Phylogenetic analysis and possible practical

applications of potentially probiotic

Lactobacillus isolates By

Richard Nyanzi

Department of Biotechnology & Food Technology

Motivation

Study objectives

Materials and methods

Results and Discussion

Conclusions

Presentation outline

Probiotics offer several potential health benefits (Nyanzi & Jooste, 2012)

Lactobacillus and Bifidobacterium are the most used genera (Bendali et al., 2011)

Previously optimised processing conditions for probiotic mageu (Nyanzi, 2007)

Excluded was precise identification, characterisation and property assessment

Health benefits are probiotic strain-specific (Vinderola & Reinheimer, 2003)

Ultimate goal: Affordable synbiotic cereal based beverage

Cereals largely consumed and suitable for the lactose intolerant (Hughson, 1995)

Masses would derive potential health benefits

Focus: Oral thrush (candidiasis) in immuno-compromised individuals

Motivation

Motivation cont’d

• Oral thrush is caused by Candida albicans in the buccal cavity

• Oral thrush is regular in the immuno-compromised

• Oral thrush: ‘disease of the diseased’ (Davies et al., 2008)

• Probiotics can be antagonistic against Candida albicans

• Synbiotic mageu beverage may ameliorate oral thrush

To investigate superiority of rpoA and pheS over 16S rRNA gene sequencing

during lactobacilli identification and phylogenetic analysis

To characterise and assess properties of potential probiotic strains

To investigate in-situ probiotic inhibition against Candida albicans growth in a

cereal medium

To determine antimicrobial and antioxidant activity of extracts from freeze-

dried probiotic cells and elucidate constituent compounds

Objectives of the study

Materials and methods

Food products (supermarkets) and probiotic supplements (pharmacies)

Isolation

Purification and preservation (Nyanzi, 2007)

Catalase, oxidase and Gram’s stain

Fructose-6-phosphate phosphoketolase activity (Orban & Patterson, 2000)

DNA isolation (Qiagen Kit)

DNA concentration (Nano drop)

Primers (Nasser et al., 2005)

(P T O)

Materials and methods Cont’d

DNA amplification (PCR thermocycler) (Nasser et al., 2005)

Sequencing (Genetic analyser ABI PRISM3100)

Sequence editing

Initial identification (NCBI: blastn search option)

Phylogenetic analysis (MEGA 5.05)

Concatenation of protein-coding genes (mafft ver. 6.857b)

Estimation of evolutionary divergence and Tajima’s neutrality test

Split decomposition analysis (SplitsTree4 v. 4.12.3 )

Assessing characteristics and properties of probiotic isolates

Probiotic isolates

Antimicrobial activity characterisation

Antibacterial activity Anti-Candida activity

Suitability of Media • Mueller Hinton agar (MHA) • Modified MHA (2% w/v dext.) • Antibiotic medium (AM) • Modified AM (2% w/v dext.) Supernatant treatments • pH unadjusted • pH neutralised • pH neutralised and filtered • MRS broth Used Agar disc diffusion method

Soft agar overlay method

Modifieddeferred cross streak method

Modified agar overlay method

Probiotic inhibitory activity against Candida strains in maize gruel (MG)

Characteristics • Acid tolerance • Bile resistance • Antibiotic resistance

Materials and methods Cont’d

Modified agar overlay method

20 mL MRS agar petri-dishes were prepared

Swab streaked probiotic strain (circle diameter of 3 cm)

Incubated anaerobically at 37 °C for 48 hours (Confluent growth)

Petri dish overlaid with 5 mL PDA tempered at 50 °C, left to solidify

Inoculated 100 µL of Candida culture: used spread plate technique

Plates kept at 4 °C for 2 hours, incubated at 37 °C for 24 hours

Petri dishes left to stand for 24 hours at room temperature

Inhibitory activity evaluation scale

- No inhibition: pathogen growth was confluent over test organism

± Doubted inhibition: Pathogen growth not confluent over test organism

+ Inhibition: no zone but no growth over test organism

++ Inhibition: Zone of inhibition with sparse pathogen colonies

+++ Total inhibition: Clear zone of inhibition without pathogen colonies

Maize gruel (MG) preparation:

Maize flour 60 g/L,

dextrose + sucrose (1:1) [2% (w/v)],

boiled for 20 minutes,

Autoclaved: 121 °C for 15 min

Treatment 1: Inoculated 100 mL of MG with

4% (v/v) 24 hour-broth probiotic culture.

Incubated at 37 °C in water bath up to 168 hours

Treatment 2:

Inoculated 100 mL of MG with

4% (v/v) 48 hour-broth Candida

culture.

Incubated at 37 °C in water bath up to

168 hours

Treatment 3:

Inoculated 100 mL of MG with

Candida + probiotic culture (1:1) 8% (v/v).

Incubated at 37 °C in water bath up to

168 hours

Fig.1: Maize gruel preparation, inoculation with microbial strains and periodic

enumeration of microbial cells

Det. pH (pH meter) and used pour plate

technique to enumerate microbial

colonies on MRS agar, PDA and RBC agar

incubated at 37 °C for 48 hours

Materials and methods Cont’d

Materials and methods cont’d

Mass probiotic cell cultivation and harvesting Ground the dried cell pellet

Inoculated MRS broth with 4% (v/v) broth culture, and analysed particle size

Incubated at 37 °C for 48 hours in a water bath

Centrifuged: Frozen at -80 °C for Freeze-dried the pellet Determined mass

6000 rpm, 10 min, 4 °C 24 hours of dried pellet

(Sorvall RC6)

Fig. 2: Mass probiotic cell cultivation, harvesting and extraction

Materials and methods cont’d

Probiotic powder sequential extraction with acetone and methanol

Ratio of powder : solvent = 1 g : 10 mL

Sonicated and Filtered

Fume cupboard drying and determination of extracted mass

Dissolved extract in 25% acetone solvent (Eloff, 1998)

Antimicrobial assays for minimum inhibitory concentrations (MICs)

Antibacterial assay (Eloff, 1998) Antifungal assay (Shai et al., 2009)

Antioxidant activity of extracts

Two-fold serial dilution of extracts and 3.7 mg/100 mL DPPH (Shikanga et al., 2010)

TLC fingerprinting: E: M: W = 10:1.35:0.5, UV light; Vanillin

Isolation of active compound (s)

3.3 Results and Discussion

Isolate

symbol

Identification claimed by supplier

or manufacturer

Strain

claimed

Identification based on API 50 CHL

biochemical results

16s rDNA sequence based

Identification (NCBI:blastn)a

U Lb. acidophilus LA5 Lb. acidophilus (99.6%) Lb. acidophilus (99%)

N Lb. acidophilus PRO Lb. acidophilus (68.6%) Lb. casei (99%)

V Lb. acidophilus NCFM Lb. acidophilus (98.4) Lb. acidophilus (99%)

Z Lb. acidophilus LAFTI L10 Lb. acidophilus (95.6%) Lb. helveticus (99%)

D Lb. acidophilus ATCC 4356 ND Lb. rhamnosus (99%)

M Lb. rhamnosus LRB Lb. rhamnosus (98.4%) Lb. rhamnosus (99%)

W Lb. rhamnosus GG Lb. rhamnosus (99.9%) Lb. acidophilus (99%)

Y Lb. rhamnosus AY675253 Lb. rhamnosus (99.9%) Lb. rhamnosus (99%)

C Lb. rhamnosus ATCC 7469 ND Lb. rhamnosus (99%)

O Lb. casei BGP93 Lb. paracasei ssp. paracasei (99.9%) Lb. rhamnosus (99%)

P Lb. casei Shirota Lb. paracasei ssp. paracasei (99.9%) Lb. casei (99%)

B Lb. casei LAFTI L26 Lb. paracasei ssp. paracasei (99.9%) Lb. casei (99%)

X Lb. reuteri - ND Lc. lactis subsp.lactis (99%)

Q Lb. paracasei BGP1 Lb. paracasei ssp. paracasei (99.9%) Lb. paracasei (99%)

R Lb. plantarum BG112 Lb. plantarum (99.9%) Lb. plantarum (99%)

S Lb. paracasei subsp. paracasei UFSBC510 Lb. paracasei ssp. paracasei (99.9%) Lb. casei (99%)

L Lb. delbrueckii subsp.lactis C09 ND Lb. plantarum (99%)

T Lb. delbrueckii subsp.lactis LMG7942T ND Lb. delbrueckii subsp. lactis (99%)

A Lb. delbrueckii subsp. delbrueckii UFSBC317 ND Lb. plantarum (99%)

Table 1: Phenotypic and genotypic identification of isolates

Fig. 3: Phylogenetic tree constructed using neighbor-joining method basing on the 16S

rDNA sequences of isolates and reference strains

Lb. casei NM140-3 [HM218558] S Lb. casei NM107-1 [HM218461] Lb. casei MGB65-2 [HM218007] Lb. paracasei subsp. paracasei KLDS1.0407 [HM067019] Lb. casei MBSL [HM188411] B Lb. paracasei F08 [GQ202836] Lb. casei ATCC 334 [AY196975] Lb. casei NWL63 [HQ293686] Lb. paracasei subsp. paracasei HB2121 [HQ615880] N P Q

Lb. casei group

Lb. casei subsp. casei ATCC 393T [AY196978] Lb. paracasei ATCC 25302 [HQ423165] C D Lb. rhamnosus NBRC 3425 [AB626049] Lb. rhamnosus GG (ATCC 53103) [GU550100] Lb. rhamnosus KLDS [GU550102] M O Y

Lb. rhamnosus group

Lb. reuteri Probio-16 [GU292563] Lb. delbrueckii subsp. lactis DSM 20072T [FR683103] T Lb. delbrueckii subsp. lactis QU 41 [AB589341] Lb. delbrueckii subsp. delbrueckii NCIB 8130T [FR683100] Lb. delbrueckii subsp. bulgaricus ATCC 11842T [FR683102] Lb. delbrueckii subsp. bulgaricus IMAU40111 [FJ749383]

Lb. delbrueckii group

Lb. helveticus KLDS 1.0601 [EU419585] Z Lb. helveticus C09 [EU377824] Lb. helveticus DSM 20075T [FR683085] Lb. acidophilus ATCC 4356T [AF429493] Lb. acidophilus VPI 6032T [FR683087] Lb. acidophilus CECT 4529 [FJ556999] Lb. acidophilus JCM 1132T [HM162411] U V W

Lb. acidophilus group

A L Lb. plantarum CAG20 [AB572045] Lb. plantarum CAG23 [AB572048] R

Lb. plantarum group

Lc. lactis subsp. lactis CAG18a [AB572041] X Lc. lactis group

Out group S. aureus RF122 [NC 007622]100

100

86

95

95

99

87

100

100

62

65

100

64

63

Fig. 4: Linearized concatenated rpoA and pheS gene phylogenetic tree constructed

using neighbor-joining method, p-distance model and the bootstrap type of phylogeny

Lb. rhamnosus YIT 0105 ( ATCC 7469) Lb. rhamnosus DSM 20021 D Y Lb. rhamnosus NBRC 3425 Lb. rhamnosus GG

M O

Lb. rhamnosus group

Q B N P S Lb. casei ATCC 334 Lb. paracasei ATCC 25302

Lb. casei group

A L R Lb. plantarum CAG20

Lb. plantarum group

T Lb. delbrueckii subsp. lactis DSM 20072T Lb. delbrueckii group

X Lacto. lactis subsp. lactis YIT 2008 ( ATCC 19435) Lc. lactis group

Lb. acidophilus ATCC 4356 U

V Z Lb. helveticus DSM 20075T

Lb. acidophilus group

Out group Pseudo. fragi JCM 5400

100

100

100

100

100

100

99

48

29

38

99

97

97

69

100

81

100

100

0.05

Gene Final

data

set

size

(bp)

Mean

Diverge-

nce

G + C

Content

(mol %)

Tajima’s Neutrality Test results

M S Ps Ѳ π D

16S# 883 0.0731 52.37 19 198 0.224236 0.064157 0.073070 0.58275

pheS 298 0.3031 47.31 19 220 0.738255 0.211225 0.313173 1.82603

rpoA 524 0.2502 43.70 17 332 0.633585 0.187412 0.250225 1.44548

C* 822 0.2720 45.03 17 552 0.671533 0.198636 0.272032 1.59743

Table 3: Evolutionary divergence estimates and application of Tajima’s neutrality

test in descriptive analysis of data used in MLSA

C*, concatenated chromosomal genes ; 16S#, 16S rRNA gene; m = number of sequences, S = Number of

segregating sites, Ps = S/m, Ѳ = Ps/a1, π = nucleotide diversity and D is the Tajima test statistic

16S rRNA gene

Fig. 5: Split graphs illustrating split decomposition analysis of gene sequences

rpoA gene 16S rRNA gene

phes gene Concatenated genes

Results: Modified agar over lay method Lb. acidophilus Lb. rhamnosus Lb. casei Bf. animalis (lactis)

Fig. 6: The population of Candida albicans strains (C1 – C7) in maize gruel incubated at

37 °C for 168 hours in the absence of probiotic strains

0.00E+00

5.00E+06

1.00E+07

1.50E+07

2.00E+07

2.50E+07

3.00E+07

0 20 40 60 80 100 120 140 160 180

Ca

nd

ida

co

un

t (c

fu/m

L)

Time of incubation (Hours)

Control

C1

C2

C3

C4

C5

C6

C7

Fig. 7: Effect of probiotic strain D on population of Candida albicans strains (C1 – C7)

in maize gruel incubated at 37 °C for 168 hours

0.00E+00

5.00E+05

1.00E+06

1.50E+06

2.00E+06

2.50E+06

3.00E+06

3.50E+06

0 20 40 60 80 100 120 140 160 180

Ca

nd

ida

co

un

t (c

fu/m

L)

Time of incubation (Hours)

MG + Lb. rhamnosus strain D

C1

C2

C3

C4

C5

C6

C7

Strains of Candida albicans

Median scores due to probiotic strains

P value

U N V Z D

C1 15 11 7 19 3 0.0683

C2 11 17 3 17 7 0.0815

C3 19 7 11 15 3 0.0683

C4 19 15 8 10 3 0.0780

C5 19 15 11 7 3 0.0683

C6 19 9 3 15 9 0.0815

C7 17 9 16.5 9 3 0.0960

Table 6: Results of Kruskal-Wallis equality-of-populations rank test on Candida

populations as influenced by probiotic bacteria strains U, N, V, Z and β after 120

hours of incubation at 37 °C

Fig. 8: Mean colony counts (n = 14) of Candida (irrespective of strain) in MG with or

without (CL = control) individual probiotic bacterial strains (U, N, V, Z & β) incubated

at 37 °C for up to 168 hours. The horizontal axis represents ‘Time of incubation

(hours)’.[NB. 1.85E+05 cfu/mL = 1.85 x 105 cfu/mL = 5.2672 log10 cfu/mL]

0 hour 48 hours 120 hours 168 hours

CL 1.85E+05 5.92E+06 1.22E+07 2.02E+07

U 1.85E+05 1.55E+06 8.14E+05 1.61E+06

N 1.85E+05 8.42E+05 1.15E+05 1.17E+06

V 1.85E+05 1.30E+06 1.32E+05 8.13E+05

Z 1.85E+05 5.13E+05 1.28E+05 1.43E+06

β 1.85E+05 5.76E+05 6.50E+04 1.30E+06

1.8

5E

+0

5 5.9

2E

+0

6 1

.22

E+

07

2.0

2E

+0

7

1.8

5E

+0

5

1.5

5E

+0

6

8.1

4E

+0

5

1.6

1E

+0

6

1.8

5E

+0

5

8.4

2E

+0

5

1.1

5E

+0

5

1.1

7E

+0

6

1.8

5E

+0

5

1.3

0E

+0

6

1.3

2E

+0

5

8.1

3E

+0

5

1.8

5E

+0

5

5.1

3E

+0

5

1.2

8E

+0

5

1.4

3E

+0

6

1.8

5E

+0

5

5.7

6E

+0

5

6.5

0E

+0

4

1.3

0E

+0

6

0.00E+00

5.00E+06

1.00E+07

1.50E+07

2.00E+07

2.50E+07

Ca

nd

ida

co

un

t (c

fu/m

L)

Fig. 9: pH of maize gruel containing Candida strains with or without (Control, CL)

probiotic strains (U, N, V, Z, β)

0

1

2

3

4

5

6

7

8

0 48 120 168

pH

of

maiz

e g

ruel (M

G)

Time of incubation (Hours)

CLU

N

V

Z

a a a a a a

b c c

d d

a

b

c c c c

a

b c c cd d

a

0

1

2

3

4

5

6

7

8

0 1 2 3 4 5 6 7 8 9 10 11 12

pH

Digits 1 - 5 & 7 - 11 represent probiotic strains U, N, V, Z & β in MG without (left-hand side) and with (right-hand side) Candida strains respectively

0 hours

48 hours

120 hours

168 hours

Fig. 10: pH of maize gruel containing probiotic strains with or without Candida strains at time intervals indicated in the legend.

Inhibitory activity of probiotic extracts against selected bacterial pathogens

Fig. 12: Minimum inhibitory concentration of extracts of strains of Lb. acidophilus

and Lb. rhamnosus after 24 hours of incubation at 37°C

E. Coli ATCC 8739 S. aureus ATCC 6358

Lb.a

cid

ophil

us

stra

ins

Lb.

rham

nosu

s st

rain

s

S. typhi ATCC 14028

Fig. 11: Fluorescing compounds in probiotic extracts

X X P P B B N N C

U V W Z M Y O D C

Fig. 12: Chromatogram of compounds in probiotic extracts

X X P P B B N N C

Table 7: MICs of probiotic extracts against bacterial pathogens

Test

organism

extract

MICs (mg/mL) against Indicator organism

E. coli ATCC S. aureus ATCC Sa. Typhi ATCC

24 hrs 48 hrs 60 hrs 24 hrs 48 hrs 60 hrs 24 hrs 48 hrs 60 hrs

U 5 5 7.5 2.5 5 10 5 5 5

V 5 7.5 10 2.5 5 10 5 5 10

W 10 10 20 2.5 10 10 5 10 15

Z 5 5 7.5 2.5 5 5 5 5 5

M 5 7.5 10 5 5 10 5 10 10

Y 2.5 5 5 2.5 5 5 2.5 5 7.5

O 5 5 7.5 5 5 10 5 7.5 10

D 2.5 5 7.5 1.25 5 5 2.5 5 5

C 5 5 5 2.5 5 5 5 7.5 10

X 20 >20 >20 10 20 >20 20 >20 >20

P 5 10 10 5 10 10 5 10 10

B 20 >20 >20 10 >20 >20 20 >20 >20

N 2.5 5 7.5 1.25 5 5 2.5 5 7.5

Table 8: Antioxidant activity (%) of selected probiotic strains’ extracts

20 10 5 2.5 1.25

Lb. acidophilus strains

U 84.5 ± 1.13 82.8 7 ± 1.37 56.62 ± 3.62 33.11 ± 2.48 18.99 ± 3.56 Y = 9.9446X + 7.235 4.3

V 86.07 ± 0.7 80.11 ± 1.52 57.02 ± 0.95 33.88 ± 2.71 21.91 ± 0.63 Y = 9.3474X + 10.34 4.24

W 81.98 ± 1.77 52.2 ± 1.53 30.32 ± 2.21 17.13 ± 0.43 11.4 ± 1.42 Y = 5.0783X + 4.805 8.9

Z 77.93 ± 1.81 71.92 ± 1.26 55.08 ± 3.75 36.96 ± 1.42 24.11 ± 3.99 Y = 8.114X + 15.05 4.31

Lb. rhamnosus strains

M 45.71 ± 3.97 44.35 ± 7.32 26.80 ± 2.99 10.65 ± 3.14 4.91 ± 1.63 Y = 5.9263X -3.165 8.97

Y 86.38 ± 0.54 63.07 ± 0.81 39.23 ± 2.3 21.32 ± 2.66 10.23 ± 1.1 Y = -7.652X+ 98.725 6.37

O 73.07 ± 1.4 47.76 ± 1.99 29.11 ± 1.28 13.13 ± 0.76 7.80 ± 3.25 Y = -5.784X + 100.19 8.7

D 66.87 ± 1.67 55.87 ± 0.77 38.36 ± 0.79 19.57 ± 0.47 11.29 ± 1.82 Y = -7.2611X + 98.11 6.63

C 79.86 ± 1.07 62.85 ± 1.11 37.51 ± 1.82 21.26 ± 1.71 14.84 ± 2.24 Y = -2.663X + 70.995 7.88

Lb. casei strains

N 45.8 ± 1.19 39.76 ± 2.87 26.64 ± 2.24 11.94 ± 1.23 6.91 ± 2.33 Y = -5.350X + 100.44 9.35

P 45.24 ± 0.82 41.64 ± 2.33 29.86 ± 1.22 15.84 ± 1.12 12.68 ± 0.98 Y = -4.72X + 94.33 9.39

B 36.92 ± 0.44 22.27 ± 1.04 10.49 ± 1.42 5.77 ± 1.51 2.63 ± 0.64 Y = -2.066X + 99.73 24.07

X 81.22 ± 2.25 70.47 ± 0.86 48.42 ± 1.01 28.25 ± 1.69 15.62 ± 2.95 Y = 8.6497X + 5.535 5.14

Control (Ascorbic acid) 90.99 ± 1.14 82.8 ± 0.55 73.46 ± 0.94 69.22 ± 0.61 50.13 ± 1.52 Y = 62.515X + 40.913 0.15

concentration of extracts (mg/mL) IC50 of extracts

(mg/mL)Linear equationSource of extracts

Lactic acid

Fig. 13: Constituents of probiotic cells and key HMBC (H C) correlations

6-O-(α-D-glucopyranosyl)-1,6-di-O-pentadecanoyl-α-D-glucopyranose

• 42% lactobacilli were incorrectly identified

• rpoA and pheS gene was 3- and 4-fold more discriminative

than 16S rRNA gene

• Concatenation of chromosomal genes improves discrimination

• Modified agar overlay method better showed anti-Candida

activity of probiotic strains

• In-situ probiotic inhibition of Candida was significant

• Probiotic extracts had antimicrobial and Antioxidant activity

• Possible use as biopreservatives and ant-oxidative stress agent

• Anti-Candida probiotic strains for synbiotic cereal beverage

Conclusions

TUT for the financial support

Staff of Biotechnology & Food Technology

Colleagues and friends

Dr M Cameron

Prof C Witthuhn

Prof S Combrink

Dr M D Awuoafack

Mr D S S Shuping

Mr F Els

Dr J Shai

Dr E A Shikanga

Dr C Zvinowanda

Dr J Wright

Dr M Kaggwa

Dr M Mujuru

Dr C Abolnik

Prof S C D Wright

Prof J N Eloff

Prof P J Jooste

Acknowledgement

Thank you