The document summarizes safety assessment procedures for probiotic bacteria intended for human use. It defines probiotics and lists common strains used. Safety assessment includes taxonomic identification, assessing absence of pathogenicity and undesirable metabolic activities, determining antibiotic resistance and potential for horizontal gene transfer, and validation in animal models. Human studies are also important to establish tolerability and identify any adverse effects. Overall, the document outlines guidelines and criteria for evaluating probiotic safety prior to human use based on established scientific principles and evidence from the literature.

1. Safety assessment of Probiotic bacteria for
Human use
DIWAS PRADHAN
Dairy Microbiology Division
N.D.R.I.

2. DEFINITION
FAO/WHO, 2002
Live microbial feed
supplements that
have beneficial effects
on the host by
improving its intestinal
microbial balance.
• Able to survive the gastro-intestinal
passage.
• Able to colonize intestinal epithelia.
• Non pathogenic and non toxic.
• Capable of exerting a beneficial effect
on the host.
• Anti-inflammatory, antimutagenic,
immunostimulatory etc.
REQUIREMENTS
PROBIOTICS
Strains used: Lactobacilli, Bifidobacteria, Saccharomyces, Bacillus, Enterococci, E. coli etc.

3. LACTIC ACID BACTERIA
Food Fermenting Bacteria
Starter Culture
 History Of Safe Use
 Traditionally used in various fermented food and dairy products
 Components of healthy gut microbiota (Commensals) Normal
inhabitants (Intestinal tract, urogenital tract etc.)

4. QPS criteria on Lactobacilli
• Includes the group of microorganisms considered as safe for consumption
• Safety assessment of a defined taxonomic group (e.g. genus or group of related species) could be
made based on four pillars (establishing identity, body of knowledge, possible pathogenicity and
end use)
• Lactobacilli -
• A number of species recommended for QPS category

5. LAB Infection
Disease Organism References
Healthy infant B. breve Hata et al., 1988
AIDS L. casei Rogasi et al., 1998
Leukemia Lactobacillus sp. Cooper et al., 1998
Acupuncture B. longum Ha et al., 1999
Colonoscopy L. rhamnosus Avlami et al., 2001
Diabetes L. casei Chanet et al., 2007
Urolithiasis L. jensenii Chazan et al., 2008
Pancreatitis L. rhamnosus Brahimi et al., 2008
Healthy senior L. rhamnosus Wolz & Schaefer 2008
Urinary tract infection L. delbrueckii Darbro et al., 2009
Endocarditis L. jensenii Garcia et al., 2011
Bacteremia L. jensenii Chazan et al., 2012
Pyelonephritis L. delbrueckii DuPrey et al., 2012
Endocarditis L. acidophilus Nishijima et al., 2012
Cholecystitis L. fermentum Chery et al., 2013

6. Lactobacillus and Bacteremia
• Identification of 85 blood isolates of LAB: L. rhamnosus (n=46), L. casei (n=12), L.
fermentum (n=12), L. jensenii (n=3), L. gasseri (n=3), L. salivarius (n=3)
Salminen et al., 2006
• Lactobacillus delbrueckii was determined to be the causative agent of bacteremia in 68-
year-old woman with urinary tract infection
DuPrey et al., 2012
• Bacteremia caused by Lactobacillus rhamnosus GG in 17-year-old boy with ulcerative
colitis
Vahabnezhad et al., 2013

7. The Acute pancreatitis study
• Clinical study assessing efficacy of a probiotic mixture in the treatment of
acute pancreatitis → more patients in critical condition compared to placebo
• Higher mortality in probiotic group (n=24/153) compared to placebo group
(n=9/145)
• Higher bowel ischemia in probiotic group (9 against 0 cases)
• Currently unclear what caused the observed effects
Reid et al., 2008
• Safety of new strains cannot be established based on history of safe use
• New strains are being isolated from gastro-intestinal tract
• Rampant horizontal gene transfer; probiotics fed in very large amounts
Also……….

8. FAO/WHO
guideline on
probiotic
evaluation

9. Taxonomy and typing
16S rDNA sequence analysis
Absence of transferable antibiotic resistance genes
Use of LSM media, Plasmid Curing.
In vitro Safety Assessment
 Absence of undesirable metabolic activities and Virulence genes
In vivo safety assessment in animal models
Endocarditis model, immunocompromised model etc. Vankerckhoven et al., 2008

10. Probiotics should not pose any risk to host
• Should not be pathogenic
• Should not possess virulence properties
 Invasion and translocation in epithelial cells
 Haemolytic and Platelet aggregation activity
 Mucin degradation
 Production of enterotoxins or cytotoxins
• Metabolic activities
 D-lactate, biogenic amines and other harmful metabolic enzymes
• Antibiotic resistance genes and its transferability
• Validation in various animal models
• Assessment of side effects in humans

11. Taxonomy and typing
• Correct identification - essential for safety assessment
– Species related scientific and technological information
• Misleading label : Bacillus coagulans vs. Lactobacillus
sporogenes
• Molecular methods as basis for identification
– 16S rRNA gene sequence analysis, PFGE and RAPD
(Kumar et al., 2011; Moyano et al., 2012; Toshimitsu et al., 2013; Tulini
et al., 2013)

12. Genetic basis of resistance • Lactobacilli - high incidences of
intrinsic resistance to Vancomycin
(Tynkkynen et al., 1999; Lavanya et al., 2011;
Hojsak et al., 2013)
• Transmissible antibiotic resistances
(Tetracycline and macrolides) in
LAB
(Flórez et al., 2008; Saarela et al., 2008)
Antibiotic resistance in LAB

13. Transferability of Antibiotic resistance
 In vitro transfer from lactobacilli to other commensal
bacteria.
(Schlundt et al., 1994; Gevers et al., 2003; Jacobsen et al., 2007; Feld et al., 2008)
 Transfer in the GIT of rodents with indigenous gut
microbiota
(Gruzza et al., 1994; Igimi et al., 1996; Jacobsen et al., 2007; Feld et al., 2008)

14. Antibiotic resistance and its transferability
1. Antibiotic susceptibility testing
2. Plasmid profiling and Curing
3. Transferability of Antibiotic Resistance

15. Antibiotic resistance pattern by disc
diffusion assay
Antibiotic susceptibility testing
Minimum inhibitory concentration by
E-test strip
Presence of any antibiotic resistance
genes will be confirmed by PCR
Klare et al., 2005
Genes Sequences Amplicon size
tet(M)
5’-AYACNCCNGGNCAYRTNGAYTT-3’
5’-CACCGAGCAGGGATTTCTCCAC-3’
1513
erm(B)
5’-CATTTAACGACGAAACTGGC-3’
5’-GGAACATCTGTGGTATGGCG-3’
405

16. Plasmid profiling and Curing
Plasmid Isolation
- Sullivan and Klaenhammer (1993)
Plasmid Curing
Observe for change in Antibiotic
Resistance

17. Gevers et al., 2003
Donor added to Recipient and filter
through a sterile filter (0.45µm)
Growing of Donor and Recipient
(separately)
Transferability Of Antibiotic Resistance
Filter Mating Experiment
Incubation of Filter in non selective
media
Plating of Mating mixtures in
Selective media
PPS washing to trap cells
PPS washing of cells
Recipients - Enterococci and
Staphylococci

18. Bover-Cid et al., 1993
Inoculation @1%
Decarboxylate media (containing
amino acid @ 1g/ml)
Observe for colour change
Quantification of Amines by Gas Liquid
Chromatography
Biogenic Amine formation
Inducer- Pyridoxal-5-phosphate

19. Zhou et al., 2001
1. Mucin Purification
Mucin Degradation Assay
2. Preparation of positive control
3. Mucin degrading assay in
Broth medium
4. SDS–PAGE analysis of
mucin glycoprotein
5. Mucin degradation assay in a
petri dish

20. Korpela et al., 1997
Collection of Blood from
Healthy individuals
Platelet aggregation and Haemolytic activity test
Preparation of platelet rich
plasma (PRP)
Measurement of platelet
aggregation
Inducer - Adrenaline
Inoculation of bacteria in
PRP
Platelet Aggregation test
Streaking with Active
Culture
Blood Agar plates (5-10%
blood)
Incubation and observe after
24 hrs
Haemolytic activity test

21. In vivo study
Safety evaluation in vivo in different mouse model
1. Oral toxicity and tolerance of high doses
• Acute toxicity
• Subacute toxicity
• Subchronic oral toxicity
2. Bacterial translocation
• Healthy animals
• Colitis mouse model
• Immunocompromised

22. Parameters
 General health and well-being
 Feed intake and growth
 Gut mucosa and internal organ histology
 Haematology
 Major blood biochemistry (total plasma protein,
albumin, plasma glucose etc.)
 Bacterial translocation (Liver, spleen, MLN etc.)

23. Human safety studies
• Lactobacillus casei Shirota preparation was well tolerated and no apparent side
effects(bacteremia) was observed in Critically ill Children
Srinivasan et al., 2006
• Dietary supplementation with Lactobacilli and Bifidobacteria well tolerated and not
associated with adverse events during late pregnancy and early infancy.
Allen et al., 2009
• Probiotic bacterial strains L. rhamnosus HN001 and B. animalis subsp. lactis HN019 was
found safe and well tolerated in infants aged 0–2 years.
Dekker et al., 2009
• Potential probiotic strain L. rhamnosus RSF-L477 well tolerated by healthy volunteers
Wind et al., 2010
• Daily treatment of healthy adults with Lactobacillus reuteri DSM 17938 for 2 months was
found safe and well-tolerated
Mangalat et al., 2012

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