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New Potentially Probiotic Strains Isolated from Humans – Comparison of Properties with Strains from Probiotic Products and ATCC Collection

Anna Zawistowska-Rojek
Anna Zawistowska-Rojek
, 
Agnieszka Kociszewska
Agnieszka Kociszewska
, 
Tomasz Zaręba
Tomasz Zaręba
 and 
Stefan Tyski
Stefan Tyski
   | Sep 24, 2022
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Article Category: Original Paper
Published Online: Sep 24, 2022
Page range: 395 - 409
Received: May 12, 2022
Accepted: Jul 14, 2022
DOI: https://doi.org/10.33073/pjm-2022-035
© 2022 Anna Zawistowska-Rojek et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Fig.1

PFGE analysis of bacteria of Lactobacillus sp. The dendrogram shows the percentage similarity of PFGE profiles after DNA digestion with two enzymes: A – SmaI, B – ApaI.
PFGE analysis of bacteria of Lactobacillus sp. The dendrogram shows the percentage similarity of PFGE profiles after DNA digestion with two enzymes: A – SmaI, B – ApaI.

Fig. 2

PFGE analysis of bacteria of Lacticaseibacillus sp. The dendrogram shows the percentage similarity of PFGE profiles after DNA digestion with two enzymes: A – SmaI, B – ApaI.
PFGE analysis of bacteria of Lacticaseibacillus sp. The dendrogram shows the percentage similarity of PFGE profiles after DNA digestion with two enzymes: A – SmaI, B – ApaI.

Fig. 3

PFGE analysis of bacteria of Lactiplantibacillus sp. The dendrogram shows the percentage similarity of PFGE profiles after DNA digestion with two enzymes: A – SmaI, B –ApaI.
PFGE analysis of bacteria of Lactiplantibacillus sp. The dendrogram shows the percentage similarity of PFGE profiles after DNA digestion with two enzymes: A – SmaI, B –ApaI.

Fig. 4

A, B. Auto-antagonism of clinical Lactobacillaceae isolates towards Lactobacillaceae isolated from probiotic products. The mean growth inhibition zone (mm) ± standard deviation. The test was performed in triplicate. * – reference strains from ATCC collection: L. acidophilus ATCC® 4356™, L. rhamnosus GG ATCC® 53103™, L. plantarum ATCC® 14917™
A, B. Auto-antagonism of clinical Lactobacillaceae isolates towards Lactobacillaceae isolated from probiotic products. The mean growth inhibition zone (mm) ± standard deviation. The test was performed in triplicate. * – reference strains from ATCC collection: L. acidophilus ATCC® 4356™, L. rhamnosus GG ATCC® 53103™, L. plantarum ATCC® 14917™

Fig. 4

C, D. Auto-antagonism of clinical Lactobacillaceae isolates towards Lactobacillaceae isolated from probiotic products. The mean growth inhibition zone (mm) ± standard deviation. The test was performed in triplicate. * – reference strains from ATCC collection: L. acidophilus ATCC® 4356™, L. rhamnosus GG ATCC® 53103™, L. plantarum ATCC® 14917™
C, D. Auto-antagonism of clinical Lactobacillaceae isolates towards Lactobacillaceae isolated from probiotic products. The mean growth inhibition zone (mm) ± standard deviation. The test was performed in triplicate. * – reference strains from ATCC collection: L. acidophilus ATCC® 4356™, L. rhamnosus GG ATCC® 53103™, L. plantarum ATCC® 14917™

Antibiotic susceptibility of Lactobacillaceae strains.

Genus Species Strain Antibiotic MIC mg/l
VA E TET CHL AMP CC CTX CAZ CRO IMP
Lactobacillus L. acidophilus ATCC® 4356™ 0.5 0.5 4 4 < 0.25 1 < 0.5 < 0.5 4 4
L. acidophilus Probiotic 1 1 0.5 4 4 0.5 4 < 0.5 4 1 8
L. acidophilus Probiotic 2.1 0.5 0.5 2 4 < 0.25 4 < 0.5 1 1 1
L. acidophilus Probiotic 3 0.5 < 0.06 0.25 4 0.5 0.125 < 0.5 2 1 0.125
L. acidophilus Probiotic 4 0.5 0.5 0.5 4 < 0.25 2 < 0.5 1 8 2
L. acidophilus Probiotic 5 < 0.25 0.25 0.25 2 < 0.25 1 < 0.5 2 < 0.5 < 0.06
L. acidophilus Probiotic 6 1 0.5 1 4 0.5 4 < 0.5 1 2 4
L. acidophilus Probiotic 7.1 0.5 0.5 1 2 < 0.25 4 2 8 4 0.125
L. ultunensis Probiotic 8 0.5 0.5 2 4 < 0.25 4 4 4 8 2
L. acidophilus Clinical 1.1 1 0.25 2 2 < 0.25 4 < 0.5 1 < 0.5 0.125
L. acidophilus Clinical 2.1 1 0.5 0.5 2 1 < 0.06 1 128 2 1
L. acidophilus Clinical 14.1 1 0.5 2 4 < 0.25 1 < 0.5 2 < 0.5 0.5
L. acidophilus Clinical 16.2 1 1 4 4 1 1 1 8 2 8
L. acidophilus Clinical 18.1 1 1 2 4 1 2 < 0.5 8 1 8
L. acidophilus Clinical 20.3 0.5 1 4 4 0.5 0.5 0.5 8 2 1
L. gasseri Clinical 12.1 0.5 1 4 4 0.5 1 1 2 2 0.5
L. acidophilus Yoghurt 1.1 1 1 4 4 0.5 4 < 0.5 4 1 8
L. acidophilus Yoghurt 2.1 0.5 1 2 4 < 0.25 4 < 0.5 1 1 1
Lacticaseibacillus L. rhamnosus ATCC® 53103™ > 256 0.5 4 4 1 2 2 16 16 > 64
L. rhamnosus Probiotic 9 > 256 0.5 2 4 0.5 2 4 2 16 > 64
L. rhamnosus Probiotic 10 > 256 0.5 1 4 1 0.25 2 8 8 32
L. rhamnosus Probiotic 11 > 256 0.5 2 4 1 1 2 4 16 > 64
L. rhamnosus Probiotic 12 > 256 > 256 1 4 0.5 > 64 8 32 32 16
L. rhamnosus Probiotic 2.2 > 256 1 4 4 0.5 2 4 8 32 > 64
L. rhamnosus Probiotic 13 > 256 1 1 4 0.5 1 2 8 16 64
L. rhamnosus Probiotic 14 > 256 1 4 4 0.5 1 4 4 16 > 64
L. rhamnosus Clinical 3.1 > 256 1 2 4 1 1 4 8 32 > 64
L. rhamnosus Clinical 1645 > 256 1 4 4 0.5 2 2 16 16 > 64
L. rhamnosus Yoghurt 3 > 256 1 1 4 0.5 1 4 8 32 32
L. casei Probiotic 7.2 > 256 0.5 0.5 4 0.5 < 0.06 1 8 16 8
L. casei Yoghurt 2.2 > 256 0.5 1 4 0.5 0.125 1 8 16 8
L. casei Yoghurt 1.2 > 256 0.5 0.5 4 0.5 0.125 1 8 16 8
L. casei Yoghurt 4 > 256 0.5 1 4 0.5 0.125 1 8 16 16
L. casei Clinical 9.2 1 0.5 4 4 1 4 2 16 2 > 64
L. paracasei Clinical 4 4 16 4 2 0.5 4 < 0.5 2 1 > 64
L. paracasei Clinical 5.2 4 > 256 4 4 0.5 4 < 0.5 2 < 0.5 > 64
Lactiplantibacillus L. plantarum ATCC® 14917™ > 256 1 32 8 > 0.25 0.5 > 0.5 1 1 > 64
L. plantarum Probiotic 15 > 256 1 16 4 < 0.25 0.125 < 0.5 < 0.5 < 0.5 16
L. plantarum Probiotic 16 > 256 1 32 4 < 0.25 4 < 0.5 2 < 0.5 > 64
L. plantarum Clinical 1876 > 256 2 32 8 < 0.25 4 128 256 128 > 64
L. plantarum Clinical 1844 1 1 2 8 1 0.125 1 8 2 2
L. plantarum Clinical 5.1 > 256 1 16 8 < 0.25 < 0.06 2 2 2 2

Pulsed field gel electrophoresis (PFGE) parameters.

Genus Time Temperature Voltage Pulse length
Lactobacillus 22 h 14°C 6 V 1–6 s
Lacticaseibacillus 22 h 14°C 5 V 1–15 s
Lactiplantibacillus 20.5 h 14°C 5.5 V 1–10 s

Survival in a low pH, in presence of bile salt, and auto-aggregation Lactobacillaceae strains.

Genus Species Strain Survivability in an acid environment Growth inhibition coefficient Auto-aggregation
Control pH 2.0 pH 3.0 0.3% bile salts 24 h
1 h 2 h 2 h
log CFU/ml log CFU/ml % log CFU/ml % log CFU/ml %
Lactobacillus L. acidophilus ATCC® 4356™ 7.05 ± 0.1 0.59 ± 0.11 < 0.01 no growth nd 6.57 ± 1.00 33.6ab 0.28 ± 0.09abcd 67.85% ± 4.70cdefg
L. acidophilus Probiotic 1 6.40 ± 0.52 4.44 ± 1.75 6.4 2.95 ± 2.62 0.6 6.24 ± 0.54 69.5cd 0.20 ± 0.04bcdef 59.21% ± 1.83abc
L. acidophilus Probiotic 2.1 6.18 ± 0.40 5.20 ± 0.78 13.9 1.97 ± 2.31 0.4 6.09 ± 0.36 81.9cde 0.22 ± 0.03bcdef 79.05% ± 6.48h
L. acidophilus Probiotic 3 8.03 ± 0.35 3.70 ± 0.33 < 0.01 0.79 ± 1.36 < 0.01 7.96 ± 0.31 86.4cde 0.32 ± 0.07abc 49.73% ± 3.38a
L. acidophilus Probiotic 4 6.39 ± 1.19 2.24 ± 1.27 0.3 0.67 ± 0.65 < 0.01 6.33 ± 1.19 87.4de 0.41 ± 0.09a 60.95% ± 3.36bcd
L. acidophilus Probiotic 5 6.15 ± 1.12 4.36 ± 1.11 2.6 2.56 ± 0.90 0.1 6.01 ± 1.09 72.7cd 0.16 ± 0.07cdef 65.58% ± 0.89cdef
L. acidophilus Probiotic 6 7.05 ± 1.56 3.77 ± 1.55 13.3 1.04 ± 0.92 < 0.01 7.03 ± 1.53 95.9e 0.28 ± 0.05abcd 82.78% ± 0.72j
L. acidophilus Probiotic 7.1 6.16 ± 0.71 1.61 ± 0.26 < 0.01 1.18 ± 0.14 < 0.01 6.04 ± 0.72 75.9cd 0.37 ± 0.01ab 72.73% ± 3.92efgh
L. ultunensis Probiotic 8 7.04 ± 0.15 4.38 ± 0.78 0.6 0.39 ± 0.26 < 0.01 6.94 ± 0.16 80.4cde 0.25 ± 0.01abcde 75.04% ± 1.29fgh
L. acidophilus Clinical 1.1 8.11 ± 0.16 1.31 ± 1.87 < 0.01 no growth nd 7.99 ± 0.20 77.0cde 0.06 ± 0.03f 63.56% ± 5.25cde
L. acidophilus Clinical 2.1 7.83 ± 0.05 0.06 ± 0.10 < 0.01 0.77 ± 0.008 < 0.01 7.75 ± 0.04 82.7cde 0.06 ± 0.02f 71.04% ± 1.96defgh
L. acidophilus Clinical 14.1 7.41 ± 0.03 2.91 ± 0.60 < 0.01 no growth nd 7.29 ± 0.01 75.3cd 0.12 ± 0.05def 52.34% ± 1.61ab
L. acidophilus Clinical 16.2 6.01 ± 0.97 3.34 ± 1.03 0.6 1.00 ± 1.73 0.1 5.90 ± 0.99 76.9cde 0.14 ± 0.08cdef 67.73% ± 3.39cdefg
L. acidophilus Clinical 18.1 6.94 ± 0.49 0.53 ± 0.50 < 0.01 0.76 ± 0.85 < 0.01 6.62 ± 0.55 47.6b 0.08 ± 0.07ef 80.77% ± 1.24i
L. acidophilus Clinical 20.3 7.21 ± 0.82 3.89 ± 1.80 1.6 no growth nd 6.61 ± 0.65 26.3a 0.18 ± 0.09cdef 69.11% ± 3.12cdefgh
L. gasseri Clinical 12.1 7.02 ± 0.20 5.53 ± 0.50 3.8 5.09 ± 0.15 1.2 7.00 ± 0.16 96.4e 0.12 ± 0.10def 77.04% ± 6.28gh
L. acidophilus Yoghurt 1.1 7.37 ± 0.97 4.52 ± 1.42 13.4 1.77 ± 2.04 0.1 7.26 ± 1.01 78.7cde 0.07 ± 0.01ef 51.78% ± 2.40ab
L. acidophilus Yoghurt 2.1 6.24 ± 0.54 3.33 ± 2.49 3.8 1.46 ± 1.40 < 0.01 6.07 ± 0.51 67.4c 0.07 ± 0.02ef 85.17% ± 0.15k
Lacticaseibacillus L. rhamnosus ATCC® 53103™ 5.65 ± 0.34 No growth nd No growth nd 5.56 ± 0.30 81.3defgh 0.15 ± 0.05abcd 81.54% ± 6.25a
L. rhamnosus Probiotic 9 7.23 ± 0.33 1.91 ± 0.76 < 0.01 0.36 ± 0.62 < 0.01 7.20 ± 0.35 94.8h 0.17 ± 0.07abcd 86.93% ± 3.89a
L. rhamnosus Probiotic 10 7.76 ± 0.14 No growth nd No growth nd 7.64 ± 0.12 76.8defgh 0.33 ± 0.10def 87.31% ± 5.28a
L. rhamnosus Probiotic 11 7.21 ± 0.36 0.61 ± 0.81 < 0.01 No growth < 0.01 7.12 ± 0.37 81.7fgh 0.18 ± 0.07abcd 66.08% ± 1.36b
L. rhamnosus Probiotic 12 7.11 ± 0.58 0.77 ± 0.68 < 0.01 0.10 ± 0.17 < 0.01 7.06 ± 0.61 89.0fgh 0.22 ± 0.02abcd 47.92% ± 2.73def
L. rhamnosus Probiotic 2.2 7.84 ± 0.05 2.14 ± 0.59 < 0.01 0.10 ± 0.17 < 0.01 7.69 ± 0.03 70.9cdef 0.48 ± 0.07f 63.49% ± 2.05bc
L. rhamnosus Probiotic 13 7.94 ± 0.09 0.75 ± 0.65 < 0.01 No growth nd 7.81 ± 0.11 73.7defg 0.21 ± 0.01abcd 59.84% ± 2.50bc
L. rhamnosus Probiotic 14 7.70 ± 0.11 1.71 ± 0.60 < 0.01 0.42 ± 0.49 < 0.01 7.62 ± 0.10 83.1defgh 0.32 ± 0.09cdef 86.56% ± 1.58a
L. rhamnosus Clinical 3.1 7.48 ± 0.22 0.33 ± 0.58 < 0.01 No growth nd 7.44 ± 0.24 90.9defgh 0.21 ± 0.05abcd 64.70% ± 4.68b
L. rhamnosus Clinical 1645 7.44 ± 0.14 1.66 ± 0.03 < 0.01 0.85 ± 0.93 < 0.01 7.40 ± 0.16 89.9gh 0.24 ± 0.09bcde 57.14% ± 1.99bcd
L. rhamnosus Yoghurt 3 7.32 ± 0.06 2.77 ± 0.15 < 0.01 No growth nd 7.24 ± 0.10 83.4fgh 0.14 ± 0.01abc 89.36% ± 3.53g
L. casei Probiotic 7.2 7.73 ± 0.04 1.61 ± 1.46 < 0.01 0.18 ± 0.31 < 0.01 7.68 ± 0.05 88.0b 0.12 ± 0.08ab 47.29% ± 2.78def
L. casei Yoghurt 2.2 7.95 ± 0.20 1.05 ± 1.82 < 0.01 No growth nd 7.67 ± 0.17 52.7defgh 0.17 ± 0.04abcd 47.21% ± 1.25def
L. casei Yoghurt 1.2 7.97 ± 0.26 1.98 ± 1.85 < 0.01 0.57 ± 0.68 < 0.01 7.78 ± 0.25 64.8efgh 0.32 ± 0.08cdef 42.15% ± 4.76ef
L. casei Yoghurt 4 7.52 ± 0.61 1.09 ± 0.67 < 0.01 No growth < 0.01 7.45 ± 0.63 86.4bc 0.14 ± 0.03abcd 39.88% ± 3.88f
L. casei Clinical 9.2 7.27 ± 0.44 5.09 ± 1.37 2.1 1.77 ± 1.56 < 0.01 6.97 ± 0.38 50.4a 0.05 ± 0.06a 77.29% ± 3.43a
L. paracasei Clinical 4 7.42 ± 0.39 No growth nd No growth nd 5.08 ± 0.52 0.7bcde 0.41 ± 0.05ef 65.16% ± 2.57b
L. paracasei Clinical 5.2 8.12 ± 0.75 No growth nd No growth nd 7.95 ± 0.76 67.7bcd 0.28 ± 0.00bcde 52.85% ± 3.85cde
Lactiplantibacillus L. plantarum ATCC® 14917™ 6.96 ± 1.22 1.00 ± 1.73 < 0.01 No growth nd 6.91 ± 1.18 91.0a 0.14 ± 0.04a 59.80% ± 1.63a
L. plantarum Probiotic 15 6.79 ± 0.18 1.65 ± 0.13 < 0.01 0.26 ± 0.24 < 0.01 6.72 ± 0.14 85.7a 0.14 ± 0.02a 37.23% ± 1.75c
L. plantarum Probiotic 16 7.87 ± 0.30 0.38 ± 0.66 < 0.01 0.20 ± 0.35 < 0.01 7.85 ± 0.29 93.9a 0.24 ± 0.05a 52.77% ± 3.41ab
L. plantarum Clinical 1876 7.89 ± 0.31 2.17 ± 0.28 < 0.01 0.85 ± 1.47 < 0.01 7.84 ± 0.32 89.5a 0.13 ± 0.02a 57.17% ± 2.46ab
L. plantarum Clinical 1844 7.64 ± 0.36 2.67 ± 0.16 < 0.01 No growth nd 7.44 ± 0.40 62.1b 0.42 ± 0.05b 39.86% ± 5.04c
L. plantarum Clinical 5.1 7.62 ± 0.14 0.10 ± 0.17 < 0.01 0.10 ± 0.17 < 0.01 7.60 ± 0.15 95.2a 0.15 ± 0.08a 50.70% ± 2.16b

Lactobacillaceae strains derived from probiotic products.

Source of isolation Lactobacillaceae species declared by the manufacturer Lactobacillaceae species identified by API/MALDI-TOF MS Strain
Dietary supplement L. acidophilus L. acidophilus Probiotic 1
Dietary supplement L. acidophilusL. rhamnosusL. casei L. acidophilusL. rhamnosus Probiotic 2
Dietary supplement L. acidophilus L. acidophilus* Probiotic 3
Dietary supplement L. acidophilus L. acidophilus Probiotic 4
Food for special medical purposes L. acidophilusL. delbrueckii subsp.bulgaricus L. acidophilus Probiotic 5
Dietary supplement L. acidophilusL. paracasei L. acidophilus Probiotic 6
Dietary supplement L. caseiL. acidophilus L. caseiL. acidophilus Probiotic 7
Dietary supplement L. acidophilus L. ultunensis* Probiotic 8
Dietary supplement L. rhamnosus L. rhamnosus* Probiotic 9
Food for special medical purposes L. rhamnosus L. rhamnosus* Probiotic 10
Food for special medical purposes L. rhamnosus L. rhamnosus* Probiotic 11
Medical device L. rhamnosus L. rhamnosus Probiotic 12
Dietary supplement L. rhamnosus L. rhamnosus Probiotic 13
Dietary supplement L. rhamnosusL. plantarum L. rhamnosus Probiotic 14
Dietary supplement L. fermentumL. gasseriL. plantarumL. rhamnosus L. plantarum Probiotic 15
Dietary supplement L. plantarum L. plantarum* Probiotic 16
Yoghurt L. acidophilusL. casei L. acidophilusL. casei Yoghurt 1
Yoghurt L. acidophilusL. casei L. acidophilusL. casei Yoghurt 2
Yoghurt L. acidophilusL. paracasei L. rhamnosus Yoghurt 3
Yoghurt L. casei L. casei Yoghurt 4

Lactobacillaceae strains isolated from clinical material.

Source of isolation Lactobacillaceae strains identified by API/MALDI-TOF MS Strain
L. acidophilus* Clinical 1.1
L. acidophilus Clinical 2.1
L. acidophilus Clinical 14.1
Cervical swabs L. acidophilus Clinical 16.2
L. acidophilus Clinical 18.1
L. acidophilus Clinical 20.3
L. gasseri* Clinical 12.1
L. rhamnosus* Clinical 3.1
Rectal swabs L. rhamnosus* Clinical 1645
L. casei Clinical 9.2
Cervical swabs L. paracasei Clinical 4
L. paracasei Clinical 5.2
Rectal swabs L. plantarum* Clinical 1876
L. plantarum Clinical 1844
Cervical swabs L. plantarum* Clinical 5.1

Antagonism of Lactobacillaceae strains towards strains of pathogenic species (n = 132).
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