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Biologia

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Multifaceted toxin profile of Bacillus probiotic in newly isolated Bacillus spp. from soil rhizosphere

  • Milad Mohkam
  • Navid Nezafat
  • Aydin Berenjian
  • Mozhdeh Zamani
  • Fatemeh Dabbagh
  • Reyhaneh Bigharaz
  • Younes GhasemiEmail author
Original Article
  • 2 Downloads

Abstract

Probiotic bacteria mainly Lactic Acid Bacteria (LAB) demonstrate to equilibrate the intestinal microflora. Strains of the Bacillus species have been extensively used as probiotics for human beings, animal feed as well as plant promoting bacteria. Attempts were made to characterize and assess the safety of Bacillus strains for their putative virulence factors. The present study includes screening the isolates by hemolysis on blood agar, DNase, lecithinase and cytotoxic activity as well as detection of toxins in the culture medium. PCR technique was used to detect genes encoding enterotoxin T (bceT), cytotoxin K (CytK), enterotoxin FM (EntFM), non-hemolytic enterotoxin (NheA, NheB), haemolysin BL (HblA, HblC, HblD) and emetic toxin (ces). Among seven Bacillus isolates, only isolates B. cereus sp. M14, B. subtilis sp. M12 and B. methylotrophicus sp. M8 showed weak hemolysis on blood agar. DNase and lecithinase activity were not observed for all isolates. However, isolates B. subtilis sp. M12 and B. cereus sp. M14 indicated low cytotoxic effects on HepG2 cell line. Using two commercial immunoassay kits, no enterotoxin was detected for all isolates. Nhe and Hbl genes were detected in isolates B. methylotrophicus sp. M8 and B. cereus sp. M14, respectively. In contrast, none of the Bacillus isolates harbored bceT, entFM and ces genes in their chromosomes. Taking together, among the evaluated Bacillus isolates, strains B. pumilus sp. M17 and B. pumilus sp. S10 found to be the most promising candidates to fulfill the safety assessments of putative virulence factors suitable for human consumption.

Keywords

Bacillus Probiotics Enterotoxins Emetic toxin Safety assessment 

Abbreviations

LAB

Lactic Acid Bacteria

PCR

Polymerase Chain Reaction

bceT

enterotoxin T

CytK

cytotoxin K

EntFM

enterotoxin FM

Nhe

non-hemolytic enterotoxin

Hbl

haemolysin BL

ces

emetic toxin

GI

GastroIntestinal

rpoB

RNA polymeraseβ subunit

recA

Recombinase A

RPMI 1640

Roswell Park Memorial Institute 1640

BHI

Brain Heart Infusion

GRAS

generally recognized as safe

EFSA

European Food Safety Authority

WHO

World Health Organization

FAO

Food and Agriculture Organization

Notes

Acknowledgments

This work was supported by a grant from the Research Council of Shiraz University of Medical Sciences, Shiraz, Iran.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Human and animal rights

This manuscript does not contain any studies including human or animals participants done by any of the authors.

Supplementary material

11756_2019_357_Fig1_ESM.png (4.1 mb)
Fig. 1

PCR pattern of hblA gene toxin producing Bacillus isolates from rhizosphere environment. C1 and C2 are positive control of B. cereus PTCC 1047 and 1015, respectively. M is 100 bp DNA ladder. (PNG 4204 kb)

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High resolution image (TIF 1144 kb)
11756_2019_357_Fig2_ESM.png (3.9 mb)
Fig. 2

PCR pattern of hblC gene toxin producing Bacillus isolates from rhizosphere environment. C1 and C2 are positive control of B. cereus PTCC 1047 and 1015, respectively. M is 100 bp DNA ladder. (PNG 3985 kb)

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High resolution image (TIF 1091 kb)
11756_2019_357_Fig3_ESM.png (4 mb)
Fig. 3

PCR pattern of hblD gene toxin producing Bacillus isolates from rhizosphere environment. C1 and C2 are positive control of B. cereus PTCC 1047 and 1015, respectively. M is 100 bp DNA ladder. (PNG 4094 kb)

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High resolution image (TIF 1120 kb)
11756_2019_357_Fig4_ESM.png (4.2 mb)
Fig. 4

PCR pattern of nheA gene toxin producing Bacillus isolates from rhizosphere environment. C1 and C2 are positive control of B. cereus PTCC 1047 and 1015, respectively. M is 100 bp DNA ladder. (PNG 4291 kb)

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High resolution image (TIF 1178 kb)
11756_2019_357_Fig5_ESM.png (4 mb)
Fig. 5

PCR pattern of nheB gene toxin producing Bacillus isolates from rhizosphere environment. C1 and C2 are positive control of B. cereus PTCC 1047 and 1015, respectively. M is 100 bp DNA ladder. (PNG 4108 kb)

11756_2019_357_MOESM5_ESM.tif (1.1 mb)
High resolution image (TIF 1135 kb)
11756_2019_357_Fig6_ESM.png (4.2 mb)
Fig. 6

PCR pattern of cytK gene toxin producing Bacillus isolates from rhizosphere environment. C1 and C2 are positive control of B. cereus PTCC 1047 and 1015, respectively. M is 100 bp DNA ladder. (PNG 4281 kb)

11756_2019_357_MOESM6_ESM.tif (1.1 mb)
High resolution image (TIF 1154 kb)
11756_2019_357_Fig7_ESM.png (4 mb)
Fig. 7

PCR pattern of ces gene toxin producing Bacillus isolates from rhizosphere environment. C1 and C2 are positive control of B. cereus PTCC 1047 and 1015, respectively. M is 100 bp DNA ladder. (PNG 4141 kb)

11756_2019_357_MOESM7_ESM.tif (1.1 mb)
High resolution image (TIF 1127 kb)
11756_2019_357_Fig8_ESM.png (1.7 mb)
Fig. 8

PCR pattern of bceT gene toxin producing Bacillus isolates from rhizosphere environment. C1 and C2 are positive control of B. cereus PTCC 1047 and 1015, respectively. M is 100 bp DNA ladder. (PNG 1781 kb)

11756_2019_357_MOESM8_ESM.tif (1.9 mb)
High resolution image (TIF 1944 kb)
11756_2019_357_Fig9_ESM.png (1 mb)
Fig. 9

PCR pattern of entMF gene toxin producing Bacillus isolates from rhizosphere environment. C1 and C2 are positive control of B. cereus PTCC 1047 and 1015, respectively. M is 100 bp DNA ladder. (PNG 1042 kb)

11756_2019_357_MOESM9_ESM.tif (1.7 mb)
High resolution image (TIF 1694 kb)

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Copyright information

© Institute of Molecular Biology, Slovak Academy of Sciences 2019

Authors and Affiliations

  • Milad Mohkam
    • 1
    • 2
  • Navid Nezafat
    • 1
    • 2
    • 3
  • Aydin Berenjian
    • 4
  • Mozhdeh Zamani
    • 5
  • Fatemeh Dabbagh
    • 2
    • 3
    • 6
  • Reyhaneh Bigharaz
    • 1
  • Younes Ghasemi
    • 2
    • 3
    Email author
  1. 1.Biotechnology Research CenterShiraz University of Medical SciencesShirazIran
  2. 2.Pharmaceutical Sciences Research CenterShiraz University of Medical SciencesShirazIran
  3. 3.Department of Pharmaceutical Biotechnology, School of PharmacyShiraz University of Medical SciencesShirazIran
  4. 4.School of Engineering, Faculty of Science & EngineeringThe University of WaikatoHamiltonNew Zealand
  5. 5.Colorectal Research CenterShiraz University of Medical SciencesShirazIran
  6. 6.Faculty of Pharmacy and Pharmaceutical SciencesHormozgan University of Medical SciencesBandar AbbasIran

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