PROBIOTICS: THE NEW

BACTERIOTHERAPY
 INTRODUCTION
 Antibiotics, which mean to destroy life,

 Probiotics literally means life giving.

WHO (2001) defn of Probiotics:

Probiotics are: ‘Live microorganisms which when administered in
adequate amounts confer a health benefit on the host.’

 Lactic acid bacteria (LAB) are the most common type of microbes used.
 convert sugars (including lactose) and other carbohydrates into lactic acid
 lowering the pH ↓ no. of microorganisms thus prevents GIT infections.
[Nichols, Andrew W. (2007).]

 Strains of the genera Lactobacillus and Bifidobacterium, are the most

widely used probiotic bacteria.
[ Tannock G (2005).]

 Probiotic bacterial cultures are intended to assist the body's naturally

occurring gut flora, an ecology of microbe.

 Claims are made that probiotics strengthen the immune system to combat
allergies, excessive alcohol intake, stress, exposure to toxic substances,
and other diseases.
[ Nichols, Andrew W. (2007).]
 HISTORY
 Probiotics have been used for centuries as natural components in health-
promoting foods.
 Metchnikoff, E. 1907 – 1st scientist to observe the positive role played by
certain bacteria.
Possibility of modify the gut flora and to replace harmful microbes
by useful microbes.
 Henry Tissier, also from the Pasteur Institute, was the first to isolate a Bifid
bacterium.
Who named it Bacillus bifidus communis.[ Tissier, H. 1900].

 Nissle, in 1917 isolated a strain of Escherichia coli – used in Rx of acute

intestinal diseases.
Escherichia coli is still in use and is one of the few examples of a
non-LAB probiotic.
 In the 1960s the dairy industry began to promote fermented milk products
containing Lactobacillus acidophilus.

 Later other strains are developed such as Lactobacillus rhamnosus,

Lactobacillus casei, and Lactobacillus johnsonii, because they are
intestinal species with beneficial properties.[ Tannock, G.W. 2003]

 The past few years probiotics have also been investigated in the oral
health perspective.
 Dental caries and periodontal diseases occur in nearly 95% of the general
public.
Although fluoride and other preventive efforts have led to a
dramatic decline in dental caries, the ability to control the actual infection
has been limited .
(Rolla and Ogaard, 1991; Reich, 2001; Kargul et al, 2003).

 The concept of microbial ecological change as a mechanism for preventing

dental change is an important one.

 The oral cavity is a complex ecosystem in which a rich and diverse micro
biota has evolved.
 Oral infectious disease may be a consequence of changes in the microbial
ecology.

 If the local environment is disturbed, then potential pathogens may gain a

competitive advantage and, under appropriate conditions, reach numbers
that predispose a site to disease.

 Regarding elimination of pathogenic members of the oral cavity a new

method such as probiotic approach (i.e. whole bacteria replacement
therapy) can be used.
The ideal probiotic microorganism should have the following characteristics:

 1. Non pathogenicity to humans.

 2. High tolerance to bile and gastric acidity.
 3. Production of L-(+) lactic acid only during fermentation (since the D-(-)
optical isomer of lactic acid has been associated with metabolic acidosis).
 4. Capability for easy proliferation in vivo.
 5. Capability for easy proliferation in vitro.
 6. High survival rate through processing conditions (during harvesting,
drying etc.)
 7. High stability at room temperature separately or when mixed with other
ingredients.
 8. Lack of potential to develop virulence.
 PROBIOTICS AND GENERAL HEALTH
 Research conducted by various individuals during the last century has
shown that lactic acid producing bacteria have many beneficial effects to
promote human health.

 In the course of their proliferation and survival in the gastrointestinal tract,

these probiotics produce metabolites such as lactic acid and antibiotic-like
substances called bacteriocins that suppress the growth of putrefactive
microorganisms.
A number of potential benefits arising from the use of probiotics has been
proposed, including

 Rotavirus diarrhea (Gorbach SL. 2002)

 Reduction of antibiotic-associated side effects (Gorbach SL. 2002)
 Managing Lactose Intolerance (Sanders ME 2000)
 Cholesterol Lowering (Sanders ME 2000)
 Improving Mineral Absorption (Famularo G 2005)
 irritable bowel syndrome (Saggioro A, 2004; Fan, YJ et al, 2006),
 allergic conditions (Saavedra, M.,2007, Abrahamsson TR, 2007),
 skin health maintenance (Thestrup-Pedersen K. 2003),
 dental health maintenance (Meurman JH, et al., 2007),
 in supporting healthy blood pressure levels (Aihara, K. et al.;2005),
 immune modulatory functions (Liong, MT, 2007; Trois, L et al.; 2007) and
 and in supporting cardiovascular health and wellness (Agerholm-LL et al.,
2000; Naruszwicz, M et al.; 2002).
 Recent research also reveals potential benefits in obesity management
(Ali, AA et al; 2005).
 liver functions (Bongaerts, G et al.; 2005),
 in the management of vaginal infections (Uehara, S et al; 2006),
 pain relief support (Gawronska, A et al.,2007),
 as anti-inflammatory agents (Tok, D et al, 2007),
 IMMUNOMODULATORY EFFECTS

 Probiotics may promote nonspecific stimulation of the host immune

system, such as -
Immune Cell Proliferation,
Enhanced Phagocytic Activity And
Increased production of secretory immunoglobulin A
(lgA)

 The presence of bacteria in the mucosa and its epithelial cell adherence
produce a variety of chemo­attractants and cytokines that can pass on
signals to mucosal immune cells.

 On the humoral side, Lactobacillus GG significantly increases in IgG, IgA,

and IgM secretion from circulating lymphocytes.
 some probiotics help to control the inflammatory response and indirectly
inhibit metalloproteinase activity. (McGeehan et al. )
Probiotics are provided in products in one of four basic ways:

– As a culture concentrate added to a beverage or food (such as fruit juice),

– inoculated into prebiotic fibers,

– inoculated into a milk-based food (dairy products such as milk, milk drink,
yoghurt, yoghurt drink, cheese, kefir, biodrink) and

– As concentrated and dried cells packaged as dietary supplements (non-dairy

products such as powder, capsule, gelatin tablets).
PREBIOTICS:
 Some dietary substances, the so-called ‘prebiotics’ can favor the growth of
these beneficial bacteria over that of harmful ones.

 Prebiotics are non-digestible food ingredients. Thus these include inulin,

fructo-oligosaccharides (FOS), galactooligosaccharide and lactulose
(Gibson et al, 1995; Guigoz et al, 2002).

 Generally prebiotic ingestion is characterized by changes in microbial

population density
(Bertelsen, 2001).
PROBIOTIC STRAINS IN THE ORAL CAVITY
 An essential requirement for a microorganism to be an oral probiotic is its
ability to adhere to and colonize surfaces in the oral cavity.

 Total species diversity in the oral cavity ranges between 500 and 700
species.
( Kazor et al 2003)

 Lactobacilli make approximately 1% of the cultivable oral microflora

(Marsh and Martin, 1999).
 The most common lactobacilli species recovered from saliva in a study by
Teanpaisan and Dahlen (2006) were L. fermentum, L. rhamnosus, L.
salivarius, L. casei, L. acidophilus and L. plantarum.

 Three of them are probiotic strains used in dairy products.

 Koll-Klais et al (2005) found no differences in salivary lactobacilli counts

between chronic periodontitis and healthy patients.

 Higher prevalence of homofermentative lactobacilli in healthy mouths

compared to samples from patients with chronic periodontitis
(Koll-Klais et al, 2006).
 These findings indicate that lactobacilli as members of resident oral
microflora could play an important role in the microecological balance in
the oral cavity.

 One mechanism of action of probiotics is suggested to be their modulation

of host immune response.

 Immune inductive sites in the oral cavity are within the diffuse lymphoid
aggregates of the Waldeyer’s ring.
The role of these anatomic structures as inductive sites of
mucosal immunity has been shown by intranasally delivered vaccines.
(Wu et al, 1997).
 Dendritic cells scattered in mucosal surfaces - antigen presentation and
activation of T-cell responses.
Depending on the signals from dendritic cells either immune
tolerance or active immune response toward a specific antigen may occur
(Banchereau and Steinman, 1998).
A marked production of interleukin-10 by dendritic cells in gut
mucosa has been registered after administration of a probiotic mixture
(Hart et al, 2004).

 However, more studies on activation of the oral immune inductive sites

after probiotic administration are needed before further conclusions can
be drawn.
PROBIOTIC ACTIVITY IN THE ORAL CAVITY
Attachment, adhesion, and oral colonization of probiotics:

 For the long-term probiotic effect of the microorganisms ,the mechanism

of adhesion to oral surfaces is an issue of importance.

 Yli-Knuuttila et al (2006) assessed colonization of L. rhamnosus GG (LGG)

in the oral cavity of healthy students.
After the 14-day trial period, the occurrence of LGG in the oral
cavity decreased gradually, indicating that no permanent colonization had
occurred and that the oral persistence of LGG was only temporary.
 Fusobacterium nucleatum plays an important role as a bridge-organism
that facilitates the colonization of other bacteria by co-aggregation
(Kolenbrander, 2000).

 Kang et al (2005) reported that W. cibaria efficiently co-aggregated with F.

nucleatum.

 Heat-resistant components firmly attached to the cell surface of W. cibaria

were responsible for the co-aggregation with F. nucleatum.
 Many authors have reported that the co-aggregation abilities of
lactobacilli species might enable them to form a barrier that prevents
colonization of pathogenic bacteria (Reid et al, 1988; Boris et al, 1997),
due to the production of a microenvironment around these pathogens in
which inhibiting substances were generated by Lactobacillus species.
 The S-layer proteins of the bacterial cell wall may play an important role in
the adherence of W. cibaria to the epithelial cells .
(Kang et al 2005).

 All test strains demonstrated 24-h survival rates in saliva but with great
variations among the strains in their binding capacity to the saliva-coated
surfaces.
(Haukioja et al 2006 )

 Lactobacilli showed better adherence than bifidobacteria. Thus,

lactobacilli may compete for the same binding sites on saliva coated
hydroxylapatite with F. nucleatum which explains their lower colonization
capacity.
 This phenomenon indicates that probiotics might affect the formation of
oral biofilms and modify resident microflora.

 Haukioja et al (2006a,b) defined a novel mechanism whereby lactobacilli

and B. lactis Bb12 affected the composition of salivary pellicle on
hydroxyapatite and thereby inhibited S. mutans adherence in vitro.
 STUDIES ON PROBIOTICS AND DENTAL
CARIES
 The impact of oral administration of probiotics on dental caries has been
studied in several experiments utilizing different test strains.

 Lactobacillus rhamnosus GG (Meurman et al, 1994; Na¨ se et al, 2001;

Ahola et al, 2002) and L. casei (Busscher et al, 1999) have proved their
potential to hamper growth of these oral streptococci.

 A statistically significant reduction in salivary mutans streptococci was

observed when Bifidobacterium DN-173 010 strain tested.

 definite S. mutans count reduction after a 2-week consumption of yoghurt

containing L. reuteri. (Caglar et al 2006)
PROBIOTICS AND PERIODONTAL DISEASE
 Chronic periodontitis, could also benefit from orally administered
probiotics.

 The presence of periodontal pathogens could be regulated by means of

antagonistic interactions.

 The effect of probiotics to the normalization of microflora was found to be

higher in comparison with Tantum Verde, particularly in the cases of
gingivitis and periodontitis
(Grudianov et al, 2002).
 A decrease in gum bleeding and reduced gingivitis has been observed by
Krasse et al (2006) with the application of L. reuteri.

 Koll-Klais et al (2006) reported that resident lactobacilli flora inhibits the

growth of Porphyromonas gingivalis and Prevotella intermedia in 82% and
65%, respectively.

 Probiotic strains (L. casei) included in periodontal dressings at optimal

concentration of 108 CFU/ ml were shown to diminish the number of most
frequently isolated periodontal pathogens: Bacteroides sp., Actinomyces
sp. And S. intermedius, and also C. albicans
(Volozhin et al, 2004).
 The routine intake of lactic acid foods may have a beneficial effect on
periodontal disease.
(Yoshihiro Shimazaki 2008).

 Guiding Periodontal Pocket Recolonization (GPR):

Streptococcus crista strains inhibited Porphyromonas gingivalis epithelial

colonization (W. Teughels 2004)

Analysis of the data showed, in a beagle dog model, that when

probiotics were applied in periodontal pockets adjunctively after root
planing, subgingival recolonization of periodontopathogens was delayed
and reduced, as was the degree of inflammation, at a clinically significant
level .
(W. Teughels 2007.)
 PROBIOTICS AND IMBALANCED ORAL
ECOSYSTEM
 Halitosis, the oral malodor, is a condition normally ascribed to disturbed
commensal microflora equilibrium.

 Shown a definite inhibitory effect on the production of volatile sulfur

compounds (VSC) by F. nucleatum after ingestion of Weissella cibaria
both in vitro and in vivo
(Kang et al 2006) .

 In children, a marked reduction in the levels of H2S and CH3SH by

approximately 48.2% (P < 0.01) and 59.4% (P < 0.05), respectively, was
registered after gargling with W. cibaria containing rinse.
 The possible mechanism in the VSC reduction is the hydrogen peroxide
generated by W. cibaria that inhibits the proliferation of F. nucleatum.

 Streptococcus salivarius, also a possible candidate for an oral probiotic,

has demonstrated inhibitory effect on VSC by competing for colonization
sites with species causing an increase in levels of VSC.
(Burton et al, 2005, 2006a,b).

 Burton et al (2006a,b) further reported that S. salivarius strain K12

produced two antibiotic bacteriocins, compounds that are inhibitory to
strains of several species of gram-positive bacteria implicated in halitosis.
PROBIOTICS AND FUNGAL INFECTIONS
 Candida albicans is among the most common infectious agents in the oral
cavity.
 The incidence of yeast infections is higher at older age and under
conditions of impaired immunity.

 Testing the pattern of colonization of L. acidophilus and L. fermentum,

Elahi et al (2005) showed a rapid decline in C. albicans in mice after the
intake of probiotic strains.

 Continuous consumption of probiotics led to almost undetectable

numbers of fungi in the oral cavity, maintaining the protective effect for a
prolonged period after cessation of application.
 The capacity of different lactobacilli to stimulate cellular and humoral factors
of mucosal protection varies particularly in terms of salivary nitrous oxide
and γ-interferon levels.

 Elahi et al (2005) have observed a correlation between the highest peak of

interleukin-4 secretion and complete eradication of C. albicans.

 A reduction in the prevalence of C. albicans in the elderly after consumption

of probiotic cheese containing L. rhamnosus GG and Propionibacterium
freudenreichii ssp. has been reported by Hatakka et al (2007)

 A concomitant feature of the probiotic activity observed in this study was

the diminished risk of hyposalivation and the feeling of dry mouth of the
subjects.
ADMINISTRATION OF PROBIOTICS
 Dairy products supplemented with probiotics are a natural means of oral
administration and easily adopted in dietary regime.

 However, for the purposes of prevention or treatment of oral diseases,

specifically targeted applications, formulas, devices, or carriers with slow
release of probiotics might be needed.

 Montalto et al (2004) administered probiotic mix both in capsules and in

liquid form without observing statistically significant difference, in the S.
mutans counts between the two test groups.
 A specially designed straw with a reservoir containing probiotics has also
been presented by Caglar et al (2006) who compared the effect of two
non-dairy delivery methods, a Life top straw (BioGaia AB, Stockholm,
Sweden) and a lozenge on the effectiveness of L. reuteri to reduce the
number of S. mutans .
Both means of administration showed significant reduction in
salivary S. mutans levels in half of the patients when compared with
subjects who received placebo.
 A recent invention for caries prophylaxis is a chewing gum containing L.
reuteri Prodentis. Consumed twice daily to regulate S. mutans counts in
the oral cavity
 Reductions in Streptococcus mutans, P. gingivalis and Campylobacter
rectus, were observed with subjects after only one to two weeks of
probiotic mouth rinse use containing S. uberis and S. oralis.
(Hillman
2006)
 Lozenges with L. reuteri inhibited S. mutans and P. gingivalis significantly….
(Approved by BDHF)
 SAFETY ASPECTS
From the safety point of view, the putative probiotic microorganisms
 should not be pathogenic,
 should not have any growth-stimulating effects on bacteria causing
diarrhea, and
 should not have an ability to transfer antibiotic resistance genes.
 The probiotics should rather be able to maintain genetic stability in oral
microflora
(Grajek et al, 2005).
 The increased probiotic consumption inevitably leads to increased
concentrations of these species in the host organism. Lactobacillus
bacteremia is a rare entity, and data on its clinical significance are mainly
found through case reports.

 For the last 30 years there have been approximately 180 reported cases
(Boriello et al, 2003).
Clinical characteristics of Lactobacillus bacteremia are highly
variable, ranging from asymptomatic to septic shock-like symptoms.
 Any viable microorganism is capable of causing bacteremia, however,
especially in patients with severe underlying diseases or in
immunocompromised state.

 the present literature supports the conclusion that the incidence of

Lactobacillus bacteremia is unsubstantial and that all the cases where it
has been registered are individuals with other systemic diseases
(Husni et al, 1997; Cannon et al, 2005).

 In a controlled study exposing 35 HIV-positive patients to L. reuteri, no

clinically significant side effects were noted.
(Wolf et al, 1998).
 Salminen (2006) has reported no adverse effects caused by LGG ingestion,
or LGG treatment in general, on HIVpositive patients.

 The absence of acquired antibiotic resistances is another safety criterion

to be tested in potential probiotic candidates.

 Several results from antibiotic susceptibility tests claim that the tet(W)
and tet(S) genes in some probiotic lactobacilli and bifidobacteria strains
are responsible for gentamycin, sulfamethoxazole, polymyxin B, and
tetracycline resistance
(Huys et al, 2006; Masco et al, 2006).
 CONCLUSIONS AND RECOMMENDATIONS
FOR FUTURE RESEARCH

 Similar to their better known actions in the gastrointestinal tract,

probiotics exert their effects in many ways also in the oral cavity.

 However, data on ‘oral probiotics’ are yet insufficient, and it is not known
whether the putative probiotic strains could modulate the mucosal
immune response in oral cavity.

 Studies of the probiotic effect on the balance of the oral ecosystem would
also be needed.
 There are no data as to whether probiotics exert any effect on oral
manifestations of autoimmune diseases.
In this regard it might be interesting to conduct studies on
patients with lichen planus, pemphigus vulgaris, cicatricial pemphigoid.

 Finally, possibilities to genetically modify or engineer potential probiotic

strains, and new vehicles may offer totally new visions and need to be
studied.