RESEARCH LETTER

Consumption of Camembert cheese stimulates commensal enterococci in healthy human intestinal microbiota ´ Gerard ´ Olivier Firmesse, Sylvie Rabot, Luis G. Bermu´dez-Humaran, Corthier & Jean-Pierre Furet Unite´ d’Ecologie et Physiologie du Syste`me Digestif, INRA, Jouy-en-Josas, France

Correspondence: Jean-Pierre Furet, Unite´ d’Ecologie et Physiologie du Syste`me Digestif, INRA, 78352 Jouy-en-Josas, France. Tel.: 133 1 34 65 29 29; fax: 133 1 34 65 24 62; e-mail: [email protected] Received 29 March 2007; accepted 28 August 2007. First published online October 2007. DOI:10.1111/j.1574-6968.2007.00933.x Editor: Wolfgang Kneifel Keywords Enterococcus sp.; Camembert cheese; gastrointestinal tract; real-time quantitative PCR.

Abstract Enterococci are natural inhabitants of the human gastrointestinal tract and the main Gram-positive and facultative anaerobic cocci recovered in human faeces. They are also present in a variety of fermented dairy and meat products, and some rare isolates are responsible for severe infections such as endocarditis and meningitis. The aim of the present study was to evaluate the effect of Camembert cheese consumption by healthy human volunteers on the faecal enterococcal population. A highly specific real-time quantitative PCR approach was designed and used to type enterococcal species in human faeces. Two species were found, Enterococcus faecalis and Enterococcus faecium, and only the Enterococcus faecalis population was significantly enhanced after Camembert cheese consumption, whereas Escherichia coli population and the dominant microbiota remained unaffected throughout the trial.

Introduction The human intestinal tract harbours a complex and dynamic microbiota representing about 1011 bacteria g
1 of stools. In healthy individuals, the predominant groups are Clostridium leptum, Clostridium coccoides, Bacteroides and, to a lesser extent, bifidobacteria (Matsuki et al., 2002; Rigottier-Gois et al., 2003; Lay et al., 2005). Together with these dominant populations, some groups such as enterobacteria, enterococci and lactobacilli are also frequently present, but at lower levels (Finegold et al., 1983; Guarner & Malagelada, 2003). They represent the subdominant populations. Their roles in microbiota equilibrium and health are not well established. Are these subdominant populations limited by the dominant microbiota or by nutrient requirements? Some species of these groups are provided by the diet and, among them, some could exert a positive effect on human health and are therefore called probiotics (mostly the lactobacilli). However, most of ingested bacteria are not detectable after product consumption except in a few cases (Alander et al., 1999; Fujiwara et al., 2001; Collins et al., 2002). So far, few data are available on the consequences of the ingestion of fermented products on intestinal homeostasis FEMS Microbiol Lett 276 (2007) 189–192

(Garrido et al., 2005). In this regard, the purpose of this work was to determine whether consumption of French Camembert cheese could modify the levels of two subdominant bacterial populations, namely enterobacteria and enterococci. The dietary intervention consisted in a total exclusion of fermented products, followed by consumption of Camembert cheese, which does not contain enterobacteria or enterococci (Lay et al., 2004), as the sole fermented product. To assess the subdominant microbiota a real-time quantitative PCR (Q-PCR) was developed. This technique allows the determination of the composition of the subdominant bacterial community of the intestinal microbiota (Matsuki et al., 2004; Rinttila et al., 2004).

Materials and methods Study design Twelve healthy human volunteers were included in the study, according to a selection process approved by the Institutional Ethics Committee (CCP, Hospital Necker, Paris). During the intervention period, the use of antibiotics and consumption of fermented products was prohibited. The protocol is shown in Fig. 1. Briefly, a 2-week run-in 2007 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

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period week 1 Fecal sample

Table 1.

Consumption of Camembert

Run-in

I

Wash out

3

7

9

Ex

C

W

Fig. 1. Protocol of the human study. Four faecal samples were collected: at initial time (I), at the end of the run-in period with exclusion of fermented products (Ex), at the end of the consumption period (C) and at the end of the wash-out period (W).

16S rRNA gene-targeted primers for real-time PCR

PCR assay

Primers

Sequence (5 0 –3 0 )

Reference

Bacteria

BACT1369F PROK1492R Probe TM1389F Ecoli F Ecoli R Efs 03 Efs 04 Efm 07 Efm 08

CGG TGA ATA CGT TCC CGG TAC GGC TAC CTT GTT ACG ACT T CTT GTA CAC ACC GCC CGT C CAT GCC GCG TGT ATG AAG AA CGG GTA ACG TCA ATG AGC AAA CTG TTG TTA GAG AAG AAC AAG GAC GT GGA CAA CGC TTG CCA CCT A AAG TCG AAC GCT TCT TTT TCC A CCA AGT GTT ATC CCC TTC TGA TG

Suzuki et al. (2000)

Escherichia coli Enterococcus faecalis Enterococcus faecium

Penders et al. (2005) This study This study

Modified from reference.

period under fermented products exclusion was followed by a 4-week consumption period and a 2-week wash-out period. A 2-week run-in period was chosen because several works focused on the analysis of probiotics survival have shown that less than 10% of ingested bacteria are present in faecal samples 1 day after their consumption. A 2-week wash-out period corresponds to the standard duration of a wash-out phase, after which detectable bacteria return to the initial level as estimated by Q-PCR analyses (Firmesse et al., 2007). Throughout the consumption period, subjects consumed 40 g of Camembert cheese twice daily. Faecal samples were collected at inclusion (I), at the end of the run-in period under exclusion diet (Ex), at the end of the 4 weeks of Camembert consumption (C) and at the end of the wash-out period (W). Fresh faecal aliquots of 0.2 g were divided and stored at
80 1C until analyses.

Identification of microbiota in human faeces Estimation of enterococcal population in faeces was performed by Q-PCR with 16S rRNA gene-targeted enterococcispecific primers (Table 1). Q-PCR analyses were carried out as described by Furet et al. (2004) and adapted by Firmesse et al. (2007). For each set of primers, the threshold cycle (Ct) of each sample was compared with a standard curve made from bacterial genomic DNA of reference strains. Real-time PCR of Escherichia coli population in faeces was assessed by Q-PCR with the specific primers described by Penders et al. (2005) (Table 1). The Q-PCR reactions (25 mL) were carried out with the SYBR-Green PCR master mix kit (AppliedBiosystems, Foster City, CA) in accordance with the manufacturer’s instructions. The results were expressed as a numerical value of CFU-equivalent g
1 of stool (see identification of enterococcal microbiota). Real-time PCR was 2007 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

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performed as described recently (Firmesse et al., 2007) for ‘all bacteria’ and the following dominant groups: Bacteroides, C. leptum and C. coccoides. The results were expressed as a numerical value of CFU-equivalents g
1 of stool and plotted as boxes and whiskers. Data were analysed using a one-way s ANOVA (JMP Software, Abacus Concepts, Berkeley, CA). When ANOVA indicated significant differences, treatments were compared in pairs using the Tukey–Kramer test. Statistical significance was accepted at P o 0.05.

Results Detection and quantification thresholds of bacterial species were established in the faecal microbiota. The quantification limit was 6.0 log CFU-equivalents g
1 of stool. No significant differences were observed for either total bacterial, which remained at a constant level of 10.9 log CFU-equivalents g
1 of stool throughout the trial (Fig. 2a), or for the main dominant groups of the microbiota, Bacteroides, C. leptum and C. coccoides (data not shown). Escherichia coli population was observed at a subdominant level and remained stable throughout the trial at a median level of 8.4 log CFUequivalents g
1 of stool (Fig. 2b). At inclusion time (I), Enterococcus faecalis was detected in the majority of faecal samples, reaching a median value of 6.3 log CFU-equivalent g
1 of stool. Enterococcus faecium was only detected in the faeces from five out of 12 volunteers at levels between 6.5 and 7.7 log CFU-equivalent g
1 of stool (Fig. 3). During the following 2 weeks of fermented products exclusion (Ex), the two populations of Enterococcus decreased. They were rarely observed and were at low levels. After 4 weeks of Camembert consumption (C), both of them increased. The Enterococcus faecalis population was found to be significantly increased, reaching a median value FEMS Microbiol Lett 276 (2007) 189–192

191

Stimulation of intestinal enterococci by Camembert cheese

11

11 log CFU-eq/g stool

(b) 12

log CFU-eq/g stool

(a) 12

10 9 8

10 9 8 7

7

6

6

I

Ex

C

W

I

Ex

C

W

Fig. 2. CFU-equivalent (CFU-Eq) of total microbiota (a) and Escherichia coli (b) in faeces of the 12 subjects at the initial time (I) and at the end of the periods of exclusion (Ex), consumption (C) and wash-out (W), as determined by real-time Q-PCR. The plots show the median (horizontal line), the interquartile range (boxes contain 50% of all values) and the 10th and 90th percentiles (whiskers).

11

11

log CFU-eq/g stool

(b) 12

log CFU-eq/g stool

(a) 12

10

**

9 8

*

7

10 9 8 7 6

6 I

Ex

C

W

I

Ex

C

W

Fig. 3. CFU-equivalent (CFU-Eq) of Enterococcus faecalis (a) and Enterococcus faecium (b) in faeces of the 12 subjects at the initial time (I) and at the end of the periods of exclusion (Ex), consumption (C) and wash-out (W), as determined by real-time Q-PCR. The plots are as in Fig. 2. A value of P o 0.05 was considered to be significant when comparing Ex with I and W (). A value of P o 0.05 was considered to be significant when comparing C with I, Ex and W ().

of 7.6 log CFU-equivalent g
1 of stool. The level decreased rapidly after discontinuation of Camembert cheese consumption (W) to reach the level observed during the runin period. In the case of Enterococcus faecium, no significant changes occurred over the duration of the trial. However, during the consumption period, an increasing trend was observed, leading to a median value of 6.1 log CFU-equivalent g
1 of stool. Using Q-PCR as described, no Enterococcus could be detected in the Camembert cheese.

Discussion Food products such as meat and diary products may contain Enterococcus (Giraffa, 2003; Hugas et al., 2003). However, Camembert cheese does not contain either enterococci (or below 2 log CFU g
1) (Lay et al., 2004) or enterobacteria, but this study suggests that, in humans, its consumption leads to a significant increase of the Enterococcus faecalis population, while the Escherichia coli population and the dominant microbiota remain unaffected throughout the trial. The technique used (Q-PCR) includes detection of living and dead microorganisms which may have different effects on the cross-talk with the human host. Enterococci are GramFEMS Microbiol Lett 276 (2007) 189–192

positive cocci present in the human intestinal tract, where they must be considered as natural inhabitants. In particular, previous studies have shown that Enterococcus faecalis and Enterococcus faecium are the most common Enterococcus species in human faeces (Devriese & Pot, 1995). In this study, the prevalence of Enterococcus faecalis is confirmed, even when consumption of diary products was excluded. Different species of enterococci are found in a variety of fermented foods such as cheese, fermented sausages, vegetables and dairy starter cultures (Cogan et al., 1997; Mannu & Paba, 2002; Giraffa, 2003; Hugas et al., 2003; FoulquieMoreno et al., 2006). Some isolates are the cause of a variety of infections such as endocarditis and meningitis (Megran, 1992; Barker et al., 1994). In addition, the ability of enterococci to exchange extra-chromosomal elements (e.g. plasmids) that encode antibiotic resistance genes, particularly vancomycin, has been of particular concern during the last few years (Bensoussan et al., 1998; Mater et al., 2005). The present data indicate that the resident populations of enterococci are influenced by consumption of Camembert cheese at dietary levels. The mechanism is unknown. It could be a ‘prebiotic-like’ effect of the cheese chemical components, as observed with bifids and non digestible 2007 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

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sugars extracted from ‘chicory’ (Roberfroid, 2005). Alternatively, there may be a direct or an indirect effect of the bacteria and yeast populations contained in the Camembert cheese. The fact that the resident enterococcal population can be influenced by moderate dietary changes is to be considered in terms of safety and health.

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