The document summarizes Giuseppe Mazzola's Ph.D thesis on the role of bifidobacteria in newborn health and intestinal microbial balance. The thesis involved developing a probiotic/synbiotic product containing Bifidobacterium breve for infants, and studying the impact of intrapartum antibiotic prophylaxis on newborn gut colonization. Key findings included that B. breve B632 showed potential as a probiotic, and that intrapartum antibiotics reduced bifidobacteria and increased Enterobacteriaceae in infant guts.

1. Alma Mater Studiorum - University of Bologna
Ph.D Course in Agricultural, Environmental and Food Science and Technology
Curriculum: Microbial Ecology and Plant Pathology - Cycle XXVII
The role of bifidobacteria in newborn health and the intestinal
microbial balance
Presented by: Giuseppe Mazzola
Advisor: Prof. Diana Di Gioia
Co-Advisor: Dr. Irene Aloisio 8th
May 2015

2. Gut microbial colonization in the early stage of life
 Birth
• Sterility?
 First days
• Enterobacteria
• Enterococci
• Bifidobacteria
• Bacteroides
• Clostridia
 Pre-weaning
• Bifidobacteria
• Enterobacteria
• Enterococci
• Bacteroides
• Clostridia
 Post-weaning to adult
• Bifidobacteria
• Enterobacteria
• Enterococci
• Bacteroides
• Clostridia
• Anaerobic cocci
Increasing biodiversity

3. Which factors determine the microbial composition of the human gut?

4. Goals of the work and research activities
A. Development of a probiotic /synbiotic product for infants and its potential applications
- Survival under gastro-intestinal conditions
- Stimulatory activity of commercial fibers
- Anti-colics activity of a Bifidobacterium strain
- Anti-Streptococcus activity of Bifidobacterium strains
- Impact of intrapartum antibiotic prophylaxis (IAP) on the newborn gut colonization
B. Achievements of new insights on early gut microbial colonization

5. A. Development of a synbiotic product and its potential applications:
in vitro studies

6. A. Development of a synbiotic product and its potential applications:
in vitro studies
Antimicrobial activity against:
Enterobacteriaceae
Streptococcus agalactiae
Survival under
gastro-intestinal
conditions
Stimulatory activity
of commercial fibers
B. breve B632
B. breve B2274
B. breve B7840
B. longum B1975

7. Survival under gastro-intestinal conditions
- Plate counts after 0, 30 and 60 or 120 min of contact with the solutions
Gastric tract
• pH 2.5
• pH 4 (newborn)
Duodenal tract
Human gastric juice
Porcine bile salts
• 1g L-1
- pH 7.0

8. Survival under gastro-intestinal conditions: plate counts
Rapid decline
≥ 4 Log CFU ml-1
No or negligible
survival
Complete
survival
≤ 1 Log CFU ml-1
reduction
Good survival
over time
≈ 1 Log CFU ml-1
reduction
Complete
survival
Complete
survival
Good survival
over time
≤ 1.5 Log CFU ml-1
reduction
Good survival
over time

9. Stimulatory activity of commercial fibers
Escherichia coli ATCC 25645
Klebsiella pneumoniae GC23a
Enterobacter cloacae GC6a
Commercial name
(10 g l-1
)
DP Carbohydrate
Vivinal®
n.a. GOS
CUP-Oligo®
3 to 6 GOS
Actilight®
950P 2 to 5 FOS
Orafti®
HSI < 10 Inulin
Orafti®
Synergy1 n.a. Inulin
Orafti®
RaftilineHP > 23 Inulin
Frutafit®
9 to 12 Inulin
BioEcolians®
> 5 α-glucooligosaccharide
Arabinex®
n.a. Arabinogalactan
Benefibra®
n.a. PHGG
Potential pathogenic strains
Clostridium difficile M216
(A620 of Bifidobacterium on the fiber
after 24 h / A620 of Bifidobacterium
on glucose after 24 h)
(A620 of pathogen on the fiber after
24 h / A620 of pathogen on glucose
after 24 h
Prebiotic
Score (PS)
−=

10. Stimulatory activity of commercial fibers : probiotic score (PS)
Enteric mixture
C. difficile
PS AVERAGE:
- Vivinal®=1.18
- Actilight®950P =1.39
- Orafti®HSI =1.52
PS AVERAGE:
- Vivinal®=1.23
- Actilight®950P =1.43
- Orafti®HSI =0.6
- BioEcolians® =↓
- Frutafit® =↓
- Arabinex® =↓
- Benefibra® =↓1.67

11. Potential applications of the bifidobacteria strains: anti-colics activity
SINGLE-STAGE CONTINUOUS CULTURES IN BENCH-TOP BIOREACTORS:
-Fresh feces from a colicky infant (breast-fed, natural delivered, not antibiotic/probiotic)
-Fecal cultures were performed in a Microbiota Medium (6g l-1
GOS/FOS 9:1)
-Inoculum preparation was performed in an anaerobic cabinet (85% N2, 10% CO2, 5% H2)
Assessment of fermentation products:
1.Microbial quantification (FISH and q-PCR)
2.Survival of the inoculated strain (RAPD-PCR)
Anti-colics effect of B. breve B632
against Enterobacteriaceae
PMC = Probiotic-
supplemented
microbiota cultures 107
CFU ml-1
MC = control microbiota
cultures. 5mL of fecal
suspension

12. T0 = B632 colonies were 85% of the
total bifidobacterial colonies
T6 = B632 colonies were 73%
T24 = B632 colonies were 64%
Anti-colics activity
B632 did not influence
the total bacteria
growth in PMC
Enterobacteriaceae
were affected in
PMC
Bifidobacteria resulted
more abundant in PMC
than MC
E. coli did not
decrease in PMC
FISH:
MC PMC
q-PCR:
RAPD-PCR

13. Potential applications of the bifidobacteria strains: anti-Streptococcus
Antimicrobial activity of the Bifidobacterium
spp. strains against Streptococcus agalactiae
Antagonistic strains:
-S. agalactiae DSM2134T
-3 S. agalactiae strains
(from vaginal swabs)
Antagonistic strains:
-S. agalactiae DSM2134T
-3 S. agalactiae strains
(from vaginal swabs)
Bifidobacterium
overnight-cultures
Inibition
evaluation
(halo’s radius)
SPOT AGAR TEST:
Radius measured ≥ 1cm
Radius measured ≤ 0.5cm

14. Conclusion
The first part of this work pointed out:
1. B. breve B632 strain is a good candidate for pediatric use;
2. The fructooligosaccharide (Actilight®950P) and the galactooligosaccharide (Vivinal®) are able
to stimulate selectively the growth of B. breve B632 and are appropriate for a synbiotic
product targeted to infants.
Next goal:
Validation in vivo of the effects of the selected probiotic (ON-GOING)
Preparation of the synbiotic product and validation in vivo

15. B. Achievements of new insights on early gut microbial colonization

16. Impact of intrapartum antibiotic prophylaxis (IAP) on the
newborn gut colonization: study design
Illumina MiSeq SystemTarget microorganisms:
•Total bacteria
•Bifidobacterium spp.
•Lactobacillus spp.
•Bacteroides fragilis
•Clostridium difficile
•Escherichia coli
Biodiversity:
•bifidobacteria
population
DNA processingCharacteristics:
•Control (not IAP
mothers)
•Treated (IAP
mothers)
•7 and 30 days old
•Breast/formula/
mixed feed
•Healty
•At term
•Vaginally delivered
•Not antibiotics
•Not pro/prebiotics
M

17. Infants at 7 days of life: microbial quantification by q-PCR
• Intrapartum antibiotic prophylaxis (IAP)
plays an important part in the
prevention of GBS infections
• Infant recruitment:
- 26 newborns born to GBS positive/IAP
treated mothers (IAP group)
- 26 newborns born to GBS negative
mothers (Control group)
Aloisio et al., 2014
Decrease of Bidobacterium after IAP: 7.29 Log (CFU/g) in
control samples vs.5.85 Log (CFU/g) in IAP samples (P<0.05)
No differences for Lactobacillus, E. coli, C. difficile,
Bacteroides fragilis
Great variability in microbial composition in newborns

18. Infants at 7 days of life: PCR-DGGE assay
PCR amplification:
•Bifidobacterium genus-specific PCR primers Bif164-F and
Bif662-R targeted on the 16S rRN
Denaturing gradient gel electrophoresis:
•DGGE gel 7% (w/v) polyacrylamide in 1X Tris-acetate-EDTA (TAE)
•Denaturing gradient of 50 to 55% urea-formamide
Reference
ladder
B. breve
B. bifidum
B. longum subsp. infantis /longum
B. pseudocatenulatum
DGGE analysis:
•Identification of unknown bands by comparing
the migrant distances;
•Identity was confirmed or detected by cutting
and sequencing of some of the bands
Exemplificative
fingerprinting
 Lower level of diversity in
IAP with respect to
Control

19. Infants at 7 days of life: PCR-DGGE assay
 B. pseudolongum, B. pseudocatenulatum,
B. longum were not influenced by IAP
 Strong decrement in frequency of B. breve
and B. bifidum (50% Control vs 25% IAP)
 Decrement in frequency of
B. dentium (38% Control vs 13% IAP)
IAP
- - - - -
Control

20. Infants at 7/30 days of life: q-PCR assay
Target microorganisms:
•Total bacteria
•Bacteroides fragilis
•Bifidobacterium spp.
•Lactobacillus spp.
Decrement in both BF-IAP and
MF-IAP infants compared to
Control infants
Increment in both BF-IAP and
MF-IAP infants

21. 16S rRNA sequencing of the variable V3 and V4 region
• Primers with overhang adapters
• Multiplexing indices
• Normalization and pooling
• Loading on the Illumina MiSeq platform
• Sequencing: recording the synthesis of DNA
strands in clusters of sample templates. Each
newly attached base liberates a fluorescent dye
that is excited by diode lasers (530 & 660 nm)
and imaged using two digital cameras.
16S Amplicon Sequencing Workflow
> 11 milion raw sequences
≈ 200,000 reads per sample

22. High-throughput pyrosequencing: results
 Significant lower diversity in BF-IAP infants compared with BF-C
 Lower diversity in BF-IAP infants compared with MF-IAP (not statistically significant)
Alpha diversity at 7 days
 Higher abundance of Enterobacteriaceae and lower
of Bifidobacteria in BF-IAP compared to BF-C
 Enterobacteriaceae continue to dominate in BF-IAP infants
compared to BF-C

23. Conclusion

24. General Conclusion
Main achievements:
 B. breve B632, as lyophilized strain or in a microencapsulated form, is a
good candidate for a probiotic product for the prevention or relieve of
infant disorders such as gas colics or streptoccoccal infections
 the FOS Actilight®950P and the GOS Vivinal® are capable of selectively
stimulating the probiotic B. breve B632 strain and are therefore suitable
ingredients for a synbiotic product targeted to infants
 Intrapartum antibiotic prophylaxis (IAP) has a negative impact toward the
early intestinal colonization, in particular towards Bifidobacterium spp.

25. Papers related to my Ph.D work:
1.M Simone, C Gozzoli, A Quartieri, G Mazzola, D Di Gioia, A Amaretti, S Raimondi, and M Rossi (2014) The probiotic
Bifidobacterium breve B632 inhibited the growth of Enterobacteriaceae within colicky infant microbiota cultures. BioMed
Research International Vol. 2014, Article ID 301053, 7 p.;
2.D Di Gioia, I Aloisio, G Mazzola, B Biavati (2014) Bifidobacteria: their impact on gut microbiota composition and their
applications as probiotics in infants. Applied Microbiology and Biotechnology 98:563–577;
3.I Aloisio, G Mazzola, L T Corvaglia, G Tonti, G Faldella, B Biavati, D Di Gioia (2014) Influence of intrapartum antibiotic prophylaxis
against group B Streptococcus on the early newborn gut composition and evaluation of the anti-Streptococcus activity of
Bifidobacterium strains. Applied Microbiology and Biotechnology 98:6051-6060;
4.G Mazzola, I Aloisio, D Di Gioia (2014) Infant development, currently the main applications of probiotics and prebiotics? In:
Venema K. and do CarmoA. P. (eds) Probiotics and Prebiotics: Current Research and Future Trends. Caister Academic Press,
Wageningen, chapter 26;
5.G Mazzola, I Aloisio, B Biavati, D Di Gioia / Development of a synbiotic product for newborns and infants / Submitted to Food
Science and Technology;
6.L Corvaglia, G Tonti, S Martini, A Aceti, G Mazzola, I Aloisio, D Di Gioia, G Faldella / Influence of intrapartum antibiotic
prophylaxis for Group B Streptococcus and type of feeding on gut microbiota during the first month of life / Under submission;
7.G Mazzola, K Murphy, R P Ross, D Di Gioia, L Corvaglia, G Faldella, C Stanton / Early gut microbiota perturbations following
intrapartum antibiotic prophylaxis to prevent group B streptococcal disease/ Under submission
Other papers:
1.G Mazzola, I Stefanini, I Nikodinoska, B Melero, J Rovira, T Langerholc, M Rossi, B Biavati, D Di Gioia / Use of lactic acid bacteria
as protective cultures in pork meat batter to prevent Clostridium spp. growth / Work in preparation;
2.G Totaro, L Paltrinieri, G Mazzola, M Vannini, La Sisti, C Gualandi, A Ballestrazzi, S Valeri, A Pollicino, A Celli, D Di Gioia, M L
Focarete / Electrospun fibers containing bio-based poly(ricinoleic acid): effect of ricinoleic acid units amount and distribution on
antibacterial properties / Work in preparation;
3.G Totaro, L Cruciani, M Vannini, G Mazzola, D Di Gioia, A Celli, L Sisti (2014) Synthesis of castoroil-deliveredpolyesters with
antimicrobial activity. European Polymer Journal 56:174-184
List of Pubblications

26. Acknowledgements