probiotic feeding trial after S. Enteritidis S1400 sentinel bird challenge

The approach used followed that of Clifton-Hadley et al. (2002) with several

modifications. Day-old birds were randomly assorted into six groups of birds with a total of

28 birds per group (Groups A-E) and one group of 30 birds (sentinel bird group: Group F)

and housed in Wey-isolators. Four of the five experimental groups were dosed by oral gavage

with 1x109 cfu of a combined 50:50 preparation of L. salivarius 59 and E. faecium PXN33 in

100µl 0.1M PBS (pH 7.2) (Table 1). The dosing regimes were as follows; Group A received

no probiotic (control group), Group B received a dose at age 1 day, Group C at age 1 & 12

days, Group D at age 12 & 20 days and Group E at age 12 days (Table 1).

Sentinel birds (Group F) were dosed with S. Enteritidis S1400 by oral gavage and

subsequently introduced to the experimental groups as follows. Birds in the sentinel group

were dosed with 5x104 cfu of S. Enteritidis S1400 in 100µl 0.1M PBS (pH 7.2) at 1 and again

at 12 days of age. At 12 days of age 6 birds from the sentinel group were introduced into each

of the remaining 5 trial groups (3 sentinel birds and 14 experimental birds per isolator)

(detailed in Table 1).

At 16, 23, 30 and 43 days of age 3 birds from each isolator (which were not sentinel

birds) were euthanized and subjected to post-mortem examination. The ileum, caecum and

colon were removed. Bacteriological enumeration of S. Enteritidis S1400, enterococci and

lactobacilli were performed as described above.

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Pulse Field Gel Electrophoresis for probiotic strain identification

PFGE was performed as previously described with modifications (Jacobsen et al.,

1999). Bacterial pellets harvested from 16hr cultures were washed once in 1ml of SE buffer

(75mM NaCl and 25mM EDTA pH 7.4). Bacterial pellets were imbedded in tempered 2%

Seakem Gold (Cambrex, East Rutherford, N.J.) and lysed in lysis buffer (50mM EDTA pH

8.2, 0.05% N-lauroyl sarcosine, 2mg/ml lysozyme and 3U/ml of mutanolysin) at 37oC for

16hrs. Plugs were incubated for 16hrs at 53°C in 10mM Tris, 0.5 M EDTA (pH 8.5), 1%

sodium dodecyl sulphate (SDS), and 2 mg of proteinase K per ml and washed (x6) with SE

buffer (Jacobsen et al., 1999). Plugs were digested with 25 units of SmaI (Promega,

Southampton, United Kingdom) for 2hrs at 25°C. Pulsed-field gel electrophoresis (PFGE)

was performed on a CHEF DRIII system (Bio-Rad, Hercules, Calif.) in 0.5% TBE extended-

range buffer (Bio-Rad) and resolved in 0.8% SeaKem Gold. DNA from Salmonella

Braenderup H9812 cleaved with XbaI was used as a size marker. Restriction fragments were

resolved under the running conditions: Block 1, 200 V, initial time, 3.5 s, final time, 25 s,

12hrs; block 2, 200 V, initial time, 1 s, final time, 5 s; 8hrs; total time, 20hrs. The gels were

visualised using ethidium bromide (Sigma, Aldrich).

Probiotic conditioned medium and disc diffusion assays

For conditioned media assays L. salivarius 59, E. faecium PXN33 and S. Enteritidis

S1400 was grown overnight in Brain Heart Infusion Broth (BHIB). Probiotic supernatants

were collected and filter sterilized using 0.2µm filters. Two sets of cell free supernatant were

used: pH adjusted to pH 7.2 and unadjusted. Duplicates of the conditioned medium were

inoculated with ~1x105 cfu/ml of S. Enteritidis S1400 and incubated in 96 well plates for

24hrs. Controls included S. Enteritidis S1400 in unconditioned BHIB media and BHIB media

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only. Optical density readings were measured at 600nm using a Flurostar Optima®.

Experiments were conducted on three separate occasions.

For disc diffusion assays semi-confluent lawns of S. Enteritidis S1400 were prepared

as previously described (Andrews, 2006). S. Enteritidis S1400 was inoculated onto Iso-

Sensitest agar (Oxoid, Cheshire, United Kingdom) to give semi-confluent lawn of growth.

Six mm diameter BD blank paper discs were inoculated with 10µl, 15µl and 20µl of E.

faecium PXN-33 and L. salivarius 59 overnight cultures grown in MRS broth. A bank disc

and disc of containing 30µg of amoxycillin/clavulanic acid was used as a negative and

positive control, respectively. Plates were incubated at 37oC for 24 hrs and the zones of

inhibition were measured. The assay was performed in triplicate and a Student’s unpaired T-

test used to compare zones of inhibition.

Statistical analysis

Statistical analysis of the data was evaluated using StatXact, Unistat and GraphPad

Prism 4, (GraphPad Software Inc.) software. Data comparisons for ex vivo analysis were

One-way ANOVA and significant differences were further analysed using Bonferroni's

Multiple Comparison Test post-test to compare the treatment groups to controls. In vivo

transformed log10 colonisation data was analysed in StatXact system using a Two-way

ANOVA General Linear Model (GLM) where the values of the control were compared to the

probiotic treated group. The swabbing data from the in vivo studies was analysed using a

Kruskal-Wallis, where significant differences were observed further analysis was conducted

using Dunnett’s post-test to compare the treatment groups to the control group. P values of

<0.05 were considered statistically significant.

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Results

Adherence of probiotic bacteria to poultry derived IVOC gut tissue and human cell

lines

E. faecium PXN-33 adhered to crop tissue with significantly higher numbers (circa 1

log higher) than the duodenum, jejunum, ileum and colon (3.90x105 cfu/g) (Figure 1A)

(p<0.01). No significant difference was observed between the counts for the remaining

tissues (p>0.05) (Figure 1A). L. salivarius 59 adhered to the crop tissue at significantly

higher levels than the duodenum, jejunum, ileum, caecum and colon (Figure 1B) (circa 1 to 2

logs 4.26x106 cfu/g) (p<0.01). No significant differences were observed between the

remaining tissues (p>0.05) (Figure 1B).

E. faecium PXN-33 associated to HEp-2 cells and CaCo-2 cells at 3.87x106 cfu (3.7%

of input) and 8.54x106 cfu (4.8% of input) with no significant difference between the cell

lines (p=0.1325) (Figure 1C and D). L. salivarius 59 associated to HEp-2 cells and CaCo-2

cells at 8.26x105 cfu (1.2% of input) and 5.77x105 cfu (0.7% of input), respectively. No

significant difference between the cell lines was observed (p=0.1534) (Figure 1C and D).

E. faecium PXN-33 associated in greater numbers than L. salivarius 59 to both HEp-2

and CaCo-2 cells (p<0.0001 and p=0.0016, respectively); E. coli 0127:H6 and E. coli 0111

was used as positive adhesion controls (Figure 1C and D). To gain some insight into the

distribution of adhesion of the probiotic bacteria, electron microscopy was performed. These

studies revealed that E. faecium PXN33 bound as single or short chains of bacterial cocci

whereas L. salivarius 59 formed dense clusters of bacterial rods (Figure 1E and 1F).

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In vivo evaluation of the effect of probiotic bacteria on weight gain, GI integrity and

mucin production in SPF White Leghorn chickens

At age 5, 15 and 22 days the average weight of the birds in the control group and

probiotic group were 53.3g (SD± 3.6g), 144.7g (SD± 9.3g)and 232.2g (SD± 28.9g) & 52.0g

(SD± 2.8g), 139.5g (SD± 6.4g) and 186.5g (SD± 81.8g), respectively. No significant

difference observed between groups (p>0.05). Birds were observed at regular intervals

throughout the study; no morbidity or mortality was observed in the control and probiotic

treated group. Tissues were evaluated for by Alcian blue and PAS staining for acidic and

neutral mucins; no significant difference in mucin production was observed between the

control and probiotic treated birds when compared to the untreated control group.

(Supplemental Figure 1A to E, respectively).

Probiotic feeding trial following direct oral challenge with S. Enteritidis S1400

No significant difference were observed in S. Enteritidis S1400 colonisation in birds

treated with the E. faecium PXN-33 or L. salivarius 59 as single preparations at any time

point tested when compared to the control (p>0.05) (data not shown). However, at age 43

days a 2 log reduction of S. Enteritidis S1400 colonisation was observed in the ileum, caecum

and colon when treated with the combined probiotic preparation; the difference between the

combined probiotic treated group compared to the control group at age 43 days was

significant (p<0.001) (Figure 2A).

Recovery of probiotic isolates from the S. Enteritidis S1400 direct oral challenge study

Prior to in vivo studies E. faceium PXN33 and L. salivarius 59 were speciated by PCR

(Supplemental Figure 2A and B, respectively). Strain specific PFGE patterns for L.

salivarius 59 and E. faecium PXN33 were confirmed using 5 additional E. faecium and L.

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salivarius strains (Supplemental Figure 2C and 2D, respectively). At 43 days of age all 10

enterococci isolates recovered from the ileum and caeca of the group treated with a combined

probiotic group were identical (Figure 2B and C), but that the profile did not match the

patterns seen for E. faecium PXN-33 (Figure 2B) which was not the anticipated result. In

contrast all 10 Lactobacillus isolates recovered from the ileum and caeca in the combined

probiotic group matched the PFGE profile described for L. salivarius 59 (Figure 2C).

Probiotic feeding trial after S. Enteritidis S1400 sentinel bird challenge study

In the group treated with the combined probiotic at 1 day of age significant

differences in the numbers of S. Enteritidis S1400 recovered from tissues compared to the

control group (p=0.0357 and p=0.0041, respectively) were observed at 16 and 23 days of age

(Figure 3A and B respectively). However, no significant reduction in S. Enteritidis S1400

colonisation was observed at 30 and 43 days of age (p>0.05) (Figure 3C and 3D).

Probiotic treatment at 1 and 12 days significantly reduced S. Enteritidis S1400 at 23

days of age compared to the control group (p=0.0373) (Figure 3B). There was no significant

reduction in the numbers of S. Enteritidis S1400 in the treatment and control group at 16, 30

and 43 days of age (p=0.5984, p=0.3816 and p=0.1404, respectively) (Figure 3A to 3C,

respectively). Birds treated with probiotic at 12 days of age showed no significant reductions

in S. Enteritidis S1400 at 16, 23 and 43 days of age (p>0.05) (Figure 3A to 3C). There was

however a significant reduction at 43 days of age (p=0.0166) (Figure 3D).

In the group treated with the combined probiotic at 12 and 20 days of age significant

reductions in the number of S. Enteritidis S1400 recovered from tissues was observed at age

26 and 43 day (p=0.0012 and p=0.0021, respectively) (Figure 3B and 3D, respectively). No

significant reductions in the number of S. Enteritidis S1400 recovered from tissues was

observed at day 16 or day 30 (p>0.05) (Figure 3A and 3C, respectively). It was noted that

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giving a probiotic dose at day 12 & 20 resulted in nearly a 10 fold greater reduction in S.

Enteritidis S1400 numbers in the ileum as compared to dosing birds at day 12 only (3.75x104

cfu/g and 1.89x105 cfu/g of S. Enteritidis S1400, respectively) (Figure 3D).

Conditioned culture medium and disc diffusion assays

There was evidence of some suppressive effects of the probiotics on the ability of S.

Enteritidis S1400 to colonise the chick and preliminary studies to define the possible

inhibitory mechanism(s) were undertaken. Using the disc diffusion assay following the

methods of Tsai et al. (see M&M) both E. faecium PXN-33 and L. salivarius 59 inhibited S.

Enteritidis S1400 growth on solid media and the zones of inhibition were similar (14mm)

showing no statistically significant differences (data not shown). No inhibition was observed

with the MRS negative control or the blank disc on either probiotic treated plate.

The data from the disc diffusion assays showed a diffusible chemical was responsible

for inhibition. This is likely to be either a pH effect or some other antibacterial. To assay pH

effects conditioned media assays (CFCS) were performed with unadjusted conditioned media

or using pH7.2 adjusted media. The average pH of unadjusted CFCS was pH 5.4 for both E.

faecium PXN-33 and L. salivarius 59. Using the time point at which S. Enteritidis S1400

growth reached an optical density of 0.08 at 600nm we demonstrated that S. Enteritidis S1400

growth was completely mitigated in E. faecium PXN33 and inhibited by L. salivarius 59

CFCS without pH adjustment (Figure 4C). However, pH adjustment of CFCS from both

probiotic bacteria mitigated inhibition of S. Enteritidis S1400 growth (Figure 4C and 4D).

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Discussion

Our work demonstrated that the oral administration of a combined probiotic

comprising L. salivarius 59 and E. faecium PXN33 resulted in reduced colonisation of

poultry by S. Enteritidis S1400. Of particular relevance to reducing the entry of this pathogen

into the human food chain we observed the greatest reduction in Salmonella colonisation as

the birds achieved at slaughter age (Metzier-Zebeli et al., 2016). Our results are consistent

with and further contribute to previous findings that L. salivarius and E. faecium reduce

Salmonella infection in poultry (Pascual et al., 1999; Carina Audisio et al., 2000). It is often

difficult to compare directly the efficacy of probiotics across studies due to differing

management systems, feed regimes, challenge strains, poultry type, genotype and other

variables. However, the work described here demonstrated that combining L. salivarius 59

and E. faecium PXN33 reduced colonisation of the chick by S. Enteritidis S1400 more

effectively than single strain probiotic treatment, a finding that has been reported previously

(Timmerman et al., 2004; Revolledo et al., 2009; Chapman et al., 2011). Whilst we did not

observe significant inhibitory effects with individual probiotic preparations, it should be

noted that birds were housed in experimental conditions and thus the probiotics may still

prove to be effective at Salmonella CE in a commercial environment. We also investigated

whether the number of probiotic administrations and the age at which treatment was given

affected the reduction of colonisation. We demonstrated that probiotic administrations at day

12 was more effective at reducing S. Enteritidis S1400 colonisation near slaughter age.

However, in contrast to day 12 administrations, administration of probiotic at day 1 resulted

in reduced Salmonella burden at age 16 days. Therefore, multiple probiotic administration

may provide continued S. Enteritidis S1400 suppression throughout the poultry growth cycle.

However, further large scale field trials are required to determine optimal probiotic

administration regimes. It should also be noted that no significant difference in Salmonella

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colonisation was observed between the treatment and control groups at day 30. This is likely

due to the cyclic nature of Salmonella colonisation and shedding patterns that are observed

within poultry flocks (Cooper et al., 1994). The bird’s immune system starts maturing at two

to three weeks of age and this raises the possibility that part of the effect seen against S.

Enteritidis S1400 may have an immune dimension, whereby the probiotic bacteria alter the

host immune system as it develops. Previous studies have shown that a probiotic product

comprising of four lactobacillus species, including Lactobacillus salivarius, reduced

inflammation and modulate T-cell responses of the host (Penha et al., 2015). Additionally, S.

Enteritidis has been shown to increase gut permeability which may contribute to colonisation

of poultry (Prado-Rebolledo et al., 2016). The administration of probiotic cultures has been

shown to prevent S. Enteritidis colonisation of poultry by increasing intestinal integrity

(Prado-Rebolledo et al. 2016). Although we did not observe any gut integrity changes by

histology, it is possible these changes were missed due to the sensitivity of the techniques

utilised and thus this warrants further investigation. It is also possible that any interaction

between L. salivarius 59 and E. faecium PXN33 and the gut epithelium, especially those

interactions leading to immune stimulation, may not occur immediately after hatch.

A direct oral gavage was used for initial studies. However, a ‘sentinel bird’ model was

used to simulate likely natural Salmonella infection of poultry, as has been modelled by

previously (Weinack et al., 1979; Bailey et al., 1998). It was suggested that sentinel bird

models are less sensitive than direct oral administration studies (Weinack et al., 1979), but

this is contrary to our findings in this study and previously (Clifton-Hadley et al., 2002). The

similarities in the pattern of S. Enteritidis S1400 colonisation in the control groups of our two

studies suggest that the oral gavage and sentinel bird models may correlate, with the main

difference for the in-contact birds being colonisation occurring over a period of hours or even

days whereas a direct gavage is immediate.

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The interaction and/or adhesion of probiotics to the intestinal mucosa of target host

animals is thought to be a major contributing factor to their beneficial effects (Ouwehand et

al., 1999) and, as already mentioned, for these probiotic strains this may be delayed or only

effective when the immune system is maturing. However, this raises the question whether the

probiotic bacteria themselves actually interact/adhere to the gut epithelium. Using PFGE we

demonstrated that L. salivarius 59 colonised poultry until 43 days of age. We need to be

careful assuming that the L. salivarius 59 we observed was the specific strain administered as

PFGE is a relatively crude differential technique. However, the profiles were identical by eye

and this is compelling evidence for the ability of this strain to colonise long term. In contrast,

none of the enterococci isolated were E. faecium PXN33.

It is interesting that the dual probiotic was more effective than either probiotic strain

alone, but that E. faecium PXN33 was not isolated sometime after inoculation. This may have

occured due to PFGE not being sensitive enough to detect the full range of E. faecium strain

types and thus E. faecium PXN33 may be present, but below the detection limit. It is also

plausible that the probiotic is transient during poultry colonisation. This merits further

investigation as our results suggest the potential for transient colonisation of probiotics may

elicit longer-term benefits for the organism, possibly through beneficial immune effects. Both

probiotic bacteria showed a tissue tropism to the crop, which is consistent with previous

findings by Brooke and Fuller (1975). The preferential adhesion of both bacteria to the crop

may explain their probiotic effect through re-seeding of the GI tract. The persistence of L.

salivarius 59 in vivo, in contrast to the transient colonisation of E. faeceium PXN33, may

reflect increased adhesion to the crop as was demonstrated in our IVOC gut tissue studies. It

should be noted that E. faecium PXN33 associated to human HEp-2 and CaCo-2 cells at

significantly higher levels than L. salivarius 59. The disparity in the IVOC and human tissue

culture assays is likely due to E. faecium species preferentially colonising humans whereas L.

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salivarius is well documented as an avian commercial bacterial species (Fuller, 1977; Garriga

et al., 1998; Dunne et al., 2001). A further consideration is that E. faecium PXN33 may be

less able to colonise but was able to alter the local environment transiently, acidifying the

crop for example, to enhance those E. faecium strains that are better adapted to colonise

poultry. If this is the case these strains may be effective probiotics also.

We investigated the potential mechanism of inhibition of S. Enteritidis S1400 by the

probiotic strains using CFCS and disc diffusion assays as has been described previously

(Fayol-Messaoudi et al., 2007). Disc diffusion and CFCS demonstrated that L. salivarius 59

and E. faecium PXN33 inhibited S. Enteritidis S1400 and that this effect was in part pH

dependent. These findings are consistent with previous research demonstrating that L.

salivarius and E. faecium species inhibition is in part due to lactic acid and lowered pH

(Fuller, 1977; Garriga et al., 1998; Makras et al., 2005). Interestingly, E. faceium PXN33

CFCS inhibition of S. Enteritidis S1400 was greater than L. salivarius 59 CFCS inhibition,

even though the average CFCS pH was equivalent. Furthermore, whilst the additive effect of

using the L. salivarius 59 and E. faecium PXN33 is not entirely explained by the pH

dependent in vitro assays, it is suggestive that reduced pH in the poultry digestive system is a

contributing factor in reducing colonisation of poultry by S. Enteritidis S1400. However,

there were no health implications suggesting the pH effects were not tolerated by the birds

and no dysbiosis was induced: gut microbial profiling may be a useful future study to assess

the wider impacts of the probiotic treatments. Whilst our study demonstrates that probiotic

reduction of pH is likely to contribute to Salmonella inhibition, other mechanisms may also

contribute to colonisation in vivo. Bacterial colonisation and the establishment of a niche

within the GI tract drives the development of phenotypes that exclude potential competitors

within the local environment. Probiotic bacteria attempt to establish this niche dominance

through mechanisms that also benefit the host, including the exclusion of pathogens.

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Probiotic bacteria may suppress or out compete potential pathogens directly by competing

with host binding sites, the production of inhibitory metabolites such as volatile fatty acids,

the production of bacteriocins or competition for nutrients (Carter et al., 2009). More recent

studies have demonstrated that these inhibitory mechanisms may be more complex than

previously thought. For example recent studies by Tanner et al. has demonstrated that

Bifidobacterium thermophilum modulates S. Typhimurium virulence factor expression in co-

culture resulting in Salmonella inhibition, thus targeting pathogen specific responses required

for host colonisation. Furthermore, probiotic bacteria can lead to pathogen inhibition through

indirect mechanisms including immunomodulation. Probiotic effects on the host immune

system have been shown to establish balanced GI tract microflora and improve innate and

humoral responses to pathogens through the regulation of cytokine pathways and antibody

production (Tellez et al., 2012). Furthermore, studies have indicated that Lactobacillus

isolates inhibit Salmonella colonisation of poultry by modulating pro-inflammatory and T-

cell dependent cytokine production (Hu et al., 2015). L. salivarius 59 and E. faecium PXN33

warrant further detailed study to establish potential beneficial host-probiotic interactions in

poultry beyond the scope of this initial study.

We also demonstrated that administration of the probiotic bacteria to poultry did not

result in weight loss or damage to the mucosa suggesting that both probiotic bacteria are not

detrimental to the host. Thus, from the data collectively, the evidence is compelling that the

two probiotics in combination were effective in reducing colonisation of birds by S.

Enteritidis S1400 and our in vitro data suggests a potential pH dependent mechanism.

Importantly, we suggest the subtle differences in the effect on the colonisation of the birds by

S. Enteritidis S1400 reflect probiotic administration, which was given as two separate bolus

administrations, and perhaps continuous administration would be more efficacious. Also,

why a combined probiotic appears more effective needs interrogation.

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Acknowledgements: The authors acknowledge the provision of probiotic strains including

support for this study from Probiotics International Ltd, Lopen Head, Somerset, TA13 5JH,

UK and colleagues in the animal services facilities at the APHA for managing the facilities

during the studies. Special thanks to Bill Cooley (APHA) for assistance with EM studies and

Alex Nunez (APHA) for assistance with histopathology.

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