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

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

Which factors determine the microbial composition of the human gut?

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

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

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 B632B. breve B2274B. breve B7840B. longum B1975

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

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

Stimulatory activity of commercial fibers

Escherichia coli ATCC 25645Klebsiella pneumoniae GC23aEnterobacter 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 -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)

−=

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

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

T0 = B632 colonies were 85% of the total bifidobacterial coloniesT6 = B632 colonies were 73%T24 = B632 colonies were 64%

Anti-colics activityB632 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

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

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

B. Achievements of new insights on early gut microbial colonization

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

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

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

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

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

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

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

Conclusion

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.

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

Acknowledgements