Phylogenetic analysis and possible practical applications ... · Modified agar overlay method 20 mL...
Transcript of Phylogenetic analysis and possible practical applications ... · Modified agar overlay method 20 mL...
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