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In Vivo Efficacy of Bacillus velezensis Isolated from Korean Gochang Bokbunja Vinegar against Carbapenem-Resistant Klebsiella pneumoniae Infections

Fatemeh Ghorbanian
Fatemeh Ghorbanian
, 
Hoonhee Seo
Hoonhee Seo
, 
Hanieh Tajdozian
Hanieh Tajdozian
, 
Youngkyoung Lee
Youngkyoung Lee
, 
MD Abdur Rahim
MD Abdur Rahim
, 
Sukyung Kim
Sukyung Kim
, 
Il-Yun Jung
Il-Yun Jung
, 
Saebim Lee
Saebim Lee
 and 
Ho-Yeon Song
Ho-Yeon Song
   | Dec 12, 2022
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Article Category: Original Paper
Published Online: Dec 12, 2022
Page range: 553 - 562
Received: Sep 09, 2022
Accepted: Nov 03, 2022
DOI: https://doi.org/10.33073/pjm-2022-051
Keywords
© 2022 Fatemeh Ghorbanian et al. published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Fig. 1

Bacterial growth and pH measurement in food grade medium with time. The probiotic strain was inoculated and cultivated. Its A) growth and B) pH were checked every 2 h.
Bacterial growth and pH measurement in food grade medium with time. The probiotic strain was inoculated and cultivated. Its A) growth and B) pH were checked every 2 h.

Fig. 2

Effect of B. velezensis on a clinical isolate of CRKP in the preventive infection murine model. A) Body weight and B) illness severity were observed during the experimental period. Illness severity score was evaluated as (1 - healthy, 2 - minimally ill, 3 - moderately ill, 4 - severely ill, 5 - dead). C) Survival rate was observed during the entire experimental period. D) CRKP treated group (left image), CRKP + B. velezensis treated group (right image). Statistical significance with the control was analyzed using unpaired Student’s f-test (*** p<0.001; **p<0.01; * p<0.05).
Effect of B. velezensis on a clinical isolate of CRKP in the preventive infection murine model. A) Body weight and B) illness severity were observed during the experimental period. Illness severity score was evaluated as (1 - healthy, 2 - minimally ill, 3 - moderately ill, 4 - severely ill, 5 - dead). C) Survival rate was observed during the entire experimental period. D) CRKP treated group (left image), CRKP + B. velezensis treated group (right image). Statistical significance with the control was analyzed using unpaired Student’s f-test (*** p<0.001; **p<0.01; * p<0.05).

Fig. 3

Effect of B. velezensis on a clinical isolate of CRKP in a therapeutic infection murine model. A) Body weight, B) severity of illness, and C) survival rate were observed during the experimental period. Illness severity score was evaluated (1 - healthy, 2 - minimally ill, 3 - moderately ill, 4 - severely ill, 5 - dead). D) CRKP treated group (left image), and CRKP + B. velezensis treated group (right image). Statistical significance with the control was analyzed using unpaired Students f-test (*** p <0.001; ** p< 0.01; * p <0.05).
Effect of B. velezensis on a clinical isolate of CRKP in a therapeutic infection murine model. A) Body weight, B) severity of illness, and C) survival rate were observed during the experimental period. Illness severity score was evaluated (1 - healthy, 2 - minimally ill, 3 - moderately ill, 4 - severely ill, 5 - dead). D) CRKP treated group (left image), and CRKP + B. velezensis treated group (right image). Statistical significance with the control was analyzed using unpaired Students f-test (*** p <0.001; ** p< 0.01; * p <0.05).

Fig. 4

Two-week repeated oral dose toxicity test of B. velezensis using guinea pigs. The body weights of guinea pigs were measured on days 1, 3, 5, 7, 9, 10, and 14. The candidate probiotic was orally administered at 2 x 10s CFU/mice/day at 200 pi per animal once a day, every day, for 2 weeks.
Two-week repeated oral dose toxicity test of B. velezensis using guinea pigs. The body weights of guinea pigs were measured on days 1, 3, 5, 7, 9, 10, and 14. The candidate probiotic was orally administered at 2 x 10s CFU/mice/day at 200 pi per animal once a day, every day, for 2 weeks.

The ability of the candidate probiotic strain to dehydrate carbon sources.

No. Type of test Candidate strain Bacillus velezensis KCTC 13417 Bacillus amyloliquefaciens KCTC 3002 Bacillus licheniformis KCTC 1659
24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h
0 Control - - - - - - - -
1 Glycerol + + + + + - + +
2 Erythritol - - - - - - - -
3 D-arabinose - - - - - - - -
4 L-arabinose - + + + + + - +
5 D-ribose + + + + + + + +
6 D-xylose - + + + + + - -
7 L-xylose - - - - - - - -
8 D-adonite - - - - - - - -
9 Methyl-pD-xylopyranoside - - - - - - - -
10 D-galactose + + + + - - - -
11 D-glucose + + + + + - + -
12 D-fructose + + + + + - + -
13 D-mannose + + + + + - + -
14 L-sorbose - - - + - - - -
15 L-rhamnose + + + + - - - -
16 Dulcitol - - - - - - - -
17 Inocitol + + + + + + - -
18 D-mannitol + + + + + - + -
19 D-sorbito + + + + + - + +
20 Methyl-aD-mannopyranoside - - - - - - - -
21 Methyl-aD-glucopyranoside - + + + + + + +
22 N-acetylglucosamine + + - - + - + -
23 Amygdaline + + + + - + + -
24 Arbutine + + + + + - + -
25 Esculine + + + + + + + +
26 Salicin + + + - + + + -
27 D-Cellibiose + + + + + + + +
28 D-Maltose + + + + + + + +
29 D-Lactose + + - - + + + +
30 D-Melibiose - + - + + + - +
31 D-Sacharose + + + - + + + -
32 D-Trehalose + + + + + + - -
33 Inulin - - - + - - - -
34 D-Melezitose + - - - - - - -
35 D-Raffinose + + + + + + + +
36 Amidon + + + - + - + -
37 Glycogen - + + - + - + -
38 Xylitol - - - - - - - -
39 Gentibiose + - - - - - + -
40 D-Turanose - - + + - - + +
41 D-Lyxose - - - - - - - -
42 D-Tagatose + + + - - - - -
42 D-Fucose - - - - - - - -
44 L-Fucose - - - - - - - -
45 D-arabitol - - - - - - - -
46 L-arabitol - - - - - - - -
47 Potassium gluconate + - - - - - - -
48 Potassium 2 ketogluconate - - - - - - - -
49 Potassium 5 ketogluconate - - - - - - - -

Identification of isolated bacterial strain based on 16S rRNA gene sequence analysis.

NCBI references Organisms Length Score Identities Gaps
NR_075005.2 Bacillus velezensis strain FZB42 1,550 2,728 bits (1,477) 1,492/1,499 (99%) 2/1,499 (0%)
NR_116240.1 Bacillus velezensis strain CBMB205 1,445 2,615 bits (1,416) 1,427/1,432 (99%) 2/1,432 (0%)
NR_112685.1 Bacillus amyloliquefaciens strain NBRC 15535 1,475 2,699 bits (1,461) 1,470/1,475 (99%) 0/1,475 (0%)
NR_041455.1 Bacillus amyloliquefaciens strain NBRC 15535 1,472 2,697 bits (1,460) 1,468/1,472 (99%) 0/1,472 (0%)
NR_102783.2 Bacillus subtilis subsp. subtilis strain 168 1,550 2712 bits (1,468) 1,489/1,499 (99%) 2/1,499 (0%)
NR_118996.1 Bacillus licheniformis strain DSM 13 1,545 2,579 bits (1,396) 1,466/1,500 (98%) 4/1,500 (0%)
NR_116023.1 Bacillus licheniformis strain BCRC 11702 1,468 2,545 bits (1,378) 1,439/1,469 (98%) 2/1,469 (0%)

Identification of the isolated bacterial strain by qRT-PCR.

Species-specific primer for- Oligo name Amplification results
Candidate probiotic strain B. velezensis KCTC 13417 B. atnyloliquefaciens KCTC 3002 B. subtilis KCTC 3135 B. lichenifortnis KCTC 1659
Bacillus velezensis Bvel + + - - -
Bacillus subtilis YtcP - - - + -
Bacillus atnyloliquefaciens spBamyphes + + + - -
Bacillus lichenifortnis Blich - - - - +
Macrolactin Min + + - - -
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