RESEARCH LETTER
Lactobacillus plantarum DSM 2648 is a potential probiotic that enhances intestinal barrier function Rachel C. Anderson1, Adrian L. Cookson1, Warren C. McNabb1,2, William J. Kelly1 & Nicole C. Roy1,2 1
Food, Metabolism and Microbiology Section, Food and Textiles Group, AgResearch Grasslands, New Zealand; and 2Riddet Institute, Massey University, Palmerston North, New Zealand
Correspondence: Rachel C. Anderson, Food, Metabolism and Microbiology Section, Food and Textiles Group, AgResearch Grasslands, Private Bag 11008, Palmerston North 4442, New Zealand. Tel.: 164 6 351 8231; fax: 164 6 351 8001; e-mail:
[email protected] Received 2 April 2010; revised 26 May 2010; accepted 3 June 2010. Final version published online 2 July 2010. DOI:10.1111/j.1574-6968.2010.02038.x Editor: Ezio Ricca
MICROBIOLOGY LETTERS
Keywords transepithelial electrical resistance; gut function; leaky gut; tight junctions.
Abstract The aim of this research was to identify bacterial isolates having the potential to improve intestinal barrier function. Lactobacillus plantarum strains and human oral isolates were screened for their ability to enhance tight junction integrity as measured by the transepithelial electrical resistance (TEER) assay. Eight commercially used probiotics were compared to determine which had the greatest positive effect on TEER, and the best-performing probiotic strain, Lactobacillus rhamnosus HN001, was used as a benchmark to evaluate the isolates. One isolate, L. plantarum DSM 2648, was selected for further study because it increased TEER 135% more than L. rhamnosus HN001. The ability of L. plantarum DSM 2648 to tolerate gastrointestinal conditions and adhere to intestinal cells was determined, and L. plantarum DSM 2648 performed better than L. rhamnosus HN001 in all the assays. Lactobacillus plantarum DSM 2648 was able to reduce the negative effect of Escherichia coli [enteropathogenic E. coli (EPEC)] O127:H6 (E2348/69) on TEER and adherence by as much as 98.75% and 80.18%, respectively, during simultaneous or prior coculture compared with EPEC incubation alone. As yet, the precise mechanism associated with the positive effects exerted by L. plantarum DSM 2648 are unknown, and may influence its use to improve human health and wellness.
Introduction Probiotics are defined as ‘live microorganisms which, when administered in adequate amounts, confer a health benefit onto the host’ (Guarner & Schaafsma, 1998). Most probiotics belong to the genera Lactobacillus and Bifidobacterium, and are often selected for their ability to grow in dairy products, survive gastrointestinal conditions and adhere to intestinal epithelial cells (Dunne et al., 2001; Delgado et al., 2008). Although these properties are important to the delivery of viable probiotics to the site of action, greater emphasis should be placed on selecting probiotics based on their specific health benefits to target particular consumer groups or health ailments (Gueimonde & Salminen, 2006). Probiotics can have a number of different mechanisms by which they are proposed to improve health, such as inhibition of pathogenic bacteria, improving epithelial and mucosal barrier function and altering the host’s immune response. 2010 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved
c
Despite the known association between impaired intestinal barrier function, gastrointestinal disorders (Barbara, 2006; Bruewer et al., 2006; Guttman et al., 2006) and illnesses in other parts of the body (Liu et al., 2005; Maes, 2008; Maes & Leunis, 2008; Sandek et al., 2008; Vaarala et al., 2008), few studies have focused on selecting probiotics based on their ability to enhance intestinal barrier function. However, using the transepithelial electrical resistance (TEER) assay as a measure of the integrity of the tight junctions between intestinal epithelial cells, studies have shown that some bacteria can enhance intestinal barrier function. A number of these studies used strains of Lactobacillus plantarum. For example, L. plantarum CGMCC 1258 was able to lessen the negative impact of enteroinvasive Escherichia coli ATCC 43893 serotype O124:NM on TEER (Qin et al., 2009), L. plantarum 299v mitigated the TNF-ainduced decrease in TEER (Ko et al., 2007) and L. plantarum MF1298 attenuated the decrease in TEER induced by Listeria monocytogenes 6896 (Klingberg et al., 2005). FEMS Microbiol Lett 309 (2010) 184–192
185
Lactobacillus plantarum enhances intestinal barrier function
Materials and methods
acid bacteria. Cultures were diluted in phosphate-buffered saline (PBS, pH 7.2), plated onto Rogosa agar and incubated in 5% CO2 at 37 1C for 48 h to select for lactobacilli. Putative L. plantarum strains with large white colonies similar to those of known L. plantarum strains were subcultured onto fresh Rogosa agar and incubated at 37 1C (5% CO2) for 48 h. Sample colonies were stored as glycerol stocks at 85 1C. Isolates were identified based on their 16S rRNA gene sequences. From the isolated genomic DNA, 16S rRNA gene was amplified using FD1 (5 0 -AGAGTTTGATCCTGGCT CAG-3 0 ) and RD1 (5 0 -AAGGAGGTGATCCAGCC-3 0 ) primers and PCR Supermix (Invitrogen) using a Thermo Hybaid PX2 thermocycler, and purified using a QIAquick PCR purification kit. Sequencing was performed at the Allan Wilson Centre Genome Service (Massey University, Palmerston North, New Zealand), and traces were aligned using CONTIGEXPRESS VECTOR NTI and the 16S rRNA gene sequences were compared with known bacterial sequences using the NCBI BLAST database. The EPEC O127:H6 (E2348/69) was obtained from Dr Roberto La Ragione at Veterinary Laboratories Agency, Weybridge, UK.
Bacterial strains
TEER assay
The source of the bacterial strains used in this study is described in Table 1. Eight commercially used probiotics were chosen on the basis that there were published data showing their efficacy in various in vitro and in vivo models (Table 1). Further strains were either L. plantarum obtained from the Deutsche Sammlung von Mikroorganismen (DSM) or human oral lactobacilli isolates. Human oral isolates were obtained from the mouth lining, tongue and teeth of volunteers using sterile tooth picks, which were incubated individually in 10 mL of Man, Rogosa and Sharpe (MRS) broth overnight at 37 1C (5% CO2) to select for lactic
Caco-2 cells (human colorectal adenocarcinoma cell line; ATCC HTB-37) were used as a model of the intestinal epithelial barrier because they differentiate spontaneously into polarized intestinal cells possessing apical brush borders and tight junctions. Caco-2 cells were seeded onto collagen membrane inserts (CellagenTM Discs CD-24, MP Biomedicals, OH) and incubated in 12-well plates in M199 with 10% v/v foetal bovine serum, 1% v/v nonessential amino acids (MEM nonessential amino acids 100 solution and 1% v/v penicillin–streptomycin) (10 000 U penicillin G sodium salt and 10 000 mg streptomycin sulphate in
The aim of this research was to identify lactobacilli isolates, with an emphasis on L. plantarum, that enhance TEER and therefore have the potential to be used as probiotics targeted at improving intestinal barrier function. Eight commercially used probiotics were compared to determine which had the greatest positive effect on TEER across intestinal epithelial cell layers, and then the best probiotic was used as a benchmark to evaluate several isolates, including four L. plantarum strains and 15 human oral isolates. The oral cavity was chosen as a source of potential probiotics because evidence suggests that lactobacilli found in human faeces, and therefore present in the intestines, originate from the oral cavity (Dal Bello & Hertel, 2006; Maukonen et al., 2008). The isolate with the greatest positive effect on TEER was further investigated to evaluate its suitability for use as a probiotic, including its ability to tolerate gastrointestinal conditions, to adhere to intestinal epithelial cells and affect adherence and TEER of enteropathogenic E. coli (EPEC) O127:H6 (E2348/69), a known enteric pathogen (Baldini et al., 1983), during coculture.
Table 1. Summary of the effect of commercially used probiotics (lactobacilli and bifidobacteria) on TEER across confluent undifferentiated Caco-2 monolayers (5 days old) over 12 h Mean change in TEER ( SEM) % compared with the control media (n = 4) expressed relative to L. plantarum MB 452 (100%)
Probiotic strain
Company
References for efficacy
Lactobacillus rhamnosus HN001 Lactobacillus plantarum 299 Bifidobacterium lactis Bb12 Lactobacillus plantarum MB 452 Lactobacillus plantarum 299v Lactobacillus casei Shirota Medium 199 control Lactobacillus rhamnosus GG Bifidobacterium lactis HN019
Danisco Probi AB Chr Hansen Biosystem VSL Pharmaceuticals Probi AB Yakult Control medium Valio Danisco
Gill et al. (2000), Cross et al. (2002) Adlerberth et al. (1996) Ruiz et al. (2005) Unpublished data Mangell et al. (2002), Schultz et al. (2002) Her´ıas et al. (2005)
222 (19),w 158 (33)w 148 (31)w 100 66 (32) 14 (57) 0 Gosselink et al. (2004), Roselli et al. (2006) 11 (79) Gill et al. (2000), Shu et al. (2001) 115 (34),w
P value o 0.05 compared with Lactobacillus plantarum MB 452. w
P value o 0.05 compared with the control medium.
FEMS Microbiol Lett 309 (2010) 184–192
2010 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved
c
186
R.C. Anderson et al.
0.85% v/v saline). Caco-2 cells were grown at 37 1C in 5% CO2 for 5 days until confluent (undifferentiated) for the screening assays. Undifferentiated Caco-2 cells were used for the initial screening because of the ease of preparing undifferentiated Caco-2 cells compared with differentiated Caco-2 cells. This was necessary because of the high volume of assays that were carried out during the screening. The TEER assay measures the integrity of the tight junctions between epithelial cells, and as these tight junctions are already formed when Caco-2 cell monolayers reach confluence (5 days), undifferentiated Caco-2 cells are often used to assess tight junction integrity. An additional TEER assay was carried out using differentiated Caco-2 cells (18 days old) to confirm the positive effects of the best selected isolates. Caco-2 monolayers were prepared the day before the TEER assay by removing the media, washing with PBS (pH 7.2) and adding M199 with 1% v/v nonessential amino acids (without foetal bovine serum and penicillin–streptomycin). In each experiment, control media (M199 with 1% nonessential amino acids) and a positive bacterial strain (either L. plantarum MB452 for commercially used probiotic strain testing or Lactobacillus rhamnosus HN001 for isolate testing) were included as controls. Overnight cultures of bacterial cells (MRS broth, 37 1C, 5% CO2) were collected by centrifugation (20 000 g for 5 min) and resuspended in M199 with 1% v/v nonessential amino acids to an OD600 nm of 0.9. After the initial resistance readings, the media were removed from the Caco-2 monolayers and replaced with treatment solutions. Each bacterial strain was tested in quadruplicate. The resistance across each cell monolayer was measured every 2 h in an electrode chamber (ENDOHM-12 tissue culture chamber; World Precision Instruments, FL) using a voltohmmeter (EVOM Epithelial Tissue Voltohmmeter; World Precision Instruments). The TEER was calculated from the resistance using Eqn.(1), where the background resistance was 14 O and the membrane area was 1.54 cm2. The change in TEER for each insert was calculated using Eqn.(2). Treatments were compared in GENSTAT (version 11) using residual maximum likelihood (REML) analysis with an unstructured covariance model to take into account the repeated measures. TEER ¼ ðresistance background resistanceÞ membrane area Change in TEER ¼ ðTEERCinitial TEERÞ 100
ð1Þ ð2Þ
Acid and bile tolerance Bacterial cultures were grown overnight in MRS broth at 37 1C with 5% CO2. Each culture was vortexed, and separate 10-mL aliquots were collected by centrifugation (3800 g for 20 min). Cell pellets were suspended to an approximate cell 2010 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved
c
concentration of 108–1010 CFU mL1 in the following test solutions: MRS broth control, MRS broth adjusted to pH 2.0, MRS broth adjusted to pH 4.0, 0.5% w/v bile and 1% w/v bile. The time points (2 and 4 h) were chosen to represent the time it takes to pass through the human upper gastrointestinal system to the lower intestinal tract. The concentrations of bile (0.5% and 1%) and pH values (pH 2.0 and 4.0) were chosen to represent the range of these variables found in the human stomach. Bacterial viability was assessed after 2 and 4 h with triplicate 20-mL dilution spots on Luria–Bertani (LB) agar plates. Values were log-transformed before REML analysis using an unstructured covariance model.
Bacterial cell adherence to Caco-2 epithelial cells Quantitative analysis of bacterial adherence to both confluent undifferentiated (5 days) and differentiated (18 days) Caco-2 cells was tested as described previously (Donnenberg & Nataro, 1995). Bacterial strains were grown overnight in MRS broth and approximately 107 CFU (10 mL) were added to each well, with each strain being assessed for adherence (3 and 6 h) in triplicate. Lactobacilli were enumerated on LB agar plates as described previously. Values were log-transformed before ANOVA analysis.
Lactobacillus plantarum DSM 2648 and EPEC coculture The effect of coculture of L. plantarum DSM 2648 and EPEC O127:H6 (E2348/69) was examined in both the TEER and the cell adherence assay. The TEER assay was performed with two hourly readings for 10 h as described previously with overnight cultures of L. plantarum DSM 2648 prepared from MRS broths. The EPEC strain was grown aerobically overnight at 37 1C in LB broth, with shaking at 100 r.p.m. EPEC cells were collected by centrifugation (20 000 g for 5 min) and suspended in M199 with 1% v/v nonessential amino acids to an OD600 nm of 0.1. TEER coculture experiments also included both bacterial strains individually to assess separate effects for control purposes. For adherence to Caco-2 cell monolayers, both the L. plantarum DSM 2648 and the EPEC strain were grown in MRS and LB broth and inoculated into tissue culture wells containing undifferentiated Caco-2 cells as described previously. The EPEC strain was incubated alone or simultaneously cocultured with L. plantarum DSM 2648 for 3 or 6 h. The effects of a 3-h preincubation of L. plantarum DSM 2648 with Caco-2 cells before the addition of the EPEC strain and a 3-h preincubation of the EPEC strain with Caco2 cells before the addition of L. plantarum DSM 2648 were also evaluated. EPEC were enumerated selectively on sorbitol MacConkey agar plates incubated aerobically at 37 1C for 18 h. EPEC adherence during coincubation with L. plantarum DSM 2648 was calculated as a percentage of the adherence of the EPEC strain during 3- and 6-h incubations, respectively. FEMS Microbiol Lett 309 (2010) 184–192
187
Lactobacillus plantarum enhances intestinal barrier function
Treatments were compared using a paired-samples t-test (two tails).
Results and discussion Identification of bacterial strains The activity of four L. plantarum strains obtained from DSM and 15 human oral lactobacilli isolates was compared
with eight commercially used probiotics chosen on the basis of published data showing their efficacy in various in vitro and/or in vivo models. Fifteen human oral bacteria were isolated with the intention of obtaining novel L. plantarum strains; however, based on 16S rRNA gene sequencing, only one was L. plantarum (Table 2). The most commonly isolated species were L. rhamnosus and Lactobacillus fermentum, of which four and five strains were isolated, respectively. The other isolates were strains of Lactobacillus
Table 2. Summary of the effect of Lactobacillus isolates on the TEER across confluent undifferentiated Caco-2 monolayers (5 days old) over 12 h
Experimental strain
Source
L. plantarum DSM 2648 L. fermentum AGR1485
Silage Human oral isolate
L. rhamnosus HN001 L. rhamnosus AGR1500
Best commercially used Human oral isolate
L. plantarum DSM 20205 L. paracasei AGR1491
Corn silage Human oral isolate
L. oris AGR1493 L. plantarum DSM 20246 L. rhamnosus AGR1499
Human oral isolate Unknown Human oral isolate
L. rhamnosus AGR1514
Human oral isolate
L. plantarum DSM 12028 L. helveticus AGR1517
Dry fermented sausage Human oral isolate
L. plantarum AGR1492
Human oral isolate
L. gasseri AGR1515
Human oral isolate
L. rhamnosus AGR1523
Human oral isolate
Medium 199 L. fermentum AGR1512
Control medium Human oral isolate
L. fermentum AGR1502
Human oral isolate
L. jensenii AGR1519
Human oral isolate
L. fermentum AGR1489
Human oral isolate
L. fermentum AGR1487
Human oral isolate
Best strain matches to the 16S rRNA gene sequence
Mean change in TEER ( SEM) % compared with the control media (n = 4) expressed relative to L. rhamnosus HN001 (100%) 235 (14)w,z 114 (9)z
99% Lactobacillus fermentum SFCB2-6c (DQ486144)
100 100% Lactobacillus rhamnosus GG ATCC 53103 (AY370682)
62 (3)w,z 56 (18)w,z 49 (12)w,z
99% Lactobacillus paracasei (DQ199664) 99% Lactobacillus oris (X94229) 99% Lactobacillus rhamnosus GG ATCC 53103 (AY370682) 99% Lactobacillus rhamnosus MCRF-412 (AY299488)
48 (16)w,z 47 (28)w,z 45 (3)w,z 42 (8)w,z 32 (21)w
99% Lactobacillus suntoryeus LH5 (AY675251)‰ 98% Lactobacillus plantarum L5 (DQ239698) 99% Lactobacillus gasseri ATCC 33323 (AF519171) 99% Lactobacillus rhamnosus MCRF-412 (AY299488) 99% Lactobacillus fermentum SFCB2-6c (DQ486144) 99% Lactobacillus fermentum SFCB2-6c (DQ486144) 99% Lactobacillus jensenii KC36b (AF243159) 99% Lactobacillus fermentum (AF302116) 99% Lactobacillus fermentum (AF302116)
31 (24)w 24 (16)w 14 (8)w 5 (38)w 0 11 (38)w 60 (11)w,z 166 (80)w,z 231 (17)w,z 268 (22)w,z
Results are given as percentage match, strain name (GeneBank accession number). w
P value o 0.05 compared with Lactobacillus rhamnosus HN001. P value o 0.05 compared with the control medium. ‰ Lactobacillus suntoryeus was reclassified to Lactobacillus helveticus (Naser et al., 2006). z
FEMS Microbiol Lett 309 (2010) 184–192
2010 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved
c
188
(b)
80 Control medium L. plantarum 299 L. rhamnosus HN001 *** B. lactis Bb12
Change in TEER (%)
60 40
***
160 Control medium L. rhamnosus HN001 L. plantarum DSM 2648
*** 120
Change in TEER (%)
(a)
R.C. Anderson et al.
** *
20 0
* *
40 * 0
–20
–40
–40 0
2
4
6
8
10
12
0
2
4
6
Time (h) (d)
160
Change in TEER (%)
100
10
12
Control medium L. plantarum DSM 2648 E.coli O127:H6 E. coli O127:H6 + L. plantarum * DSM 2648 *
Control medium L. rhamnosus HN001 L. plantarum DSM2648
120
8
Time (h)
** **
**
80 ** 40
0
–40
Change in TEER (%)
(c)
*
80
50
* * 0
*
–50
*
*
*
**
–100 0
2
4
6
8
10
12
Time (h)
0
2
4
6
8
10
Time (h)
Fig. 1. Change in the TEER across confluent Caco-2 monolayers over time. The change in TEER is the percentage change compared with the initial TEER for each monolayer. The values plotted are the means for four monolayers and the error bars show the SEM. P o 0.05 compared with the control media. (a) Confluent undifferentiated Caco-2 cells in the presence of the three commercially used probiotics that had the greatest positive effect on TEER. (b) Confluent undifferentiated Caco-2 monolayers in the presence of Lactobacillus plantarum DSM 2648 and Lactobacillus rhamnosus HN001. (c) Differentiated Caco-2 monolayers in the presence of L. plantarum DSM 2648 and L. rhamnosus HN001. (d) Confluent undifferentiated Caco-2 monolayers in the presence of L. plantarum DSM 2648 and Escherichia coli O127:H6 alone and in combination.
paracasei, Lactobacillus oris, Lactobacillus helveticus, Lactobacillus gasseri and Lactobacillus jensenii.
Effect of commercially used probiotics on TEER The commercially used probiotics were screened in the TEER assay to assess their effect on the integrity of the tight junctions between the intestinal confluent undifferentiated Caco-2 monolayers (5 days old). Lactobacillus plantarum MB452 was used to normalize between assays, because it has a consistently positive effect on TEER (unpublished data). 2010 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved
c
Lactobacillus plantarum 299, L. rhamnosus HN001 and Bifidobacterium lactis Bb12 were the three commercially used probiotics that had the greatest positive effect on TEER measurements and induced increases compared with the control media of 158%, 222% and 148%, respectively (Table 1). Only L. rhamnosus HN001 positively enhanced the overall TEER more than L. plantarum MB452 (P o 0.05 compared with L. plantarum MB452). Lactobacillus rhamnosus HN001 was selected as the benchmark for isolate comparison because it had the greatest positive effect on TEER at all time points and the smallest FEMS Microbiol Lett 309 (2010) 184–192
189
Lactobacillus plantarum enhances intestinal barrier function
(b)
12 10 8 Control pH4 pH2
6 4
* 2
L. rhamnosus HN001 (log CFU/mL)
L. rhamnosus HN001 (log CFU/mL)
(a)
*
Control 0.5% bile 1% bile
10 8
*
*
6 4
* *
2 0
0 0
2 Time (h)
4
0
2
4
Time (h) 12
12
** 10 8
Control pH4 pH2
6 *
4
*
2 0
L. plantarum DSM 2648 (log CFU/m L)
L. plantarum DSM 2648L (log CFU/m L)
12
10 8
*
6 4 Control 0.5% bile 1% bile
2 0
0
2
4
0
2
4
Time (h)
Time (h)
Fig. 2. Viable cell counts of Lactobacillus plantarum DSM 2648 and Lactobacillus rhamnosus HN001 in the presence of (a) acid and (b) bile over time. The values plotted are means of triplicates and the error bars show the SEM. P o 0.05 compared with bacteria in the control media.
variation between replicates (Fig. 1a). Lactobacillus rhamnosus HN001 reduces the severity of pathogen infections (Gill et al., 2001; Shu & Gill, 2002) and stimulates the immune response in rodents (Gill et al., 2000; Gill & Rutherfurd, 2001a, b; Cross et al., 2002), and this study shows that it is also able to enhance tight junction integrity.
Effect of isolates on TEER The 19 bacterial isolates were screened in the TEER assay using confluent undifferentiated Caco-2 monolayers (5 days old) to determine whether any isolates were able to enhance TEER to a greater extent than the commercially used probiotic benchmark, L. rhamnosus HN001. Nine isolates positively enhanced TEER compared with the control media (Table 2; P o 0.05). Of these, one isolate, L. plantarum DSM 2648, caused a 235% increase in TEER, which was greater than the benchmark probiotic (P o 0.05 compared with the control media and L. rhamnosus HN001) (Fig. 1b). Lactobacillus plantarum DSM 2648 also had a similar effect on TEER when tested using differentiated Caco-2 monolayers (18 days old) (Fig. 1c). FEMS Microbiol Lett 309 (2010) 184–192
This study demonstrates the strain-dependent effects of lactobacilli on intestinal barrier function and that all strains of the same species should not be assumed to have similar healthpromoting properties. Lactobacillus plantarum are effective in enhancing TEER, with three out of the five L. plantarum isolates tested having a positive effect on TEER compared with the control media. A number of human oral isolates were also effective in enhancing TEER compared with the control media. Three out of four L. rhamnosus isolates, the L. paracasei isolate and the L. oris isolate had a positive effect on TEER. However, several of the human oral isolates had a negative effect on TEER; three out of five L. fermentum isolates and the L. jensenii isolate induced a decrease in TEER compared with the control media. In contrast, one isolate of L. fermentum induced an increase in TEER compared with the control media.
Ability of L. plantarum DSM 2648 to tolerate gastrointestinal conditions and adhere to intestinal cells Lactobacillus plantarum DSM 2648 was chosen for further investigation because it had a greater positive effect on TEER 2010 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved
c
190
Fig. 3. Viable cell counts of Lactobacillus plantarum DSM 2648 and Lactobacillus rhamnosus HN001 adhered to confluent undifferentiated (5 days old) and differentiated (18 days old) Caco-2 monolayers over time. The values plotted are the means of triplicates and the error bars show the SEM. P o 0.05 compared with L. rhamnosus HN001.
compared with the benchmark, L. rhamnosus HN001, over the 12-h test period. Acid and bile tolerance (2 and 4 h) of L. plantarum DSM 2648 was compared with that of L. rhamnosus HN001 (Fig. 2). Both bacterial strains were able to tolerate acidic conditions (pH 4 for 4 h) without the loss of cell viability; however, both strains had a reduced viability of 6–7 log units under conditions of pH 2 for 4 h. The viability of L. rhamnosus HN001 decreased by 2 log units in the presence of 0.5% bile and by 5 log units in the presence of 1% bile, whereas the viability of L. plantarum DSM 2648 only reduced by 2 log units by 1% bile. The ability of L. plantarum DSM 2648 to adhere to intestinal cells (3 and 6 h) was also compared with that of the benchmark strain, L. rhamnosus HN001 (Fig. 3). Lactobacillus plantarum DSM 2648 adhered in higher numbers (10 times more) to both confluent undifferentiated and differentiated Caco-2 cells compared with L. rhamnosus HN001 (P o 0.05 at 3 and 6 h). Lactobacillus plantarum DSM 2648 displayed better in vitro tolerance to gastrointestinal conditions compared with L. rhamnosus HN001, which has been detected in human faeces after ingestion (Tannock et al., 2000); thus, it is possible that L. plantarum DSM 2648 may also survive passage through the human gastrointestinal tract. 2010 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved
c
R.C. Anderson et al.
Fig. 4. Adhesion of EPEC O127:H6 E2348/69 to Caco-2 cells and the effect of preincubation or simultaneous coculture with Lactobacillus plantarum DSM 2648. Solid bars are the number of adherent EPEC in the absence of L. plantarum DSM 2648. The second and fifth bars show the number of adherent EPEC during a simultaneous coculture with L. plantarum DSM 2648 for 3 and 6 h, respectively. The third bar shows the number of adherent EPEC after 6 h when L. plantarum DSM 2648 was added 3 h before EPEC, and the sixth bar shows the number of adherent EPEC after 6 h when EPEC was added 3 h before L. plantarum DSM 2648. The values plotted are the means of four replicates and the error bars show SEM. P o 0.05 compared with the 3- or the 6-h control value, respectively.
Action of L. plantarum DSM 2648 on EPECinduced TEER changes and bacterial adherence Lactobacillus plantarum DSM 2648 was also able to prevent the deleterious EPEC-induced TEER changes observed when the EPEC strain was incubated alone (Fig. 1d); however, the action of L. plantarum DSM 2648 was transient, lasting for at most 8 h. The action of L. plantarum DSM 2648 on EPEC interactions with Caco-2 cells was further explored using coculture adherence experiments. Lactobacillus plantarum DSM 2648 was found to have the greatest inhibitory effect on the adherence of EPEC (80.18% reduction) to Caco-2 cells when added before the enteric pathogen, but had no effect when added 3 h after the addition of the EPEC strain (Fig. 4). In contrast to the study by Michail & Abernathy (2002), where coincubation of the L. plantarum 299v with the EPEC strain did not result in any statistically significant reduction in EPEC adherence, when the L. plantarum DSM 2648 was added simultaneously with the EPEC in this study, EPEC adherence to the Caco-2 cells was reduced by 65.5% (3 h) and 55.9% (6 h), respectively (Fig. 4). FEMS Microbiol Lett 309 (2010) 184–192
191
Lactobacillus plantarum enhances intestinal barrier function
Conclusions This study showed that L. plantarum DSM 2648 has a number of characteristics desirable for a probiotic selected specifically for its ability to enhance intestinal barrier function. These data warrant further investigation to determine whether the promising in vitro results correspond with in vivo efficacy and to understand the mechanism by which it exerts the positive effects on Caco-2 cells alone and a reduction in the deleterious effects of EPEC during coincubation. The ability of L. plantarum DSM 2648 to survive passage through the gastrointestinal system could be investigated by monitoring viability in the faeces of humans consuming the bacterium. If proven to be effective, L. plantarum DSM 2648 could be used as a probiotic to benefit humans with a range of conditions as well as for general well-being.
Acknowledgements This work was funded by the AgResearch PreSeed Fund (contract #118). R.C.A. was supported by a Foundation of Research, Science and Technology Postdoctoral Fellowship (AGRX0602). The authors acknowledge the contributions of Kate Broadley, Michelle Kirk and Kelly Armstrong (cell culture), Diana Pacheco (16S rRNA gene sequencing), Rechelle Perry (tolerance assays), Caroline Thum (adherence assays) and Jason Peters and Steven Trask (TEER assays).
References Adlerberth I, Ahrne S, Johansson ML, Molin G, Hanson LA & Wold AE (1996) A mannose-specific adherence mechanism in Lactobacillus plantarum conferring binding to the human colonic cell line HT-29. Appl Environ Microb 62: 2244–2251. Baldini MM, Kaper JB, Levine MM, Candy DC & Moon HW (1983) Plasmid-mediated adhesion in enteropathogenic Escherichia coli. J Pediatr Gastr Nutr 2: 534–538. Barbara G (2006) Mucosal barrier defects in irritable bowel syndrome. Who left the door open? Am J Gastroenterol 101: 1295–1298. Bruewer M, Samarin S & Nusrat A (2006) Inflammatory bowel disease and the apical junctional complex. Ann NY Acad Sci 1072: 242–252. Cross ML, Mortensen RR, Kudsk J & Gill HS (2002) Dietary intake of Lactobacillus rhamnosus HNOO1 enhances production of both Th1 and Th2 cytokines in antigen-primed mice. Med Microbiol Immun 191: 49–53. Dal Bello F & Hertel C (2006) Oral cavity as natural reservoir for intestinal lactobacilli. Syst Appl Microbiol 29: 69–76. Delgado S, O’Sullivan E, Fitzgerald G & Mayo B (2008) In vitro evaluation of the probiotic properties of human intestinal
FEMS Microbiol Lett 309 (2010) 184–192
Bifidobacterium species and selection of new probiotic candidates. J Appl Microbiol 104: 1119–1127. Donnenberg MS & Nataro JP (1995) Methods for studying adhesion of diarrheagenic Escherichia coli. Method Enzymol 253: 324–336. Dunne C, O’Mahony L, Murphy L et al. (2001) In vitro selection criteria for probiotic bacteria of human origin: correlation with in vivo findings. Am J Clin Nutr 73: 386S–392S. Gill HS & Rutherfurd KJ (2001a) Immune enhancement conferred by oral delivery of Lactobacillus rhamnosus HN001 in different milk-based substrates. J Dairy Res 68: 611–616. Gill HS & Rutherfurd KJ (2001b) Viability and dose–response studies on the effects of the immunoenhancing lactic acid bacterium Lactobacillus rhamnosus in mice. Brit J Nutr 86: 285–289. Gill HS, Rutherfurd KJ, Prasad J & Gopal PK (2000) Enhancement of natural and acquired immunity by Lactobacillus rhamnosus (HN001), Lactobacillus acidophilus (HN017) and Bifidobacterium lactis (HN019). Brit J Nutr 83: 167–176. Gill HS, Shu Q, Lin H, Rutherfurd KJ & Cross ML (2001) Protection against translocating Salmonella typhimurium infection in mice by feeding the immuno-enhancing probiotic Lactobacillus rhamnosus strain HN001. Med Microbiol Immun 190: 97–104. Gosselink MP, Schouten WR, van Lieshout LM, Hop WC, Laman JD & Ruseler-van Embden JG (2004) Delay of the first onset of pouchitis by oral intake of the probiotic strain Lactobacillus rhamnosus GG. Dis Colon Rectum 47: 876–884. Guarner F & Schaafsma GJ (1998) Probiotics. Int J Food Microbiol 39: 237–238. Gueimonde M & Salminen S (2006) New methods for selecting and evaluating probiotics. Digest Liver Dis 38 (suppl 2): S242–S247. Guttman JA, Li Y, Wickham ME, Deng W, Vogl AW & Finlay BB (2006) Attaching and effacing pathogen-induced tight junction disruption in vivo. Cell Microbiol 8: 634–645. Her´ıas MV, Koninkx JF, Vos JG, Huis in’t Veld JH & van Dijk JE (2005) Probiotic effects of Lactobacillus casei on DSS-induced ulcerative colitis in mice. Int J Food Microbiol 103: 143–155. Klingberg TD, Pedersen MH, Cencic A & Budde BB (2005) Application of measurements of transepithelial electrical resistance of intestinal epithelial cell monolayers to evaluate probiotic activity. Appl Environ Microb 71: 7528–7530. Ko JS, Yang HR, Chang JY & Seo JK (2007) Lactobacillus plantarum inhibits epithelial barrier dysfunction and interleukin-8 secretion induced by tumor necrosis factor-alpha. World J Gastroentero 13: 1962–1965. Liu Z, Li N & Neu J (2005) Tight junctions, leaky intestines, and pediatric diseases. Acta Paediatr 94: 386–393. Maes M (2008) The cytokine hypothesis of depression: inflammation, oxidative & nitrosative stress (IO&NS) and leaky gut as new targets for adjunctive treatments in depression. Neuroendocrinol Lett 29: 287–291.
2010 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved
c
192
Maes M & Leunis JC (2008) Normalization of leaky gut in chronic fatigue syndrome (CFS) is accompanied by a clinical improvement: effects of age, duration of illness and the translocation of LPS from gram-negative bacteria. Neuroendocrinol Lett 29: 902–910. Mangell P, Nejdfors P, Wang M, Ahrne US, Westrom B, Thorlacius H & Jeppsson B (2002) Lactobacillus plantarum 299v inhibits Escherichia coli-induced intestinal permeability. Digest Dis Sci 47: 511–516. Maukonen J, Matto J, Suihko ML & Saarela M (2008) Intraindividual diversity and similarity of salivary and faecal microbiota. J Med Microbiol 57: 1560–1568. Michail S & Abernathy F (2002) Lactobacillus plantarum reduces the in vitro secretory response of intestinal epithelial cells to enteropathogenic Escherichia coli infection. J Pediatr Gastr Nutr 35: 350–355. Naser SM, Hagen KE, Vancanneyt M, Cleenwerck I, Swings J & Tompkins TA (2006) Lactobacillus suntoryeus Cachat and Priest 2005 is a later synonym of Lactobacillus helveticus (Orla-Jensen 1919) Bergey et al. 1925 (Approved Lists 1980). Int J Syst Evol Micr 56: 355–360. Qin H, Zhang Z, Hang X & Jiang Y (2009) L. plantarum prevents enteroinvasive Escherichia coli-induced tight junction proteins changes in intestinal epithelial cells. BMC Microbiol 9: 63. Roselli M, Finamore A, Britti MS & Mengheri E (2006) Probiotic bacteria Bifidobacterium animalis MB5 and Lactobacillus rhamnosus GG protect intestinal Caco-2 cells from the inflammation-associated response induced by enterotoxigenic Escherichia coli K88. Brit J Nutr 95: 1177–1184.
2010 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved
c
R.C. Anderson et al.
Ruiz PA, Hoffmann M, Szcesny S, Blaut M & Haller D (2005) Innate mechanisms for Bifidobacterium lactis to activate transient pro-inflammatory host responses in intestinal epithelial cells after the colonization of germ-free rats. Immunology 115: 441–450. Sandek A, Rauchhaus M, Anker SD & von Haehling S (2008) The emerging role of the gut in chronic heart failure. Curr Opin Clin Nutr 11: 632–639. Schultz M, Veltkamp C, Dieleman LA, Grenther WB, Wyrick PB, Tonkonogy SL & Sartor RB (2002) Lactobacillus plantarum 299V in the treatment and prevention of spontaneous colitis in interleukin-10-deficient mice. Inflamm Bowel Dis 8: 71–80. Shu Q & Gill HS (2002) Immune protection mediated by the probiotic Lactobacillus rhamnosus HN001 (DR20) against Escherichia coli O157:H7 infection in mice. FEMS Immunol Med Mic 34: 59–64. Shu Q, Qu F & Gill HS (2001) Probiotic treatment using Bifidobacterium lactis HN019 reduces weanling diarrhea associated with rotavirus and Escherichia coli infection in a piglet model. J Pediatr Gastr Nutr 33: 171–177. Tannock GW, Munro K, Harmsen HJ, Welling GW, Smart J & Gopal PK (2000) Analysis of the fecal microflora of human subjects consuming a probiotic product containing Lactobacillus rhamnosus DR20. Appl Environ Microb 66: 2578–2588. Vaarala O, Atkinson MA & Neu J (2008) The ‘perfect storm’ for type 1 diabetes: the complex interplay between intestinal microbiota, gut permeability, and mucosal immunity. Diabetes 57: 2555–2562.
FEMS Microbiol Lett 309 (2010) 184–192
