Basic Aspects and Pharmacology of Pro Bio Tics an Overview of Pharmacokinetics Mechanisms of Action...

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Basic aspects and pharmacology of probiotics:

an overview of pharmacokinetics, mechanisms

of action and side-effects

Philippe Marteau* MD, PhD

Professor

Department of Gastroenterology, European Hospital Georges Pompidou, Assistance Publique des Hopitaux de Paris

and Paris V University, France

Fergus Shanahan MD

Professor

Department of Medicine, University College Cork, National University of Ireland, Cork, Ireland

Probiotics have been defined as non-pathogenic micro-organisms that, when ingested, exert apositive influence on host health or physiology. Their pharmacology is more complex than that ofinert drugs but is now being studied in detail. Some strains have a high survival capacity until theyreach the faeces, whereas others are rapidly killed by acid and bile (a characteristic that can beused for the delivery of active intracellular components). Potential translocation and permanentcolonization are rare but possible events; and should come under closer scrutiny. Mechanisms ofaction can be direct or indirect through modifications of the endogenous flora or throughimmunomodulation. The active components are poorly known but include bacterial formylatedpeptides, peptidoglycan cell wall constituents and nucleotides. Although the safety of commercialprobiotics is excellent, this aspect should be studied in more detail, especially inimmunocompromised hosts.

Key words: probiotics; pharmacology of probiotics; survival of probiotics; side-effects ofprobiotics; adhesion.

Probiotics have been defined as non-pathogenic micro-organisms that, when ingested,exert a positive influence on host health or physiology.1 They consist of bacteria(especially lactic acid bacteria and bifidobacteria) and yeasts (especially Saccharomyces),and may be present either in food (especially fermented milks), food supplements ordrugs. Before randomized controlled trials were performed, many physicians weresceptical on their efficacy. A pharmacological approach is now used to assess the effectsand pharmacokinetics of probiotics. Both vary not only between species, but even

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Best Practice & Research Clinical GastroenterologyVol. 17, No. 5, pp. 725–740, 2003doi:10.1016/S1521-6918(03)00055-6, www.elsevier.com/locate/jnlabr/ybega

* Corresponding author. Address: Service d’Hepato-Gastroenterologie, Hopital Europeen GeorgesPompidou, 20 rue Leblanc, 75908 Paris Cedex 15, France. Tel.: þ33-1-5609-3551; Fax: þ33-1-5609-3554.E-mail address: [email protected] (P. Marteau).

http://www.elsevier.com/locate/ybega

between strains, and many strains are effective, as shown in double-blind, randomizedcontrolled trials. We summarise here the current knowledge, ideas and questionsrelating to the mechanisms of action, pharmacokinetics and safety of probiotics in man.

MECHANISMS OF ACTION

Probiotics differ greatly. The clinical conditions in which efficacy has been reportedrange from infectious, allergic and inflammatory to neoplastic, suggesting that a singlemechanism of action for all probiotics and all effects is unlikely. The final effects onhealth or physiology may be either direct or indirect through modification of theendogenous ecosystem or the immune response. Multiple microbe–microbe andmicrobe–host interactions probably account for the versatility of probiotic action.Mutual competitive interactions, competition for essential nutrients and the productionof antimicrobial factors, such as bacteriocins, protect the host from overgrowth withpathogens.2 Probiotics may also promote host defence by priming and conditioning themucosal immune response.3 Other actions include an influence on intestinal barrierfunction4, and multiple mechanisms have been proposed to explain the apparent anti-cancer effect in the context of colon cancer.5 The anti-inflammatory effects ofprobiotics in the context of Crohn’s disease and ulcerative colitis may involve signallingwith the epithelium and mucosal immune system. Since the indigenous commensal floraexert a regulatory influence on epithelial and subepithelial structures within the gut6,7,it seems likely that probiotics have a similar effect.

The molecular details of host–flora interactions are poorly understood; microbialsignals that have been identified include bacterial formylated peptides such as f-met-leu-phe8, lipopolysaccharide (LPS), peptidoglycan cell wall constituents and nucleotides.The host’s innate immune response distinguishes signals from pathogens and fromcommensals by pattern recognition receptors or Toll-like receptors (TLRs), which areexpressed differentially between immune cells and by intestinal epithelial cells. Immunecells use multiple TLRs to detect several features of a microbe simultaneously. TLR2recognizes lipoproteins and peptidoglycans, and triggers the host response to Gram-positive bacteria and yeast; TLR4 mediates responses to LPS primarily from Gram-negative bacteria; TLR1 and TLR6 participate in the activation of macrophages byGram-positive bacteria; and TLR5 and TLR9 recognize flagellin and bacterial (CpG)DNA respectively.9 Bacterial DNA and oligonucleotides containing unmethylated CpGdinucleotides stimulate lymphocytes, whereas eukaryotic DNA and methylatedoligonucleotides do not.10–12 The stimulation of dendritic cells by CpG DNA isassociated with the production of T-helper type 1 (TH1)-like cytokines such asinterleukin-12 (IL-12).

Rachmilewitz et al recently showed that administration of immunostimulatory DNAwas beneficial for the colonic mucosa of mice with chemically induced colitis.13 Theythen showed that the beneficial effect of the probiotic mixture VSL#3 on this model ofcolitis was derived from its DNA as VSL#3 genomic unmethylated DNA was effectivewhereas VSL#3 methylated DNA and calf thymus DNA were ineffective.14 Otherauthors have recently shown that bacterial VSL#3 DNA downregulates pro-inflammatory cytokine secretion by attenuation of the nuclear factor-kB (NF-kB)pathway in intestinal epithelial cells.15 The transcription factor NF-kB is a centralregulator of epithelial responses to pathogens such as invasive Salmonella.16 Non-pathogenic components of the flora, on the other hand, have been reported to

726 P. Marteau and F. Shanahan

attenuate pro-inflammatory responses by blocking degradation of the counter-regulatory factor IkB.17

It is unlikely that probiotic bifidobacteria and lactobacilli use the same mechanism,and other signal transduction pathways are likely to emerge to account for their anti-inflammatory effects. In vitro experiments using either isolated Caco-2 cells or thesame cells together with immunocompetent cells have shown that the micro-organismsdo not all produce the same cytokine secretion pattern and that bacterial signalling atthe mucosal surface is dependent on a network of cellular interactions.18 Thedownregulation of tumour necrosis factor-alpha production in Crohn’s disease has alsobeen demonstrated with probiotics ex vivo.19 In addition to a direct impact on epithelialand cytokine responses, probiotics may also influence the development and activity ofregulatory T-cells.20–22 This would account for their apparent clinical efficacy inimmunologically polarized disorders involving TH1 and TH2 immunological processes.Mucosal regulatory T-cells appear to be similar to regulatory T-cells within mostperipheral tissues that are responsible for preventing auto-immunity to tissue-specificself-antigens.

Direct effects of probiotics via some of their enzymes are easier to study andpredict. The probiotics that contain lactase have, for example, been shown to helplactose digestion23, and yeast containing sucrase aids in sucrose digestion.24 Finally, themechanism of action of probiotics can be predetermined by modifying bacteria toproduce biotherapeutic molecules. This has been achieved with Lactococcus lactis, whichwas engineered to deliver the anti-inflammatory cytokine IL-10 or lipase to the gut.25,26

The first strategy was as efficaceous as corticosteroid therapy in two animal models ofinflammatory bowel disease and the second helped lipid digestion in pigs withpancreatic insufficiency.25 Before genetically modified probiotics can be introduced inhumans, a reliable mechanism of controlling and eliminating the genetically modifiedorganism is, however, required to ensure that there is no risk to public health.27

PHARMACOKINETICS

Probiotics or biotherapeutic agents are an original way of delivering active constituentsto targets in the gastrointestinal tract.28 These active constituents include enzymes,immunomodulatory components (from the cell wall or DNA), vaccinal epitopes (in thefuture) and components possessing antagonistic activities against other micro-organisms. Probiotics protect these activities against acid in the stomach and candeliver them to the target site. To see any progress in the development of probiotics,we must increase our knowledge of the active constituents responsible for each effect,the target sites and the pharmacokinetics of probiotics. Most pharmacokinetic studieshave described the fate of probiotics (i.e. usually their ‘survival’) in the gastrointestinaltract. Indeed, the active constituents are seldom known, and their pharmacokinetics,except for those of lactase from yoghurt bacteria23, cannot therefore be assessed.

Methods of studying the pharmacokinetics of probiotics

In vitro models

Some in vitro models have been used to predict the survival of probiotics in vivo ortheir adherence to the intestinal epithelium.29 Many authors have studied the sensitivityof strains to pH or bile in static conditions. Multicompartmental dynamic models have

Basic aspects and pharmacology of probiotics 727

also been developed in which a computer program reproduces the dynamics ofsecretion and transit of the chyme.29,30 Adhesion of the strains to the mucus orepithelium is usually studied in vitro using animal or human mucus and cell cultures suchas Caco-2.31,32 The experimental conditions are critical and sometimes differ from whatcan be expected in vivo, so the reliability of such models in predicting the in vivosituation is debatable.

In vivo techniques, markers and expression of the results

The best way to establish the pharmacokinetics of a probiotic in the gastrointestinaltract is to measure it in vivo. The majority of studies have assessed the survival ofprobiotics before the stage of the faeces. It is, however, also important for some specificeffects to determine the survival of probiotics in other parts of the tract. Researchersinterested in the effects of probiotics in the small bowel have also studied thepharmacokinetics in the terminal ileum.29 Two techniques have been used to obtainsamples from the gut lumen: the collection of faeces or stomal effluent, and intestinalintubation. The use of perfusion techniques allows a determination of not only theconcentrations of probiotics, but also their flow rates.29 Whatever technique is used tosample the chyme, markers should be used if one wants to determine the potentialcolonization property of a probiotic. Colonization can be defined as the potency of aprobiotic to persist in the body for a longer period than an inert marker ingested at thesame time. Spores of Bacillus stearothermophilus are often used as a transit marker asthey do not grow, are not destroyed in the gastrointestinal tract and are easilyenumerated on agar Plate at 658C, at which no intestinal bacteria grow. After theiringestion, these spores are eliminated in the faeces in an exponential manner, andbecome undetectable is subjects with ‘normal’ intestinal transit within 5–9 days.

The sensitivity of bacteriological techniques to detect and identify the probioticwithin the endogenous flora is also critical in survival studies. A clear identification ofthe strain using specific traits or probes is always best. To increase the detection limit ofthe probiotic within the endogenous ecosystem, several studies have been performedusing probiotic variants with antibiotic resistance, especially to streptomycin andrifampicin.29,33 Another method is the use of control periods, but it is more difficult tointerpret such experiments.34 An increase in lactobacilli in the faeces after the ingestionof lactobacilli may, for example, be due either to the passage of the ingested strain upuntil the faeces or to an increased excretion of endogenous lactobacilli. The mucosa-adherent and luminal flora differ at the different levels of the gastrointestinal tract.35

The colonization of the mucosa can be studied from biopsies, and the conditions usedto prepare the sample should be mentioned as they may theoretically influence theresults. This has not yet, however, been studied thoroughly, and whether the mucosa-adherent ecosystem is a more relevant target for clinical end-points than the luminalflora is also currently purely speculative.

It is important to consider the expression of the results. The best criterion isprobably the concentration of probiotic (or of its active ingredient) at the target site.The concentration of probiotics needed to obtain a clinical effect is often quoted as$106 colony-forming units/ml (cfu) in the small bowel and $108 cfu/g in the colon.28

The scientific basis for this is, however, weak. The small bowel concentration has beenproposed because it leads to a clinical effects (diarrhoea) in subjects with bacterialcolonization of the small bowel; the concentration in the colon has been proposed as itrepresents the lower limit of the dominant flora. The percentage survival allows thecomparison of different probiotics. Dose–reponse studies are, however, scarce, and it


is not known whether the percentage survival is stable for various ingested doses. Slightdifferences in the percentage survival of various products are therefore probably notclinically relevant.

Factors influencing the pharmacokinetics of probiotics

The survival of probiotics depends on their intrinsic resistance, on host factors and onthe vehicle in which they are ingested. Gastric acid constitutes a major defencemechanism. Pedrosa et al, for example, showed that the survival of Lactobacillus gasseriADH was high in subjects with hypochlorhydria36, the resistance to acid differing widelybetween probiotics.29 Bile salts are the second important factor.30 In addition,experimental data suggest that pancreatic juice can exert some antimicrobial activity.37

The influence of other digestive secretions, for example mucus and enteric secretionssuch as defensins, is as unknown yet. Motility also constitutes a defence mechanism, apathological slowing of peristalsis favouring chronic bacterial colonization of the smallintestine. The equilibrium between the flora and the fate of probiotics also depends onmicrobial interactions, including competition for substrates and adhesion sites, andmodifications of the environment (through bacterial byproducts or bacteriocins).Several studies have, for example, demonstrated that the endogenous flora influencesthe pharmacokinetics of the probiotic yeast Saccharomyces boulardii. Indeed, the faecalelimination of the yeast was faster than that of spores of B. stearothermophilus, and thesuppression of the endogenous flora by antibiotics enhanced the survival ofSaccharomyces boulardii in rat colon.38,39 A quicker faecal elimination than that of amarker has also been observed for Lactococcus lactis.40 Finally, the immune system isalso involved in the control of the flora and thus probably in the pharmacokinetics ofprobiotics, although we are unaware of any studies in this area.

Probiotics may be ingested in various conditions, for example while fasting, during ameal or protected in capsules or by microencapsulation.41 The fall in pH and the lengthof time that food remains in the stomach differ greatly with different meals. As aconsequence, the pharmacokinetics of probiotics can be influenced by the vector withor in which they are consumed. Saxelin et al have shown that the survival of Lactobacillusstrain GG differed when it was ingested in tablets, gelatin capsules, fermented milks or awhey drink.42,43

Pharmacokinetic data

Survival of ingested probiotics in the gastrointestinal tract and faecal colonization

Some probiotics are destroyed in the stomach whereas others have a high survival untilreaching the faeces (Figure 1). Yoghurt bacteria, i.e. Lb. delbruecklii subsp. bulgaricus andStreptococcus thermophilus, have a poor intrinsic resistance to acid.44 Pochart et alobserved that the concentration of viable yoghurt bacteria reaching the duodenumafter the ingestion of yoghurt containing 107 cfu/ml was at its peak around 105 cfu/ml.45

Using a dynamic in vitro model, we observed that 26% of ‘ingested’ Lb. bulgaricussurvived passage through the stomach.30 Pettersson et al reported that viable yoghurtbacteria reached the ileum in a quarter of their subjects, and Lindwall and Fondenreported that, after consumption of a yoghurt containing 109 Lb. bulgaricus organismspre gram, the concentration of lactobacilli in the ileostomy bag was 105–106 cfu/ml.46,47

Lactococcus lactis MG1363 and L. fermentum KLD also had a rather low survival in theileum. These bacteria are also very sensitive to bile, and because they release their


intracellular contents in the presence of bile, they have been suggested as vectors todeliver metabolic activity to the duodenum (Figure 2).37

The survival capacities of various strains of Lb. acidophilus, Lb. reuteri, Lb. rhamnosus,Lb. plantarum, Lb. salivarius, Lb. casei and Lb. johnsonii in acid conditions are higher thanthat of Lb. bulgaricus.28 Approximately 1–10% of Lb. acidophilus ingested in fermentedproducts were found to survive until the ileum in several human studies using intestinalintubation techniques.28,48 In one of our studies, the concentration of lactobacilli

Oral dose

Stomach

Duodenum

Ileum

Colon

Stools

A B C D M

Figure 1. Some ingested probiotics are rapidly destroyed in the stomach (A), whereas others survive betterbeyond the stomach but are destroyed by bile (B) or by the endogenous flora (C). Some probiotics have a highsurvival through the gastro intestinal tract (D), close to that of a marker (M).

To 1h 2h 3h 4h 5h 6h 7h 8h

Time

MarkerResistant strain

Sensitive strain

Ileal

flow

rat

e of

pro

biot

ics

orm

arke

r

Figure 2. Schematic pharmacokinetic profile of sensitive and resistant strains of probiotic in the small bowel.The kinetics of the inert maker are influenced by gastric emptying and intestinal transit. The kinetics of theresistant strain are similar to that of the marker. The kinetics of the sensitive strain are, however, close to thatof the marker during the initial period, but as the strain is rapidly killed by acid in the stomach and bile in theintestine, the peak is smaller and the duration of the passage of living bacteria in the ileum is shorter. Some ofthese ‘sensitive’ strains can be used to deliver intra-cellular enzymes to the small bowel.


flowing through the ileum after the ingestion of a cup of milk product containingLb. acidophilus Yoplait-A1 strain was 100 times higher than the concentration seen afterthe ingestion of a control meal.48 Lactobacillus plantarum NCIB 8826, Lb. salivarius433118 and some Bifidobacterium spp. demonstrated a very high survival capacity. Theirconcentration in the ileum reached 108 and 107 cfu/ml respectively after a single dose;they passed through the ileum at a concentration above 105 cfu/ml for more than5 hours. No small bowel colonization was observed.

Some Bifidobacterium spp. from fermented dairy products and Lb. plantarum NCIB8826 exhibited a high survival in the whole gastrointestinal tract; 25–30% of theingested bacteria being recovered from the faeces (Table 1).29,49,50 Faecal concen-trations reached 108 cfu/g, and these bacteria did not colonize the gut. Other studies inhealthy volunteers with different probiotic preparations showed that the faecalconcentrations of ingested Lb. acidophilus, Lb. reuteri, Lb. salivarius UCC118 and Lb.rhamnosus strain GG reached around 106 cfu/g.28,29,42,43 Human studies withSaccharomyces boulardii (which has proven efficacy in antibiotic-associated diarrhoeaand Clostridium difficile colitis) showed that 60% of the ingested yeast was recovered asdead cells in the faeces and that concentration of living yeasts was above 105 cfu/g in thecolon.51,52

Probiotics are usually excreted within a few days of their ingestion in faeces at thesame rate as or even more quickly than a transit marker.28,40,49,51,52 Some authors havehowever, observed the persistence of some probiotic strains such as Lactobacillus GG inthe faeces of a few subjects for longer periods than can be expected from a ‘normalpersistence’.53

Table 1. Examples of faecal recovery of living probiotics after their ingestion.

Probiotic

Faecal

recovery Reference

Bifidobacterium sp. 30% Gastroenterology 1992; 102: 875–878

Lb. plantarum NCIB 8826 25% Alimentary Pharmacology and Therapeutics 2000; 14: 823–828

Lb. acidophilus 2–5% Journal of Dairy Science 1978; 61: 1–10

Lb. rhamnosus GG 1–5% Digestive Diseases and Sciences 1992; 37: 121–128

In fermented milk 1% Microbial Ecology in Health and Disease 1993; 6: 119–122

In capsules 2% International Journal of Food Microbiology 1995; 25: 199–203

Lactococcus lactis TC165.5 0.1–2.0% Applied and Environmental Microbiology 1995; 61: 2771–2774

Saccharomyces boulardii 0.36% Biopharmaceutics and Drug Disposition 1989; 10: 353–364

Lb. reuteri 0.01% Microbial Ecology in Health and Disease 1995; 8: 41–50

B. longum SBT2928

(BL2928SR)

þ Journal of Applied Microbiology 2001; 90: 43–52

B. animalis DN-173 010 þ Journal of Applied Microbiology 2000; 88: 1019–1027

Lb. paracasei LTH 2579 þ Systemic Applied Microbiology 2000; 23: 260–266

Lb. casei Shirota þ International Journal of Food Microbiology 1999; 48: 51–57

Lb. Plantarum DSM 9843 þ International Journal of Food Microbiology 1998; 42: 29–38

Lb. helveticus rifampicin

-resistant CP53

þ Letters in Applied Microbiology 2001; 32: 108–113

Lb. salivarius UCC118 þ American Journal of Clinical Nutrition 2001; 73(supplement 2):

386S–392S


Adhesion to the intestinal epithelium or mucus and colonization of the mucosa

Many probiotic strains adhere to intestinal cell lines such as Caco-2 or HT29 and/or tothe intestinal mucus.54–59 This property differs between strains, and its predictabilityfor competitive exclusion properties and immunomodulatory activities in vivo shouldbe studied. Johansson et al were the first to demonstrate the possibility of colonizationof the intestinal mucosa by probiotics.60 They administered a soup containing 19different lactobacilli to human volunteers and searched on biopsies for colonization ofthe intestinal and rectal mucosa using bacterial probes. Two strains of Lb. plantarum(Lp299 and Lp299v) were found in biopsies taken 11 days after ingestion. The first wasof human origin, and the other had initially been isolated from a sour dough. Alanderet al gave Lb. rhamnosus GG (6 £ 1010 cfu per day) to human volunteers and searchedfor its presence in colonic biopsies that were taken 12 days after stopping the probiotic.The strain was not recovered from the faeces but was found in biopsies in two subjectsout of seven.61 The relevance of such results for predicting efficacy or the danger ofprobiotic strains is so far unknown.

Pharmacokinetics of active components vehiculated by probiotics

We assessed the pharmacokinetics in the small bowel of the lactase contained inyoghurt bacteria using intestinal perfusion.23 Eight lactase-deficient subjects ingested400 g yoghurt and on another day the same quantity of heated yoghurt in which thebacteria and their lactase content had been destroyed. Chyme was collectedcontinuously in the terminal ileum for 8 hours after these two meals, and the ilealflow rates of lactose and lactase were assessed. As shown in Figure 3, the ingestion ofyoghurt increased lactase activity in the intestine. About one-fifth of the lactase presentin the yoghurt reached the terminal ileum, and the quantity of lactose that remainedundigested after yoghurt ingestion was significantly lower than that reported afterheated yoghurt. Lactase activity was not increased when the samples were sonicated,which indicates that the yoghurt bacteria had delivered the lactase outside their cellmembrane. Yoghurt bacteria are easily lysed by bile. Interestingly, other probioticbacteria with a high lactase content but with higher intrinsic resistance to bile provedless efficient in helping lactose digestion in lactase-deficient subjects. This led to

Yogurt

Heated Yogurt

Lactase from yogurt

0 2 4 6 8Time (h)

Ileal

lact

ase

flow

rat

e

Figure 3. The Ileal flow rate of lactase after the ingestion of yoghurt or heated yoghurt in eight lactase-deficient subjects. Based on Marteau et al.23


the concept that bile-sensitive probiotics could be used for the delivery of intracellularenzymes or other active components to the gastrointestinal tract.26,37

Safety

The safety of the current products is excellent, but probiotics may, as living micro-organisms, theoretically be responsible for four types of side-effect in susceptibleindividuals: infections, deleterious metabolic activities, excessive immune stimulationand gene transfer.

Theoretical and observed adverse events

Infections

Probiotics are not selected among pathogens, and the theoretical risk of infection isthus very low. The risk of their passage in blood, eventually by translocation, is,however, important to determine. Bacterial translocation is defined as the passage ofmicro-organisms from the gastrointestinal to extra-intestinal sites such as themesenteric lymph nodes, liver, spleen and bloodstream. Indigenous bacteria arecontinuously translocating in low numbers but are rapidly killed in the lymphoid organs.Bacterial translocation is a major cause of severe infection in immunosuppressed,trauma and post-surgical patients. This may result from three mechanisms: intestinalbacterial overgrowth, increased permeability or damage of the intestinal mucosalbarrier, and immunodeficiency.

Rare cases of infection, including septicaemia and endocarditis caused by lactobacilli,bifidobacteria or other lactic acid bacteria, have been reported.62 Enterococcus faeciumand E. faecalis are more frequently involved in clinical infection, and there is concernover the emergence of vancomycin-resistant strains. In most cases of infection, theorganism appeared to have come from the patient’s own microflora, but, in a few cases,the recent consumption of probiotics was proposed as a potential cause. About 30cases of fungaemia have been reported in patients treated with Saccharomycesboulardii63,64, and two cases of infection have been traced back to food-borne Lb.rhamnosus.65,66 Nearly all subjects who had fungaemia involving Saccharomyces boulardiihad an indwelling vascular catheter.63,64 Contamination of the air, environmentalsurfaces and the hands of the nurses after opening the probiotic packets was found tobe the source of catheter contamination. It is therefore recommended, for hospitalizedpatients, that packets or capsules of probiotics should be opened with gloves andoutside the patients’ room.63 Another potential mechanism is translocation of theyeast67, but this has not been observed in patients with intestinal ulceration in clinicaltrials.68,69 The risk for immunosuppressed patients is unclear; indeed, Saccharomycesboulardii has been shown to protect immunodeficient mice significantly againstpathogens, but yeast infection has also been reported in immunosuppressedsubjects.70–72

Infection caused by Lb. rhamnosus similar to the probiotic GG strain was observed ina 74-year-old woman with non-insulin-dependent diabetes.65 She suffered from a liverabscess, which proved to contain Lb. rhamnosus, and pleuropulmonary infection. Nocause for this infection was found, but the woman reported a regular consumption ofdairy drinks containing Lb. rhamnosus GG. The clinical strain appeared to beindistinguishable from the GG strain. The other case of infection occured in a 67-year-old man with a mild mitral valve regurgitation who habitually chewed a probioticmixture. He had carious teeth to be removed and suffered after a few days from


an endocarditis. Lactobacillus rhamnosus was isolated from his blood, further analysisshowing that it was indistinguishable from one of the organisms present in the probioticpreparation.66

Saxelin and colleagues73,74 studied the prevalence of bacteriaemia caused byLactobacillus species in Southern Finland and compared the characteristics of the bloodculture isolates and the probiotic dairy strains. In their first study, lactobacilli wereidentified in eight of 3317 blood culture isolates, none of the isolates corresponding to adairy strain. In the second study, 5912 blood cultures were analysed, none of the 12lactobacilli isolated being identical to any of the commercial Lactobacillus strains. Tosummarise, there is no evidence that ingested probiotic lactobacilli or bifidobacteriapose any greater risk of infection than do commensal strains, but there is insufficientknowledge on the risks or benefits of probiotics in immunodeficiency. Other riskfactors for opportunist infection, such as extremes of age, pregnancy and digestivelesions, have not been identified as risk factors for probiotic infections.

Metabolic effects

If one accepts that probiotics can as a vehicle for or promote metabolic activities in thegastrointestinal tract that may have a positive effect on health, one also has to acceptthat they may induce metabolic activities detrimental to the host. During bacterialcolonization of the small bowel, the micro-organisms present in high number in thesmall bowel can induce diarrhoea and intestinal lesions, especially via the deconjugationand dehydroxylation of bile salts.75 One study has drawn attention to the potential riskof excessive deconjugation or dehydroxylation of bile salts in the small bowel byprobiotics. Indeed, it showed in patients with ileostomy that ingested Lb. acidophilus andBifidobacterium sp. could transform conjugated primary bile salts into free secondarybile salts. Excessive degradation of the intestinal mucus layer by probiotics maytheoretically be detrimental. Some endogenous bacteria, including lactobacilli and somestrains of bifidobacteria, have the ability to degrade mucus. Ruseler-van Embden et alstudied the mucus-degrading properties of three probiotic strains (Lb. acidophilus,Bifidobacterium sp. and Lb. rhamnosus GG) contained in fermented milks. No mucusdegradation was observed in vitro or in gnotobiotic rats associated with one of thesestrains.76

Immunological adverse events

When administered parenterally, bacterial cell wall components such as peptide-glycan-polysaccharides from different Gram-positive bacteria including lactobacilli can induceside-effects such as fever, arthritis and auto-immune disease.77 These side-effects aremediated by cytokines, and it is now well established that cytokine secretion is elicitedby some probiotics. The oral administration of high doses of lactic acid bacteria did notinduce immunological side-effects in mice.78 However, a systemic uptake of cell wallpolymers from the intestinal lumen—hence the immunological side-effects—has beenobserved in rats with colonic injury.79 To our knowledge, no immunological side-effectof a probiotic has been reported in man, except one case of auto-immune hepatitis thatmight have been enhanced by the ingestion of very large doses of yoghurt.80 Thepotential enhancement of auto-immune diseases by probiotic consumption should bestudied.


Gene transfer of antibiotic resistance

Some antibiotic resistance genes can be transferred from ingested lactobacilli to theendogenous flora in the mouse gastrointestinal tract.81 The probability of gene transferdepends on the nature of the genetic material to be transferred (plasmids, transposons,etc.), on the nature of the donor and recipient strains, on their concentrations and onselection pressure (especially the presence of antibiotics). It is difficult to assess in vivoand in vitro, and it is even more difficult to state what probability level of gene transfer isacceptable. The antibiotic resistance of probiotics is not in itself a hazard unless it wouldrender the probiotic untreatable in cases of infection or if it could be transferred topotential pathogens and resistance could have therapeutic consequences. Infectionscaused by vancomycin-resistant organisms, including staphylococci and enterococci, is aserious clinical problem. The safety and long-term effects on antibiotic resistance ofE. faecium strains used as a probiotic therefore clearly need careful assessment. Thesituation is not the same for lactic acid bacteria, despite the fact that many of them arenaturally resistant to vancomycin. Indeed, this natural intrinsic resistance of lactobacilli,leuconostocs and pediococci is chromosomally encoded and not inducible ortransferable, and these micro-organisms are sensitive to many other antibiotics.

Assessment of safety

Many intrinsic properties, such as an excessive deconjugation of bile salts ordegradation of mucus, can be studied in vitro. Of the pharmacological characteristics,translocation and permanent colonization are probably those, which most urgentlyneed to be studied. A rather large body of data from short-term clinical trials on healthyvolunteers attests to the safety of probiotics. In a few studies, biological parameterswere analysed because it was thought that the probiotic might influence them. Severalauthors, for example, looked at intestinal permeability and showed that it was notsignificantly increased by various probiotics.4,82,83 In other cases, the probiotic safetywas studied following the same rules as used for chemicals and assessinghaematological, renal and hepatic parameters. As the risks are obviously very lowwith the available products, pre-marketing studies will not be sufficient to measurethem, and vigilance studies are needed. The studies by Saxelin et al described above73,74

provide an example of epidemiological surveillance. The recent consumption ofprobiotics should be clarified in every new case of severe infection caused byenterococci, lactobacilli, bifidobacteria or yeast, and the strains involved should becompared with the probiotic ones.

SUMMARY

Randomized trials have demonstrated the efficacy of some probiotics in specific clinicalsituations. Important progress is to be expected as the flora and local immune systemplay an important role in many intestinal diseases and can be influenced by probiotics.The development of probiotic use in clinical practice will depend on the availability ofpreparations and on studies testing them in the real clinical conditions in terms ofproducts, formulations and doses. Our knowledge of pharmacokinetics is increasing asa result of improvements in in vitro models, sampling techniques in the gut and tools forthe reliable identification of probiotics within complex ecosystems. Strains differ intheir survival capacity at different levels of the gastrointestinal tract and in their


adhesion to epithelial cells. Studies are needed to investigate the adhesion of probioticsto the epithelium in vivo, potential colonization and the influence of the vehicle in whichthey are ingested. Dose–response studies should also be carried out. All these efforts,which are also needed for safety reasons, should help to confirm or refute currentpotential hypotheses such as ‘probiotics should be of human origin, should have a highsurvival capacity, should adhere to the intestinal epithelium’. Last but not least, researchshould provide an answer to the questions of how much probiotic should be consumed,how often and for how long, and what concentrations should be present in thecommercial preparations. Although the current products seem very safe, the questionof safety should not be overlooked but assessed in the pre-marketing and post-marketing phases of development (pharmacovigilance).

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Practice points

† double-blind randomized controlled trials have shown that a number ofprobiotics are effective in several well-defined clinical situations

† it is unlikely that there is a single mechanism of action all underlying probioticsand their effects

† probiotics differ greatly in their characteristics† the survival of probiotics in the gastrointestinal tract varies greatly between

strains† some probiotic strains have a very high survival capacity in the gastrointestinal

tract† the safety of currently used probiotic strains is excellent

Research agenda

† there is a need to establish:– the active ingredients that are responsible for the clinical effects of

probiotics– the target sites of probiotics in the body– dose–response effects– whether probiotics translocate and whether this is a positive, neutral or

negative trait† the development of genetically engineered probiotics should include initial

testing in animal models and studies of their ecological consequences


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Basic Aspects and Pharmacology of Pro Bio Tics an Overview of Pharmacokinetics Mechanisms of Action...

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