BACKGROUND
lactobacillus reuteri is a heterofermentative lactic acid bacteria that belongs to the authochtonous microbiota of humans and animals  <cit> .
animal studies have shown that certain lb.
reuteri strains used as probiotics can provide protection against detrimental effects of certain microbiological, chemical and physical stressors; lower cholesterol; modulate immune responses; and improve the development of ileal tissue  <cit> .
lb.
reuteri sd <dig>  has been shown to reduce the incidence and severity of diarrhoea of different origins in children and adults  <cit> , and to survive the passage through the stomach and upper intestine and transiently colonize the human gastrointestinal tract  <cit> .
indeed, this strain has been used for more than ten years as probiotic and/or starter culture in food and health care products  <cit> .
in the presence of glycerol lb.
reuteri can synthesize 3-hydroxypropionaldehyde .
3-hpa is excreted into the medium where it forms together with the hydrate and the dimer , a dynamic multi-component equilibrium   <cit> .
reuterin is a potent antimicrobial agent active against gram positive and gram negative bacteria, as well as yeasts, moulds and protozoa  <cit> .
reuterin is synthesized in vitro under ph, temperature and anaerobic conditions similar to those of the gastrointestinal tract  <cit> .
in vivo, active reuterin synthesis could occur in the colon via lb.
reuteri metabolism, if sufficient amounts of glycerol become available as a product of luminal microbial fermentations, digestion of luminal fats, sloughed mucus and desquamated epithelial cells, and intestinal clearing of endogenous plasma glycerol  <cit> .
to our knowledge, there are presently no data concerning the amount of glycerol available for bacterial conversion to reuterin in the human intestine.
reuterin is water soluble, effective in a wide range of ph, resistant to proteolytic and lipolytic enzymes and has been therefore studied as a food preservative or auxiliary therapeutic agent  <cit> .
reuterin is also believed to play a role in the probiotic effects of lb.
reuteri sd <dig>  but its mode of action on microbial growth has not been elucidated due to the high complexity of the hpa-system chemistry  <cit> .
it has been postulated that reuterin might inhibit the activity of bacterial ribonucleotide reductase, an enzyme catalyzing the first step in dna synthesis, by competition  with ribonucleotides for binding sites or by reaction  with unstable sulfhydryl groups of ribonucleotide reductase or with thioredoxin which is required for enzymatic activity  <cit> .
the inhibition of the conversion of ribonucleotides to deoxyribonucleotides would explain the broad-spectrum activity of reuterin  <cit> .
it has also been suggested that the mechanism of action of reuterin might be directed towards sulfhydryl enzymes, and that the tolerance of bacteria to reuterin might be associated with intracellular levels of lmw-sh such as glutathione .
reduced glutathione , is a cysteine-containing tripeptide, mostly found in gram negative bacteria such as purple and cyanobacteria.
the highest gsh concentrations are observed in aerobes or facultative anaerobes, whereas strict anaerobes are generally deprived of gsh but possess other soluble thiol compounds  <cit> .
however, some faculative anaerobic gram positive bacteria  can synthesize significant amount of gsh, whereas others  can import gsh from the growth medium, but this faculty is strain dependent  <cit> .
gsh plays important functions in bacteria including control of redox potential, protection against oxidative stress, detoxification of endogenously and exogenously derived toxins, protein folding and storage and transport of organic sulfur  <cit> .
this potent antioxidant is also believed to play a key role in detoxification of reactive aldehydes, such as acrolein and protects cells from oxidative damage such as produced by h2o <dig>  <cit> .
recently, glutathione reductase has been shown to contribute to lactobacillus sanfranciscensis oxygen tolerance  <cit> .
various studies have reported the mic of reuterin against pathogens and lactic acid bacteria  <cit> .
however, the activity of pure reuterin against intestinal bacteria has never been reported, even though this data is important for understanding the probiotic effects of lb.
reuteri in vivo when administered to humans and animals.
the aim of this study was therefore to determine the in vitro antibacterial activity of pure reuterin on a representative panel of human intestinal bacteria.
the intracellular concentration of lmw-sh and the sensitivity of intestinal bacteria to hydrogen peroxide were also recorded to tentatively correlate reuterin sensitivity to intracellular lmw-sh such as gsh and oxygen tolerance.
RESULTS
production and purification of reuterin
approximately  <dig> mm reuterin was produced by lb.
reuteri sd <dig> from  <dig> mm glycerol.
after purification of  <dig> ml of this solution, about  <dig> g of pure reuterin was recovered, corresponding to a yield of  <dig> %.
a high purity of reuterin, free from contaminants such as glycerol and  <dig>  propanediol, was obtained after the purification process, as shown by hplc analysis .
antimicrobial activity
the mic and mbc of the tested bacteria are presented in table  <dig>  as expected, lb.
reuteri was the most resistant species, with mic and mbc ranges for both tested strains of 30– <dig> mm and 60– <dig> mm, respectively, followed by other lactobacilli .
the mic and mbc for the sensitive strain escherichia coli were in the range from  <dig> to  <dig> mm and from  <dig> to  <dig> mm, respectively.
adsm: deutsche sammlung von mikroorganismen und zellkulturen, braunschweig, germany atcc: american type culture collection, rockville, maryland, usa hpb: health protection branch, health and welfare canada, ottawa, ontario, canada eth-z:culture collection of the food biotechnology laboratory, eth zürich, switzerland lmg: belgian co-ordinated collections of micro-organisms, gent, belgium sd <dig> = biogaia, stockholm, sweden
bmic and mbc data are expressed as the range obtained from at least three independent repetitions for each species
clmw-sh concentrations are expressed as mean ± sd of at least three independent repetitions
dnot determined
among the  <dig>  tested species,  <dig> showed mic values below that measured for the indicator e. coli strain with mic equal or below  <dig>  mm.
the most sensitive strains to reuterin were bacteroides vulgatus and clostridium difficile , followed by bacteroides thetaiotaomicron, eubacterium eligens, bifidobacterium longum var infantis, eubacterium biforme, bifidobacterium longum and bifidobacterium catenulatum .
the four most resistant non lactobacilli species were clostridium clostridioforme  followed by ruminococcus productus, bifidobacterium breve and listeria innocua .
intracellular lmw-sh concentration including gsh
intracellular concentrations of lmw-sh in tested bacteria are reported in table  <dig>  lmw-sh were detected in all bacteria and was in the range from  <dig>  to  <dig>  μmol/ <dig> colony forming unit ) for many bacteria.
the highest concentrations were found in cl.
difficile  followed by collinsella aerofaciens, lactobacillus salivarius, lactobacillus acidophilus and streptococcus salivarius .
e. coli, bacteroides fragilis, lb.
reuteri ,l. innocua, b. longum var infantis and enterococcus faecium showed intermediate values ranging from  <dig> to  <dig> μmol/ <dig> cfu).
sensitivity to h2o2
the sensitivity of several intestinal bacteria to h2o <dig> is shown in table  <dig>  for all tested bacteria, except for e. faecium, exposure to  <dig>   <dig> and  <dig> g l- <dig> h2o <dig> caused a decrease in viable cell counts compared to the control medium without h2o <dig>  among the  <dig> tested, the  <dig> most resistant species were e. faecium, lb.
acidophilus, lb.
reuteri  and s. salivarius with survival rates to  <dig> g l- <dig> h2o <dig> ranging from  <dig>  to  <dig> %.
the second group, b. longum var infantis and lactobacillus fermentum survived to  <dig> g l- <dig> h2o <dig>  whereas a third group with four bifidobacteria and three lactobacilli species showed survival rates to  <dig> g l- <dig> h2o <dig> ranging from  <dig>  to  <dig>  %.
the most sensitive group with complete growth inhibition with  <dig> g l- <dig> of h2o2included  <dig> species belonging to known strict anaerobe genera from the human gut .
surprisingly, listeria spp.
and e. coli, classified as facultative anaerobes, were also completely inhibited with  <dig> g l- <dig> h2o <dig>
1no growth
different letters in a line indicate significant differences with the tukey's test
discussion
in this study, the mic and mbc of pure reuterin for different intestinal bacteria were determined using a serial dilution microtiter plate assay.
with the exception of dobrogosz et al.
<cit> , who express the inhibitory activity of reuterin with arbitrary units defined, after analysis with 14c-labeled glycerol, as  <dig> u equal  <dig> μg reuterin/ml, most of the authors have used poorly defined arbitrary units to express the inhibitory concentration of reuterin  <cit> .
therefore, a direct comparison of published data for antimicrobial activities of reuterin against different bacteria is difficult.
another problem of these studies is that the molar concentration of reuterin was calculated using the molecular mass of the dimer form  <cit> , although reuterin occurs in its monomeric form at the low concentrations used  <cit> .
moreover arbitrary units are changed by many experimental factors as shown for bacteriocins, such as variation of sensitivity among indicator strains of the same species, medium and incubation conditions for the activity tests and concentrations of antimicrobial compounds in tested preparations  <cit> .
for data comparison, test conditions must be standardized and mic data expressed in μg ml- <dig> or mm.
in this study the molar concentration of reuterin was determined by a method originally developed for acrolein quantification, using the acid-catalyzed formation of a colored complex between acrolein/hpa and tryptophan  <cit> , which has been adopted by several other authors to correctly quantify reuterin  <cit> .
furthermore, all bacteria were tested under controlled test conditions and the same medium for the activity test.
our data showed that reuterin is an effective inhibitor for most tested intestinal bacteria, with the exception of lactobacilli.
among the  <dig> species non belonging to the lactobacillus genus,  <dig> were inhibited and/or killed by reuterin concentrations equal or below  <dig>  mm.
they exhibited higher sensitivity than e. coli, the usual sensitive indicator used to test reuterin  <cit> .
dobrogosz et al.
<cit>  and chung et al.
<cit>  reported, using a two-fold dilution test, mic for e. coli k <dig> of 4– <dig> u/ml ,  <dig> u/ml  for lb.
reuteri and ranging from  <dig> to  <dig> u/ml  for different lactobacilli.
our data show the same trends; mic values for lactobacilli were at least  <dig> times higher than for e. coli .
however, under our test conditions, mic measured for lactobacilli were approximately  <dig> times higher than reported in previous studies.
dobrogosz et al.
<cit>  found that listeria monocytogenes showed a similar low sensitivity to reuterin as for lactobacilli, with mic of 4– <dig> u/ml , which was different from our data for l. ivanovii and l. innocua, which were much more sensitive to reuterin than lactobacilli.
these differences for mic measured in different studies can be due to various factors of the arbitrary activity tests, including ph, culture media, incubation temperature, concentration and physiological state of the tested bacteria which affect both the growth  <cit>  of the tested strains and the activity of reuterin  <cit> .
as well, a strict criteria for mic calculation  was used in our study.
the aldehyde function of 3-hpa is highly reactive due to its β-hydroxy moiety and therefore the molecule spontaneously reacts with available amino- and sulfhydryl-functional groups present in compounds of the growth medium and bacteria  <cit> .
according to lüthi-peng et al.
<cit> , only  <dig> % of reuterin remains free after  <dig> h of incubation at 37°c in a complex medium.
the lactobacilli were also tested in the lamvab growth medium  <cit> , which is used as selective medium to enumerate lactobacilli in fecal samples, at ph  <dig> .
with this medium mic were 2– <dig> times lower than the mic obtained with supplemented bhi .
this result clearly shows that the comparison of data from different studies on reuterin activity is difficult, but comparison within one study is much more valid.
this limitation should also be taken into account when sensitivity data to antimicrobial compounds such as reuterin obtained with in vitro tests, are extrapolated to other in vitro systems or in vivo conditions such as in the gastrointestinal tract.
to tentatively explain the different behavior of intestinal bacteria toward reuterin activity, we analyzed their intracellular concentration of lmw-sh including gsh, which has been shown to be involved in aldehyde detoxification mechanisms  <cit> .
with this intention, we used the ellmann's reagent that specifically reacts with thiol groups.
to make the distinction between lmw-sh, and thiol groups of proteins, the concentration of intracellular lmw-sh was determined in the cell-free supernatant of bacterial crude extracts after protein elimination by precipitation with sulfosalicylcylic acid.
although, all lmw-sh and not only gsh were measured in the present study, this test gives a good estimation of gsh concentration because other lmw-sh occur only in low concentration  <cit> .
lmw-sh were present in all tested bacteria.
the highest intracellular concentrations  were measured in two gram positive strict anaerobic species  and one facultative anaerobic bacteria .
although glutathione appears to be mostly present in cyanobacteria and purple bacteria  <cit> , high intracellular glutathione concentrations have already been measured in gram positive closdriales, including strict anaerobes such as clostridium perfringens  <cit>  and lactobacillus spp.
<cit>  and bifidobacterium spp.
<cit> .
it is usually suggested that gram positive organisms do not produce gsh but accumulate it from the growth medium.
however, other evidences indicate that some gram positive bacteria  can synthetize gsh  <cit> .
moreover, fernandes and steele  <cit>  showed that intracellular gsh levels in lactic acid bacteria varied widely between organisms and were very dependent on strains tested as well as growth conditions, including growth medium composition  and aeration, as well as bacterial growth phase.
very few data are reported on the capacity of intestinal bacteria to synthesize or harvest gsh from the growth medium.
it has been shown that some probiotic lactobacilli strains can produce and even release high amount of gsh  <cit> .
in our study, the growth medium was deprived of cystein, which is a major precursor for gsh synthesis.
however, gsh from yeast extract was likely available for bacterial uptake in the growth medium  <cit> .
we recently observed that external addition of gsh could conteract the toxicity of reuterin  toward e. coli k <dig>  which suggests a correlation between induced toxicity of 3-hpa on e. coli k <dig> and depletion of gsh .
however, in this study, the lack of correlation between intracellular gsh concentration and reuterin sensitivity suggests that other detoxification mechanisms prevent 3-hpa toxicity, such as  <dig> -propanediol-dehydrogenase, which converts 3-hpa to the unreactive  <dig> -propanediol.
no inhibitory activity of  <dig> -propanediol against intestinal bacteria is known until now.
gsh is considered to play a key role in protecting cells against oxidative stress.
it is usually the most abundant intracellular, low-molecular-weight thiol in e. coli  <cit>  and might contribute to protect e. coli cells  <cit>  as well as gram positive bacteria  <cit>  from the lethal effects of oxidative damage.
it is known that gsh can reduce hydrogen peroxide in exchange for oxidation of gsh to glutathione disulfide   <cit> .
the tolerance of intestinal bacteria to hydrogen peroxide was then determined to assess a possible correlation between their sensitivity to reuterin and oxygen detoxification.
oxygen toxicity is considered to result from the effects of activated compounds including superoxide, hydrogen peroxide and hydroxyl radicals  <cit> .
facultative anaerobic bacteria decompose and detoxify these activated oxygen metabolites enzymatically with superoxide dismutase and catalase  <cit> .
most of the intestinal bacteria, classified as strict anaerobic organisms, are devoid of these enzymes and therefore their growth can be inhibited when h2o <dig> is present  <cit> .
indeed, the most oxygen sensitive species tested, which belongs to the orders bacteroidales  and clostridiales  were completely inhibited with  <dig> g l- <dig> hydrogen peroxide.
five bifidobacteria among the six tested, were not inhibited by  <dig> g l- <dig> hydrogen peroxide although they are classified as a strict anaerobic bacteria.
using the same technique, shimamura et al.
<cit>  reported variations in h2o <dig> sensitivity of bifidobacterium spp., and found similar results with b. infantis being the least sensitive to h2o <dig>  followed by b. breve, b. adolescentis and b. longum.
the facultative anaerobic bacteria tested were found to largely differ in their sensitivities to hydrogen peroxide.
among lactobacilli, lb.
acidophilus and lb.
reuteri dsm  <dig> were the most tolerant.lb.
acidophilus and bifidobacterium spp.
have nadh oxidase and nadh peroxidase to scavenge hydrogen peroxide and prevent cell death in the presence of oxygen  <cit> .
moreover, it is known that some lb.
acidophilus strains produce hydrogen peroxide and peroxidase  <cit> .
in addition, certain lactobacilli produce superoxide dismutase  <cit>  and a non-heme pseudocatalase  <cit> .
surprinsigly, e. coli and listeria spp.
also classified as facultative anaerobes and which possess a catalase were inhibited by  <dig> g l- <dig> hydrogen peroxide.
but it has also been shown that the high h2o <dig> sensitivity of e. coli can be related to dna damage caused by hydrogen peroxide  <cit> .
in this study, we did not establish any correlation between bacterial hydrogen peroxide sensitivity and intracellular lmw-sh concentration or reuterin sensitivity.
however, the wide range of organisms tested an the multiplicity of oxygen detoxification mechanisms as well as the wide functions of lmw-sh and gsh in microorganisms  <cit>  suggest that strain specific mechanisms are likely to be involved rather than a general mechanism applicable to all bacteria.
CONCLUSIONS
this study reports for the first time the antimicrobial activity of reuterin on various intestinal bacteria.
large differences among species were observed.
with the exception of lactobacillus strains which were more resistant, most intestinal bacteria were inhibited by reuterin concentration below  <dig>  mm.
moreover, the mbc  recorded for the two listeria spp.
tested were higher than for the main intestinal commensals.
the most sensitive species were inhibited by reuterin concentration around  <dig> mm, which suggests that low concentration of reuterin production in situ  seems to be sufficient to alter the intestinal bacterial growth.
however caution should be applied when extrapolating these results obtained with in vitro activity tests to specific in vivo conditions, where other important factors such as nutrient competition and antagonistic and/or synergistic relationships between the intestinal microbiota can largely influence the inhibition effects of antimicrobials such as reuterin.
complementary experiments are therefore needed in more complex in vitro systems, such as in vitro intestinal fermentation models, as in vivo experimentation is very difficult.
no correlation was found between bacterial sensivity to reuterin and intracellular lmw-sh and/or hydrogen peroxide sensitivity.
our data suggest that protection via lmw-sh such as gsh is not a general protection mechanism toward reuterin toxicity.

 lactobacillus reuteri is a heterofermentative lactic acid bacteria that belongs to the authochtonous microbiota of humans and animals. animal studies have shown that certain lb. reuteri strains used as probiotics can provide protection against detrimental effects of certain microbiological, chemical and physical stressors; lower cholesterol; modulate immune responses; and improve the development of ileal tissue. lb. reuteri sd has been shown to reduce the incidence and severity of diarrhoea of different origins in children and adults, and to survive the passage through the stomach and upper intestine and transiently colonize the human gastrointestinal tract. indeed, this strain has been used for more than ten years as probiotic and/or starter culture in food and health care products. in the presence of glycerol lb. reuteri can synthesize 3-hydroxypropionaldehyde. 3-hpa is excreted into the medium where it forms together with the hydrate and the dimer, a dynamic multi-component equilibrium. reuterin is a potent antimicrobial agent active against gram positive and gram negative bacteria, as well as yeasts, moulds and protozoa. reuterin is synthesized in vitro under ph, temperature and anaerobic conditions similar to those of the gastrointestinal tract. in vivo, active reuterin synthesis could occur in the colon via lb. reuteri metabolism, if sufficient amounts of glycerol become available as a product of luminal microbial fermentations, digestion of luminal fats, sloughed mucus and desquamated epithelial cells, and intestinal clearing of endogenous plasma glycerol. to our knowledge, there are presently no data concerning the amount of glycerol available for bacterial conversion to reuterin in the human intestine. reuterin is water soluble, effective in a wide range of ph, resistant to proteolytic and lipolytic enzymes and has been therefore studied as a food preservative or auxiliary therapeutic agent. reuterin is also believed to play a role in the probiotic effects of lb. reuteri sd but its mode of action on microbial growth has not been elucidated due to the high complexity of the hpa-system chemistry. it has been postulated that reuterin might inhibit the activity of bacterial ribonucleotide reductase, an enzyme catalyzing the first step in dna synthesis, by competition with ribonucleotides for binding sites or by reaction with unstable sulfhydryl groups of ribonucleotide reductase or with thioredoxin which is required for enzymatic activity. the inhibition of the conversion of ribonucleotides to deoxyribonucleotides would explain the broad-spectrum activity of reuterin. it has also been suggested that the mechanism of action of reuterin might be directed towards sulfhydryl enzymes, and that the tolerance of bacteria to reuterin might be associated with intracellular levels of lmw-sh such as glutathione. reduced glutathione, is a cysteine-containing tripeptide, mostly found in gram negative bacteria such as purple and cyanobacteria. the highest gsh concentrations are observed in aerobes or facultative anaerobes, whereas strict anaerobes are generally deprived of gsh but possess other soluble thiol compounds. however, some faculative anaerobic gram positive bacteria can synthesize significant amount of gsh, whereas others can import gsh from the growth medium, but this faculty is strain dependent. gsh plays important functions in bacteria including control of redox potential, protection against oxidative stress, detoxification of endogenously and exogenously derived toxins, protein folding and storage and transport of organic sulfur. this potent antioxidant is also believed to play a key role in detoxification of reactive aldehydes, such as acrolein and protects cells from oxidative damage such as produced by h2o. recently, glutathione reductase has been shown to contribute to lactobacillus sanfranciscensis oxygen tolerance. various studies have reported the mic of reuterin against pathogens and lactic acid bacteria. however, the activity of pure reuterin against intestinal bacteria has never been reported, even though this data is important for understanding the probiotic effects of lb. reuteri in vivo when administered to humans and animals. the aim of this study was therefore to determine the in vitro antibacterial activity of pure reuterin on a representative panel of human intestinal bacteria. the intracellular concentration of lmw-sh and the sensitivity of intestinal bacteria to hydrogen peroxide were also recorded to tentatively correlate reuterin sensitivity to intracellular lmw-sh such as gsh and oxygen tolerance.  production and purification of reuterin approximately mm reuterin was produced by lb. reuteri sd from mm glycerol. after purification of ml of this solution, about g of pure reuterin was recovered, corresponding to a yield of %. a high purity of reuterin, free from contaminants such as glycerol and propanediol, was obtained after the purification process, as shown by hplc analysis. antimicrobial activity the mic and mbc of the tested bacteria are presented in table as expected, lb. reuteri was the most resistant species, with mic and mbc ranges for both tested strains of 30– mm and 60– mm, respectively, followed by other lactobacilli. the mic and mbc for the sensitive strain escherichia coli were in the range from to mm and from to mm, respectively. adsm: deutsche sammlung von mikroorganismen und zellkulturen, braunschweig, germany atcc: american type culture collection, rockville, maryland, usa hpb: health protection branch, health and welfare canada, ottawa, ontario, canada eth-z:culture collection of the food biotechnology laboratory, eth zürich, switzerland lmg: belgian co-ordinated collections of micro-organisms, gent, belgium sd = biogaia, stockholm, sweden bmic and mbc data are expressed as the range obtained from at least three independent repetitions for each species clmw-sh concentrations are expressed as mean ± sd of at least three independent repetitions dnot determined among the tested species, showed mic values below that measured for the indicator e. coli strain with mic equal or below mm. the most sensitive strains to reuterin were bacteroides vulgatus and clostridium difficile, followed by bacteroides thetaiotaomicron, eubacterium eligens, bifidobacterium longum var infantis, eubacterium biforme, bifidobacterium longum and bifidobacterium catenulatum. the four most resistant non lactobacilli species were clostridium clostridioforme followed by ruminococcus productus, bifidobacterium breve and listeria innocua. intracellular lmw-sh concentration including gsh intracellular concentrations of lmw-sh in tested bacteria are reported in table lmw-sh were detected in all bacteria and was in the range from to μmol/ colony forming unit ) for many bacteria. the highest concentrations were found in cl. difficile followed by collinsella aerofaciens, lactobacillus salivarius, lactobacillus acidophilus and streptococcus salivarius. e. coli, bacteroides fragilis, lb. reuteri,l. innocua, b. longum var infantis and enterococcus faecium showed intermediate values ranging from to μmol/ cfu). sensitivity to h2o2 the sensitivity of several intestinal bacteria to h2o is shown in table for all tested bacteria, except for e. faecium, exposure to and g l- h2o caused a decrease in viable cell counts compared to the control medium without h2o among the tested, the most resistant species were e. faecium, lb. acidophilus, lb. reuteri and s. salivarius with survival rates to g l- h2o ranging from to %. the second group, b. longum var infantis and lactobacillus fermentum survived to g l- h2o whereas a third group with four bifidobacteria and three lactobacilli species showed survival rates to g l- h2o ranging from to %. the most sensitive group with complete growth inhibition with g l- of h2o2included species belonging to known strict anaerobe genera from the human gut. surprisingly, listeria spp. and e. coli, classified as facultative anaerobes, were also completely inhibited with g l- h2o 1no growth different letters in a line indicate significant differences with the tukey's test discussion in this study, the mic and mbc of pure reuterin for different intestinal bacteria were determined using a serial dilution microtiter plate assay. with the exception of dobrogosz et al., who express the inhibitory activity of reuterin with arbitrary units defined, after analysis with 14c-labeled glycerol, as u equal μg reuterin/ml, most of the authors have used poorly defined arbitrary units to express the inhibitory concentration of reuterin. therefore, a direct comparison of published data for antimicrobial activities of reuterin against different bacteria is difficult. another problem of these studies is that the molar concentration of reuterin was calculated using the molecular mass of the dimer form, although reuterin occurs in its monomeric form at the low concentrations used. moreover arbitrary units are changed by many experimental factors as shown for bacteriocins, such as variation of sensitivity among indicator strains of the same species, medium and incubation conditions for the activity tests and concentrations of antimicrobial compounds in tested preparations. for data comparison, test conditions must be standardized and mic data expressed in μg ml- or mm. in this study the molar concentration of reuterin was determined by a method originally developed for acrolein quantification, using the acid-catalyzed formation of a colored complex between acrolein/hpa and tryptophan, which has been adopted by several other authors to correctly quantify reuterin. furthermore, all bacteria were tested under controlled test conditions and the same medium for the activity test. our data showed that reuterin is an effective inhibitor for most tested intestinal bacteria, with the exception of lactobacilli. among the species non belonging to the lactobacillus genus, were inhibited and/or killed by reuterin concentrations equal or below mm. they exhibited higher sensitivity than e. coli, the usual sensitive indicator used to test reuterin. dobrogosz et al. and chung et al. reported, using a two-fold dilution test, mic for e. coli k of 4– u/ml, u/ml for lb. reuteri and ranging from to u/ml for different lactobacilli. our data show the same trends; mic values for lactobacilli were at least times higher than for e. coli. however, under our test conditions, mic measured for lactobacilli were approximately times higher than reported in previous studies. dobrogosz et al. found that listeria monocytogenes showed a similar low sensitivity to reuterin as for lactobacilli, with mic of 4– u/ml, which was different from our data for l. ivanovii and l. innocua, which were much more sensitive to reuterin than lactobacilli. these differences for mic measured in different studies can be due to various factors of the arbitrary activity tests, including ph, culture media, incubation temperature, concentration and physiological state of the tested bacteria which affect both the growth of the tested strains and the activity of reuterin. as well, a strict criteria for mic calculation was used in our study. the aldehyde function of 3-hpa is highly reactive due to its β-hydroxy moiety and therefore the molecule spontaneously reacts with available amino- and sulfhydryl-functional groups present in compounds of the growth medium and bacteria. according to lüthi-peng et al., only % of reuterin remains free after h of incubation at 37°c in a complex medium. the lactobacilli were also tested in the lamvab growth medium, which is used as selective medium to enumerate lactobacilli in fecal samples, at ph. with this medium mic were 2– times lower than the mic obtained with supplemented bhi. this result clearly shows that the comparison of data from different studies on reuterin activity is difficult, but comparison within one study is much more valid. this limitation should also be taken into account when sensitivity data to antimicrobial compounds such as reuterin obtained with in vitro tests, are extrapolated to other in vitro systems or in vivo conditions such as in the gastrointestinal tract. to tentatively explain the different behavior of intestinal bacteria toward reuterin activity, we analyzed their intracellular concentration of lmw-sh including gsh, which has been shown to be involved in aldehyde detoxification mechanisms. with this intention, we used the ellmann's reagent that specifically reacts with thiol groups. to make the distinction between lmw-sh, and thiol groups of proteins, the concentration of intracellular lmw-sh was determined in the cell-free supernatant of bacterial crude extracts after protein elimination by precipitation with sulfosalicylcylic acid. although, all lmw-sh and not only gsh were measured in the present study, this test gives a good estimation of gsh concentration because other lmw-sh occur only in low concentration. lmw-sh were present in all tested bacteria. the highest intracellular concentrations were measured in two gram positive strict anaerobic species and one facultative anaerobic bacteria. although glutathione appears to be mostly present in cyanobacteria and purple bacteria, high intracellular glutathione concentrations have already been measured in gram positive closdriales, including strict anaerobes such as clostridium perfringens and lactobacillus spp. and bifidobacterium spp.. it is usually suggested that gram positive organisms do not produce gsh but accumulate it from the growth medium. however, other evidences indicate that some gram positive bacteria can synthetize gsh. moreover, fernandes and steele showed that intracellular gsh levels in lactic acid bacteria varied widely between organisms and were very dependent on strains tested as well as growth conditions, including growth medium composition and aeration, as well as bacterial growth phase. very few data are reported on the capacity of intestinal bacteria to synthesize or harvest gsh from the growth medium. it has been shown that some probiotic lactobacilli strains can produce and even release high amount of gsh. in our study, the growth medium was deprived of cystein, which is a major precursor for gsh synthesis. however, gsh from yeast extract was likely available for bacterial uptake in the growth medium. we recently observed that external addition of gsh could conteract the toxicity of reuterin toward e. coli k which suggests a correlation between induced toxicity of 3-hpa on e. coli k and depletion of gsh. however, in this study, the lack of correlation between intracellular gsh concentration and reuterin sensitivity suggests that other detoxification mechanisms prevent 3-hpa toxicity, such as -propanediol-dehydrogenase, which converts 3-hpa to the unreactive -propanediol. no inhibitory activity of -propanediol against intestinal bacteria is known until now. gsh is considered to play a key role in protecting cells against oxidative stress. it is usually the most abundant intracellular, low-molecular-weight thiol in e. coli and might contribute to protect e. coli cells as well as gram positive bacteria from the lethal effects of oxidative damage. it is known that gsh can reduce hydrogen peroxide in exchange for oxidation of gsh to glutathione disulfide. the tolerance of intestinal bacteria to hydrogen peroxide was then determined to assess a possible correlation between their sensitivity to reuterin and oxygen detoxification. oxygen toxicity is considered to result from the effects of activated compounds including superoxide, hydrogen peroxide and hydroxyl radicals. facultative anaerobic bacteria decompose and detoxify these activated oxygen metabolites enzymatically with superoxide dismutase and catalase. most of the intestinal bacteria, classified as strict anaerobic organisms, are devoid of these enzymes and therefore their growth can be inhibited when h2o is present. indeed, the most oxygen sensitive species tested, which belongs to the orders bacteroidales and clostridiales were completely inhibited with g l- hydrogen peroxide. five bifidobacteria among the six tested, were not inhibited by g l- hydrogen peroxide although they are classified as a strict anaerobic bacteria. using the same technique, shimamura et al. reported variations in h2o sensitivity of bifidobacterium spp., and found similar results with b. infantis being the least sensitive to h2o followed by b. breve, b. adolescentis and b. longum. the facultative anaerobic bacteria tested were found to largely differ in their sensitivities to hydrogen peroxide. among lactobacilli, lb. acidophilus and lb. reuteri dsm were the most tolerant.lb. acidophilus and bifidobacterium spp. have nadh oxidase and nadh peroxidase to scavenge hydrogen peroxide and prevent cell death in the presence of oxygen. moreover, it is known that some lb. acidophilus strains produce hydrogen peroxide and peroxidase. in addition, certain lactobacilli produce superoxide dismutase and a non-heme pseudocatalase. surprinsigly, e. coli and listeria spp. also classified as facultative anaerobes and which possess a catalase were inhibited by g l- hydrogen peroxide. but it has also been shown that the high h2o sensitivity of e. coli can be related to dna damage caused by hydrogen peroxide. in this study, we did not establish any correlation between bacterial hydrogen peroxide sensitivity and intracellular lmw-sh concentration or reuterin sensitivity. however, the wide range of organisms tested an the multiplicity of oxygen detoxification mechanisms as well as the wide functions of lmw-sh and gsh in microorganisms suggest that strain specific mechanisms are likely to be involved rather than a general mechanism applicable to all bacteria.  this study reports for the first time the antimicrobial activity of reuterin on various intestinal bacteria. large differences among species were observed. with the exception of lactobacillus strains which were more resistant, most intestinal bacteria were inhibited by reuterin concentration below mm. moreover, the mbc recorded for the two listeria spp. tested were higher than for the main intestinal commensals. the most sensitive species were inhibited by reuterin concentration around mm, which suggests that low concentration of reuterin production in situ seems to be sufficient to alter the intestinal bacterial growth. however caution should be applied when extrapolating these results obtained with in vitro activity tests to specific in vivo conditions, where other important factors such as nutrient competition and antagonistic and/or synergistic relationships between the intestinal microbiota can largely influence the inhibition effects of antimicrobials such as reuterin. complementary experiments are therefore needed in more complex in vitro systems, such as in vitro intestinal fermentation models, as in vivo experimentation is very difficult. no correlation was found between bacterial sensivity to reuterin and intracellular lmw-sh and/or hydrogen peroxide sensitivity. our data suggest that protection via lmw-sh such as gsh is not a general protection mechanism toward reuterin toxicity. 
hydrogen peroxide sensitivity and intracellular lmw-sh concentration were also analysed. this suggests that detoxification by lmw-sh such as glutathione is not a general mechanism and that other mechanisms are probably involved in bacterial tolerance to reuterin and hydrogene peroxide. reuterin produced from glycerol by lactobacillus reuteri, a normal inhabitant of the human intestine, is a broad-spectrum antimicrobial agent. no correlation between bacterial intracellular concentrations of lmw-sh, including glutathione, and reuterin or hydrogen peroxide sensitivities were found. our data showed that intestinal bacteria were very sensitive to reuterin and that their intracellular concentration of lmw-sh was not directly linked to their capacity to resist reuterin or hydrogen peroxide. the aim of this work was to test the activity of reuterin against a representative panel of intestinal bacteria and to study a possible correlation between intracellular low molecular weight thiols such as glutathione, hydrogen peroxide and/or reuterin sensitivity. glutathione, a powerful antioxidant, which also plays a key role in detoxifying reactive aldehydes, protects certain bacteria from oxidative stress, and could also be implicated in resistance to reuterin. lactobacilli and clostridium clostridioforme were more resistant with mic ranging from to mm. reuterin is active toward enteropathogens, yeasts, fungi, protozoa and viruses, but its effect on commensal intestinal bacteria is unknown. our data showed that most tested intestinal bacteria showed mic below that for a sensitive indicator escherichia coli.
 reuterin produced from glycerol by lactobacillus reuteri, a normal inhabitant of the human intestine, is a broad-spectrum antimicrobial agent. it has been postulated that reuterin could play a role in the probiotic effects of lb. reuteri. reuterin is active toward enteropathogens, yeasts, fungi, protozoa and viruses, but its effect on commensal intestinal bacteria is unknown. moreover reuterin's mode of action has not yet been elucidated. glutathione, a powerful antioxidant, which also plays a key role in detoxifying reactive aldehydes, protects certain bacteria from oxidative stress, and could also be implicated in resistance to reuterin. the aim of this work was to test the activity of reuterin against a representative panel of intestinal bacteria and to study a possible correlation between intracellular low molecular weight thiols such as glutathione, hydrogen peroxide and/or reuterin sensitivity. reuterin was produced by lb. reuteri sd in pure glycerol solution, purified and used to test the minimal inhibitory and minimal bactericidal concentrations. hydrogen peroxide sensitivity and intracellular lmw-sh concentration were also analysed.  our data showed that most tested intestinal bacteria showed mic below that for a sensitive indicator escherichia coli. lactobacilli and clostridium clostridioforme were more resistant with mic ranging from to mm. no correlation between bacterial intracellular concentrations of lmw-sh, including glutathione, and reuterin or hydrogen peroxide sensitivities were found.  our data showed that intestinal bacteria were very sensitive to reuterin and that their intracellular concentration of lmw-sh was not directly linked to their capacity to resist reuterin or hydrogen peroxide. this suggests that detoxification by lmw-sh such as glutathione is not a general mechanism and that other mechanisms are probably involved in bacterial tolerance to reuterin and hydrogene peroxide. 