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

Zawistowska-Rojek, Anna; Kociszewska, Agnieszka; Zaręba, Tomasz; Tyski, Stefan

doi:10.33073/pjm-2022-035

Figures & Tables

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

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™

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
Genus	Species	Strain	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
			Control	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	33.6^ab	0.28 ± 0.09^abcd	67.85% ± 4.70^cdefg
	L. acidophilus	Probiotic 1	6.40 ± 0.52	4.44 ± 1.75	6.4	2.95 ± 2.62	0.6	6.24 ± 0.54	69.5^cd	0.20 ± 0.04^bcdef	59.21% ± 1.83^abc
	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	81.9^cde	0.22 ± 0.03^bcdef	79.05% ± 6.48^h
	L. acidophilus	Probiotic 3	8.03 ± 0.35	3.70 ± 0.33	< 0.01	0.79 ± 1.36	< 0.01	7.96 ± 0.31	86.4^cde	0.32 ± 0.07^abc	49.73% ± 3.38^a
	L. acidophilus	Probiotic 4	6.39 ± 1.19	2.24 ± 1.27	0.3	0.67 ± 0.65	< 0.01	6.33 ± 1.19	87.4^de	0.41 ± 0.09^a	60.95% ± 3.36^bcd
	L. acidophilus	Probiotic 5	6.15 ± 1.12	4.36 ± 1.11	2.6	2.56 ± 0.90	0.1	6.01 ± 1.09	72.7^cd	0.16 ± 0.07^cdef	65.58% ± 0.89^cdef
	L. acidophilus	Probiotic 6	7.05 ± 1.56	3.77 ± 1.55	13.3	1.04 ± 0.92	< 0.01	7.03 ± 1.53	95.9^e	0.28 ± 0.05^abcd	82.78% ± 0.72^j
	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	75.9^cd	0.37 ± 0.01^ab	72.73% ± 3.92^efgh
	L. ultunensis	Probiotic 8	7.04 ± 0.15	4.38 ± 0.78	0.6	0.39 ± 0.26	< 0.01	6.94 ± 0.16	80.4^cde	0.25 ± 0.01^abcde	75.04% ± 1.29^fgh
	L. acidophilus	Clinical 1.1	8.11 ± 0.16	1.31 ± 1.87	< 0.01	no growth	nd	7.99 ± 0.20	77.0^cde	0.06 ± 0.03^f	63.56% ± 5.25^cde
	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	82.7^cde	0.06 ± 0.02^f	71.04% ± 1.96^defgh
	L. acidophilus	Clinical 14.1	7.41 ± 0.03	2.91 ± 0.60	< 0.01	no growth	nd	7.29 ± 0.01	75.3^cd	0.12 ± 0.05^def	52.34% ± 1.61^ab
	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	76.9^cde	0.14 ± 0.08^cdef	67.73% ± 3.39^cdefg
	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	47.6^b	0.08 ± 0.07^ef	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	26.3^a	0.18 ± 0.09^cdef	69.11% ± 3.12^cdefgh
	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	96.4^e	0.12 ± 0.10^def	77.04% ± 6.28^gh
	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	78.7^cde	0.07 ± 0.01^ef	51.78% ± 2.40^ab
	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	67.4^c	0.07 ± 0.02^ef	85.17% ± 0.15^k
Lacticaseibacillus	*L. rhamnosus*	ATCC^® 53103™	5.65 ± 0.34	No growth	nd	No growth	nd	5.56 ± 0.30	81.3^defgh	0.15 ± 0.05^abcd	81.54% ± 6.25^a
	L. rhamnosus	Probiotic 9	7.23 ± 0.33	1.91 ± 0.76	< 0.01	0.36 ± 0.62	< 0.01	7.20 ± 0.35	94.8^h	0.17 ± 0.07^abcd	86.93% ± 3.89^a
	L. rhamnosus	Probiotic 10	7.76 ± 0.14	No growth	nd	No growth	nd	7.64 ± 0.12	76.8^defgh	0.33 ± 0.10^def	87.31% ± 5.28^a
	L. rhamnosus	Probiotic 11	7.21 ± 0.36	0.61 ± 0.81	< 0.01	No growth	< 0.01	7.12 ± 0.37	81.7^fgh	0.18 ± 0.07^abcd	66.08% ± 1.36^b
	L. rhamnosus	Probiotic 12	7.11 ± 0.58	0.77 ± 0.68	< 0.01	0.10 ± 0.17	< 0.01	7.06 ± 0.61	89.0^fgh	0.22 ± 0.02^abcd	47.92% ± 2.73^def
	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	70.9^cdef	0.48 ± 0.07^f	63.49% ± 2.05^bc
	L. rhamnosus	Probiotic 13	7.94 ± 0.09	0.75 ± 0.65	< 0.01	No growth	nd	7.81 ± 0.11	73.7^defg	0.21 ± 0.01^abcd	59.84% ± 2.50^bc
	L. rhamnosus	Probiotic 14	7.70 ± 0.11	1.71 ± 0.60	< 0.01	0.42 ± 0.49	< 0.01	7.62 ± 0.10	83.1^defgh	0.32 ± 0.09^cdef	86.56% ± 1.58^a
	L. rhamnosus	Clinical 3.1	7.48 ± 0.22	0.33 ± 0.58	< 0.01	No growth	nd	7.44 ± 0.24	90.9^defgh	0.21 ± 0.05^abcd	64.70% ± 4.68^b
	L. rhamnosus	Clinical 1645	7.44 ± 0.14	1.66 ± 0.03	< 0.01	0.85 ± 0.93	< 0.01	7.40 ± 0.16	89.9^gh	0.24 ± 0.09^bcde	57.14% ± 1.99^bcd
	L. rhamnosus	Yoghurt 3	7.32 ± 0.06	2.77 ± 0.15	< 0.01	No growth	nd	7.24 ± 0.10	83.4^fgh	0.14 ± 0.01^abc	89.36% ± 3.53^g
	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	88.0^b	0.12 ± 0.08^ab	47.29% ± 2.78^def
	L. casei	Yoghurt 2.2	7.95 ± 0.20	1.05 ± 1.82	< 0.01	No growth	nd	7.67 ± 0.17	52.7^defgh	0.17 ± 0.04^abcd	47.21% ± 1.25^def
	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	64.8^efgh	0.32 ± 0.08^cdef	42.15% ± 4.76^ef
	L. casei	Yoghurt 4	7.52 ± 0.61	1.09 ± 0.67	< 0.01	No growth	< 0.01	7.45 ± 0.63	86.4^bc	0.14 ± 0.03^abcd	39.88% ± 3.88^f
	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	50.4^a	0.05 ± 0.06^a	77.29% ± 3.43^a
	L. paracasei	Clinical 4	7.42 ± 0.39	No growth	nd	No growth	nd	5.08 ± 0.52	0.7^bcde	0.41 ± 0.05^ef	65.16% ± 2.57^b
	L. paracasei	Clinical 5.2	8.12 ± 0.75	No growth	nd	No growth	nd	7.95 ± 0.76	67.7^bcd	0.28 ± 0.00^bcde	52.85% ± 3.85^cde
Lactiplantibacillus	*L. plantarum*	ATCC® 14917™	6.96 ± 1.22	1.00 ± 1.73	< 0.01	No growth	nd	6.91 ± 1.18	91.0^a	0.14 ± 0.04^a	59.80% ± 1.63^a
	L. plantarum	Probiotic 15	6.79 ± 0.18	1.65 ± 0.13	< 0.01	0.26 ± 0.24	< 0.01	6.72 ± 0.14	85.7^a	0.14 ± 0.02^a	37.23% ± 1.75^c
	L. plantarum	Probiotic 16	7.87 ± 0.30	0.38 ± 0.66	< 0.01	0.20 ± 0.35	< 0.01	7.85 ± 0.29	93.9^a	0.24 ± 0.05^a	52.77% ± 3.41^ab
	L. plantarum	Clinical 1876	7.89 ± 0.31	2.17 ± 0.28	< 0.01	0.85 ± 1.47	< 0.01	7.84 ± 0.32	89.5^a	0.13 ± 0.02^a	57.17% ± 2.46^ab
	L. plantarum	Clinical 1844	7.64 ± 0.36	2.67 ± 0.16	< 0.01	No growth	nd	7.44 ± 0.40	62.1^b	0.42 ± 0.05^b	39.86% ± 5.04^c
	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	95.2^a	0.15 ± 0.08^a	50.70% ± 2.16^b

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

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

Figures & Tables

Fig.1

Fig. 2

Fig. 3

Fig. 4

Fig. 4

Antibiotic susceptibility of Lactobacillaceae strains_

Pulsed field gel electrophoresis (PFGE) parameters_

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

Lactobacillaceae strains derived from probiotic products_

Lactobacillaceae strains isolated from clinical material_

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

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